Adeeb Shehzad Editor

Cancer
Biomarkers in
Diagnosis and
Therapeutics
Cancer Biomarkers in Diagnosis
and Therapeutics
Adeeb Shehzad
Editor

Cancer Biomarkers
in Diagnosis
and Therapeutics
Editor
Adeeb Shehzad
Department of Biomedical Engineering
and Sciences, School of Mechanical and
Manufacturing Engineering (SMME)
National University of Sciences and
Technology
Islamabad, Pakistan

ISBN 978-981-16-5758-0 ISBN 978-981-16-5759-7 (eBook)

https://doi.org/10.1007/978-981-16-5759-7

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Preface

Despite the recent decline in cancer incidence rates, long-term mortality rates remain
unchanged. One of the most important factors for increased survival of cancer is
detection at an early stage. Clinical assays that detect the early events of cancer
through the use of molecular signatures or biomarkers offer an opportunity to
intervene and prevent cancer progression. Molecular signatures of the phenotype
of a cell that aids in early cancer detection and risk assessment will likely play an
important role in screening and early detection. Although new information and
technologies are clearly the driving force in biomarker discovery, translating new
findings into clinical application remains a major challenge.
Tumor development is a complex process requiring coordinated interactions
between numerous proteins, signaling pathways, and cell types. As a result of exten-
sive studies on the molecular pathogenesis of cancer, several novel regulatory
pathways and networks have been identified. The steps in these pathways have
delineated a number of unique events in cells, marked by morphological and histolog-
ical changes and altered expression of genes and proteins. During the transformation
of a normal cell into a cancer cell, the cell signature changes, and these changes
become unique signals of their presence and inherent features. By reading these
signals accurately, we can improve the early detection and diagnosis of individual
cancers. After decades of using basic research in an attempt to unravel the underlying
cellular and molecular mechanisms of cancer, the scientific community has uncovered
novel candidate targets for the early detection of cancer. By the time a tumor is
detected, several molecular changes have already occurred. Diagnostic assays to detect
these changes using BIOMARKERS have considerable potential for early detection.
Discovering cancer biomarkers is a relatively easy process based on the number
of papers published every year on this subject. However, translating these
discoveries into useful clinical assays will be very difficult. To date, fewer than 25
cancer biomarkers have been approved by the US Food and Drug Administration
(FDA) and most of these are for monitoring the response to therapy. In the field of
biomarkers, much of the biomarker research remains “stuck” at the discovery phase.
A number of explanations have been given for the lack of cancer biomarkers being
moved into clinical use. These include the high-performance standards needed to
make a biomarker clinically useful, the complex biology of tumors, a flawed
discovery process, lack of validation or a validation process that is cumbersome

v
vi Preface

and expensive, regulatory requirements, and an academic system that does not
reward translational research.
Biomarker research requires a knowledge-driven environment in which
investigators generate, contribute, manage, and analyze data available from a variety
of sources and technological platforms. The goal is a continuous feedback loop to
accelerate the translation of data into knowledge. Collaboration, data sharing, data
integration and standards are integral to achieving this goal. Only by seamlessly
structuring and integrating data sources will the complex and underlying causes and
outcomes of cancers be revealed, and effective prevention, early detection, and
personalized treatments be realized. There is a general consensus that if markers
from the early stages of the tumor may be identified, then treatment is likely to be
more successful.
Screening tools are needed that exhibit the combined requirements of high
SENSITIVITY and high SPECIFICITY for early-stage cancers which are widely
accepted, affordable, and safe. Significant improvement in our basic understanding
of the biology of cancer initiation and progression has shown that oncogenes and
tumor suppressor gene mutations can be identified in bodily fluids that drain from an
organ affected by the tumor. In this book, a number of chapters discuss the various
aspects of biomarkers, from discovery to development to validation to clinical utility
and clinical use. Clinical utility refers to the ability to make clinical decisions and
improve outcomes.
The chapters in the book are organized to shed lights on biomarker discovery,
development, and validation. We also highlight clinical needs in early detection, the
natural history of the disease and associated evidence in support of biomarker
research, an overview of the current state of the art in biomarker research, current
progress toward bringing these biomarkers into clinical use, and the future of
biomarker-based screening and early detection for the respective organ types. This
book also addresses the use of nanotechnology in the current screening strategies and
discusses deficiencies with the present practice and identifies clinical needs that may
benefit from biomarker-based approaches.
It is hoped that readers will appreciate the complexities of biomarker research,
especially for detection and screening, and be inspired or inspire others to take the
challenging tasks of biomarker discovery, development, and validation. In the era of
precision medicine, biomarkers have an important role to play by only not
identifying disease-specific molecular changes, but also in identifying targets for
precision treatments. Early detection and precision medicine are inseparable
approaches and will jointly inform the future course of actions in the clinical
management of diseases. The use of biomarkers (for instance in triaging patients
who are likely to benefit and who will not benefit from subsequent diagnostic
workups) will not only benefit clinical management but will also be an important
part of health economics in which the cost to the society and medical care will be
optimized, reduced, and improved.

Islamabad, Pakistan Adeeb Shehzad

Contents

1 Introduction to Cancer Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . 1

Musawira Iftikhar, Aroosa Younis, Young Sup Lee,
and Adeeb Shehzad
2 Technologies for Identification and Validation of Cancer
Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Aneela Javed, Hamza Sher, Zilli Huma, and Ishaq N. Khan
3 Biomarkers for Cancer Drug Development . . . . . . . . . . . . . . . . . . . 65
Gauhar Rehman
4 Clinical Proteomics: Diagnostics and Prognostic Markers
of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Saima Zafar, Aniqa Saeed, and Saadia Zahid
5 Microbiome as Cancer Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . 101
Bianza Moise Bakadia, Sehrish Manan, Mazhar Ul-Islam,
Biampata Mutu Mukole, Ajmal Shahzad, Ahmed M. E. Abdalla,
Muhammad Wajid Ullah, and Guang Yang
6 Predictive Biomarkers for Anticancer Drugs . . . . . . . . . . . . . . . . . . 149
Nosheen Fatima Rana and Tahreem Tanweer
7 Biomarkers in Cancer Survival and Drug Resistance . . . . . . . . . . . 177
Muhammad Ikram and Zia Uddin
8 Biomarkers in Tumor Recurrence and Metastasis . . . . . . . . . . . . . . 201
Dilawar Khan and Mudassir Khan
9 Biomarkers for Cancer Immunotherapy . . . . . . . . . . . . . . . . . . . . . 229
Haseeb Ahsan, Salman Ul Islam, Muhammad Bilal Ahmed,
Young Sup Lee, Mughal Qayum, and Jong Kyung Sonn
10 Role of Biomarkers in Personalized Medicine . . . . . . . . . . . . . . . . . 249
Salman Ul Islam, Muhammad Bilal Ahmed, Haseeb Ahsan,
and Young Sup Lee

vii
viii Contents

11 Development of Novel Cancer Biomarkers for Diagnosis and

Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Kholood Abid Janjua, Raheem Shahzad, and Adeeb Shehzad
12 Nanotechnology for Cancer Biomarkers . . . . . . . . . . . . . . . . . . . . . 345
Abdul Muhaymin, Uzma Azeem Awan, Adnan Haider,
and Muhammad Naeem
Editor and Contributors

About the Editor

Adeeb Shehzad is currently serving as Assistant Professor in the Department of

Biomedical Engineering and Sciences at the School of Mechanical and Manufacturing
Engineering, National University Sciences and Technology, Islamabad, Pakistan. He
completed his MS (2008–2010) and PhD (2010–2014) from the School of Life
Sciences at Kyungpook National University, Daegu, South Korea. He has earlier
served as a Research Professor in the School of Life Sciences at Kyungpook National
University, Daegu, South Korea (2014–2015) and Visiting Professor at Institute for
Research and Medical Consultations (IRMC), Imam Abdulrahman bin Faisal Univer-
sity (IAU), Dammam, Saudi Arabia (2019–2021). His research interest is mainly
oriented to the screening and development of plant-isolated compounds as anticancer
agents, as well as the in vitro and in vivo studies of synthetic drugs on modulation of
different signaling pathways in cancers. He has been conferred with various
scholarships, notably Kyungpook Honor Scholarship and Higher Education Commis-
sion awards for MS and PhD studies. He is also serving as a referee for well-known
international research journals. He has more than 10 years of teaching experience in
Cancer Biology, Pharmacology, and Biotechnology.
He has also published more than 70 research articles in peer-reviewed interna-
tional journals and authored or coauthored numerous book chapters. He is a member
of many international scientific societies and organizations, importantly Korean
Society of Molecular and Cellular Biology, South Korea, honorary member of
Drug Regulatory Authority of Pakistan (DRAP), and Pakistan Pharmacist Associa-
tion, Pakistan.

Contributors

Ahmed M. E. Abdalla Department of Biochemistry, College of Applied Science,

University of Bahri, Khartoum, Sudan
Muhammad Bilal Ahmed School of Life Sciences, BK21 FOUR KNU Creative
BioResearch Group, Kyungpook National University, Daegu, Korea

ix
x Editor and Contributors

Haseeb Ahsan School of Life Sciences, BK21 FOUR KNU Creative BioResearch
Group, Kyungpook National University, Daegu, Korea
Department of Pharmacy, Faculty of Life and Environmental Sciences, University of
Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan
Uzma Azeem Awan Department of Biological Sciences, National University of
Medical Sciences, Rawalpindi, Punjab, Pakistan
Bianza Moise Bakadia Department of Biomedical Engineering, College of Life
Science and Technology, Huazhong University of Science and Technology, Wuhan,
PR China
Adnan Haider Department of Biological Sciences, National University of Medical
Sciences, Rawalpindi, Punjab, Pakistan
Zilli Huma Neurooncology & Oncomedicines Research Group, Institute of Basic
Medical Sciences, Khyber Medical University, Peshawar, Pakistan
Musawira Iftikhar Advanced Membrane Technology Research Centre
(AMTECH), Faculty of Chemical and Energy Engineering, University of Tkenologi
Malaysia, Skudai, Johor, Malaysia
Muhammad Ikram Department of Pharmacy, COMSATS University Islamabad,
Abbottabad, KP, Pakistan
Salman Ul Islam School of Life Sciences, BK21 FOUR KNU Creative
BioResearch Group, Kyungpook National University, Daegu, Korea
Department of Pharmacy, Cecos University, Peshawar, Pakistan
Kholood Abid Janjua Department of Biomedical Engineering and Sciences,
School of Mechanical and Manufacturing Engineering (SMME), National Univer-
sity of Sciences and Technology, Islamabad, Pakistan
Aneela Javed Atta ur Rahman School of Applied Biosciences, National University
of Sciences and Technology, Islamabad, Pakistan
Neurooncology & Oncomedicines Research Group, Institute of Basic Medical
Sciences, Khyber Medical University, Peshawar, Pakistan
Dilawar Khan Department of Healthcare Biotechnology, Atta-ur-Rahman School
of Applied Biosciences (ASAB), National University of Sciences and Technology
(NUST), Islamabad, Pakistan
Ishaq N. Khan Neurooncology & Oncomedicines Research Group, Institute of
Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan
Cancer Cell Culture & Precision Oncomedicine Lab, Institute of Basic Medical
Sciences, Khyber Medical University, Peshawar, Pakistan
Mudassir Khan Department of Healthcare Biotechnology, Atta-ur-Rahman
School of Applied Biosciences (ASAB), National University of Sciences and Tech-
nology (NUST), Islamabad, Pakistan
Editor and Contributors xi

Young Sup Lee School of Life Sciences, Kyungpook National University, Daegu,
South Korea
School of Life Sciences, BK21 FOUR KNU Creative BioResearch Group,
Kyungpook National University, Daegu, Korea
Sehrish Manan Department of Biomedical Engineering, College of Life Science
and Technology, Huazhong University of Science and Technology, Wuhan, PR
China
Abdul Muhaymin CAS Key Laboratory for Biomedical Effects of Nanomaterials
and Nanosafety & CAS Center for Excellence in Nanoscience, National Center for
Nanoscience and Technology of Chin, Beijing, PR China
Biampata Mutu Mukole National Institute for Biomedical Research, Ministry of
Health, Kinshasa, Democratic Republic of the Congo
Muhammad Naeem Department of Biological Sciences, National University of
Medical Sciences, Rawalpindi, Punjab, Pakistan
Mughal Qayum Department of Pharmacy, Kohat University of Science & Tech-
nology, Kohat, Khyber Pakhtunkhwa, Pakistan
Nosheen Fatima Rana Department of Biomedical Engineering and Sciences,
School of Mechanical & Manufacturing Engineering, National University of
Sciences & Technology, Islamabad, Pakistan
Gauhar Rehman Department of Zoology, Abdul Wali Khan University Mardan,
Mardan, Pakistan
Aniqa Saeed Biomedical Engineering and Sciences Department, School of
Mechanical and Manufacturing Engineering (SMME), National University of
Sciences and Technology (NUST), Islamabad, Pakistan
Ajmal Shahzad Department of Biomedical Engineering, College of Life Science
and Technology, Huazhong University of Science and Technology, Wuhan, PR
China
Raheem Shahzad Department of Horticulture, University of Haripur, Pakistan
Adeeb Shehzad Department of Biomedical Engineering and Sciences, School of
Mechanical and Manufacturing Engineering (SMME), National University of
Sciences and Technology, Islamabad, Pakistan
Hamza Sher Atta ur Rahman School of Applied Biosciences, National University
of Sciences and Technology, Islamabad, Pakistan
Jong Kyung Sonn School of Life Sciences, BK21 FOUR KNU Creative
BioResearch Group, Kyungpook National University, Daegu, Korea
Tahreem Tanweer Department of Biomedical Engineering and Sciences, School
of Mechanical & Manufacturing Engineering, National University of Sciences &
Technology, Islamabad, Pakistan
xii Editor and Contributors

Zia Uddin Department of Pharmacy, COMSATS University Islamabad,

Abbottabad, KP, Pakistan
Mazhar Ul-Islam Department of Chemical Engineering, College of Engineering,
Dhofar University, Salalah, Oman
Muhammad Wajid Ullah Department of Biomedical Engineering, College of Life
Science and Technology, Huazhong University of Science and Technology, Wuhan,
PR China
Guang Yang Department of Biomedical Engineering, College of Life Science and
Technology, Huazhong University of Science and Technology, Wuhan, PR China
Aroosa Younis Department of Biomedical Engineering and Sciences, School of
Mechanical and Manufacturing Engineering (SMME), National University of
Sciences and Technology, Islamabad, Pakistan
Saima Zafar Biomedical Engineering and Sciences Department, School of
Mechanical and Manufacturing Engineering (SMME), National University of
Sciences and Technology (NUST), Islamabad, Pakistan
Saadia Zahid Department of Healthcare Biotechnology, Atta-ur-Rahman School
of Applied Biosciences (ASAB), National University of Sciences and Technology
(NUST), Islamabad, Pakistan
Introduction to Cancer Biomarkers
1
Musawira Iftikhar, Aroosa Younis, Young Sup Lee,
and Adeeb Shehzad

Abstract

The global increase in cancer rates and mortality warrants the identification of
biomarkers that are mechanism-and-disease-specific for the detection, diagnosis,
disease progression, and development of new regimes for treatment. Cancer
biomarkers are biologically active molecules including proteins (enzymes or
receptors), nucleic acids (coding and noncoding RNAs), immunoglobulins, or
shorter chains of amino acids or peptides. A biomarker can also be used for the
detection of modifications in gene expression or protein activity and epigenetic
changes or productions of stimuli-induced antibodies by either tumor or healthy
cells under normal or pathological conditions. These biomarkers carry a unique
and identifiable molecular structure, such as extent and activities of the genome,
polypeptides, or epigenetic alterations in circulatory fluids (whole blood, serum,
or plasma), excretory fluids (stool, urine, sputum, or milk), and tissues, providing
great potential for early diagnosis, monitoring, and selecting a suitable drug for
patients with cancer. This chapter underpins the recent findings of cancer
biomarkers concerning their expression pattern, molecular and biochemical char-
acterization, diagnostic and therapeutic utilization, and translation into the clinics
for the therapeutic intervention of patients with cancer. Several studies have

M. Iftikhar
Advanced Membrane Technology Research Centre (AMTECH), Faculty of Chemical and Energy
Engineering, University of Tkenologi Malaysia, Skudai, Johor, Malaysia
A. Younis · A. Shehzad (*)
Department of Biomedical Engineering and Sciences, School of Mechanical and Manufacturing
Engineering (SMME), National University of Sciences and Technology, Islamabad, Pakistan
e-mail: [email protected]
Y. S. Lee
School of Life Sciences, Kyungpook National University, Daegu, South Korea

# The Author(s), under exclusive license to Springer Nature Singapore Pte 1

Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_1
2 M. Iftikhar et al.

reported various prognostic and predictive cancer biomarkers, although few have
been commercialized. However, large multicenter validation studies are required
to elucidate their effectiveness and role in translation to the cancer clinics for
development into personalized medicines for the management of patients with
cancer. Finally, we discuss the potential role of nanotechnology in the develop-
ment and validation of future prospective cancer biomarkers. In this chapter, we
summarize the processes for discovery and development of important diagnostic
biomarkers with clinical utility, informing clinical monitoring to improve
outcomes of patients with cancer.

Keywords
Cancer · Biomarkers · Molecular markers · Prognosis · Diagnosis · Proteomics

1.1 Introduction

In recent years, great effort has been made in the early detection, typing, and staging
of cancer, which consequently has impacted patient management, duration, and
selection of treatment. Traditionally, Chinese, Ayurvedic, and Egyptians have used
a variety of substances to identify and differentiate between breast cancer and
mastitis as well as markers for severe and acute malignancies. The first fluid test
and examination were reported by Bence-Jones in 1847, wherein he identified a
cancer-associated immunoglobulin G (IgG) protein, which is light chain antibody
protein produced by the patients of multiple myeloma and can be detected in the
urine of patients due to the heat denaturation and coagulation properties of IgG. In
1986, Sinclair et al. also identified and confirmed that patients of multiple myeloma
have Bence-Jones protein in their sera (Dimopoulos et al. 2007). There is evidence
that the Federal Drug Authority (FDA) approved and validated the use of Bence-
Jones protein for the detection and confirmation of various cancers including
multiple myeloma and leukemia. In 1867, Michael Foster evaluated the pathology
and physiology of the pancreas and observed an increase in the levels of urinary
amylase in patients indicated that pancreatic cancer originated from the ductal cells
and not from the acinar cells (Shehzad et al. 2021). The use of polyclonal antibodies
by Yalow and Berson in 1950 allowed the use of cancer biomarkers and caused a
major shift in the diagnostic strategy and evaluation for precise cancers (Shehzad
et al. 2021). In 1965, Joseph Gold reported the use of carcinoembryonic antigen
(CEA) for the detection of colon cancer as it was usually only expressed in fetal
tissue (Poole 2016). In the early 1970s, a serum diagnostic test had been developed
and was commercially available for the detection of various cancers, such as the
CEA immunoassay additional biomarkers that were developed in 1975 were mono-
clonal antibodies and in 1982, CA 19-9, CA 15-3, and CA-125 were developed for
colorectal cancer (CRC), breast, and ovarian cancer, respectively. These markers are
used in routine practice as reliable indicators in patients with cancer (Hou et al.
2012). Moreover, many studies have successfully translated the use of cancer
1 Introduction to Cancer Biomarkers 3

biomarkers into clinical use. The electrophoretic peaks of urine and monoclonal
serum are used for myeloma detection, human chorionic gonadotropin and
α-fetoprotein is used for germ cell cancers, and alkaline phosphatase is used in
bone tumors and prostate-specific antigen for the detection of prostate cancer (Kalof
et al. 2010; Shehzad et al. 2018a, b). Despite these recent advances in molecular
techniques and the discovery of various cancer biomarkers, very few have been
approved by the FDA for diagnostic and therapeutic purposes in the clinic. High-
throughput genomic and proteomic studies are urgently required for the identifica-
tion and understanding of the putative functions of cancer biomarkers for their
diagnostic and therapeutic applications in the management of patients with cancer
(Shehzad et al. 2020).
There has been a dramatic increase in the incidence and mortality rates of cancer
in recent years, and cancer has been declared a major health problem. According to
the World Health Organization, cancer is expected to be the leading cause of death,
with an estimated 10 million deaths and 18.1 million incidences globally in 2020
(Ferlay et al. 2020; Shehzad et al. 2019). Extensive knowledge of the underlying
mechanisms of cancer development, its early detection and identification using
cancer biomarkers, suitable treatment with proper regimes, and patient monitoring
with follow-up measures could certainly decrease cancer incidence and burden
around the globe (Arbyn et al. 2020). The definition of biomarkers according to
the National Cancer Institute is a biological molecule or a substance that is present in
bodily fluid, tissues, or blood, which indicates a conventional, aberrant, or patho-
physiological process of any specific disease, such as cancer. Generally, biomarkers
can differentiate between healthy and infected tissue at the early stages of the disease
or can indicate the risk of developing, or the presence of, disease. The number of
biomarkers being discovered is growing continuously, and a wide range of
biomarkers can include polypeptides (enzyme or receptor), RNA (coding and
noncoding RNAs), antibodies, and peptides concentrations Shehzad (2021).
Biomarkers are present and detected in the bloodstream (whole blood, serum, or
plasma), secretary fluid (stool, urine, sputum, or milk), or tissue-derived, which need
special biochemical techniques for its evaluation, such as biopsy and special imaging
including mass spectroscopy of low-molecular-weight plasma peptides for surface-
enhanced laser desorption/ionization to characterize the ovarian tumor cells and can
be removed from the surrounding healthy cell populations (Kohler et al. 2011;
Shehzad et al. 2013d). Bioinformatics studies and peptide assays describe markers
that differentiate between tumor and nontumor patients, independent of the insight of
disease the patterns represent. Cancer biomarkers can be indicators of modifications
or alterations present in gene or protein expression, epigenetic changes, or
dysregulated metabolism. Some biomarkers may be inherited from ancestors, and
their mutation in sequence can be detected in both germline and tumor-derived DNA
(AlDubayan et al. 2018).
The term cancer biomarker refers to a biological substance that is produced in
response to an infection, which can be used as an indicator to differentiate between
the normal process or carcinogenic processes at different cellular stages; tumor-
associated antigens are one such biomarker that is a clinically significant marker for
4 M. Iftikhar et al.

the detection of tumor (Davis et al. 2019). Cancer biomarkers are efficient diagnostic
tools to calculate risk, screen, and detection of cancer, and they can differentiate
between benign and malignant tumors and predict and monitor the status and overall
outcome of therapeutic intervention and patient management in a clinical setting
(Kalinke et al. 2020; Shehzad et al. 2017). In line with this, many processes are
required for approval, validation, and translation of biomarkers into the clinical
setting for their use in the early detection and treatment of cancer. In this chapter,
we detail the various processes required for the identification and validation of
important diagnostic biomarkers that have clinical use and inform the clinical
monitoring for improved outcomes for patients with cancer.

1.1.1 Clinical Pathology of Cancer and Biomarkers

Cancer is a broad term that refers to a group of diseases identified by an abnormal

increase in the proliferation of cells. Cancer arises and progresses due to the
accumulation of genetic and epigenetic alterations in the cell, which ultimately
leads to disruption in the cell cycle and enhances the role of regulatory proteins
triggering the proliferation of cells and reducing the function of proteins that
typically prevent cell proliferation (Shehzad et al. 2016, 2017). Successive mutations
in oncogenes and truncation or deletion in the coding sequences of tumor suppressor
genes contribute to the formation of cancer. In particular, cancer is caused by two
types of genomic mutations: chromosomal instability (CIN) and microsatellite
instability (MIS) (Shehzad et al. 2013a). CIN is characterized by abnormal chromo-
somal segregation and accumulation of abnormal DNA content, whereas MIS results
in the loss of gene function for proteins that are normally involved in the repair of
DNA during DNA replication in dividing cells (Shehzad et al. 2013b). There is
compelling evidence that suggests that epigenetic changes that occur due to methyl-
ation and/or acetylation of the promoter genes, and histone modifications, can also
trigger the development of cancer through condensation and alteration of the chro-
matin (Shehzad et al. 2014a). Generally, tumors begin and extend with abnormal
proliferation of cells (hyperplasia) to form an uneven morphology and acquire an
adequate supply of nutrients and oxygen through the growth of new blood vessels
(angiogenesis). Consequently, it can reach secondary sites where it can develop new
tumors (metastasis). Cancer development is also mediated by the overexpression of
survival genes, growth factors, antiapoptotic genes, and those genes participating in
drug resistance (Shehzad et al. 2013c). Globally, cancer is a devastating public
health burden, and its early detection and identification can decrease its morbidity
and mortality rates. Genetic alterations of cancer cells and dysfunction of the signal
transduction networks of various proteins, enzymes, receptors, and growth factors
misregulate cellular processes and cell-to-cell communications, which can be
detected by the release or expression of biomarkers in several patients with cancer.
Therefore, a thorough understanding of these biomarkers may provide early assess-
ment in multiple cancers, including type and stage of cancer, the differentiation
between a benign and malignant tumor, determining prognosis and predictive factors
1 Introduction to Cancer Biomarkers 5

for patients with cancer, monitoring the recurrence and drug resistance, and response
to therapeutic interventions within clinical settings (Lánczky et al. 2016). A thor-
ough understanding of the underlying molecular mechanisms, cellular processes,
and dysregulated signaling networks of cancer tumors, as well as advancing high-
throughput and biochemical analysis may identify cancer biomarkers and determine
their clinical use in patients with cancer. The development of advanced assays is
required for the comparison of the therapeutic efficacy of biomarkers with cancer
pathology, followed by their association with other cellular markers for early
detection, disease nature, and therapeutic intervention for particular cancers. A
significant factor for oncology research is the development of precise and
standardized tools for measuring the type and stage of cancer against different
currently known biomarkers (Shehzad et al. 2014b). A deep understanding of normal
variations, preneoplastic status, grade of neoplasm, and stage will allow cancer
biomarkers to be useful for the treatment and prevention of cancer, as well as for
better management of patients with cancer.

1.2 Serum, Biological Fluid, and Tissue Cancer Biomarkers

The release of CB in tumors cells, blood cells, or other bodily fluids can allow the
understanding of the underlying mechanisms of carcinogenesis. CB molecules are
detected during the initiation, proliferation, progression, or metastasis of cancer. CB
molecules can be present in biological fluid as a result of three possible mechanisms:
epigenetic changes, amplification of a gene product, or overexpression of a gene. In
ovarian cancer, human epididymal secretory protein 4 (HE4) has been identified as a
biomarker that is overexpressed and can be detected in serum (Al-Amodi and Kamel
2016).
Understanding carcinogenic components can help elucidate the formation and
secretion of CB in blood, bodily fluids, and infected cells and understand their
progressive increase during malignant growth, progression, and metastasis. The
increase in CB expression levels in various samples can be identified in three
ways. First is the overexpression of the biomarker in the sample as a result of an
increase in the epigenetic modifications, such as methylation of DNA, which results
in the presence of CB, such as HE4 in ovarian malignancy. HE4 overexpression in
ovarian carcinoma can be easily identified in serum (Harmsma et al. 2013). HE4
overexpression is also present in endometrial, breast, and bronchial adenocarcinoma
(Harmsma et al. 2013; Mahmoud et al. 2014). The second indication is the increase
in serum biomarkers. One such example of a serum biomarker includes alpha-
fetoprotein (AFP), a single-peptide oncofetal protein, utilized for patients suffering
from hepatocellular carcinoma. Another serum biomarker is HER2-neu, a cell layer
surface-bound tyrosine kinase, which is increased in the serum of patients with
breast cancer, as a result of it undergoing proteolysis (Jain 2017). The FDA specifies
that HER2-neu should be evaluated for the presence of breast cancer metastasis. The
third indication evaluates the presence of invasive cancer cells and angiogenesis, and
prostate-specific antigen (PSA) can be utilized in this manner in patients with
6 M. Iftikhar et al.

prostate cancer (Osborne et al. 2011). An elevation in PSA levels can occur due to a
misshaped cellular layer of the prostatic cells and/or lymphangiogenesis (Shehzad
et al. 2013a). The clinical utilization of CB, particularly circling proteins, is
progressing due to an improvement in the “omics” discoveries of disease biomarkers
present in bodily fluids that are allowing the development of novel and sensitive
diagnostic tools for the early development of malignant growth. Of significance are
those malignant growths that are not currently effectively diagnosed, such as naso-
pharyngeal, ovarian, and pancreatic diseases (Thurnham and Northrop-Clewes
2016; Shehzad et al. 2013b). CB can be distinguishable among the malignant tissues,
cells in the lymph system, bone marrow, or circulating cancer cells (Comen et al.
2018). CB can be detected in natural bodily fluids including, serum, plain liquid,
pleural liquid, or urine, which are all noninvasive samples for analyzing. Cerebro-
spinal fluid is suitable for the cerebrum and CNS malignancy. Urine is among the
most promising samples for identifying bladder malignancy, as well as patient
observation (Dube et al. 2019). Likewise, prostate disease antigen 3 (PCA3) is
another promising biomarker for the diagnosis and development of malignancy in
the prostate (Grambergs et al. 2019). Stool samples are utilized for colorectal
malignancy, and areola suction liquid, ductal lavage, and pimple liquid are different
liquid types for breast cancer that can be utilized for evaluation of CB (Grambergs
et al. 2019).

1.3 Clinical Applications and Performance Indications

of Cancer Biomarkers

The clinical usefulness of CBs was previously limited as an effective tool for patient
diagnosis and treatment. The fifth International Conference arranged on the topic of
Human Tumor Markers clearly defined the definition of tumor markers (Gyani et al.
2014). In 1988, in Stockholm, Sweden, biochemical tumor markers were defined as
substances that were formed in cancer cells and discharged in body liquids where
their quantification can be obtained through nonobtrusive examinations. The corre-
lation between marker levels and dynamic tumor mass, and tumor markers are useful
for the management of malignancy patients (Rizza et al. 2018; Shehzad et al. 2012).
Markers, accessible in most disease cases, are a significant tool for the prediction of
patient outcomes, although they are by and by inappropriate for screening. Sero-
symptomatic estimations of markers should accentuate relative patterns rather than
be utilized as outright cutoff levels. However, CB can be utilized additionally for the
screening of all-inclusive communities, progression risk, differential determination,
and clinical arranging of patients with cancer (Abuawad et al. 2021). Moreover, CB
can be utilized to understand the tumor risk and indicate the progression and clinical
endpoint (Wiley 2020). Those biomarkers that are commonly used in clinical
practice include PSA, AFP, CA125, and CEA. Among all those serum biomarkers,
PSA is utilized to evaluate the risk and development of prostate disease. Disease
antigen CA-125 is a biomarker of ovarian malignancy, although it has low sensitiv-
ity and specificity. CEA is another biomarker that is elevated in patients with
1 Introduction to Cancer Biomarkers 7

colorectal, breast, lung, or pancreatic malignant growth (Garg et al. 2021). Other
promising biomarkers include survivin and HER2-neu (Lianidou et al. 2014). A
significant development would be to progress a reliable CB test that allows the
detection of patients with cancer and tailor treatment options based on their CB
expression levels to improve patient outcomes.

1.3.1 Sensitivity and Specificity for the Evaluation of the Accuracy

of CB

Tumors release CBs and can therefore be identified in body liquids, discharges, or
cancer tissues and cells (Garcia-Cao et al. 2012). CB can be identified in circulatory
fluids, such as blood, plasma, or serum, as well as in discharges, such as sputum,
urine, or CSF. Therefore, CB evaluation can be conducted in a noninvasive way. The
assessment of malignant growth biomarkers present in cells or tissues requires
different methods, such as tissue biopsy, which is a more intrusive method than
analysis of serum biomarkers. Hereditary biomarkers can be identified in the DNA of
tumor tissue, buccal mucosa cells, or blood (Joshi et al. 2019). Assessment of
the symptomatic estimation of any biomarker is typically performed concerning
the sensitivity and specificity of that particular biomarker. Specificity indicates the
ability of that biomarker to distinguish between healthy and diseased patients,
whereas sensitivity alludes to the capacity of that test to identify a particular
biomarker in those patients with cancer (Garcia-Cao et al. 2012). At complete
biomarker cutoff levels, the biomarker values may be over the positive cutoff
range, however not all of those patients who fall above the positive cutoff value
are diseased patients and are therefore known as false positives. Therefore, sensitiv-
ity is determined by the proportion of all positive cases over the positive cutoff value,
against the absolute number of genuine positive cases; sensitivity is the genuine
positive rate (TPR) (Aada and Tiwari 2019). Essentially, by applying a similar cutoff
level for similar tests, few people with ordinary outcomes underneath the cutoff
values are typical (genuine adverse), and not every one of them is diseased (false
antagonistic). Subsequently, the genuine negative rate or specificity can be identified
as the proportion of all negatives identified underneath the cutoff value compared
with the number of genuine negative patients (Black 2018). Assuming that the
sensitivity of a given CB is 100%, this implies that it will identify all disease patients,
and if another CB has 90% sensitivity, it will only be able to identify 90% of patients
with a malignant growth (genuine positives) but will miss 10% of malignant patients
(false negatives) (Wu 2020). Therefore, sensitivity and specificity could be applied
across all conceivable cutoff limits and both are equally identified with one another
(Naqi et al. 2019).
8 M. Iftikhar et al.

1.3.2 Receiver Operating Characteristic (ROC) Curve Examination

The investigation of various sensitivities and specificities at varying limits would be

useful to develop the precision of diagnostic tests. During World War II, the ROC
curve was presented by the British to identify exact radar indicators and later utilized
for assessing radiological tests (Bleibel et al. 2006). ROC curve can be utilized to
evaluate a biomarker by plotting its sensitivity along the Y-axis and its specificity or
false-positive rate (FPR) along the X-axis to evaluate its diagnostic ability in
separating healthy and unhealthy subjects (Duev et al. 2019). ROC curve has been
broadly utilized for evaluating the precision of a diagnostic test and can provide
important data on its use. Such data that the ROC curve provides includes the area
under the curve (AUC) and indicates the normal sensitivity levels for all conceivable
specificity values and includes the entire area under the whole ROC curve (Lin et al.
2018; Ponnibala et al. 2021). The AUC will have values in the range of one to zero as
the estimations of X and Y presumably also have values going from zero to one. The
closer the estimated value of AUC is to 1, then the better the clinical execution of that
test where it can distinguish between positive and negative cases (Zheng 2018). The
test with a greater AUC value is generally of better execution (Liu et al. 2014),
whereas comparing the two tests if both AUC regions are equivalent indicates the
same diagnostic ability of the two tests (Isomaa et al. 2013).

1.3.3 Ideal Biomarkers

Biomarkers that fall outside of the cutoff points will result in patients being identified
as a false-negative; however, this will diminish the number of false positives, which
indicates the specificity of the biomarker (Ceccarini et al. 2015). Likewise, if the
cutoff point is low, it will have good sensitivity but low specificity, as there are fewer
false-negative subjects but more false-positive subjects. In line with this, sets of
sensitivities and specificities may portray the preciseness of the biomarker and its
capacity to segregate between healthy and unhealthy (Ceccarini et al. 2015). If the
cutoff of the sensitivity is 100% or less, then we must consider the implications of
what that value means for the patient (Ray et al. 2010). The cutoff values will predict
whether that patient undergoes further examinations between tests or not (Ballman
2015). An ideal biomarker should be able to differentiate between tumor and benign
cases and aggressive tumors from nonaggressive ones, and it must be of high
specificity and sensitivity. Moreover, the isolation of that biomarker must be nonin-
vasive and economical (Hayes 2015). The ideal biomarker needs to require as little
hands-on approach as possible in its evaluation, and the CB needs to satisfy the
following listed general properties to be an ideal candidate. Currently, no biomarker
meets these prerequisites; however, these models should be considered for the
identification of a diagnostic biomarker (Andre et al. 2011).

• High clinical sensitivity: identified in all patients with a particular disease

(100% TPR).
1 Introduction to Cancer Biomarkers 9

• High clinical specificity: low false-negative rate (100% true negative).

• Organ- or tissue-specific.
• Proportionality to tumor weight or volume: quantitative proportionately to tumor
volume or illness movement.
• Shorter half-life: rapidly reflecting early changes in tumor progression for legiti-
mate observation of treatment.
• Present (assuming any) in the serum of healthy people and patients with infection
at low levels.
• Clear metastasis segregation.
• Exists in measures, such as quantitative, normalized, reproductive, and approved
measures.
• Inexpensive to obtain and analyze.
• Obtained in a noninvasive way—identified in serum, body fluids, or effectively
open tissue.

1.4 Clinical Uses and Limitations of Cancer Biomarkers

Tumor biomarkers or CB are usually either proteins or glycoproteins, which are

presumed not to be directly involved in carcinogenesis or advancement of the
disease but rather a by-product of the cellular dysregulation (Pan et al. 2015). Low
subatomic weight and small particles or markers of nucleic acids, lipid metabolites,
proteins, and peptides are all promising biomarkers and have recently been evaluated
for their use. The use of CB relies upon various elements including their synthetic
nature, proposed systems for their delivery, and applications. Six years ago, Mishra
and Varma proposed the characterization and use of CB in a clinical setting
(Kuspinar and Mayo 2014). They grouped CB into recognition biomarkers such as
atoms of RNA, diagnostic biomarkers such as proteins, and visualization biomarkers
such as glycol biomarkers. The clinical application of CB is for the early identifica-
tion, confirmation of the symptoms, evaluation of the predicted therapeutic response,
and disease monitoring (Kuspinar and Mayo 2014; Ward 2019). CBs can also be
used to indicate those patients at high risk of progression and those that would
benefit from early interventions (Ward et al. 2015). Biomarkers can indicate
aggressiveness, oxidative pressure, single-nucleotide polymorphisms, and other
mutations (Ward 2019). As confirmed by the FDA, CBs can have clinical signifi-
cance in a range of disease types.

1.4.1 Screening/Early Identification

Screening is characterized as the orderly utilization of a test to recognize those

subjects who are at adequate danger of developing a particular problem to profit by
additional examination or direct preventive activity, among people who have not
looked for clinical consideration by virtue of side effects of that issue (Lorincz
2016). In asymptomatic patients, earlier therapy interventions have a much more
10 M. Iftikhar et al.

favorable outcome compared with interventions at a later stage where the tumor is
progressing. Data suggests that there was a fall in the 5-year survival rate in patients
with breast cancer from ~90% in patients with early localized breast cancer to ~60%
in local metastasis, and 30% in distant metastasize (Zhao et al. 2018). Screening for
CB would allow the identification of malignant growth at the early or asymptomatic
stage and will result in an increment in the survival rate. A screening test should have
high specificity to limit the false positives as much as possible (Sicsic and Franc
2014; Shehzad et al. 2011). Having high specificity reduces the FPR and ultimately
reduces the need for unneeded and further intrusive diagnostic techniques. An ideal
screening program must be noninvasive, economical, and unquestionably leads to an
evident decrease in mortality rates and subsequently increases the survival rate. As a
rule, all screening programs are coordinated for malignant growths, and further
treatment and follow-up are required (Sicsic and Franc 2014; Zhao et al. 2018).
One restriction with screening for CBs is that they may not be elevated until later in
disease progression. However, there are not many biomarkers that are used as
screening biomarkers, such as AFP, which is used in the screening of hepatocellular
malignant growth in those patients who are considered high risk. PSA is used for
screening for the development of prostate disease, CA125 in detecting ovarian
malignant growth, and fecal occult blood testing (FOBT) for the screening of
colorectal tumors (CRC), and vanillyl mandelic corrosive (VMA) for the screening
of neuroblastoma in infants (Prensner et al. 2012). The approval of PSA as a
screening biomarker for prostate malignant growth has been informed by the
FDA; however, levels of PSA can increase in patients without cancer as a result of
benign prostatic hyperplasia and prostatitis, resulting in false positives (Prensner
et al. 2012). The use of PSA screening for reducing the mortality rate is controversial
(Amaro et al. 2014).

1.4.2 Identifying/Differential Determination

A diagnostic biomarker can be used for at risk patients as well as for those already
symptomatic. An ideal diagnostic biomarker must have similar attributes as those
needed for screening. The vast majority of established biomarkers used for screening
can also be used as diagnostic markers, such as PSA, which is a very well-
established model. PSA, together with a digital rectal examination, is the most
generally utilized diagnostic tool for prostate malignancy (Baker 2009). The
constraints for symptomatic accessible biomarkers include low symptomatic sensi-
tivity and specificity; however, diagnostic biomarkers need to have high sensitivity
to be a suitable diagnostic biomarker (Baker 2019; Shehzad et al. 2010). For
instance, the Bence-Jones protein in urine is one of the most established symptom-
atic markers of various myelomas (Rundle et al. 2012). In any case, some CB are
valuable in confirming analysis, frequently used with a suite of different markers,
particularly to distinguish metastatic tumors alongside other clinical imaging
instruments (Simon 2011). Utilization of a series of CB to improve sensitivity and
specificity of diagnosis has been conducted for the analysis of specific malignant
1 Introduction to Cancer Biomarkers 11

growths (Huang et al. 2017a, b). It has been reported that a panel of four biomarkers
comprising leptin, osteopontin, prolactin, and insulin-like development factor 2 alto-
gether have a sensitivity and specificity of 95% for the detection of ovarian malig-
nant growth (Bergman et al. 2013). Furthermore, two other biomarkers, when used
with the aforementioned biomarkers, CA125 and macrophage inhibitory factor, will
increase the sensitivity from 95% to 99.4% for the detection of ovarian cancer
(Munksgaard and Blaakaer 2012). Different studies have aimed to improve the
detection sensitivity and specificity using a mix of CA125 with ultrasonography
for the diagnosis of ovarian malignant growth (Bell et al. 2013).

1.4.3 Prognosis/Estimation

Prognosis is the likelihood of cure or the outcome for any patient. A prognostic
marker is obtained from a patient at diagnosis to indicate the disease stage and
likelihood of favorable outcome independent of treatment. In the interim, a predic-
tive biomarker allows the understanding of the need for various treatment types;
subsequently, a present biomarker is fundamental for customizing treatments
(Griffiths et al. 2002). The extent of the CB levels increases usually reflects the
weight or mass of the tumor; subsequently, high CB levels generally can indicate a
poor prognosis. As indicators of tumor stage, CB can also indicate the arranging
framework for malignant growth or tumor-node-metastasis (TNM) characterization
for tumor development. For instance, tumors in testicular germ cells that have
elevated levels of a CB, such as AFP, LDH, and HCG-β, may indicate the aggressive
nature of the disease with a poor prognosis. Therefore, such biomarkers can be used
for the staging of the TNM framework set up with a site-specific factor, called a
prognostic factor. LDH alone has been used for staging lymphoma tumors (Dalle
Grave 2012). Nevertheless, the precision of the marker in determining tumor stage is
poor. Estrogen receptor (ER) is a prognostic and predictive tissue biomarker, which
has been utilized for deciding on which patients will respond to hormonal treatment.
Along these lines, patients with ER-positive tumors will generally respond favorably
with ER modulators or aromatase inhibitors, independent of the stage of the breast
cancer. ER is considered as a prognostic marker because when tumors are
ER-negative, it indicates a poor prognosis, and ER-positive patients will have a
better prognosis (Osborne et al. 2011). In a similar setting, high levels of HER2 in
the serum of patients with breast cancer have a poor prognosis. Treatment for HER-2
positive breast malignant growth patients includes trastuzumab (Herceptin), which is
a recombinant monoclonal immunizer. Herceptin has been used in females suffering
from metastatic breast malignant growth that overexpresses HER2 and is the first-
line chemotherapy used in patients. KRAS is a biomarker that is used for colorectal
malignancy, and patients with substantial changes in KRAS have a poor response
against epidermal development factor receptor (EGFR)-mediated treatments (Hartig
2011; Stolow 2020).
12 M. Iftikhar et al.

1.4.4 Therapeutic Monitoring/Follow-Up/Evidence of Metastasis

or Recurrence

Clinically valuable biomarkers normally vary as per the tumor status, size, or burden
changes, and incremental changes of CB can be associated with the advancement of
the diseases or indications of remission. CB levels tend not to change with a stable
tumor. The recurrence of a disease can be identified biochemically by monitoring
any elevation in the levels of CB in asymptomatic patients and prior to clinical
assessment. Continuous monitoring of the patient during and posttreatment will
indicate the successfulness of the treatment provided based on the levels of
CB. However, increasing CB values from the basal level indicates the reappearance
of the disease. Numerous CB can be used to monitor the treatment, identification of
recurrence, or metastasis; for example, CEA in colorectal cancer, cancer antigen
125 (CA 125) in ovarian tumors, and PSA for prostatic tumors, respectively
(Al-Amodi and Kamel 2016).
In a few patients wherein the treatment strategies are not working, they will
encounter escalating levels of CB, and depending on patient prognosis, the elective
treatment should be stopped. Assessment of CB as a screening and predictive
biomarker should be diagnostically accurate and specific to tailor viable treatments,
continue the beneficial treatments, or early termination/substitution of ineffective
treatment for those cancers. Carbohydrate antigen 19-9 (CA19-9) is a CB that is used
in pancreatic CRC (Mystkowska 2015). The FDA endorsed the use of CA19-9 in
2002 as a biomarker of pancreatic cancer. However, it is not recommended to be
used as a screening biomarker (Mystkowska 2015; Wang et al. 2011). Monitoring
biomarkers have been broadly utilized in clinical practice, although when used as a
detection biomarker, there are issues such as poor accuracy of the results (Ishiura
et al. 2019).

1.5 Uses of CB in Malignant Cancers

Cancer is a global problem, and The American Cancer Society has stated that cancer
is one of the primary causes of death, and causes one in four deaths in the United
States. According to The American Cancer Society, it has been estimated that there
was a total of 1,898,160 (970,250 men and 927,910 women) new cancer cases in
2021, and 608,570 deaths (319,420 men and 289,150 women) in the United States.
A recent report from the International Agency for Research on Cancer, the
GLOBOCAN recognized that the types of tumor among men were lung, prostate,
colorectal, liver, and urinary bladder; whereas breast malignancy, liver, lungs, and
ovarian diseases were the most widely found in females worldwide. In the most
recent decade, there has been a large increase in the cost of medical care, combined
with the restricted viability of single malignant treatment therapies, which has
indicated the importance of biomarkers. CB is a compelling apparatus in clinical
practices for the management of patients with cancer. Therefore, currently used or
future CB may be used for the risk assessment of disease, screening among
1 Introduction to Cancer Biomarkers 13

asymptomatic individuals, confirming detection, differentially segregating between

benign and metastatic tumors, calculating the expected outcome or prognosis, and
evaluating the effectiveness of treatment (Hirschberg et al. 2016).

1.5.1 Breast Malignant Growth

Breast cancer is the most common malignancy among females and is the leading
cancer cause of death worldwide. Its prevalence is increasing compared with other
types of cancers in females (Westen and Morrison 2001). Therefore, it is important
to use all accessible tools for its early diagnosis and appropriate management of
patients with cancer. Clinically, the pathophysiology of breast cancer includes
irregular lump formation, areola release, skin, or phenotypic changes. The screening
instructions defined by The American Cancer Society indicate that women over the
age of 40 years should have a mammogram and a clinical breast test either yearly or
every other year (Heru 2013). Detection of breast cancer usually relies on patient
discovery; nonetheless, the use of CB in breast malignancy is used to indicate
prognosis, observing treatment success, and for follow-up. In particular, CB do
not have a particular use for early analysis or diagnosis (Krishna and Rajabhushnam
2019). The evaluation of ER and progesterone receptors in the tissue can be used for
the detection of breast cancer as indicated by the European Society of Medical
Oncology. They can also be used for predicting the response to hormone therapy
in the early stages of breast cancer and to manage advanced breast cancer disease
(Krishnavenia et al. 2020). Another prognostic marker is HER-2, generally used in
identifying patients with early or metastatic breast malignancy, and is used to
identify patients who require therapy with trastuzumab (Herceptin) (Zebari et al.
2019) or tamoxifen treatment at the early stage of breast cancer development (De and
Biswas 2020). Identifying those patients at a high risk and therefore should be
included in a screening program utilizes the genetic mutations of BRCA1 or
BRCA2 genes, which are present in up to 5% of breast malignant cases. Because
of their high vulnerability to breast and ovarian cancer development, it is unequivo-
cally suggested that females with BRCA1 or BRCA2 mutations should be routinely
screened (Ceccarini et al. 2015; De and Biswas 2020). It is suggested that low levels
of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1
(PAI-1) correlate to a decreased risk of breast cancer recurrence and demonstrated to
be a self-regulating prognostic element of a recently analyzed lymph node-negative
breast cancer (Gowthaman 2019). The use of serum biomarkers is the most appro-
priate for monitoring during the treatment process, and to a lesser degree, they also
function as prognostic markers, such as CA15.3, CEA, and BR (Wu et al. 2014).
They are used in combination with different radiological and clinical applications to
monitor chemotherapy progress in breast cancer cases. The rise in serum levels of
these biomarkers may indicate a recurrence or cancer development (Fu and Li 2016).
14 M. Iftikhar et al.

1.5.2 Prostate Cancer

Prostate malignant growth (PC) is a common disease in men and is the leading cause
of cancer-related death (Mohler et al. 2010). The PSA test has dramatically improved
screening and diagnosis of prostate malignancy, and since its implementation, PSA
has resulted in an increase in the number of detected cases at an early stage, thereby
decreasing the mortality rate of prostate malignancy (Ilic et al. 2013). Numerous
studies have improved upon the sensitivity of PSA as an asymptomatic marker by
using PSA and its isoforms (pro-PSA) and comparing the percentage rate of
pro-PSA with PSA; this may separate benign and malignant prostatic tumor presence
in patients with PSA values from 4 to 10 μg/L (Mottet et al. 2014). Other novel and
promising biomarkers under investigation include human kallikrein type 2, prostate
cancer-specific antigen 3 (PSA 3), and prostate stem cell antigen (Andriole et al.
2012). Evaluating PCA 3 in urine samples may also be used for the detection of
identifying prostate malignancy. Increased levels of metalloproteinase 2 and
9 (MMP-2 and MMP-9), members of the protease family, are associated with
prostate malignant growth (Djulbegovic et al. 2010). MMPs have been investigated
as biomarkers for therapeutic assessment and monitoring in prostate malignancy
(Markt et al. 2015).

1.5.3 Ovarian Malignancy

A large proportion of patients with ovarian cancer present late in tumor growth and
have usually progressed to stage III or IV, meaning poor survival rates. Therefore,
there is an urgent need for a sensitive and specific diagnostic biomarker (Moore et al.
2011). CA 125 is the CB most often used for ovarian cancer. It is used as a screening
biomarker for women with a family history of the disease or who are at high risk of
developing an ovarian disease. CA125 is used in conjunction with vaginal ultraso-
nography as a reliable, diagnostic biomarker (Aziz et al. 2020). CA125 has addi-
tionally been used as a diagnostic biomarker, which diminishes with chemotherapy
or medical procedures. Monitoring of CA125 14 days prior to the beginning of any
therapeutic intervention and subsequent follow-up required for the continual moni-
toring of the disease are needed (Irungu 2016). Other biomarkers are currently under
investigation for use as a biomarker of ovarian disease including kallikreins (Deo
et al. 2019), hCG, interleukin-6 (IL-6), prostasin, TPA, lysophosphatidic corrosive,
plasminogen activator inhibitor-1(PAI-1), Her-2/neu, tumor-related inhibin, CEA,
and trypsin inhibitor (Anderson 2010; Deo et al. 2019; Huang et al. 2011).

1.5.4 Colorectal Malignant Growth

CRC is the third most common cancer worldwide. According to The American
Cancer Society, a total of 149,500 new cases and 52,980 deaths related to colorectal
cancer are predicted to occur in the United States in 2021 (Siegel et al. 2021). For
1 Introduction to Cancer Biomarkers 15

colorectal carcinoma, the most common site for malignancy is the rectum with 38%
of all cases developing here, followed by sigmoid accounting for 29% of the cases
(Chalya et al. 2013). The screening program for CRC should be provided to all
asymptomatic people over the age of 50 years (Wolf et al. 2018), according to the
National Academy of Clinical Biochemistry (NACB). Several screening techniques
are available as previously described. Fecal occult blood test (FOBT) is the most
widely used stool CB (Westwood et al. 2017). Testing for blood in the stool involves
either identifying globin, which is a part of the blood (hemoglobin), using a fecal
immunochemical test or the guaiac test that estimates pseudoperoxidase action of the
heme portion of hemoglobin. CEA was brought into clinical practice in 1965 (Bevan
and Rutter 2018), and it is generally used as an all-inclusive or non-organ, non-
tissue-specific tumor marker. CEA is not used in the screening of CRC as it has low
sensitivity, and there is a low prevalence of CRC among asymptomatic individuals.
Nonetheless, CEA is a proficient prognostic and treatment monitoring biomarker
(Spada et al. 2014). CEA testing is recommended at the start of treatment and
subsequently every 1–3 months during the therapeutic routine; it is also used for
deciding on treatment options for metastatic CRC cases (Brenner and Tao 2013).
CA19-9 has been used as a prognostic marker to monitor CRC after surgical
resection and as a monitoring marker for therapeutic interventions (De Wijkerslooth
et al. 2012). CA242 and the tissue inhibitor of metalloproteinase type 1 (TIMP-1) are
two more CBs that are under investigation, and both have been used in addition to
CEA for the detection of patients with colorectal cancer (Chiu et al. 2013).

1.6 New Biomarkers/Approval/Advancements

In the last 20 years, the FDA has only approved a few of the large number of
malignancy biomarkers identified for detecting, monitoring, or indicating the recur-
rence of cancer (Frisoni et al. 2017). The biomarkers need to have significant use in
the clinical setting to enhance the outcome of patients with cancer, as well as have
potential in diagnosis and therapeutics (Javitt et al. 2020). At first, CB needs to
identify individuals with the disease and then used in follow-up to plan for treatment.
In line with this, numerous biomarkers do not meet these ideal characteristics in the
clinical settings as they are either unable to precisely indicate the therapeutic
strategies, or their sensitivity and specificity are not as high as required (Wallentin
et al. 2014). In reality, the proportion of effective clinical interpretations of
biomarkers was low (0.1%) (Allinson 2018). Studies have shown that CB develop-
ment is a multistep process, which starts with identifying the biomarker, developing
a measuring approach for assessment, evaluating its clinical potential for beginners,
normalizing the test, and finally approving the biomarker for clinical use (Bonora
et al. 2019). In line with this, a well-established and organized stage model for the
advancement of the assessment and interpretation of biomarkers for a clinical setting
is warranted. This model is in agreement with other models commonly used in
medication advancement procedures and comprises preclinical exploratory
examinations, clinical measures and accuracy, large-scale longitudinal repository
16 M. Iftikhar et al.

studies, advancement in screening assessments, and finally malignant growth control

considerations. In addition, the biomarker must be evaluated for its clinical use for
providing state-of-the-art patient management, as well as support the therapeutic
benefits of cancer therapy in patients (Fiserova et al. 2020).

1.6.1 Challenges for the Investigation of Novel Biomarkers

Improvements in biomarkers for the screening of malignancies, early detection, and

observation of the treatment are hindered by biological and monetary difficulties.
Most of the available diagnostic methods used are generally for detecting late-stage
or fully developed malignant growth. However, premalignant or early neoplasm is
amenable to surgery and prevention. Therefore, screening tests could recognize
malignancy at the preclinical stage, although they may not identify micrometastasis,
thereby restricting the advantage of early recognition and treatment (Goel et al.
2021). It is also challenging that in many organs, such as the prostate and colon,
paraneoplastic injuries are much more typical than malignant cancers (Mathur et al.
2020). Therefore, screening techniques simply detect early cancers and do not
necessarily dissect the underlying mechanisms and malignant behaviors of the
tumors (Sureshkumar et al. 2020). The advancement of CB must consider the
heterogeneity of the tumor; it contains numerous naturally interconnecting networks
with various responses to therapeutic interventions. The improvement of biomarkers
may be hampered by this heterogeneity, and as a result, the development of
biomarkers using genomic and proteomic methods may be used to cautiously
address these heterogeneity concerns (Fuchs and Buhmann 2011). Definite and
extensive information on cancer at the molecular level has developed drastically in
the last 20 years and has brought about critical improvement in the description of
human tumors, which has been shifted toward the advancement of focused
treatments, allowing customized treatment options (Shi et al. 2018). Hence, it is
proposed that the development of profoundly ground-breaking technologies, such as
genomics, epigenomic, transcriptomic, proteomic, and metabolomic studies, can
advance scientific discovery and optimization with high accuracy over currently
available biomarkers (Stewart et al. 2017). The aforementioned technologies have
increased the number of potential biomarkers, such as DNA, RNA, and other protein
biomolecules, that have potential in the clinical setting (Zoppi 2020). The previous
idea of a single biomarker has recently been substituted with the discovery of
multibiomarkers of genes or proteins, raising the question of whether heterogeneous
and multifactorial malignant growth may have a single distinct mark (Myal et al.
2010).

1.6.2 Genomic Advancements

Genomic advancements have been used broadly for the interpretation of tumors at
the submolecular level, which subsequently has given a better understanding of
1 Introduction to Cancer Biomarkers 17

malignancy and may provide researchers the essential tools to develop drugs that
could target specific molecules (Jin et al. 2020). The US National Cancer Institute
defined customized medication as a type of medication that utilizes data about an
individual’s phenotype and genotype, proteins, and assessment tools to prevent,
diagnose, analyze, and treat the particular disease (Fancello et al. 2019). Genomic
changes that might be related to malignant growth include gene modification and
amplification, transformation, chromosomal mutations, and atypical methylation.
These genomic changes are characterized by DNA double-strand breaks, transcrip-
tion errors in mRNA or microRNA, translation of proteins, or the regulation of
various metabolites at the cellular level. Genotyping changes can be assessed using
genome sequencing innovations or microarrays (Hebbring 2019). Mutation screen-
ing may be evaluated via a sequencing procedure, whereas DNA microarrays and
PCR can be used to assess DNA microarrays and DNA expression levels (Fancello
et al. 2019). Genomic microarrays are an exceptionally ground-breaking and sensi-
tive method; they can predict the clinical behavior of tumors (Fancello et al. 2019).
For biomarker discovery and implementation, genomics has been widely used. The
accessibility of biological strategies allows the oncologist to have an insight deep
into the underlying mechanism of cancer progression, in terms of planning for the
synthesis of biological medication with a better understanding of
pharmacogenomics. In this way, biomarkers allow the impact of hereditary varia-
tion, providing new techniques for treating patients at a personalized level.
Customized medicine is the product of such investigations (Logotheti et al. 2019).

1.6.3 Epigenomics

Epigenetics refers to the heritable changes in the expression level of DNA rather than
the changes that normally occur in the sequence of DNA. Epigenetic changes
include DNA methylation, histone alterations, and noncoding RNAs. These changes
are present extensively in various human malignancies, and their regulation may be
associated with early disease improvement. In this manner, they are a useful source
of potential markers with expansive applications in diagnostics (Bernstein et al.
2010). Methylated DNA is a distinct stable epigenetic mechanism that is associated
with tumor growth and progression. Furthermore, the primary advantage of
detecting methylation is due to the inherent stability of DNA. Methylated genes in
tumors can be quantified to diagnose disease (Horvath and Raj 2018). The assess-
ment of DNA methylation can be conducted using a range of techniques using
various types of biological material, such as tissue, plasma, serum, sputum, and urine
for a variety of cancers (Kelley and Fishel 2016). The techniques for evaluating
DNA methylation have improved considerably over time. An epigenetic change
including bisulfate transformation of DNA, followed by PCR amplification, allows
the evaluation of gene-specific methylation, which uses probes and primers specifi-
cally designed for the detection of methylated DNA (Olejniczak et al. 2010). The
identification of DNA methylation areas is now possible because of this break-
through. These areas may be converted into algorithms of ones and zeros, resulting
18 M. Iftikhar et al.

in a sophisticated activity that depicts hereditary changes in each cell or tissue,

independent of whether it is functioning normally or abnormally. A large genome-
wide screening effort has investigated over 200 such biomarkers for various human
malignancies for DNA methylation biomarkers in fluids, tissue, and blood
(Mockenhaupt 2015).

1.6.4 Proteomics

The proteomics-based methodology is one of the most dynamic and innovative tools
for the identification of various diseases with a wide range to confirm, complement,
or provide more detailed information compared with other high-throughput
approaches, such as genomic, transcriptomic, and epigenetics. Notwithstanding
genomic expression profiling, it is a profoundly solid technique for malignancy
characterization and diagnosis (Sallam 2015). Studies that focus on differential
expression levels of mRNA have been very descriptive and normally do not tend
to correspond to the level or activity of functional proteins. Proteomic technologies
that correspond to the protein expression profiles with cancer are essential for a
detailed and comprehensive understanding of the malignant growth mechanism.
Besides, focusing on a specific protein pathway associated with tumorigenesis
presents a valuable approach in malignant growth treatment, as proteins mediate
their effect through specific pathways rather than functioning separately (Janjua et al.
2020; Liang et al. 2012). Macromolecules including proteins are exceptionally
unique and very dynamic regulatory bodies. It is well known that proteins are
modulated by the regulation of several processes including posttranslational modifi-
cation, proteolytic degradation, and interaction with a complex network (Huang
et al. 2017a, b).
Proteomics studies are associated with the whole structure and expression of a
protein in biological fluids, in cells and tissues, an organelle, or the whole body. In
this way, knowing the concentration of proteomic profiles of different proteins
obtained from biological samples of patients with cancer can provide an in-depth
understanding of tumor pathogenesis, observation, the outcome of malignancy
treatment, and management of patients with cancer. Several different proteins are
associated or released by the tumor cells into the biological fluids, due to the
activation complex networking of inflammation, cell death, and necrosis (Batta
et al. 2012). Cellular events taking place during the abnormal cell cycle or cell
death change the expression levels of proteins. Abrupt signal transduction and
expression of proteins can be detected and easily correlated with healthy individuals
for early diagnosis of patients with cancer. Secreted proteins are investigated by
assessment of secretory pathways utilizing secretomic approaches and have
advanced the understanding and clinical application of cancer biomarkers.
Proteomic advancements can also be used for improving gene annotations based
on proteogenomic data. Cross-examination and comparative analysis of the genome
and the proteome encourage the discovery of posttranslational changes and proteo-
lytic episodes (Batta et al. 2012; Indovina et al. 2011).
1 Introduction to Cancer Biomarkers 19

1.6.5 Metabolomics

A cancer biomarker can be a secreted metabolite, which is released by tumor cells,

during metabolic pathways, or biological processes, and can be used for the analysis
of cancer and monitor patient response toward treatments (Johnson et al. 2012).
Although the key tumor markers are proteins, they can be sorted as cellular,
biochemical, physiological, or anatomical. These markers might be used for the
diagnosis (to distinguish between the stages of cancer), prognosis (survey lethality),
and assessment of the patient’s response to cancer therapy. The markers can be
detected in body fluids (blood, urine, serum, stool, saliva), or tissues samples or
biopsies of the cancer. In addition, it has been demonstrated recently that cancer
volatile organic compounds markers can be detected in the breath (Beger 2013). In
any case, recognition of these markers is a mechanistic process, and metabolomics is
one of the omics innovations and is a better representation of the phenotypic changes
in patients with cancer (Liesenfeld et al. 2013). Investigating the cancer metabolome
is an effective and powerful method to study the phenotypic changes, which are
tumor-associated. Screening biomarkers by selecting a variety of analytical
procedures have been emphasized (Armitage and Ciborowski 2017). It is believed
that the pattern of several metabolites is more accurate and precise and indicative of
cancer status than a single metabolite. Metabolomic approaches allow the detection
of a variety of metabolites in a single examination. The foremost tools utilized for
metabolome investigation are mass spectrometry and nuclear magnetic resonance
spectroscopy (Wang et al. 2016).

1.7 Conclusion

Biomarkers for various cancers play a significant role in the field of oncology
and clinical practice for the risk assessment, screening, and diagnosis, especially
when coordinated with other diagnostic techniques to determine prognosis
and the response to treatment. Cancer biomarkers can facilitate a deep understanding
of the development of cancer. Clinicians and scientists need a thorough understand-
ing of the underlying molecular aspects, clinical use, and reliability of biomarkers
to determine if they are suitable for patient use, or whether additional testing is
required before incorporation into routine clinical practices. The challenges
and future potential biomarkers will play a significant role in advancing personalized
medication when combined with current therapeutics and diagnostics.

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Technologies for Identification
and Validation of Cancer Biomarkers 2
Aneela Javed, Hamza Sher, Zilli Huma, and Ishaq N. Khan

Abstract

With the recent advances in genomic profiling, high-throughput technologies and

improved treatment strategies based on personalized medcines, biomarkers
have emerged with an important role in the early detection and clinical manage-
ment of cancer patients. Genetic-based biochemical analysis has developed to
examine specific molecular pathways with abnormal expression of regulatory
proteins and has been evaluated as potential predictive biomarkers for therapeutic
decision in various cancer treatments. Genome-based prognostic biomarkers can
measure and detect the risk of developing cancer in various tissues or, alterna-
tively, assess the progression of cancer following clinical staging or potential
response to the available therapeutic strategies. The development of novel cancer

A. Javed (*)
Atta ur Rahman School of Applied Biosciences, National University of Sciences and Technology,
Islamabad, Pakistan
Neurooncology & Oncomedicines Research Group, Institute of Basic Medical Sciences, Khyber
Medical University, Peshawar, Pakistan
H. Sher
Atta ur Rahman School of Applied Biosciences, National University of Sciences and Technology,
Islamabad, Pakistan
Z. Huma
Neurooncology & Oncomedicines Research Group, Institute of Basic Medical Sciences, Khyber
Medical University, Peshawar, Pakistan
I. N. Khan
Neurooncology & Oncomedicines Research Group, Institute of Basic Medical Sciences, Khyber
Medical University, Peshawar, Pakistan
Cancer Cell Culture & Precision Oncomedicine Lab, Institute of Basic Medical Sciences, Khyber
Medical University, Peshawar, Pakistan

# The Author(s), under exclusive license to Springer Nature Singapore Pte 27

Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_2
28 A. Javed et al.

biomarkers for clinical utilization including diagnosis, prognosis, and drug ther-
apy response is hindered by various challenges including scientific validation,
regulatory, and legislation for the efficient translation to the clinics. This chapter
underpins the different stages of biomarker development, identification and
validation of cancer biomarkers, and successful implementation in the cancer
management. With challenges, time is no far when biomarkers will shape the
future of personalized medicine and cancer therapy.

Keywords
Cancer · Biomarker · Identification · Prognosis · Validation · Response

2.1 Cancer Biomarkers

Cancer is a cluster of diseases, responsible for the death of about nine million
individuals and almost one-sixth of global mortality. The rapidly increasing number
of cancer cases has been greatly affecting the health sector. The study forecasts that
over the next 20 years the cases may increase by 70%. This disease burden can be
reduced effectively by the application of cancer biomarker for predictive measures,
early detection, and appropriate therapy followed by routine checkup. The US Food
and Drug and Administration (FDA) define biomarker in the following context “Any
biological molecule that can be used as diagnostic indicator to measure the risk and
presence of disease” (Ilyin et al. 2004; World Health Organization 2017). It can be
enzyme, cell, gene, protein, nucleic acids which can be detected in blood, urine,
tissues, and body fluid, etc. Cancer biomarkers (CB) are biological substances
secreted by tumors or other cells, that can be utilized as an indicative tool to detect,
prognose and diagnose cancer and can be used to distinguish the subpopulation of
patients’ response to a therapy (Goossens et al. 2015; Rhea and Molinaro 2011).

2.2 Types of Cancer Biomarkers

Cancer biomarkers can be categorized into the following classes based on their
usage:

2.2.1 Screening Biomarkers

Screening biomarkers are the first type of cancer biomarkers that can be utilized for
early detection of cancer: it is used to identify those individuals that are at danger of
developing a specific disease or to detect a disease when the individuals having it are
asymptomatic which is different from the diagnosis of symptomatic individuals.
This results in increased survival rate and reduces other complications and morbidity
(Weigelt et al. 2005). Example of screening biomarkers includes APF which is used
2 Technologies for Identification and Validation of Cancer Biomarkers 29

in screening for hepatocellular cancer in high-risk individuals, CA125, in screening

for ovarian cancer, for prostate cancer PSA is used as screening biomarker and in
screening for colorectal cancers, fecal occult blood testing (FOBT) is used (Duffy
2015).

2.2.2 Predictive Biomarkers

Predictive biomarker, another type of cancer biomarker used to detect/predict the

response of cancer cells to specific therapy or drug, i.e., the HER2 activation in
breast cancer in response to trastuzumab or the prediction of mutated KRAS activa-
tion resistance to EGFR inhibitor cetuximab in colorectal cancer (Cameron et al.
2017; Romond et al. 2005; Slamon et al. 2001; Van Cutsem et al. 2009).

2.2.3 Prognostic Biomarkers

Prognostic biomarkers can be used to provide information regarding the disease

recurrence or progression, but not linked directly with therapeutic interventions, i.e.,
21-gene recurrence score in breast cancer, used to predict the cancer recurrence in
tamoxifen-treated node-negative breast cancer (Paik et al. 2004).

2.2.4 Diagnostic Biomarkers

Diagnostic biomarkers, another type of cancer biomarker utilized to detect the

presence or absence of a particular disease in a patient. Stool cancer DNA in
colorectal cancer surveillance is used as diagnostic biomarker lately (Imperiale
et al. 2014).

2.2.5 Monitoring Biomarkers

The biomarkers used for the monitoring or prediction of cancer recurrence post
therapy is known as Monitoring biomarkers. The level of these biomarkers increase
above the basal level in cancer recurrence can be predicted biochemically prior to
any clinical or radiological evidence, i.e., carbohydrate antigen CA19-9, used as
monitoring biomarker in pancreatic cancer and is FDA approved since 2002 (Bast
et al. 2001; Koprowski et al. 1979; Rosty and Goggins 2002; Sharma 2009).
30 A. Javed et al.

2.3 Discovery of CBMs

The discovery of cancer biomarkers employs numerous routes that includes the
coverage of several disciplines ranging from high-throughput data initiation to
generation of big-data and utilization of machines learning algorithms to the valida-
tion of biomarkers in different preclinical and clinical trials. These comprehensive
steps involved in the cancer biomarker discovery has depicted in the Fig. 2.1.

Fig. 2.1 Depiction of numerous technologies for identification and validation of cancer
biomarkers. High-throughput technologies have generated huge bulk of big-data that is being
deciphered by data mining for the generation of meaningful information. This data-mining results
in the identification of novel targets that goes on becoming a cancer biomarker following different
approaches such as support vector machine learning and analysis of integrated databases. In the
meantime, the potential biomarker-specific inhibitors’ hunt also begins that employ different
computed techniques such as cheminformatics to identify potential functional groups for having
binding affinity with the identified biomarkers. The potential cancer biomarker is being validated in
preclinical studies employing in silico, in vitro, microfluidics and in vivo approaches. This leads to
the developmental validation of cancer biomarker in human population following its comprehen-
sive journey in clinical trials, with ultimate success of biomarker approval for cancer clinics
2 Technologies for Identification and Validation of Cancer Biomarkers 31

2.3.1 Preclinical Studies

2.3.1.1 In-Silico Studies

The integration, evaluation, and analysis of gene banks from huge databases present
in gene expression profiling repositories can be done through sets of tools termed as
“Bioplat (biomarker platform)”. The core purpose of user-friendly Bioplat software
is to aid in early diagnosis and prognosis of cancer patients by means of functional
genomic data. Along with “in-silico identification” of new cancer biomarkers it is
also helpful in extracting data from gene repositories as well as gene expression
analysis.
Bioplat plays a significant role in edition of gene and creation of biomarkers with
the help of identifiers in the embedded database, named Gene name, Entrez,
Ensembl and Probe IDs. Additionally, Bioplat can also integrate gene data by
means of online available resources including DAVID (Database for Annotation,
Visualization and Integrated Discovery), STRING (Search Tool for the Retrieval of
Interacting Genes/Proteins), Enrichr, Expression Atlas, RNA-seq Atlas and Gene
Cards.
The gene signature optimization process is the prominent step in the Bioplat
software development. The significant processes of Bioplat comprises of “blind
search” and “particle swarm optimization (PSO)” helps in hitting the right optimum
gene in less time (Butti et al. 2014).
However, another study encompasses some other approaches for in-silico identi-
fication of cancer biomarkers includes Panther, UniProtKB, NetOGlyc, NetNGlyc,
Oncomine, and Cytoscape (Azevedo et al. 2018).

2.3.1.2 In Vitro
The use tissue culture paved a promising path towards the discovery of cancer
biomarkers. The tissue cultures are rich in tumor cell lines and hence, wide spectrum
of candidate biomarkers (Minamida et al. 2011). The limitation in the accessibility of
patient tissue sample leads towards the transition to use tumor cell lines as second
option for the discovery of potential biomarker.
The major ingredient of Conditioned media (CM) is secretory proteins that plays
the major role in the identification of biomarkers with greater efficacy (Xu et al.
2010).
The traditional 2D (two-dimensional cell cultures) are replaced by 3D (three-
dimensional cell culture) for the exclusive representation of homeostasis during
in vitro analysis. The 3D cultures resemble tissue engineering models which helps
in the understanding of gene expression and molecular mutated pathways of cancer
(Lenas et al. 2009; Martin et al. 2008).
Among several techniques for better understanding of biomarkers, mass spec-
trometry has got the central focus. Through minimal number of sample, mass
spectrometry has the significance to calculate accurate molecular mass with preci-
sion (Boja and Rodriguez 2012). Two broad categories of mass spectrometry for the
identification of biomarkers are gel-based (2-DE and 2D-DIGE) and gel-free
(SILAC, iTRAQ) techniques (Leong et al. 2012).
32 A. Javed et al.

Additionally, gel-free techniques are also emerged as promising technique for the
discovery of biomarkers. In tissue culture-model system, Stable isotope labelling by
amino acids in cell culture (SILAC), that includes the integration of amino acid
within stable isotope nuclei are now considered as method of choice. iTRAQ
(Isobaric tags for relative and absolute quantitation) can also be used as alternate
method (Mann 2006).

2.3.1.3 Microfluidics Chip Technology

Microfluidic chip technology utilizes an approach that can control fluids on a
microscale, thus manipulating the cell-culture-related parameters in a comprehensive
way to mimic the microenvironment of a malignant tumor in vivo (Xu et al. 2016).
The microfluidic chip has strongly emerged as a biochip that can assimilate numer-
ous fields, including cell biology, oncology, pathology, physiology, biophysics,
biomechanics, bio-printing, motorized design, and so forth (Chaudhuri et al. 2016;
Rosenbluth et al. 2008). In the recent decades, the application of biochip technology
has displayed remarkable potential in the field of cancer treatment. A number of
science validation techniques such as 2D and 3D cell and tissue cultures, spheroids
and tissue organoid cultures can be performed on microfluidic biochips (Vadivelu
et al. 2017). Moreover, cancer patients’ derived cell lines and tissues can also be
cultured on microfluidic biochips in a observable, controllable, manageable, and a
high-throughput fashion that will significantly advance the progress of personalized
medicine (Mulholland et al. 2018).
The novel biomarker and drug development consist of a number of major
practices, including drug discovery, validations via preclinical trials and clinical
developmental trials. Since the initial progress in 1990s, microfluidic biochip tech-
nology has been employed in multiple research disciplines including single cell
analysis, medicinal synthesis, proteomics, tissue engineering, libraries screening,
and medical diagnosis (Yu et al. 2014). Such platforms deliver novel understandings
of biological mechanisms and endow the effective and rapid generation of novel data
analysis. The microfluidics biochip revolution escalated due to the numerous effec-
tive applications offered by system size shrinking, while in the meantime providing
high-throughput analysis, improved sensitivity, enhanced analytical potential,
multiplexing abilities, and utilizes less volume of reagents, as well as its portable
and easily fabricated (Boobphahom et al. 2020). This ultimately results in the
development of economical in vitro models for lead compounds’ identifications
that can steadfastly predict the effectiveness, cytotoxicity, and pharmacokinetics of
test compounds in humans, as well as for novel library screening analyses.

2.3.1.4 In Vivo
With the emergence of biomarkers discovery from in vivo mouse models, the
extraction of plasma from genetically modified mouse model can be an attractive
approach (Hingorani et al. 2003). Extraction of plasma from mice during stages of
pancreatic tumor development, followed by proteomic approaches helps in marking
the protein alterations (Aguirre et al. 2003).
2 Technologies for Identification and Validation of Cancer Biomarkers 33

Through comparative analysis technique, the noticeable similarity in expression

of candidate biomarkers in human and mouse models were observed. To mark out
differences in the protein concentrations, different samples are labeled with Cy dyes,
IPAS (intact-protein analysis system) is done to indicate the protein differences. On
the other hand, mass spectrometry can be helpful to highlight the gaps in protein
bands (Wang et al. 2005).
Another sera comparison between the mouse model having human A549 lung
adenocarcinoma cells with the control mouse group. The result showed very promi-
nent quantitative and qualitative alterations in “expression of protein” between two
groups. The key investigation revolves around the fact that differences in protein
expression due to acute-phase inflammatory protein responses or antibody-mediated
immune responses. Through histopathological staining techniques, it can be
concluded that protein alterations are due to secondary changes in host origin and
are not related to tumor cell derived proteins (Subramaniam et al. 2013).

2.3.2 Clinical Studies

2.3.2.1 CBMs Already in Clinics?

The EPGR (epidermal growth factor receptor) family member named as HER2
(ERBB2) is used as molecular biomarker in clinical settings. The amplification
and overexpression of HER2 shows considerable responses against monoclonal
antibodies, e.g., trastuzumab and pertuzumab. Among 20% of breast cancer patients,
the phase 3 trails reflect the appreciable results of anti-HER 2 therapy along with
better survival rates (Piccart-Gebhart et al. 2005; Romond et al. 2005).
Presently, ten HER 2 assays have been approved as companion diagnostic
devices by FDA as well as approval of three HER2 assays (nucleic-acid based
tests) are done by the Center for Devices and Radiological Health. However, other
categories of biomarkers in clinics are BCR-ABL in chronic myeloid leukemia,
KRAS mutations in colorectal cancer and multiple mutations in non-small cell lung
cancer (NSCLC) (Kalavar and Philip 2019) (Table 2.1).

2.3.2.2 CBMs Clinical Trials

To replace the invasive cancer biomarkers, significant efforts are done to introduce
predictive biomarkers. They are majorly based on single protein or gene and are
mostly in phase II or III trials for evaluation and validation along with therapeutic
targets (Tables 2.2 and 2.3).
 34

Table 2.1 List of FDA-approved protein tumor markers presently utilized in clinical practice adapted from (Füzéry et al. 2013)
Year approved
or cleared for Type of
Biomarker Clinical use the first time Specimen Methodology submission Type of cancer
Pro2PSA Differentiating between cancer 2012 Serum Immunoassay PMA Prostate
and benign disease
Free PSA Differentiating between cancer 1997 Serum Immunoassay PMA Prostate
and benign conditions
p63 protein Assist with the differential 2005 FFPE Immunohistochemistry 510(k) Prostate
diagnosis tissue
ROMA (HE4 + CA-125) Prediction of cancer 2011 Serum Immunoassay 510(k) Ovarian
OVA1 (multiple proteins) Prediction of cancer 2009 Serum Immunoassay 510(k) Ovarian
HE4 Tracking recurrence of disease 2008 Serum Immunoassay 510(k) Ovarian
progression
CA-125 Keeping track of the disease’s 1997 Serum, Immunoassay 510(k) Ovarian
course and how well it’s plasma
responding to treatment
Fibrin/fibrinogen Keeping track of the disease’s 2008 Serum Immunoassay 510(k) Colorectal
degradation product progression
(DR-70)
c-Kit Detecting malignancies and 2004 FFPE Immunohistochemistry PMA Gastrointestinal
assisting in the selection of tissue stromal tumors
patients
CA19-9 Keeping track of the disease’s 2002 Serum, Immunoassay 510(k) Pancreatic
progress plasma
Estrogen receptor (ER) Prognosis and treatment 1999 FFPE Immunohistochemistry 510(k) Breast
response tissue
Progesterone receptor Prognosis and treatment 1999 FFPE Immunohistochemistry 510(k) Breast
(PR) response tissue
A. Javed et al.
 2

CA27.29 Keeping track of the disease’s 1997 Serum Immunoassay 510(k) Breast
reaction to treatment
CA15-3 Keeping track of the disease’s 1997 Serum, Immunoassay 510(k) Breast
reaction to treatment plasma
Circulating tumor cells Predicting cancer’s progression 2005 Whole Immunomagnetic 510(k) Breast
(EpCAM, CD45, and prognosis blood capture, immune-
cytokeratins 8, 18+, 19+) fluorescence
HER-2/neu Assessment in the context of 1998 FFPE Immunohistochemistry PMA Breast
therapy tissue
AFP-L3% Risk assessment for disease 2005 Serum HPLC, microfluidic 510(k) Hepatocellular
development capillary
electrophoresis
Technologies for Identification and Validation of Cancer Biomarkers
35
36 A. Javed et al.

Table 2.2 List of CBMs in Phase 1–Phase 4 clinical trials adapted from (Goossens et al. 2015)
Organ Cancer Biomarker Associated drug
Breast Breast BRCA1/2 Olaparib
HER2 (Tumor is Lapatinib
negative, but CTCs are
positive)
TOP2A (in people who Anthracycline-based
have HER2 neoadjuvant
overexpression) chemotherapy
CTCs that are HER2 Trastuzumab—
positive Emtansine
Gastrointestinal Colorectal BRAF LGX818, BYL719
RAS (type of mutation) FOLFOXIRI and
bevacizumab
Biomarkers that are new Cetuximab
(unspecified)
Esophago-gastric HER2 Trastuzumab and
afatinib
Hematological Cutaneous and GATA-3 MLN9708
peripheral T-cell
lymphomas
Head and neck Squamous cell HER and KRAS HM781-36B
carcinoma
Lung NSCLC BRAF V600E Trametinib, dabrafenib
ROS1 Crizotinib
Skin Melanoma BRAF V600E/K Binimetinib, trametinib

2.4 Technologies That Lead to CBMs Discovery

2.4.1 Genomics (Nuclear and Mitochondrial CBMs)

2.4.1.1 Next-Generation Sequencing (DNA and RNA seq)

Genomic alterations are under study for most major tumors using sequencing
techniques (Brooks 2012). Maxam Gilbert and Sanger laid the basis for next-
generation sequencing through their cleavage method and dideoxy synthesis respec-
tively (Maxam and Gilbert 1980; Sanger and Coulson 1975; Sanger et al. 1977).
Next-generation sequencing, deep or massively parallel sequencing can sequence an
entire genome in a single day which is extremely fast in comparison to Sanger
sequencing which took almost 10 years to sequence human genome (Behjati and
Tarpey 2013). Short-read whole genome sequencing and barcode linked read
sequencing are novel approaches that can be used to resolve genomic
rearrangements which can lead to tumorigenesis (Cunha 2017).
 2

Table 2.3 List of ongoing clinical trials for CBMs adapted from (Kirwan et al. 2015)
Marker Full name Cancer type Detection type Clinical applications
PSA, or Prostate-specific antigen Prostate Concentrations of proteins Screening, differentiating
Pro2PSA between cancer and benign
disease
AFP α-Fetoprotein Liver Protein concentrations and Diagnosis, staging, recurrence
fucosylation of the core (for detection, and therapy
AFP-L3) monitoring
OVA1 test Apoliprotein A1 + prealbumin + transferrin Ovarian Concentrations of proteins Prediction
(multiple (down), Β-2 Microglobulin + CA 125II (up)
proteins)
HE4 Human epididymis protein 4 Ovarian Concentrations of proteins Detecting recurrence,
monitoring therapy
CA125 Cancer antigen 125 Ovarian Concentrations of proteins Detecting recurrence,
monitoring therapy
ROMA test HE4 + CA125 Ovarian Concentrations of proteins Prediction
CA19-9 Carbohydrate antigen 19-9 Ovarian, pancreatic SLea on mucin glycoproteins Monitoring therapy
and gangliosides
hCG Human chorionic gonadotropin Ovarian, testicular Concentrations of proteins Diagnosis, staging, recurrence
detection, and therapy
monitoring
CA15-3 Cancer antigen 15-3 Breast Sialylated O-linked Monitoring therapy
Technologies for Identification and Validation of Cancer Biomarkers

oligosaccharide on MUC1
CA27-29 Cancer antigen 27-29 Breast MUC1 protein levels Monitoring therapy
HER2/neu Human epidermal growth factor receptor 2 Breast Concentrations of proteins Therapy choice
CEA Carcinoembryonic antigen Breast, lung, Concentrations of proteins Detecting recurrence,
pancreatic, gastric monitoring therapy
and colon
Tg Thyroglobulin Thyroid Concentrations of proteins Monitoring therapy
37
38 A. Javed et al.

2.4.1.2 Microarrays: Gene Expression Profiling

Microarray is basically an arrangement of nucleic acids attached to a solid surface
and it can be used to detect expression of different nucleic acids (DNA, mRNA,
miRNA, circRNA, etc.). Recently, circulator RNAs microarray was used to discover
novel circulating biomarkers for diagnosis of gastric cancer.

2.4.1.3 Genome-Wide Association Studies

Genome-wide association studies or GWAS is used to identify linkage between
genotype and phenotype and it can be used to associate a genetic variant with a
particular disease (Tam et al. 2019). This approach has proved to be effective in
particular with respect to breast cancer, where it has been used to associate many risk
factors and biomarkers to this particular disease (Walsh et al. 2016).

2.4.2 Proteomics (Cytoplasmic and Membrane CBMs)

2.4.2.1 Western Blotting

Western blotting is an important procedure for the immunodetection of proteins
particularly less abundant proteins after electrophoresis (Kurien and Scofield 2006).
Diagnostic and therapeutic biomarkers for hepatocellular carcinoma, ovarian cancer,
and breast cancer were discovered using western blotting (Cho 2007).

2.4.2.2 FACS
Fluorescence-activated cell sorting or FACS is a technique which is utilized to sort,
detect, and count fluorescently labelled cells. Recently, a better technology has been
devised, intelligent image-activated cell sorting (iLACS), which is a machine intel-
ligence technology and has the capacity to analyze fluorescence-intensity profiles as
well as multidimensional images of the cells and hence can sort cells and their
components more efficiently (Isozaki et al. 2019).

2.4.2.3 MALDI-TOF
MALDI-TOF or matrix-assisted laser desorption/ionization-time of flight is an
inexpensive technique which can be used with mass spectrometry to analyze protein
composition of a tissue and it has been proven valuable in discovering novel
biomarkers of gastrointestinal cancer, cancer of respiratory system, breast cancer,
ovarian, and has the potential of discovering many more valuable biomarkers in
other types of cancer (Rodrigo et al. 2014).

2.4.3 Bioinformatics (Predictive/Deduced CBMs)

2.4.3.1 Molecular Docking

Molecular docking is a tool which can be used to analyze interaction between two
molecules (Morris and Lim-Wilby 2008) and hence can show us whether two
molecules are likely to interact in in vivo conditions or not. Many tools are available
2 Technologies for Identification and Validation of Cancer Biomarkers 39

online to perform molecular docking, of which one is HADDOCK 2.4 (High

ambiguity driven protein–protein docking), it uses information of already identified
or predicted protein interfaces in ambiguous interaction restraints and dock proteins
accordingly (Van Zundert et al. 2016) and is different from ab-initio methods.

2.4.3.2 Simulations
Simulations or molecular dynamics (MD) simulations is a basic tool for evaluating
biomolecules and biomolecules interactions that were generated through in-silico
approach (Hansson et al. 2002). For MD simulation, many software and servers are
also available, for example, CABS-flex 2.0 which is an online server for quick
modeling of protein structural flexibility (Kuriata et al. 2018) and GROMACS which
is a software to simulate Newtonian equation of motions on particles (Van Der Spoel
et al. 2005).

2.4.3.3 Molecules-Interaction Network Analysis

TargetScan and STRING are just an example of servers that can be used to visualize
interaction of miRNAs with their targets and proteins with proteins respectively
(Agarwal et al. 2015; Szklarczyk et al. 2019). These interactions can be used to
analyze and predict biomarkers.

2.4.3.4 Support Vector Machine Learning

The support vector machine (SVM) learning, which is a supervised learning method,
utilizes a collection of labeled training data to generate input–output mapping
functions (Wang 2005), or in simple words has the advance ability to classify things
through its learning abilities. It is a powerful classification tool that can be used to
discover new biomarkers (Huang et al. 2018). ISOWN is a program based on this
approach (Kalatskaya et al. 2017).

2.4.3.5 Integrated Databases

The Cancer Genomic Atlas (TCGA) dataset contains molecular characteristics of
33 different types of over 20,000 cancer and matched normal samples. TCGA and
other similar databases are used by ISOWN. OncoMX is also a database more
focused on biomarkers which consists of literature from different databases such
as EDRN, Bgee, BioXpress, Reactome, and BioMuta (Singleton and Mazumder
2019).

2.4.4 Metabolomics

To detect cancer, predict response to different therapies and predict or monitor

cancer recurrence, metabolites released as a byproduct by any metabolic pathway
or during tumor growth can be used as a cancer biomarker. During cancer occurrence
and development, specific metabolites expression changes due to which they can be
used as biomarkers for cancer (Cardoso et al. 2018; Haukaas et al. 2017; Winter et al.
2003; Zaimenko et al. 2017). These biomarkers can be detected in circulatory fluids
40 A. Javed et al.

like blood and CSF, excretory fluids like urine, saliva and by the tissues itself
(Cavaco et al. 2018; Hadi et al. 2017; Harvie et al. 2016; Jagannathan and Sharma
2017). The exploration of the cancer metabolome appears to be an effective
approach to analyze the phenotypic variations connected with tumor proliferation
because metabolome is a strong representative of phenotype compared with genome,
transcriptome and proteome (Holmes et al. 2008). Metabolite markers are different
from traditional biomarkers (e.g., biochemical indices) and rely on various analytical
techniques with includes nuclear magnetic resonance spectroscopy and mass spec-
trometry. Various metabolite markers have been identified until now. One of them
thoroughly studied is 2-hydroxyglutarate (2-HG) which is being identified in many
types of cancer which includes breast cancer, renal cancer, papillary thyroid carci-
noma, and AML and is a product of IDH1 and IDH2 mutation (Borger et al. 2014;
Dang et al. 2009; Fathi et al. 2014; Kanaan et al. 2014; Montrose et al. 2012; Rakheja
et al. 2011; Shim et al. 2014; Wang et al. 2013).

2.4.5 Epigenetics Biomarkers

Heritable changes occurring at the molecular level in the cell are primarily due to
alterations in the nucleotide sequence, as deciphered clearly by the human genome
project. However further analysis has now led scientists to discover the importance
of the other components of the human genome that can alter how phenotypes are
expressed. These includes the epigenetic mechanisms like DNA methylation and
histone modifications as well as the role of non-coding RNA.
These changes maybe because of external (environmental effects) or internal
mutations by controlling trigger zones on the DNA, i.e., repressor proteins. These
epigenetic factors have been identified to play a major role in various malignancies
and thus maybe used as potential biomarkers for tumor identification, progression,
and recovery (Kamińska et al. 2019). Bisulfite sequencing is a valuable technique to
analyze DNA cytosine methylation. After bisulfite treatment of the sample, PCR
amplification is performed which converts unmethylated cytosines into thymine
(Xi and Li 2009).
Therefore, whatever the genetic sequence the final phenotypic expression
depends on how the mutations are translated and hence the term epimutation.
Epimutations is heritable and is associated with repression of genetic activity in
somatic and in some cases germ cells.
The Human Epigenome Project (HEP) has evolved and expanded to add data to
the ENCODE database (Encyclopedia of DNA elements) and the Cancer Genome
Atlas (TCGA) with 212 cell culture lines. Covalent modifications of DNA or its
histones (chromatin) play central role in epigenetic inheritance. This section shall
investigate epigenetic markers in the field of oncology as under:

2.4.5.1 DNA Methylation: Aberrations

Both hyper and hypomethylation of promoters can silence important tumor suppres-
sor genes. Since its first discovery in 1983 there has been immense progress in
2 Technologies for Identification and Validation of Cancer Biomarkers 41

developing in vitro diagnostic (IVD) assays for cancer screening and progress. DNA
methylation is important in reprogramming the predetermined genetic makeup. Post
fertilization there is loss of the original methylation from the paternal side and some
from the maternal, erasing epigenetic memory of the parents and then later on
re-methylation introduces a phenotype very specific and tailored to the new individ-
ual or offspring (Bradbury 2003). The two major known regions for methylation to
occur are the promotor region and the CpG-rich region (cytosine residues)
converting cytosine to 5-methylctosine. They silence the non-coding promoter
sites and attract methyl-CpG-binding domain proteins (MBD).

2.4.5.2 Histone Posttranslational Modifications

Histones are made up of amino acids and once the amino acids are changed, the
shape is modified and thus a new lineage-specific transcription is continued after cell
division. Modification of histone by methylation and acetylation lead to euchromatin
whereas, phosphorylation and deacetylation, heterochromatin that is condensed and
inactive. Global histone acetylation modifications are potential markers of tumor
recurrence with a better prognosis as compared to global methylation.
Thus based on these, patient can be classified into two subtypes, but as it is more
dangerous minute modifications such as Lys16 and Lys20 hypomethylation is
considered characteristic of human tumor cells (Shain and Pollack 2013), for
example breast cancer with these modifications has a worse prognosis (Elsheikh
et al. 2009). The presence of isoforms of histone also upsurge the tendency of cancer
as in overexpression of H2A.Z in prostate and bladder tumors (Monteiro et al. 2014).
Increased levels of circulating histones because of cancerous cell death or vigorous
release are an indication of tumor progression and are a non-invasive biomarker to
predict tumor response to chemotherapy as well. Upregulation of H3Cit histone have
been documented in predicting short-term mortality (Thålin et al. 2018).

2.4.5.3 Chromatin Spatial Modifications

One of the chromatin remodeling complex, the Switch/Sucrose Non-Fermentable
(SWI/SNF) is mutated in a wide range of cancers from ovarian, gastric to pancreatic
(Shain and Pollack 2013).

2.4.5.4 MicroRNAs
These are non-coding RNAs that regulate various biological functions and each
miRNA targets approximately 200 or so messenger RNAs (mRNAs), thus inhibiting
translation. These miRNAs are regulated by either CpG islands or histone
modifications. miRNAs act as biomarkers from both tumor tissue and body fluids
like blood, CSF, urine, and saliva. Thus, the study of circulatory miRNAs in liquid
biopsy’s samples delivers encouraging biomarkers’ platforms for non-invasive-
based diagnosis in many human cancers. The detailed role of miRNAs as prognostic,
predictive, and diagnostic factor is give in Table 2.4.
 42

Table 2.4 The predictive, prognostic, and diagnostic role of different epigenetic markers in the field of oncology
Invasive/non-
invasive Biological
Epigenetic biomarkers Prognostic Predictive diagnostics material Target cancer
Methylation
MLH1 hypomethylation +
Invasive FFPE Colorectal cancer (Jass 2007; Weisenberger et al.
2006)
MGMT hypermethylation + + Invasive FFPE Glioblastoma (Wick et al. 2012)
IDH1 p.R132H mutation and MGMT +
Invasive FFPE Glioblastoma (Roszkowski et al. 2016)
hypermethylation
RB1 hypermethylation +
Invasive FFPE Retinoblastoma (Livide et al. 2012; Ohtani-
Fujita et al. 1997)
GSTP1, RASSF1, APC methylation status +
Invasive FFPE Prostate cancer (Partin et al. 2014; Stewart et al.
2013)
SEPT9 +
Non-invasive Blood Colorectal cancer (Mikeska and Craig 2014;
Wang et al. 2018)
Lung cancer (Powrózek et al. 2014)
MGMT-STP27
+ Invasive FFPE Oligodendrogliomas and oligoastrocytomas (van
den Bent et al. 2013)
ESR1
+ Non-invasive Blood Breast cancer (Mastoraki et al. 2018)
ZNF331 +
Invasive FFPE Colorectal cancer (Vedeld et al. 2018)
SALL1
+ Invasive FFPE Head and neck cancer (Misawa et al. 2018)
Histone modifications
H3Cit +
Non-invasive Blood Advanced cancers (Thålin et al. 2018)
cf-nucleosome epitope combination +
Non-invasive Blood Colorectal cancer (Rahier et al. 2017)
H3K4me3 and Wdr82 expression +
Non-invasive Blood Colorectal cancer (Liu et al. 2018)
Chromatin conformation
ARID1 (ARID1A and ARIDB) +
Non-invasive Blood Neuroblastoma tumors (Sausen et al. 2013)
Methylation status of CTCF locus +
Invasive FFPE Colorectal cancer (Liu et al. 2017)
A. Javed et al.
 2

SMARCA4/BRG1
+ Invasive Frozen Non-small cell lung cancer (Bell et al. 2016)
tissue
miRNA
miR-21 + + Invasive/non- FFPE/ Multiple types of cancers (Jansen et al. 2005;
invasive blood Larrea et al. 2016; Leupold et al. 2007; Resnick
et al. 2009; Ryu et al. 2011)
miR-30d, miR-21 +
Invasive FFPE Non-small cell lung cancer (Czubak et al. 2015)
miR-31-3p + + Invasive FFPE Colorectal cancer (Manceau et al. 2014)
miR-106a, miR125a-5p, miR-129-3p, +
Invasive FFPE Non-small cell lung cancer (Gilad et al. 2012)
miR-205, miR-21, miR-29b, miR-375,
miR-7
miR-29a, miR-92a +
Non-invasive Blood Colorectal cancer (Huang et al. 2010)
miR-506, miR-4316 +
Non-invasive Blood Colorectal cancer (Krawczyk et al. 2017)
miR-126, miR-145, miR-210, miR-205-5p +
Non-invasive Blood Non-small cell lung cancer (Leng et al. 2017)
miR-149-3p, miR-150-5p, miR-193a-3p +
Non-invasive Blood Melanoma (Fogli et al. 2017)
miR-200 family, miR-17 family
+ Non-invasive Blood Prostate cancer (Lin et al. 2014)
miR-17, miR-155 +
Invasive FFPE Non-small cell lung cancer (Czubak et al. 2015)
Technologies for Identification and Validation of Cancer Biomarkers
43
44 A. Javed et al.

2.4.6 Microbiomics Biomarkers

Omics technologies are promising contributors towards the discovery of biomarkers.

The path towards the development of personalized medicines is paved by the
discovery of relevant biomarkers under the umbrella of omics technologies
(Quezada et al. 2017).
The microbial communities resides over and inside human body consisting of
bacteria, viruses, fungi and archaea. They are termed as “microbiota/microflora” and
encoded genes are called “microbiome” (Schwabe and Jobin 2013). Maintenance of
homeostasis and shielding effect against pathogen are highlighted roles of
microbiomes (Shreiner et al. 2015).
In 2007, Human microbiome project (HMP) brought the importance of
microbiome in limelight through bioinformatics approaches. The major outline
was to manipulate the components of microbiome to trigger immunity responses
against deadly diseases (Clemente et al. 2012).
However, the disturbances or alterations in microbiome are directly proportional
in triggering different cancer. Even a single alteration in microbiota can lead to
drastic consequences (Bultman 2014). A continuous evolving microbiome has been
recognized as playing a crucial role in carcinogenesis at a molecular level. One of the
penalties in coexisting with these bacteria, fungi and viruses is the potential silent
hazardous effect on human health. Thus, elaborating the taxonomy of theses
microbes and understanding their basic mechanisms can we shed a light on the
role they play not only in disease development but also in reversing these to become
therapeutic agents and diagnostic tools (Singh et al. 2015).
Different composition of microbiota in multiple organs in human reflects the
variability of inflammation responses and carcinogenesis in different body parts.
Additionally interpersonal alterations of microbiome compositions at various loca-
tion within the same organ can also lead towards cancer (Huttenhower et al. 2012).
The susceptibility of cancers also varies with the presence or percentage of
microbiome in multiple organs. The higher densities of microbiome in large intestine
are indicators of higher risk of cancer compared to small intestine (Breitbart et al.
2008; O’Hara and Shanahan 2006).
The variety of microbiome along with metabolites are present in body fluids, i.e.,
blood, saliva, urine, and cervicovaginal discharge is a promising factor in proving
microbiome as novel as well as non-invasive cancer biomarkers (Farrell et al. 2012).
For example, in non-small lung cancer, the higher percentage of hippuric acid
metabolite was marked in PD-1 blockade therapy responders as compare to
non-responders. Therefore, hippuric acid can act as “combinatorial biomarker” for
the screening of patients for cancer immunotherapy and others are directed towards
different therapies (Hatae et al. 2020).
The advent of next-generation sequencing technology has permitted us to further
explore the inter-relationship of the disease, host, and microbe triad especially so in
the gut microbiomes elaborating their role in cancer via direct or even immunologi-
cal mechanisms. Any imbalance of these factors or dysbiosis is then linked with a
plethora of diseases, including cancers and so these microbiomes may in future be
2 Technologies for Identification and Validation of Cancer Biomarkers 45

used as markers for cancer diagnostic. This has led to a rapid expansion of the study
of DNA of microbes or microbiomics (Feng et al. 2020).
Though many studies have identified these pathogens in different cancers it is still
not clear whether these are a cause or effect of these cancers. Do these proliferate
under the influence of the tumor cells or lead to the growth and progression of these
cancers? In either case identifying and using these as markers may help track the
prognosis of disease or even be possible routes for targeted therapies.
There are however many challenges because of the complexity of the
technologies involved for example, in case of gut microbiota, whether the sample
is from stool versus biopsy samples, correctly defining the genes and finally under-
standing the source of microbial genes because of this being a very young field
(Cong and Zhang 2018). To overcome the insufficient biomass as well as contami-
nation and variability of kits, repetition is the best possible way to validate and
substantiate the findings across labs and microbiomes.
The most studied microbiome is the gut microbiome and it has shown in some
cases that treatment with simple antibiotics can lead to reversal of tumors like
Helicobacter pylori-induced gastric mucosa-associated lymphoid tissue (MALT)
and lymphoma using lansoprazole 30 mg, amoxicillin 1 g and clarithromycin
500 mg (PREVPAC) (Stolte et al. 2002). By creating enzymatically active protein
toxins, directly inducing host cell DNA damage or interfering with critical host cell
signaling pathways of cell proliferation, apoptosis, and inflammation, certain bacte-
rial species can have a pro-tumoral effect (Fiorentini et al. 2020).
The mechanisms that the carcinogenic microbes employs are shown in Table 2.5
(Goodman and Gardner 2018).

2.4.7 Cancer Imaging Technologies

Imaging technologies are used commonly to detect and categorize cancer. Imaging is
performed widely to stage cancer, to monitor cancer therapy, to detect disease
recurrence, or for surveillance purposes (Dregely et al. 2018).
In oncology, Image Biomarkers (IBs) that are used commonly include clinical
TNM (tumor, node, metastasis) stage, objective response, and left ventricular ejec-
tion fraction. Beside these other biomarkers that are used extensively in cancer
research and drug development are MRI, CT, PET, and ultrasonography biomarkers
(O’Connor et al. 2017). In the diagnosis, staging and treatment of cancers, the
imaging modalities range from radiological X-rays, computed tomography
(CT) and magnetic resonance imaging (MRI) to ultrasound (US) and radioactive
single-photon emission computed tomography (SPECT), positron emission tomog-
raphy (PET), and optical imaging. Imaging in cancer is still poor despite advances in
other aspects of diagnostic radiology unless tumor-to-background ratio improves by
2–4 times with increase efficiency in sensitivity and contrast agent targeting
(Frangioni 2008).
46 A. Javed et al.

Table 2.5 Description of carcinogenic microbes’ mechanisms

Hallmark Microbes MOA Source cited
Tumor- Gram-negative TLR2 and TLR4 mediated Garrett (2015),
promoting strain broadly overexpression of innate Zitvogel et al.
inflammation inflammation (2016)
Initiation of IL-17/23 pathway
cytokines
Clostridium species Production of DCA from bile Yoshimoto et al.
and induction of IL-1β and (2013)
IL-6
Toxigenic Induction of IL-17 cytokines Wu et al. (2009)
Bacteroides fragilis and Th17 skewing
Fusobacterium Increase in IL-6, IL-8, IL-18 Kostic et al. (2013),
and NF-κB signaling Rubinstein et al.
(2013)
Helicobacter Increase in IL-1B, IL-8 and Peek and Blaser
NF-κB signaling through (2002)
CagA
Propionobacterium IL-6, IL-8 and IFN-γ induction Fehri et al. (2011)
acnes
Avoiding Helicobacter Induction of Tregs Peek and Blaser
immune Upregulation of epithelial (2002)
destruction PD-L1
GGT and VacA inhibition of
T-cell proliferation
Fusobacterium Induction of Gur et al. (2015),
immunosuppressive MDSCs Kostic et al. (2013)
Fap2 inhibition of TIGIT
Sustaining Fusobacterium E-cadherin binding and Rubinstein et al.
proliferative nucleatum upregulation of β-catenin (2013)
signaling signaling
P. acnes Increased proliferation and Fehri et al. (2011)
upregulation of COX-2
Helicobacter Reduction of p27 in Peek and Blaser
epithelium, increase in gastrin (2002)
Genome Escherichia coli PKS producing calobactin that Arthur et al. (2012)
instability and induces double-strand breaks
mutation Helicobacter ROS production in response to Peek and Blaser
infection that damages host (2002)
cells

For several cancers, MRI is now the main imaging evaluation method and plays a
key role in management decisions. It is the initial imaging tool for prostate cancer
and myeloma diagnosis; for rectal, cervical, and endometrial cancer staging; and for
hepatocellular cancer response evaluation. A variety of MRI biomarkers are already
identified or are well on their way to being established for oncology evaluation in
clinical practice. These MRI biomarkers include BI-RADS (Breast Imaging
Reporting and Data System), PI-RADS (Prostate Imaging Reporting and Data
2 Technologies for Identification and Validation of Cancer Biomarkers 47

System), and LI-RADS (Liver Imaging Reporting and Data System), to diagnose
breast, prostate, and hepatocellular cancers, respectively (Dregely et al. 2018).
PET (Positron Emission Technology) scans are used for the detection of cancer
and also for the examination of the effects of cancer therapy. It is used to identify
localized biochemical changes at the site of cancer. PET scans show only the
location of a molecular marker, they do not provide anatomical information. It is a
diagnostic test that requires the acquisition of physiological images that are depen-
dent on positron detection. Positrons are tiny particles which emit from a radioactive
substance when administered to the patient (Scaros and Fisler 2005).
Patient receives an injection of radioactive tracers that contain a type of sugar
attached to a radioactive isotope. When cancer cells take up the sugar and attached
isotope, positively charged, low-energy radiations known as positrons emit. The
electrons in the cancer cells react with the positrons and result in the production of
gamma rays. These gamma rays are then detected by the PET machine, which
transforms this information to the form of a picture.
For example, 18F-FDG is a commonly used tracer to detect cancer in clinical
oncology. FDG-PET is very useful in the diagnosis, staging, and monitoring cancer
therapies, particularly Hodgkin lymphoma (Zaucha et al. 2019).
The newer and improved versions of these modalities include PET radiotracers
using Gallium 68, and hyperpolarization MRI using Carbon 13 pyruvate will be
needed to increase sensitivity in diagnosis of cancers. The specificity may be
increased by using cancer-specific targeting ligands like immunoglobulin (Fass
2008).
Cancer treatment using image-guided chemotherapy by MRI, optical tomography
using radioisotopes for neoadjuvant therapies are now changing our approach to
cancer treatment (Table 2.6).

2.5 Emerging Technologies

2.5.1 Circulatory Cancer Biomarkers

2.5.1.1 Circulating Tumor Cells

It is possible to find circulating tumor cells (CTCs) in the peripheral blood of patients
with metastatic cancer. Recently, with the advent of technologies that are sufficiently
sensitive to detect very rare cells, research to enhance the detection of CTCs has
increased considerably. The development of such tools has empowered research into
defining the clinical implications of CTCs and has revealed that the levels of CTCs in
patients’ blood shows a relationship with prognostic outcomes and is a clinically
significant biomarker for patients’ prognosis with metastatic prostate, colon and
breast cancers. Several studies have shown that CTC tracking can be used to assess
patient responses to therapy and to track genetic and phenotypic tumor changes in
real time (Preedy and Patel 2015).
Because of the correspondence with traditional tumor tissue’s biopsy, the word
“liquid biopsy” for measuring the concentration of CTCs in blood was introduced
 48

Table 2.6 New imaging technologies for different cancers types and their advantages and disadvantages
Clinical Cancer/
problem discipline New technology Advantage Disadvantage Reference
Screening Breast Dedicated High resolution and increased Ionizing radiation (same as Boone et al. (2006), Boone and
CT-scan sensitivity 2-view mammogram) Lindfors (2006)
PEM High sensitivity, moderate to Requires IV injection of Weinberg (2006)
high resolution radiotracer
DCE-MRI High resolution and Requires IV injection of Dougherty et al. (2007)
specificity lanthanide chelate
Diffuse optical Non-contrast analysis, safe, Low resolution, low to moderate Carpenter et al. (2007), Hsiang
tomography 3D, quantitative, high sensitivity et al. (2005), Nioka et al. (1994)
(spectroscopy) specificity, able to combine
with MRI
Optical (photon Fast, inexpensive, safe Surface imaging only, Georgakoudi and Van Dam
scattering) interference from blood (2003), Kim et al. (2006), Lovat
et al. (2006)
Optical (multi- Fast, inexpensive, safe Surface imaging only, Perelman (2006)
wavelength interference from blood
spectroscopy)
Optical Fast, inexpensive, safe Surface imaging only, Orfanoudaki et al. (2005)
(autofluorescence interference from blood
spectroscopy)
Optical Fast, inexpensive, safe Surface imaging only, Huh et al. (2004)
(polarization interference from blood
spectroscopy)
Optical coherence Fast, high resolution, safe Limited depth to 2 mm, Bouma et al. (2000), Peter et al.
tomography endogenous contrast only (2005)
Exogenous High sensitivity and Requires IV injection of contrast DaCosta et al. (2005), Kennedy
fluorescence specificity agent et al. (1996), Wallace et al.
(2006)
A. Javed et al.
 2

Virtual CT-based Non-invasive, relatively fast Uses ionizing radiation, Alencar et al. (2007)
colonoscopy difficulty with flat lesions and
small polyps
MRI-based Non-invasive, relatively fast Difficulty with flat lesions and Pickhardt and Kim (2007)
small polyps
Staging PET Replacements for Higher sensitivity and Desired half-life not always Ajaj and Goyen (2007)
SPECT resolution available
radiotracers
Time-of-flight Twofold higher resolution or Limited availability Cherry (2006), Gabriel et al.
detection sensitivity (2007)
MRI Hyperpolarization High sensitivity possible, Relatively short relaxation times Golman et al. (2006), Surti et al.
in vivo tracking of molecule of agents tested to date (2007)
metabolism
PARACEST Higher sensitivity than Sensitivity not yet adequate for Jonischkeit et al. (2006)
traditional lanthanide imaging receptor-based imaging
All Low-molecular Rapid biodistribution and Tumor contact time often Handl et al. (2004), Humblet
weight targeting clearance inadequate et al. (2006), Kelloff et al.
ligands (2005), Mammen et al. (1998),
Misra et al. (2007), Vinogradov
et al. (2007)
Signal Improved SBR Requires endocytosis and Graff et al. (2004)
amplification/ pH-dependent activation
background
Technologies for Identification and Validation of Cancer Biomarkers

reduction
(optical)
Treatment Chemotherapy Image-guided Highly sensitive Expensive, not all tumors Kenmoku et al. (2007), Lordick
treatment FDG-avid, difficult to quantify et al. (2007), Nahmias et al.
(18FDG-PET) log kill (2007)
(continued)
49
 50

Table 2.6 (continued)

Clinical Cancer/
problem discipline New technology Advantage Disadvantage Reference
Image-guided Moderately sensitive Presently unavailable, difficult Kartachova et al. (2004), Kelloff
treatment (99mTc- to quantify log kill et al. (2007), Rottey et al. (2006)
Annexin V)
Image-guided No ionizing radiation Requires intravenous injection Kartachova et al. (2007)
treatment of lanthanide chelate, difficult to
(DCE-MRI) quantify log kill
Image-guided No ionizing radiation, fast, Low resolution, moderate Chou et al. (2007)
treatment (optical safe, quantitative, high sensitivity, difficult to quantify
spectroscopy) sensitivity and specificity log kill
Radiotherapy Ion beam-induced Near real-time feedback on Requires specialized and Cerussi et al. (2007)
PET and PET/CT dose delivery expensive infrastructure,
difficult mathematical modeling
Surgery Optical Fast, real-time, high Poor depth penetration De Grand and Frangioni (2003),
(reflectance NIR sensitivity and specificity (1–3 mm) Figueiredo et al. (2006),
fluorescence) Frangioni (2003), Horowitz et al.
(2006), Ke et al. (2003), Li et al.
(2006), Nishio et al. (2006),
Tanaka et al. (2006)
Optical Depth penetration up to Requires separate acquisition
(tomographic NIR several cm, quantitative, high and reconstruction, low to
fluorescence) specificity moderate resolution, low to
moderate sensitivity
A. Javed et al.
2 Technologies for Identification and Validation of Cancer Biomarkers 51

(Alix-Panabières and Pantel 2013). In comparison to tissue biopsy, the liquid biopsy
offers numerous advantages, for example, efficient and simple pulling out of liquid
sample from patients, cheaper and least painful procedure and low risk for patients
suffering because of its nominal invasiveness. This does not only deliver the
prospect for improved understanding of the underlying biological mechanisms
such as cells’ spreading and metastasis, but also to utilize these types of circulatory
cells as biomarkers for the detection, analysis, and treatment of complete cancer
more efficiently and successfully. Nevertheless, due to the exceptionally low levels
of CTCs in blood and mostly the missing of cancer-specific biomarkers, their
detection still poses a major challenge and holds some limitations upon their
significance in cancer diagnosis. Liquid biopsy has many advantages as compared
to tissue biopsy such as low cost, rapid extraction, and minimal invasiveness. This
not only helps in the better understanding of cancer biology but also helps in the use
of these cells as biomarkers to more effectively diagnose and analyse cancer.
Racila and colleagues described a major scientific breakthrough in 1998 to
identify the extremely rare Circulating Tumor Cells (CTCs) (Racila et al. 1998).
They used antibodies designed against epithelial cell adhesion molecules (EpCAM)
joined with ferrofluids. These were combined with flow cytometry that they
performed as immunomagnetic CTCs enrichment. This method was used for the
origination of the CellSearch® (CS) system that is currently being used frequently
and is the lone CTCs detection method approved by the US-FDA (Marcuello et al.
2019).
For detecting CTC in the peripheral blood of cancer patients, several in vitro
approaches have been reported. However, currently used in vitro techniques, they
have limitations such as less yield and sensitivity. An innovative in vivo CTC
isolation product, the GILUPI CellCollector® can isolate CTC directly from the
circulating blood. It intends to increase the yield while capturing CTC and has been
approved with a Conformité Européenne (CE) mark, for application in solid cancers
and by the China Food and Drug Administration for breast cancer. This new strategy
has been found to have high capture rates for advanced stage lung cancer and can
even detect CTC in ground glass nodule patients as well (He et al. 2020).

2.5.1.2 Circulatory DNA/RNA

The circulatory fluids such as the blood samples carries small quantities of circula-
tory tumor DNA/RNA (ctDNA/ctRNA) released from the primary and metastatic
tumors cells along with the cell-free DNA (cfDNA) from non-malignant cells,
primarily hematopoietic cells. ctDNA can provide a more detailed description of
the range of mutations that could be found in the tumor of a patient as compared to
single tissue biopsy. ctDNA can provide a potential for minimally invasive disease
course monitoring and residual disease evaluation following surgery (Marcuello
et al. 2019).

2.5.1.3 miRNA
MicroRNAs (miRNAs or miR-) are endogenous single stranded non-coding RNAs
that can post-transcriptionally control the expression of hundreds of target genes.
52 A. Javed et al.

There are two main mechanisms by which they can negatively regulate gene
expression, firstly through binding to the 30 -untranslated regions (30 -UTRs) of target
mRNAs, thus inhibiting the translation. Secondly, by binding effective complemen-
tarily to messenger RNA sequences, consequently resulting to their degradation
(Luo et al. 2013; Yang et al. 2015). On the other hand, there is also some data
present that miRNAs can also trigger translation of target mRNAs (Vasudevan et al.
2007).
The initial association between human cancer and miRNA was revealed in 2002
(Calin et al. 2002). MiRNAs can be present alone or in combination with other
proteins in the circulation. In addition, they are able to be released directly into
extracellular fluids and can also be carried with the help of microvesicles (O’Brien
et al. 2018). In 2008, Chim et al. found placental miRNAs in maternal plasma,
making it first principal research on miRNAs in biological liquids (Chim et al. 2008).
Subsequently many studies were conducted for characterization of miRNAs in fluids
as biomarkers.
MiRNAs possess many distinctive features that makes them as ultimately
non-invasive cancer biomarkers. Cancer-specific miRNAs are extra stable and
resistant to storage, their sequences are conserved throughout different species,
they can be identified by cutting-edge technologies in small amounts of samples
with high specificity and reproducibility, and are found in many biological fluids
(e.g., blood, breast milk, amniotic fluid, saliva, feces, tears, urine) that makes their
detection easy and minimal-invasive (Mitchell et al. 2008).

2.5.1.4 Exosomes
In both natural and pathological conditions, exosomes are released by cells. These
exosomes carry nucleic acids and proteins which are the indicators of the patho-
physiological conditions and hence can be used as biomarkers in clinical diagnostics.
Tumor cells release exosomes which contain tumor-specific RNAs that can serve as
potential biomarkers for cancer diagnosis. Exosomes include several proteins,
including common membrane and cytosolic proteins, as well as origin-specific
protein subsets that represent cell functions and conditions (Roldán Herrero 2021).
For example, exosomes are highly enriched with tetraspanins, a family of scaf-
folding membrane proteins. The exosomal marker CD63 is also a member of the
tetraspanin family. In 2009, Logozzi and colleagues revealed that plasma CD63+
exosomes were significantly higher in patients with melanoma relative to healthy
controls (Logozzi et al. 2009). All of these circulatory cancer biomarkers and their
promising role in cancer research are depicted in Fig. 2.2.

2.5.2 Drug Repurposing

Repurposing or repositioning involves drugs of which the mechanism of actions is

completely or partially understood. Clinical repositioning studies may also take
benefit of this information and provide predictive biomarkers from initial phase
development or trials. These biomarkers are frequently established among
 2
Technologies for Identification and Validation of Cancer Biomarkers

Fig. 2.2 Depiction of circulatory cancer biomarkers in liquid biopsies and its wide range applications in understanding cancer genomics and proteomics’
instabilities. The biological fluids from cancer patients contain large number of circulatory biomarkers that includes exosomes, circulatory tumor cells from
primary origin or metastatic site, blood cells, different types of micro RNAs, circulatory proteins such as cell surface receptors, enzymes and signaling
molecules, and cells’ free circulatory DNA and RNA released from the tumor site. These circulatory cancer biomarkers offers numerous diagnostic, prognostic
53

and therapeutic applications by analyzing the cancer cells’ chromosomal abnormalities, single cell analysis, RNA expression profile, types and levels of
miRNAs, proteins expression and its phosphorylation, in vitro and in vivo cultures assays, genes amplifications, insertions and deletions, the segments
translocations and other different types of genetic mutations
54 A. Javed et al.

molecules, which are recognized to be involved in sensitivity or resistance to the test

compound. In early drug agent testing, the use of predictive biomarkers may upsurge
the treatment efficacy of the testing agent in question by raising the efficacy of the
test agent in the favorable population of the selected biomarker. In the same way,
drug-induced cytotoxicity in the unfavorable population of the selected biomarker
can be avoided as these clinical trials-involved participants will not be exposed to the
test agent/drug (Stenvang et al. 2013).

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Biomarkers for Cancer Drug Development
3
Gauhar Rehman

Abstract

Cancer biomarkers, which are indicators of cancer cells in the body, can help with
diagnosis, prognosis, and treatment effectiveness as well as recurrence prediction.
We discuss recent advances in cancer therapeutics, as well as the use of
biomarkers and anticancer drug advancements. The discovery and application
of biomarkers will boost oncology drug development efficiency. Preclinical trials
or basic research are often used to identify potential clinical biomarkers of drug
efficacy. The approval rate for oncology drugs is poor, and most of the drugs that
did not receive approval were in late stages of development. In addition to that,
attrition rates are high. Biomarkers have been shown to increase response rates,
progression-free survival rates, and overall survival rates in drug growth. As a
result, the biomarker-based approach seems to be linked to more active drug
programmes, with a shorter time frame and a higher chance of success. The next
wave of advancements in cancer therapy will be guided by the search for novel
biomarkers, innovative designs, and delivery methods for targeted agents.

Keywords
Cancer · Biomarkers · Survival · Growth · Cells death · Diagnosis · Therapy

G. Rehman (*)
Department of Zoology, Abdul Wali Khan University Mardan, Mardan, Pakistan
e-mail: [email protected]

# The Author(s), under exclusive license to Springer Nature Singapore Pte 65

Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_3
66 G. Rehman

3.1 Introduction

Cancer is an important health problem that is the world’s second leading cause of
death. According to last year’s annual statistics of World Health Organization
(WHO), cancer patients and deaths from cancer were estimated to be 18.1 million
and 9.6 million, respectively. Similarly, one in every six women and one in every
five men will develop cancer at some point in their lives. Furthermore, one out of
every eleven women and one out of every eight men will die as a result of it. The
number of patients that were diagnosed from cancer within 5 years was reported to
be 34.8 million. The prevalence and mortality rate of cancer is higher in Asia and
Africa compared to other areas. In most countries, WHO recognizes poor prognosis
and limitations as key factors preventing timely access to diagnostic and therapeutic
agents (Bray et al. 2018). In future there will be globally predicted experience of new
cancer cases and death (Fouad and Aanei 2017). However, further improvement of
research in cancer diagnosis and therapy may help in health care management of
many types of cancers (Kourou et al. 2020). Likewise, understanding mechanisms of
cancer at the molecular and cellular levels can help in early diagnosis and precise
prediction of treatment (Zangooei and Habibi 2017). Furthermore, effective
subtyping of cancer patients into clinically relevant subtypes will aid in the devel-
opment of fair and evidence-based diagnosis and therapy (Wu et al. 2020).

3.2 Cancer Therapy

There are many cancer-fighting strategies ranging from chemoprevention (strategy

to block or slow the onset of premalignant cancers with relatively non-toxic chemical
substances to chemotherapy, radiotherapy, and ultimately surgical oncology) (Zanni
et al. 2015). Previously, major efforts have been made to classify cancers based on
biomarker expression profiles, including mRNAs and protein. (Kim et al. 2021). The
acquisition of tumor resistance to chemotherapy is found in nearly all cases, greatly
reduces its effectiveness, and remains a major challenge for advanced cancer clinical
management. Genetic alterations, improvements in the pH of the tumor microenvi-
ronment, activation of survival signalling pathways, increased drug efflux by ABC
transporter proteins, or the proliferation and development of tumor cell
subpopulations that are inherently immune can all lead to multidrug resistance
(Dlugosz and Janecka 2016).
There is a rapidly rising interest in developing molecularly focused therapeutics
that block or activate unique signalling pathways of tumor cells in order to enhance
cancer treatment outcomes. The US Food and Drug Administration (FDA) has
licenced more than 80 molecularly targeted oncology medications to treat multiple
human malignancies over the last two decades. These targeted therapies include
small molecules and monoclonal antibodies aimed at blocking specific pathways that
drive carcinogenesis and tumor development. They have different mechanisms of
action: inducing cancer cell programmed cell death (apoptosis), blocking particular
enzymes and receptors of the growth factor involved in the proliferation of cancer
3 Biomarkers for Cancer Drug Development 67

cells, or altering the role of proteins that control gene expression and other cellular
functions. Among these therapeutic targets, the signalling components of human
epidermal growth factor receptor 2 (HER2), epithelial growth factor receptor
(EGFR) and programmed death receptor-1 (PD-1) have contributed to the effective
development of cancer therapy powered by molecular markers. These targeted
therapies are promising to enhance patient outcomes by focusing on particular
oncogenic proteins, rather than interacting with all rapidly dividing cells. Therapeu-
tic targets are most likely existent in some but not all tumor cells due to the great
heterogeneity that occurs in cancers, both between and within patients (Wu et al.
2010).
Accordingly, prescient biomarkers are expected to help distinguish subset
populaces that are well on the way to encounter a positive or horrible impact from
these mediations (Nalejska et al. 2014). In clinical settings, an approved prescient
biomarker is assessed utilizing in vitro buddy indicative gadgets (IVDs) which give
data vital for the protected and compelling utilization of a relating restorative item
(Lee and Shen 2015).
Over the last few decades, there has been an explosion in anticancer drug
discovery studies, ranging from novel general cytotoxic agents that target malignant
features (such as accelerated proliferation) to the development of more focused
compounds including kinase-targeted small molecules that target addictive
oncogenes specifically (Hoelder et al. 2012).
Given the vigorous nature of this discovery campaign, and the production and
non-development of thousands of drugs, only 5 percent of leading drug candidates
end up progressing through the health center. Indeed, our capacity to foresee patient
outcomes before reaching clinical trial remains a major constraint on drug discovery
and clinical performance. The best preclinical model will be relatively inexpensive,
suitable for high-throughput screening and, most significantly, match the biology of
human tumors as accurately as possible (Dhandapani and Goldman 2017). The
accurate detection of biomarkers that influences the efficacy of a potential therapeu-
tic aids in improving sensitivity, which in turn improves overall survival rates (Jin
et al. 2019). Precision medicine or customized cancer treatment involves adjusting
antitumor therapy to patients’ specific clinical characteristics, tumor molecular
profiles and related microenvironments in order to treat cancer more efficiently
and with a reduced amount of toxicity (Massard et al. 2017). While molecular and
immunotherapy agents have revolutionized cancer care over the past decade, only a
small percentage of patients react, whereas individuals who react ultimately develop
progressive disease and gain drug resistance. There is therefore a vital need for
robust biomarkers to be identified and validated that can predict resistance or
sensitivity to such treatments. Key insights into the dynamic biology of cancer
growth and progression have been provided by the incorporation of cancer genomic
profiling into clinical practice. However, the implementation of such innovations has
also unintentionally introduced many data analysis-related problems, containing the
problems associated with the detection of driver changes, the target classification of
various levels of evidence, and the selection of rational rehabilitations for patients
with appropriate drugs at the accurate time at the person level (Johnson et al. 2015).
68 G. Rehman

In oncology, where drugs are expensive, life expectancy is limited, and the risk of
drug toxicity is always high, there is an urgent need to recognize and treat the
patients who are most likely to receive assistance from a given drug..

3.3 Biomarkers

World Health Organisation defines biomarker as any constituent, design, or succes-

sion that can be measured in the body or its product and impact or foresee the
occurrence of result or infection (Singh et al. 2020). The utilization of delicate and
explicit biomarkers for sickness determination, forecast, and checking, is an alluring
option in contrast to a significant number of the current strategies being used. The
presence and levels of certain tissue-inferred atomic markers can help recognize
subtypes in heterogeneous sicknesses, for example, malignant growth (Duffy et al.
2017). Cancer biomarkers are either delivered by the tumor or by the body in light of
the tumor (Fig. 3.1).

3.3.1 Biomarkers Discovery

3.3.2 Cancer Biomarkers Classification

Biomarkers of cancer can be classified into the subsequent classes dependent on their
use. Predictive biomarkers predict reaction to explicit remedial mediations, for
example, positive/enactment of HER2 that forecasts reaction to trastuzumab in

Phase I: Preclinical exploratory phase to help identify promising directions

Phase II: Clinical assay and validation phase necessary to evaluate the ability of the
assay to detect established disease.

Phase III: Retrospective/longitudinal phase to determine the putative biomarker’s ability

to detect preclinical disease and to define a ‘‘screen positive rule’’.

Phase IV: Development of prospective screening to identify the extent and characteristics
of disease detected by the test and false–positive rate.

Phase V: Designing of prospective randomized trials to determine the impact of screening

on reducing the burden of disease–related mortality in the general population.

Fig. 3.1 Flowchart of various phases in the biomarker discovery (Kumar et al. 2006)
3 Biomarkers for Cancer Drug Development 69

breast malignant growth (Goossens et al. 2015). Similarly, KRAS-enacting

transformations anticipate protection from EGFR inhibitors, for example, cetuximab
in colorectal malignant growth Van Cutsem et al. 2009). Prognostic biomarker, then
again, may not be straightforwardly connected to or trigger explicit helpful choices,
yet intend to advise doctors with respect to the danger of clinical results, for example,
malignant growth repeat or illness movement later on. An illustration to a prognostic
disease biomarker is the 21-quality repeat score which was prescient of breast
malignant growth repeat and in general endurance in hub negative, tamoxifen-
treated breast malignant growth (Paik et al. 2004). Diagnostic biomarker is another
class of biomarker, utilized to distinguish whether a patient has a particular infection
condition (Imperiale et al. 2014). Customized disease treatment coordinates analytic
biomarkers, prognostic biomarkers, prescient biomarkers, pharmacokinetic, pharma-
codynamic, pharmacogenomic biomarkers and substitute biomarkers show in
Fig. 3.2. (Ileana Dumbrava et al. 2018).

3.4 Biomarkers in Drug Development

Drug improvement in every helpful zone, including oncology, has entered a basic
period driven by the business need of drug organizations to make sure about key
expansions in profitability, reducing the time and cost needed for dispatching new
medications. Low efficiency in medication improvement creates costs that are
progressively dreadful in the present drugs market including the expenses of late-
stage drug steady loss in stage 2 or 3 for lacking adequacy; the expenses of
neglecting to distinguish preclinically those mixes having unmanageable dangers;
and the chance expenses of ending mixes in preclinical advancement for security
issues in light of the fact that accessible logical data is deficient to help sound danger
evaluation and the executives in the canter. Cancer drugs’ improvement has been
advancing enormously all through the past 50 years. Appreciation of sub-atomic
variations and different pathways which lead to the last occasion of harmful cells and
its outcomes has prompted a cycle called customized or accuracy oncology.
Biomarkers have arisen as demonstrative or prognostic devices, yet in addition as
prescient devices of reaction to medicines. By and large, the biomarker is a proxy for
particular focuses to cancer treatments (Lara Gongora et al. 2020).
Industry at present is occupied with a huge activity to create powerful viability
and well-being biomarkers that can be utilized with regards to new medication
improvement techniques to altogether diminish these expenses by supporting
sound “go-on” choices to end unsatisfactory mixes at the soonest conceivable
stage and educated “go” choices and danger the executive’s methodologies to
keep up great medications being developed (Floyd and Mcshane 2004).
Drug improvement dependent on biomarker evaluation is an arising and as of
now settled field of study. Focal points of this technique, for example, viability and
less ideal opportunity for the endorsement of the medication, have been talked about.
There are a few difficulties with regards to biomarkers and medication advancement.
A high steady loss rate is one of these difficulties, particularly in late phases of the
 70

Fig. 3.2 Types of biomarkers in the multistep drug development process

G. Rehman
3 Biomarkers for Cancer Drug Development 71

clinical turn of events. Exploring with recognizable proof of biomarkers should zero
the potential for treatment. Likewise, substitute endpoints must be approved which
can further improve the medication advancement technique. As an outcome,
expenses can be reduced with the identification of early medication viability
(Smith et al. 2014).
The significant difficulties in tumor growth drug improvement are separate
reactions, adequacy, and harmful results. The drug business, drug strategy
producers, and executives are continually searching for novel pharmacogenomic
or pharmacoproteomic examinations that may recognize likely biomarkers to help
take care of these issues. Without a doubt, the atomic objective of most helpful
specialists stays obscure. This has prompted costly turn of events and creation of
malignant growth drugs in light of an absence of data on targets, which can be
utilized to test the adequacy of therapeutics. Novel approaches are expected to
recognize individualized patient advantages of treatments, limit the danger of harm-
fulness, and decrease the expense of treatment. The essential test is which kind of
biomarker to use across the wide range of illness measures. Phenotypic articulation
markers (RNA/protein) shift among cell types and change after some time and show
distinctive posttranscriptional or posttranslational adjustments. Notwithstanding,
proteins are bountiful, effectively open, and show guarantee for estimating results
and contemplating changes in infection state. Another test in portraying biomarkers
is the multifaceted nature of the articulation profile of likely markers in generous
conditions near the sickness aggregates (Bensalah et al. 2007).

3.5 Biomarkers in Cancer Treatment

The significant difficulties in cancer drug improvement are separate reactions,

viability, and harmful results. The drug business, drug strategy producers, and
overseers are continually searching for novel pharmacogenomic or
pharmocoproteomic examine that may recognize likely biomarkers to help take
care of these issues. In reality, the atomic objective of most helpful specialists
stays obscure. This has prompted a costly turn of events and the creation of disease
drugs due to an absence of data on targets, which can be utilized to test the viability
of therapeutics. Novel approaches are expected to distinguish individualized patient
advantages of treatments, limit the danger of harmfulness, and diminish the expense
of treatment. The essential test is which sort of biomarker to use across the wide
range of illness measures. Phenotypic articulation markers (RNA/protein) shift
among cell types and change over the long haul and show distinctive posttranscrip-
tional or posttranslational adjustments. Nonetheless, proteins are bountiful, effec-
tively open, and show a guarantee for estimating results and contemplating changes
in the sickness state. Another test in describing biomarkers is the unpredictability of
the articulation profile of likely markers in generous conditions near the sickness
aggregates (Bensalah et al. 2007).
Presently, cancer therapy is turning into an undeniably customized clinical
intercession. There are numerous prescriptions which follow up on a particular
72 G. Rehman

Fig. 3.3 The biomarkers and responding medications in colorectal, breast and lung cancers (Kuo
et al. 2019)

objective to repress the development of malignancy cells, and the utilization of these
focused on treatments might be led by the occurrence of biomarkers. The normal
biomarkers for malignant growth treatment are introduced in Fig. 3.3. For patients
having breast malignant growth, endocrine treatment is advantageous for patients
with ER/PR articulation, while a HER2 inhibitor ought to be utilized for patients
with HER2 articulation. In like manner, in CRC (Colorectal malignancy) patients, an
EGFR inhibitor ought to be thought of, if tolerant have EGFR articulation without
RAS transformation (Douillard et al. 2013). For patients with NSCLC (Non-little
cell cellular breakdown in the lungs), different TKIs and immunotherapy are the
primary line medicines against sickness with EGFR change, ALK revamp, ROS1
reworking, or customized passing ligand 1 (PD-L1) expression (Reck et al. 2016).
The investigation led by Kuo et al. (2019) expressed that in Taiwan, the recurrence
of EGFR transformation and ALK improvement in patient with NSCLC are 55.7%
and 9.8%, respectively that biomarkers are analyzed before settling on a therapy
choice. For instance, EGFR articulation test for CRC conclusion is repaid by the
NHI, and some other biomarker tests, for example, those for EGFR transformation,
RAS change, and PD-L1 articulation, are typically supported by drug organizations.
Afterward the illness is portrayed for explicit biomarkers articulation, doctors will
settle on an educated choice on most accurate cure for patients. All in all, these
focused on treatments furnish treatment benefits in patients with a particular bio-
marker. The utilization of biomarkers for malignancy treatment choice in Taiwan is
reliable with NCCN and Pan-Asian rules (Kuo et al. 2019).
Meta-analysis of stage one contemplates distributed in a 3-year time frame
was incorporated in an aggregate of 13,203 patients. The creators showed that the
malignancy treatment on biomarker-based procedure was related with a developed
3 Biomarkers for Cancer Drug Development 73

reaction rate (30.6% vs. 4.9% p < 0.001) and a more drawn out middle PFS
(5.7 vs. 2.95 months, p < 0.001) the techniques that did not have the utilization of
a biomarker. Besides, customized arms utilizing a DNA biomarker (genomic modi-
fication) had a higher reaction rate than those with a protein biomarker
(42% vs. 22.4% p < 0.001). All things considered, the biomarker procedure was
still minority among the examinations 16% (58 of 351 arms). The middle treatment
mortality was not measurably unique among biomarker-driven methodology as
opposed to not, guaranteeing security of the system. Endurance couldn’t be gotten
to, as it was not revealed in most of the examinations. Another fascinating result was
that 8 of 9 anomaly preliminaries with reaction rates exceeding 60% was
customized, underscoring the significance of the biomarkers to the recognize
treatments that will address blockbusters in oncology (Lara Gongora et al. 2020).
In the area of cancer research and treatment, the idea of exact medication—
anticipation and management procedures that consider singular changeability—
depends on the improvement of substantial biomarkers grilling key distorted
pathways conceivably targetable with focused on immunologic treatments (Collins
and Varmus 2015). In spite of the fact that biomarkers, for example, prostate-explicit
antigen (PSA), have been known and utilized for quite a long time to endeavor to
direct prognostic and remedial choices, the new upheaval in sub-atomic science,
with the ascent of high-throughput sequencing and expanded sub-atomic portrayal of
tumor tissue has prompted an outstanding expansion in endeavours to quantify and
target distorted pathways at the sub-atomic level. By and by, there has been a huge
hole between different beginning reports of biomarkers, regularly with symptomatic
execution that can’t be duplicated in later examinations, and full clinical usage and
approval of the biomarkers because of issues in investigation, configuration, test
stages, and accessibility of examples for biomarker advancement (Tzoulaki et al.
2011). By the by, with the new development of exceptionally specific sub-atomic
focused on specialists and high-throughput genomic portrayal advances, hearty and
very much approved malignancy biomarkers are progressively required. For exam-
ple, over 90% of oncological medications that enter clinical advancement won’t
arrive at market endorsement because of disappointment of clinical preliminaries to
exhibit helpful advantage, adding to expensive and slow malignancy drug improve-
ment (Paul et al. 2010). As recognized by FDA, the sensible utilization of
biomarkers is relied upon to assume a significant part in limiting danger of clinical
preliminary disappointment by enhancing the preliminary populaces with explicit
atomic subtypes reacting better to tried treatments. (Goossens et al. 2015).
TAA (Tumor-Associated Antigens) or disease biomarker are significant targets
for malignant growth treatments. Neutralizer-based specialists focusing on malig-
nant growth biomarkers incorporate monoclonal antibodies radiolabelled MoAbs,
bispecific T-cell engagers (BiTEs), and counter acting agent drug forms (ADCs). In
the past few years, illusory antigen receptor-designed T cells (CAR - T) has become
a significant discovery in malignancy immunotherapy (June et al. 2018).
74 G. Rehman

3.6 Cancer Biomarkers Currently Available in Clinic

Overexpression/intensification of HER2 (ERBB2), a member of the epidermal

growth factor receptor (EGFR) family, predicts reaction to monoclonal antibodies
such as trastuzumab and pertuzumab in breast cancer, is an example of a molecular
biomarker in use (Goossens et al. 2015). In critical stage III breast cancer
preliminaries, it was discovered that subjects with HER2 overexpression (roughly
20% of patients) infected with anti-HER2 therapy had increased infection-free and
overall stamina (Goossens et al. 20152015). HER2 overexpression is comparatively
prescient of reaction to trastuzumab in esophago-gastric adenocarcinoma (Bang et al.
2010). Other most important prescient biomarkers, remembering BCR-ABL for
ongoing myeloid leukemia and KRAS changes in colorectal disease and various
transformations in non-little cell cellular breakdown in the lungs (NSCLC). Regard-
less of this, numerous other prognostic biomarkers are accessible over the LDT
pathway (Koscielny 2010). Another quality articulation-based test, Oncotype Dx
breast Cancer Assay estimates 21 qualities foreseeing bosom malignant growth
repeat in ladies with hub negative or hub positive, ER-positive, HER2-negative
obtrusive bosom disease (Mamounas et al. 2010). Analytic biomarkers are perhaps
the most assorted classes of biomarkers going from examines produced for malig-
nancy screening to indicative tests surveying movement of a known disease. One
ongoing illustration of a symptomatic biomarker is Cologuard, a multigene DNA
(KRAS transformations, unusual NDRG4 and BMP3 methylation) stool test joined
with fecal immunochemistry intended to screen for colorectal disease in people at
normal danger of colorectal malignancy (Imperiale et al. 2014). These urging results
prompted the endorsement of this test by the FDA in August 2014. As of late, there
has additionally been expanded interest in growing insignificantly intrusive symp-
tomatic tumor biomarkers, utilizing the estimation of flowing DNA or microRNA.
For example, another innovation named malignancy customized profiling by pro-
found sequencing (CAPP-Seq) has been tried on flowing tumor DNA in patients
with non-little cell cellular breakdown in the lungs (Newman et al. 2014).
Biomarkers approved in a particular kind of disease are going through disclosure
and approval in different malignancies (for example BRAF transformations or HER2
overexpression) hidden in certain common oncogenic drivers and less common
tumors are additionally profiting by the fast advancements in the field (Rutgers
et al. 2013).

3.6.1 FZR1 as a Probable Biomarker for NACT in Breast Cancer

In the field of bosom disease treatment, an exhaustive treatment methodology is built

up that included a medical procedure, chemotherapy, radiotherapy, endocrine treat-
ment and atomic focused on therapy (Gass et al. 2018). Careful therapy is as yet the
main methodology for bosom malignant growth. The idea of bosom moderating and
the advancement of bosom rationing a medical procedure is quite possibly the most
noteworthy accomplishments of disease treatment. Right now, Neoadjuvant
3 Biomarkers for Cancer Drug Development 75

chemotherapy (NACT) adds to bosom saving malignancy treatment that chemother-

apy medications are conveyed before a medical procedure to shrivel the tumor.
NACT is decreasing the tumor size by preceding a medical procedure causes less
harm to encompassing tissue and separate the edge of the tumor from solid tissue.
NACT is broadly utilized in breast cancer treatment to downstage privately
progressed (inoperable) sickness and make it operable, especially for huge tumors
(Mougalian et al. 2015). FZR1 is a biomarker of breast cancer NACT dependent on
the transcriptomic information examination and the atomic component examination.
FZR1 is associated with the guideline of the strength and transcriptional movement
of tumor silencer p53. The capacity of FZR1 is to weak the cell apoptosis and cell
cycle capture by chemotherapy drug acceptance. The approval with a companion of
clinical patient examples showed that the declaration of FZR1 can be a biomarker for
the adequacy of NACT. The assessment was performed by the IHC and the evalua-
tion of optical thickness score, which is achievable and appropriate for the medical
application (Liu et al. 2020).
FZR1 is a possible NACT biomarker in breast cancer, and it interacts with the
apoptosis-inducing guideline of chemotherapy drugs.
These outcomes propose that FZR1 influences bosom malignant growth cell
protection from chemotherapeutic specialists by controlling cell cycle capture. It is
accounted for that FZR1 restrains BRAF oncogenic capacities through both
APC-subordinate proteolysis and APC free interruption of BRAF dimers. FZR1 is
considered as a tumor silencer that are adversely controlled by the enactment of the
MEK/ERK oncogenic flagging cascade (Wan et al. 2017). The proof demonstrated
that FZR1 associated with PRL-3 to manage the movement of colorectal malignancy
by controlling the soundness of AURKA (Zhang and Wang 2019). These results are
consistent with our clinical findings that the outflow of FZR1 is linked to the guess
and resilience of patients with bosom disease. FZR1 has been identified as a tumor
suppressor and an oncoprotein in a variety of diseases. Deficiency of FZR1 adds to
the improvement of chemotherapy safe clones in mouse and human B cell intense
leukemia. FZR1 represses the replicative pressure and p53-subordinate cell passing
in neural forebears. Due to all these facts, this information is reliable that FZR1
balanced apoptosis through p53 strength. The FZR1 could be the biomarker that can
be used to demonstrate the efficacy of NACT in apoptosis and cell cycle arrest, that
FZR1 could contribute to recognition of drug for chemotherapy and apoptosis. Our
findings suggest that FZR1 may be a possible biomarker for NACT in breast cancer
(Liu et al. 2020).

3.7 Biomarkers for Preclinical Modelling

3.7.1 Screening Apoptosis

Though preclinical cancer models have their own set of biological challenges,
screening drugs necessitates a concerted effort to identify specific biomarkers that
indicate anticancer viability. Novel phases have been designed as a strategy to
76 G. Rehman

enhance the biomarkers predictive of clinical response or resistance. ChemoINTEL

(unique name MICK Assay), for example, measures the in vitro apoptotic response
of a patient’s tumor to chemotherapy drugs by continuously measuring multiple
biochemical and morphologic apoptotic markers within single cells over a 48-h cell
culture period. ChemoINTEL is a new classification of chemo affectability test based
on medication-induced apoptosis in cell lines rather than the traditional phenotypic
markers that have been used for a long time (Bosserman et al. 2012).

3.7.2 CD20, CD22, CD30, and CD79b as Lymphoid Malignancy

Targets

MoAbs against CD20 have been broadly utilized for lymphoid malignancies
(Marcus et al. 2017). ADCs are progressively utilized as chemoimmunotherapy.
Four new ADCs have been endorsed for the treatment of lymphoid malignancies:
brentuximab vedotin focusing on CD30, inotuzumab ozogamicin and
moxetumomab pasudotox is focusing on CD22 and polatuzumab vedotin focusing
on CD79b (Kantarjian et al. 2016). More biomarkers are being focused with ADCs
or CAR-T cells. These biomarkers incorporate CD25, CD37, CD56, CD70, CD74,
and CD138 (Yu and Liu 2019).

3.7.3 CD33, CD123 and CLL-1 as Focuses for Myeloid Malignancies

Gemtuzumab ozogamicin (GO) is an ADC against CD33 that is generally

communicated on myeloid cells. GO has been affirmed for recently analyzed just
as hard-headed/backslid (RR) intense myeloid leukemia (AML). GO might be
utilized as a solitary specialist or in blend with chemotherapy regimens. What’s
more, a few novel ADCs focusing on CD33 are under clinical turn of events. These
incorporate vadastuximab talirine (SGN-CD33A), IMGN779, and AVE9633
(huMy9–6-DM4). ADCs focusing on CD123, for example, IMGN632 and
SGNCD123A, are being tried in clinical preliminaries. Further advancement of
SGN-123A was anyway ended because of well-being concerns. Nibble and ADCs
focusing on CLL-1 are at present going through preclinical or early clinical
examinations for AML. CLL-1-directed CAR-T cells are in clinical preliminaries
for AML treatment (Liu 2019).

3.7.4 Biomarkers for Strong Tumor Immunotherapy

CD133-focused in on CAR-T cells have been utilized for strong tumors counting
cholangiocarcinoma (Glumac and LeBeau 2018). Mesothelin-zeroed in on CAR-T
cells have been represented in mesothelioma, cell breakdown in the lungs, chest
threatening development, gastric sickness and pancreatic danger (Kelly et al. 2012).
Lymphocyte receptor-engineered T cells against AFP and MAGE-A1 have been
spoken to immunotherapy of resistant tumors (Zhang and Wang 2019).
3 Biomarkers for Cancer Drug Development 77

3.7.5 Tyrosine Kinase Biomarkers as Targets of Small Molecule

Inhibitors

Inhibitors of BCR-ABL, JAK2, FLT3, Bruton tyrosine kinase have prompted a

change in perspective in the administration of leukemia. TKIs focusing on an
assortment of tyrosine kinase oncoproteins, for example, VEGFR, EGFR, FGFR,
RET, HER2, MET, MEK have particularly changed the helpful scene of such tumors
as non-little cell cellular breakdown in the lungs, bosom malignant growth, bladder
disease, liver malignancy, and renal cell carcinoma (Liu 2019).
Biomarkers of numerous non-tyrosine kinase oncoproteins are significant targets
additionally for disease treatment. Inhibitors of BCL-2, isocitrate dehydrogenases
(IDH1 and IDH2), PI3 kinase, BRAF, mTOR, PARP and CDK have immensely
extended the armamentarium against an assortment of disease types, for example,
leukemia, lymphoma, melanoma, bosom malignancy, and ovarian malignancy
(Kopetz et al. 2019).

3.7.6 Designing Biomarkers Through Systems Biology for Cancer

Treatment

CD33 could be a myeloid marker and the target of the GO ADC in AML (Hoseini
et al. 2018). In any case, off-tumor harm levels due to clarification of CD33 in
ordinary hematopoietic cells restrain the clinical applications. Right when CD33
quality was taken out from the human hematopoietic stem and ancestor cells
(HSPC), CD33-zeroing in on CAR-T cells expressly slaughtered AML cells without
myelotoxicity in receivers migrated with CD33-invalid HSPCs. Clinically, the
similar philosophy using structures science to plan HSPCs has been attempted in a
HIV+ lenient with outstandingly resolute exceptional lymphoblastic leukemia
(ALL) (Xu et al. 2019). For the present circumstance, CCR-5 of a totally HLA
facilitated allogeneic common benefactor HSPCs were taken out with CRISPR
advancement. The benefactor HSPCs with eliminated CCR-5 were migrated into
the HIV+ lenient. The ALL went into complete decrease with creativity of CCR-5
negative hematopoiesis. The ALL went into total diminish with imagination of
CCR-5 negative hematopoiesis. This framework utilizing system biology and
arranging opens up a few other seasons of fabricated hurt express biomarkers for
centered affliction treatment. Journey for new biomarkers and novel plans similar as
transport methodologies, for instance, nanotechnology of centered experts are
driving the accompanying surge of advances in threatening development treatment
(Liu 2019).
T-PLL (T-cell prolymphocytic leukemia) is a helpless predictive illness with
exceptionally restricted alternatives of productive treatments. Maximum patients
are headstrong to chemotherapies and regardless of high reaction rates after
alemtuzumab, basically all patients backslide. Along these lines, there is a neglected
clinical requirement for novel treatments in T-PLL. As the CCR7 chemokine
receptor is a particle communicated in a wide scope of malignancies and applicable
78 G. Rehman

in numerous tumor measures, the current examination tended to the biologic job of
this receptor in T-PLL. In addition, we clarified the rebellious action interceded by a
foe of CCR7 monoclonal neutralizer (mAb) and surveyed whether its foe of tumor
development would warrant progression towards clinical applications in T-PLL. Our
results appear that CCR7 could be a prognostic biomarker for the most part of
continuance in T-PLL patients and a valuable receptor locked in with the develop-
ment, assault, and continuance of leukemic cells. Focusing on CCR7 with a mAb
controlled ligand-intervened hailing pathways and affects tumor cell executing in
fundamental cases, and planning antibodies against CCR7 also significantly effec-
tive in T-cell leukemia xenograft models. Together, these disclosures make CCR7 an
engaging molecule for novel mAb-based remedial applications in T-PLL, a sickness
where late medicine screen endeavours and considers tending to unused blends have
focused in on chemotherapy or small molecules (Cuesta-Mateos et al. 2020).

3.8 Challenges of Biomarkers in Medical Revelation

The difficulties of biomarkers in medication disclosure and improvement might be

measured at 3 unique points: (1) distinguishing the correct objective of the medica-
tion such as biomarker; (2) approval of the biomarker investigation being referred
(3) advancement of coordinated biomarker-driven medicines (Fig. 3.4). Pushing
ahead, unmistakably these biomarker difficulties will be dependent upon constant
mechanical enhancements. For instance, as DNA sequencing improves and turns out
to be more savvy, entire genome sequencing could be regularly used to recognize
uncommon however exceptionally penetrant germline changes that might actually
prompt atomic screening programs and early discovery of applicable tumors. (Ileana
Dumbrava et al. 2018) (Fig. 3.4).

3.9 Future Recommendation

In the upcoming years, cooperation among clinicians, researchers and administrative

organizations is substitute for a fruitful medication improvement model. To start
with, the improvement of explicit, clinically practised and systematically approved
biomarkers is principal. A critical test is to diminish costs, when the track is
provoking progressively more express biomarkers, limited to a small portion of
patients. Second, the improvement of high viability medicines with insignificant
harmfulness stays a test. Third, old style plan of clinical investigations sequencing
ought to be returned to, to advance the expenses, which right now are impractical,
and to improve the period from medication improvement to patients’ entrance.
Eventually, the administrative offices should accept Manuscript Information Classi-
fication: General invigorate drug advancement and depend on exceptional projects to
quicken the endorsement and admittance to promising medications.
 3
Biomarkers for Cancer Drug Development

Fig. 3.4 Challenges and future perspectives in biomarkers in drug discovery and development. Adapted from Ileana Dumbrava et al. (2018)
79
80 G. Rehman

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Clinical Proteomics: Diagnostics
and Prognostic Markers of Cancer 4
Saima Zafar, Aniqa Saeed, and Saadia Zahid

Abstract

In the recent years, through the introduction of high-throughput technologies to

innumerable fields of medicine, investigation of big cohorts of data is no longer a
problem in the advanced research laboratories. However, designing of coherent
study, obtaining of high-quality data under optimal conditions, and persuasive
elucidation are critical features in ensuring the advancement of decent discipline
available of these current technologies. The emerging field of proteomics have
transformed the practices in the field of cancer biomarkers.
Currently, the application of rigorous biomarker identification and validation
is an emerging arena. These studies emphasize on the multicenter studies homog-
enous procedures for high-throughput targeted MS assays. Furthermore,
advances in MS sensitivity are accessing toward novel tumor-specific
proteoforms comprising posttranslational modifications and variants devising
from genomic anomalies. Moreover, proteomic data complementing the genomic
and transcriptomic datasets imitates the emergent field of proteogenomics, which
shows great potential to surge our understanding of cancer biology.
In this chapter, we will discuss the role of proteomics toward the diagnostic
and prognostics aspects and recent improvements in MS-based clinical

Saima Zafar and Aniqa Saeed contributed equally with all other contributors.

S. Zafar (*) · A. Saeed

Biomedical Engineering and Sciences Department, School of Mechanical and Manufacturing
Engineering (SMME), National University of Sciences and Technology (NUST), Islamabad,
Pakistan
e-mail: [email protected]
S. Zahid
Department of Healthcare Biotechnology, Atta-ur-Rahman School of Applied Biosciences
(ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan

# The Author(s), under exclusive license to Springer Nature Singapore Pte 83

Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_4
84 S. Zafar et al.

proteomics with a focus on oncology. We will deliver a detailed overview of

samples types with clinical relevance, as well as, deliberation for sample prepa-
ration strategies, protein quantitation approaches, MS configurations, and data
analysis pipelines currently available to researchers.

Keywords

Clinical proteomics · Targeted assay · Mass spectrometry · Cancer proteome ·

Diagnostics · Prognostics · Shot-gun Proteomics · Tumor pathophysiology ·
HUPO · Profiling

4.1 Clinical Proteomics

The successful understanding and development of human genome project (HGP)

and other genomic projects has led to a stupendous and massive pathway for
genomic and proteomic studies helping scientists gain a better understanding of
molecular studies (Paik et al. 2008). Clinical proteomics refers to the studies of
proteins and peptides, differing from genomic studies that involves studies of DNA.
The qualitative and quantitative filtering of dynamic protein structures circumscribe
large spectrum and preclinical diagnostic tools for cancer diseases (Apweiler et al.
2009). Eventually proteomics addresses the prerequisite of early diagnosis and the
role of identified therapeutic targets towards suitable and personalized disease
management and therapeutics. Progression of clinical proteomics leads to discovery
of disease biomarkers that becomes cardinal desire in study of proteins to evaluate
disease. However, the recent challenges are in developing exact procedures for both
clinical handling and reduction of complexity and to increase detection ability of
dynamic proteins and peptides that are present in minute amounts. Proteomics
involves the interaction between the proteins and peptides differing from the inter-
action of gene expression level, that explains what genomics is, but a clear disad-
vantage of it is not giving the information and proper expression of the genes so the
gene becomes useless and can only be started well through the proteomic studies
(Fig. 4.1a) (Paik et al. 2008).
The basic concept of a disease biomarker is shifting toward a new exemplar that
explains how a normal protein and peptide differs from the infective one leading
toward accurate diagnosis through clinical studies (Anderson 2005). Clinical prote-
omics is best accessed with fresh and newly collected samples that have proper
mapping of connective tissues and are morphologically consistent and easy to treat
with body fluids of sufficient quantity. Clinical proteomics purposely engage
proteomic methodologies, molecular structures, and vast analysis of bioinformatics
to identify dynamic patterns of protein diseases that results in better assessment for
disease prevention, early diagnosis, and proper selection of treatment methodologies
that vary from person to person but are unique and universal for protein structures
4 Clinical Proteomics: Diagnostics and Prognostic Markers of Cancer 85

Fig. 4.1 (a) Main applications of proteomics, (b) Challenges in identification of biomarkers in
proteomic studies

(Savino et al. 2012). All these steps and studies regarding clinical proteomics have
helped scientists and microbiologists to focus easily on the novel diagnostic and
therapeutic analysis of cancer diseases using biomarkers and dynamic protein
structures for the betterment of human beings.
86 S. Zafar et al.

4.2 Goals and Need for Proteomics

Basic goals of proteomics refer to categorizing presentable dynamic proteins avail-

able in a biological system at a certain point under given reliable conditions like any
drug intake, stressful environmental condition, and mutations. Advancements in
mass spectrometers and MS based methods have resulted in best quantitative
analysis of large number of the samples at least up to 8 in a very short time. This
saves the time of the patients and gives correct analysis and diagnosis on time. What
method is to be chosen depends upon the study’s preference and patient’s comor-
bidity and conditions. Some of the few important methods for proteomic studies
include SILAC, iTRAQ, TMT, and Label-free methods (Rai et al. 2002).
Proteins being the major outfit of biological system gives scientists and
microbiologists an accurate perception on how biological systems adapt under
different conditions. The details of mutations and undergoing changes in a cell,
concerning benign and malignant details of cancer cells and the differences between
cells of different tissues explaining mutations in dynamic protein structures, are all
elaborated during clinical proteomics (Paik et al. 2008). These details are helpful in
better understanding of basic biological mechanisms and undergoing cell mutations,
finding biomarkers and tumor markers that help in early diagnosis of disease and
help in good prognosis after early treatment. Major need of proteomics is in proper
and timely treatment using modalities like targeted therapies and other chemother-
apy drugs after measurements of proteins through biomarkers (Knowles et al. 2003).
Clinical proteomics is the basic need and goal for monitoring the properties and
mechanism of the whole dynamic protein structures from a cell or organism and help
in determining the pathophysiology of these proteins. Proteomics enable us to gain
better understanding of systemic and functional metabolism of proteins even in
various physiological states and conditions like changes in cell cycle and signaling
ligands. Almost all these tasks that are important for cancer diagnosis are not
detected by genomics or transcriptomics and are only observable in studies of
proteins (Zhang et al. 2004). A clear and major example of this is that only proteins
can sense extracellular signals by detecting the exact binding protein to which the
cell responds once the molecules get attached to them. Moreover, mechanisms and
changes that occur when proteins bind with other complex structures including
transport system and signals are not detected by genomic studies. Such systems
require proteomic studies for better analysis and diagnosis of cancer (Fig. 4.1b)
(Gygi et al. 1999).
Many other cellular and molecular processes involving covalent modifications
and phosphorylation of proteins is a magnificent example of why proteomic studies
ae important. Proteomic studies are the basic need when it comes to these processes
as they are invisible and not understandable in genomics and transcriptomics
(Schneider and Hall 2005). For a system to be detected by genomics, mechanism
must be very fast to be covered in milliseconds while these transcription and binding
processes are to slow that becomes the utmost reason of why genomics and
transcriptomics fails and the need for proteomic studies became very important
specially in carcinoma studies. Proteoforms, the expressed form of proteins, defining
4 Clinical Proteomics: Diagnostics and Prognostic Markers of Cancer 87

the most important physiology and disease conditions, act as molecular actors and
cellular phenotype, only accessed by proteomic details. The exemplary mechanism
of regulation of a transcribed mRNA that is further translated during many important
biological systems is also not assessed by genomic studies. These issues have
increased the need for proteomic studies and made them important and prior goal
for diagnosis of cancer studies and biomarker prognosis (Liotta et al. 2003).

4.3 Methods of Protein Measurement and Biomarker

Identification

Studies and research related to protein mutations in cancer diseases have been
studied for the past 70 to 80 years, but no extensive proteomic technique was well
known till then. Detailed proteomic studies and extensive technologies for protein
mutation and biomarker involvement for carcinomas were known since last three
decades and thus have been a source of great help for cancer diagnosis and early
treatment before the patient reaches extreme stages. Combination of clinical proteo-
mics and imaging diagnostic modalities has helped better clinical presentation of
protein biomarkers in different carcinomas like prostate cancer, breast carcinomas,
hepatocellular carcinomas, and rectosigmoid tumors (Knowles et al. 2003).
It is important to discuss the basic methods for protein measurements and
biomarkers identification here that are in best practice nowadays and have made
life easier by reducing death rates of patient suffering from carcinomas.

4.3.1 Bottom-up or Shotgun Proteomics

Bottom-up or shotgun proteomics is one of the most versatile used method of protein
analyzes. Basis of this type of MS-based studies involves isolation of proteins that
are bisected into peptides either chemically or through enzyme involvement methods
(Hoffmann et al. 2001). This involves chromatography and other separation methods
that subdivides the end product mainly through reverse phase chromatography
methods. Final analysis of these resultant peptides is done using MS-based methods
after ionizing them through ESI, electrospray ionization (Hawkridge and Muddiman
2009).
Quantification and exact visualization of these resultant dynamic peptide
structures helps in correct interpretation of disease and measurement of proteins,
thus resulting in early diagnosis of carcinomas.

4.3.2 Mass Spectrometry-Based Proteomics

It is a vast and emerging technique of proteomics that helps in identification and

exact quantification of protein structures along with necessary components that are
essential for life. For better understanding and characterization of the proteins at
88 S. Zafar et al.

different levels including the proteome and sub-proteome levels, MS-based proteo-
mics is used. MS-based proteomics (Hawkridge and Muddiman 2009) and used
chromatographic instruments are sometimes associated with other newly advanced
technologies such as separation of the ions and microchip-based proteome
measurements at microscopic levels that are highly sensitive targeted techniques
for cancer diagnosis. MS-based research and workshop was held in 2013 in National
institute of Health (NIH) in USA that focused on these newly advanced technologies
and approved them as one of the best proteomic methods for cancer diagnosis and
biomarker identifications (Maes et al. 2015).
MS-based proteomics have been widely studied and used in protein analysis.
After several advancements and research, this technique has been widely used for
diagnosis of epigenetic cancers. This is quite simple and helpful as compared to the
previous technique as it extracts the tissue or blood sample and then processes the
dynamic protein structures for further evaluation. Mass spectroscopy helps in
detailed study of the protein mutations the same way as genomics tells the details
of the DNA through microarray images, quite advanced as compared to simple
visualization of sample under microscope (Greenbaum et al. 2002). The use of
MS-based proteomics is considered as one of the best advancements toward epige-
netic biomarkers because of their exemplary involvements with chromatin structures
and histone proteins involving histone posttranslational modifications resulting in
best diagnostic and therapeutic results for cancers. Detection of proteomic
biomarkers through protein microarrays, that are closely related to cancer develop-
ment and prognosis has changed the perspective for cancer research studies. Meta-
static carcinomas and primary tumors are easily diagnosed through MS-based
techniques where all other fails to do so.

4.3.3 Polyacrylamide Gel Electrophoresis (PAGE)

This method involves extraction of proteins on the basis of their molecular masses
and is frequently used in analysis of carcinomas for more than two decades. Very
few advancements have been yet made on this but combining it with MS using
fluorescent gels have helped diagnostic methods to become easier through proteomic
studies (Gharbi et al. 2002). Proteins are separated by electrophoresis using a gel
matrix resulting in migration of smaller proteins rapidly. Rate of this migration and
molecular masses are one of the major factors effecting the results. Mostly PAGE-
SDS technique (Maurer 1971) is used where strong protein denaturing detergents are
present and binds to the protein dynamic structure in a flow. The presence of these
denaturing detergents acts as reducing agents and thus bifurcates the disulfide bonds
that helps in folding and unfolding of proteins and thus best analysis is made on the
basis of resultant linear chains. Biomarkers and protein analysis has made diagnosis
of carcinomas quite easier and faster using these MS techniques (Fig. 4.2).
4 Clinical Proteomics: Diagnostics and Prognostic Markers of Cancer 89

Fig. 4.2 Separation of samples (protein) on the molecular bases and ions processing’s. Processing
of samples (in-gel base and in-fluid base) on the polyacrylamide gels and remove the insoluble
materials by filtration. The proteomic pattern of the samples is then acquired using electrospray
ionization mass spectrometry and further identified and characterized as diagnostic and prognostic
biomarkers of cancer

4.4 Proteomics and Cancer

Cancer being a combination of genetic and environmental abnormalities is mainly

caused by uncontrolled production of cells resulting in necrosis and disrupted
cellular signals (Citti et al. 2018). Large number of pathological cells accumulate
and grow abnormally resulting in genomic and proteomic defects causing mutations
of chromosome and DNA detected by genome structures and mutations of proteins
and peptides analyzed by proteomic studies. Carcinomas are one of the most leading
cause of the death majorly breast carcinomas, hepatocellular carcinoma, prostate
90 S. Zafar et al.

cancers, and rectosigmoid cancers. Mutations in protein structures that results in

cancers are detected by proteomic studies (Somiari et al. 2003).
Carcinomas can easily be treated by preventive measures, early detection, and
adequate therapeutic modalities varying from patient to patient. Major concern of
oncologists is the early and timely diagnosis of cancer either by genomic studies or
by proteomic evaluation (Petricoin et al. 2002). There is no doubt that genomic
studies play an important role in cancer diagnosis but histone modifications,
translations, and peptide mutations are ignored by genomic evaluation and there
one needs complete study of proteomes for less false positive results and accurate
staging of cancers (Haslinger et al. 2004). Genes being the basic controller of any
cell behavior play an important role in cancer diagnosis but protein expressions and
effectiveness explaining molecular basis of any disease are not understood by
genomics. In carcinomas, almost all proteins invade, increases chances of metastases
and respond better to any targeted therapies and controllers after interaction with
surrounding cells (Verrills 2006). Proteins help in understanding the molecular
mechanism of carcinogenesis and cell cycle network involving mitosis and meiosis
thus adequately identifying the signaling network of almost all types of cancers
along with its prognosis. Alterations in normal mechanism in any stage of carcino-
genesis is analyzed best by proteomic studies that accurately enables oncologists and
cancer biologists to better understand circumstances even in all environmental and
carcinogenic changes (Apweiler et al. 2009).
Clinical proteomics concerning with cancer diagnosis is further elaborated as
“Onco-proteomics” that involves protein studies in cancer mutations and
interactions with proteomic alterations. This disciplinary branch of proteomics
encourages better understanding of caner pathology and tumor biomarkers for best
therapeutic results (Fig. 4.3a) (Sallam 2015).
One of the dormant mechanisms for the proteomic alterations in cancer is the
omnipresent aneuploidy, which is known as uncontrolled and imbalanced chromo-
somal structures. Aneuploid cells are resultant outcomes of stressful and toxic
conditions leading to defect in proteo-stasis, disturbing the equilibrium and balance
of dynamic peptides and proteins.
All these details are still under research but a large number of studies have
explained the relationship of defective proteo-stasis and gene mutations. Due to
the abnormal increase of chromosome, any induced protein expression and genetic
expression is not obligatory to be translated (Wang et al. 2004). This increases
chances of protein degradation and chronic issues in folding–unfolding of proteins.
It is known that many proteins and peptide structures, mainly kinases and multimeric
protein complexes, need more requirements for the cellular protein folding, and thus
they are more susceptive to misfolding than others.
Largely available tests for screening of cancers have low sensitivity and specific-
ity thus differentiation of benign and malignant carcinomas is not done to the best of
patient need. This leads to false positive results, (Huang et al. 2020) late diagnosis
and wrong staging resulting is poor prognosis for cancer. The emergence of onco-
proteomics is a source of hope for identification of biomarkers and tumor markers
helping obtain best results after targeted therapies and chemotherapies for cancer
4 Clinical Proteomics: Diagnostics and Prognostic Markers of Cancer 91

Fig. 4.3 (a) Proteomics in cancer research (b) Separation of proteins and usage of MS-based
techniques for the identification of cancer biomarkers. Discovery of proteomics; Plasma and fluids
(CSF and nipple discharge) for aspirations and function of dynamic proteins

patients. Best timely screening of malignancies has led to increased survival rate
after ongoing treatment through accurate and timely diagnosis with the help of
proteomics.

4.5 Early Diagnosis of Cancer

According to previous studies, proteomics deals with quantitative research and

analysis of proteins but many recent studies have concluded the role of proteomics
in structural analysis of dynamic proteins as well as leading to proper investigation
of disease on time. The main goal of any disease-related research and therapeutic
studies is the timely diagnosis of that disease leading to effective treatment
(Hawkridge and Muddiman 2009). Proteomics play a vital role in early diagnosis
of tumors giving world a new pathway in carcinogenesis.
92 S. Zafar et al.

The limitations of less sensitivity in analyzing the structures of proteins during the
study of proteomics is the major hurdle and challenge for oncologists and
bio-scientists. Furthermore, the proteins involved in cellular homeostasis, metabo-
lism and structure are abundant and are present 10 thousand to 100 thousands fold
greater than proteins involved in signaling networks in an individual cell leading
toward great challenges in cell signaling (Yates 1998). The identification of desired
biomarkers for the tumor studies in the field of proteomics have become easier
specially during aspiration studies like pleural cancers, nipple aspirations in breast
cancers and plasma studies due to new research in this field. Considering all fluid
filled structures of human body, plasma specimens are one of the deepest fluids of
human body containing large number of proteomes that help in best study and results
during aspiration while treating tumor patients (Fig. 4.3b). The human plasma
proteome promises the exemplary diagnosis of cancer by covering all major
challenges of proteomics like early detection of cancer and prognosis of disease
(Anderson 2005).
Discovery of biomarkers was initially done using different conventional methods
and proteomic studies. Major methodologies for early diagnosis of tumors were,
(Greenbaum et al. 2002)

• Protein distillation
• Enzyme Linked Immuno-Sorbent Assay
• Western Blot and Gel Electrophoresis

All these methodologies comprehend less specificity and sensitivity thus leading
towards the prerequisite of using advanced technologies. The currently used
analyzers in proteomics for biomarker identifications and early diagnosis of cancer
are, (Maurer, 1971)

• 2D PAGE (Two-dimensional polyacrylamide gel electrophoresis)

• Mass spectrometry
– MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight)
– ESI (Electrospray Ionization)
– SELDI-TOF (Surface enhanced laser desorption/ionization time-of-flight
mass spectrometry) Many relevant studies are apprehensive with the identi-
fication of antigens or biomarkers for diagnostic, prognostic, or therapeutic use
and have become supreme focus of oncologists. Genetic markers detected cyto-
genetically or through detection of changes in gene like mutations, are also now
entering clinical practice, but some changes likely to be important in carcinogen-
esis, (Knowles et al. 2003) and its diagnosis such as abnormal expression of
proto-oncogenes may not be associated with a detectable genetic injury (Fig. 4.2).
4 Clinical Proteomics: Diagnostics and Prognostic Markers of Cancer 93

4.5.1 Diagnostics of Cancer and Proteomic

Cancer proteomics circumscribes the identification and quantitative analysis of many

protein structures that are differentially expressed and damages the healthy tissues of
human body leading to different stages of carcinomas from preneoplasia to neoplasia
and necrosis resulting in malignancies (Wilson and Nock 2003).
Proteomics is of cardinal importance in the discovery of biomarkers and tumor
markers because the proteome reflects both the intrinsic genetic program of the cell
and the impact of its immediate environment. Expression of protein structures and
peptides occur through transcription as well as posttranscriptional and translational
events occurring in cells (Yeoh et al. 2002). According to previous studies, there are
more than 200 posttranslational modifications that proteins could undergo that affect
structure, and function of dynamic proteins. Interactions of protein–protein and
nuclide–protein structures, their stability and their targeting nature, all play vital
roles to a potentially large number of protein resultants from one gene. In cancer and
other mutated diseases, many changes occur at protein level and convert a normal
healthy cell into a mutated one with chances of necrosis. These neoplastic changes
occur and result in tumor necrosis leading to carcinomas. Neoplastic cells are the
resultant of protein modifications, deviant localization, and altered cell functions.
Basis of proteomics is understanding these changes occurring in cell with the help of
MS-based techniques (van der Merwe et al. 2007) and other proteomic technologies
resulting in proper identification of biomarkers helping in early diagnosis of cancers
and better prognosis of targeted treatment therapies.
Research proved that even after all proteomic and genomic efforts, some cancers
metastasize rapidly even after best therapeutic interventions and therapies. One of
the basic reasons behind this is late diagnosis of carcinomas leading toward less
revival rate. Even then, better understanding of disease through onco-proteomic can
lead to therapies that include increasing prognosis and palliation of disease (Ong
et al. 2003).

4.6 Prognostics of Cancer and Proteomics

Even after the extreme advancements in cancer studies, survival rate for most
cancers is not more, and the reason remains the same being late diagnosis and
expensive treatment therapies. The basic goal of proteomics in cancer studies
remains the same where oncologists and surgeons strive for better prognostics and
palliative therapies. Where all other treatment modalities fail, the only solution left is
palliation of disease to increase lifespan of human patients and help in good
prognosis of disease. Estimation of prognosis of the cancer diseases and tumor
patients is the basic need before the start of any treatment (Huang et al. 2020).
Many studies use RNA sequence data from the Cancer Genome Atlas (TCGA)
and Genotype-Tissue Expression (GTEx) after Human genome project (HGP) for
evidence of good prognosis of tumor markers that help in building better prognostic
models (Verrills et al. 2006). It is clear from studies that cancer is not only a
94 S. Zafar et al.

combination of genetic mutations like in DNA, but also involves modifications in

protein structures along with altered metabolic levels and protein expressions.
Mutations in cancer-associated genes can be manifested in defective protein struc-
ture. All known alterations can exert their detrimental impact by causing changes in
stability of protein making the whole structure of protein and peptides permittable
for degradation thus changing the protein’s functional site and affinity that controls
all major protein-protein interactions (Jessani et al. 2002).
Major genomic and proteomic alterations in cancer can be easily augmented by
the recently emerging field of “interactome profiling” that focusses on centering of
main network and provides tremendous data for protein-protein and protein–peptide
interactions. Gulati and his co-workers explained this concept concluding that
network related approaches have become necessary tool for interpretation of
carcinomas (Sallam 2015). They explained diagnostics of carcinomas and elaborated
different modalities that are used for diagnostics of tumor.

4.7 Recent Advances in Clinical Proteomics Methodologies

Presently, the large-scale total protein profiling is not a preferable approach. Instead,
screening of specific subsets of protein biomarkers for cancer is the utmost applica-
ble and significant strategy that may lead toward individualized patient-tailored
therapy (Verrills 2006). Moreover, a wide array of sample types analyzed by clinical
proteomics provided specific disease biomarkers. Although the importance of tissue
samples cannot be marginalized, many studies also employed a noninvasive or
minimally invasive sample collection manner (i.e., liquid biopsies) analyzing
blood (plasma, serum) and urine to reveal novel biological insights of disease
biomarkers and mechanisms. For instance, the urinary proteomics has substantially
attributed for diagnostic and prognostic biomarkers for renal diseases. The exploita-
tion of high-resolution properties of CE-MS has revealed several urinary proteins
from ureteropelvic junction (UPJ) obstruction, a common clinical problem after birth
(Chevalier 2004). Mass spectrometry-based approach offered a large dataset of
urinary proteins associated not only with urological cancers, such as prostate,
bladder, and kidney cancer but also with nonurological cancers, including breast,
gastric, lung, esophageal, cervical, endometrial, colorectal cancer, and other tumors
(Zhang et al. 2018).
Similarly, the plasma proteome, is also potentially suitable for comprehensive
biomarker discovery for various cancers. With advent of high-throughput proteo-
mics technology, the once expected outcome that is the development of panels of
biomarkers for early detection of cancer and likelihood of the probable therapeutic
response is partially achieved. However, due to complexity in plasma proteome
makes it difficult to have a comprehensive dataset of targeted proteins. Perhaps
combined methodological approach can overcomes these hurdles. A combination of
high abundance protein ultradepletion (e.g., MARS-14 and an in-house IgY deple-
tion columns) strategies, extensive peptide fractionation methods (SCX, SAX, High
pH and SEC), and SWATH-MS (Sequential Window Acquisition of all THeoretical
4 Clinical Proteomics: Diagnostics and Prognostic Markers of Cancer 95

Mass Spectra) unravel a subset of 5 proteins from plasma that may represent
different stages of colorectal cancer (Ahn et al. 2019). Also devising an integrated
quantitative proteomics pipeline combining the label-free and multiplexed-labeling
(iTRAQ and TMT) in addition to targeted quantitation of proteins through multiple-
reaction monitoring (MRM) and parallel-reaction monitoring (PRM) mass spec-
trometry is feasible for unbiased screening for plasma-based cancer biomarker
discovery (Kumar et al. 2020). Moreover, SWATH-MS approach applied for
tumor tissues also resulted in the quantitation of thousands of proteins with high
reproducibility, providing novel insights on disease mechanism and biomarker
discovery for various cancer types including soft tissue sarcomas as well (Gao
et al. 2017).
Advances in mass spectrometry-based tools markedly assisted the clinical prote-
omics workflow such as mass cytometry (CyTOF) that enabled high-dimensional
and unbiased characterization of tumor-infiltrating immune populations in cancer.
Mass cytometry, also known as cytometry by time-of-flight (CyTOF), combines the
high-throughput of flow cytometry and the fine resolution of mass spectrometry
(Stern et al. 2018). High-dimensional single-cell proteomics analysis using CyTOF
established a comprehensive dataset of immune cells and stem-like cells, providing
an insight in immunotherapy decisions and prognosis for renal tumors and colon
cancers (Li et al. 2020; Zhang et al. 2019). The high-field asymmetric ion mobility
spectrometry (FAIMS) and trapped ion mobility spectrometry (TIMS) has further
enhanced the peptide detections from routine analysis of cancer cell lines and other
clinically relevant specimens from breast, pancreatic, and ovarian cancers (Hebert
et al. 2018).
In addition, protein microarray technology also significantly contributed to cancer
biomarker discovery. The analytical protein arrays are potentially used for accurate
diagnosis and screening for high-risk individuals with pancreatic and breast cancer.
Similarly, functional protein arrays also have become a popular tool for serum,
plasma, and tissue protein profiling.
Overall, these methodological advancements and modifications enhance the
precision and accuracy of clinical proteomics in cancer research, and making it an
integral part of clinical practice both for prognostics and diagnostic determinations.

4.8 Conclusions and Future Directions

Clinical proteomics initially starts with an investment of large number of questions

and need for searching right samples clinically. New biological and pathological
outcomes of diseases are awaited when employ the modern proteomic approaches.
Timely diagnosis and better understanding of proteomes leads to better prognostic
results. Newly known and functional MS-based technologies have enabled all type
of proteomic modifications even with high specific and sensitive quantitative mea-
surement. MRM-MS is nowadays becoming a more effective introduced technol-
ogy. The technological evolution and confluence of intervention and verification of
96 S. Zafar et al.

Fig. 4.4 Advancements in cancer research by proteomics

these modalities are the main reason why prognosis of tumor patients in getting
better day by day.
Medical research and studies are benefited to a large extent by rapidly growing
use of proteomics making diagnostic and prognostic results of cancer better with
each coming era. Newly introduced therapeutic modalities and interventional
techniques have shortened the span of struggle and increased the lifespan of cancer
patients. The struggles of oncologists and researchers, along with the major role and
support of Human Proteome Organization (HUPO), have increased the better under-
standing of what actually proteomics is especially in tumor studies (Fig. 4.4).
Standard proteomics and use of main proteomic technologies in identification of
biomarkers, accurate diagnosis, better prognosis and immunological targeted
therapies have become the main need of every cancer institute in the recent era.
Moreover, the widely spreading multiplexing technological modality of protein
chips should further increase the multi-planner sources for cancer treatments of
such analytical tests in near future. With emerging recent technologies, proteomics
has the power to impose our understanding of the molecular basis of cancer disease
and to identify novel drug targeting the cells for accurate diagnosis using controllers
and proteomics. Recent advancements help researchers and oncologists to better
identify protein biomarkers and cancer tumor markers that will be used for better
patient care in the near future.
4 Clinical Proteomics: Diagnostics and Prognostic Markers of Cancer 97

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Microbiome as Cancer Biomarkers
5
Bianza Moise Bakadia, Sehrish Manan, Mazhar Ul-Islam,
Biampata Mutu Mukole, Ajmal Shahzad, Ahmed M. E. Abdalla,
Muhammad Wajid Ullah, and Guang Yang

Abstract

Cancer is the leading cause of deaths worldwide due to their complexity, diver-
sity, risk of reoccurrence, and limited therapeutic options, which are further
hindered by the potential side effects. Moreover, the limited potential of detection
tools lead to the severity of different types of cancers. An early detection
definitely increases the chances of recovery and survival rates. To this end,
microbiome, representing the collection of all microorganisms and their genes
that live on and inside the body, is recognized as an important player in the
diagnosis of different types of cancers. Different well-studied human
microbiomes mainly comprising of different bacteria have potential etiological
roles in carcinogenesis and/or modulating the individual response to therapies.
This chapter provides the current knowledge on different healthy microbiome of
different parts of the body and how it is altered during the development of
different types of cancers. Specifically, it discusses the microbiome of intestine,
oral, lung, vagina, gut, uterus, skin, etc. and their role as biomarkers for the
detection of colorectal, pancreatic, liver, breast, lung, cervical, oral and

B. M. Bakadia · S. Manan · A. Shahzad · M. W. Ullah (*) · G. Yang

Department of Biomedical Engineering, College of Life Science and Technology, Huazhong
University of Science and Technology, Wuhan, PR China
e-mail: [email protected]; [email protected]
M. Ul-Islam
Department of Chemical Engineering, College of Engineering, Dhofar University, Salalah, Oman
B. M. Mukole
National Institute for Biomedical Research, Ministry of Health, Kinshasa, Democratic Republic of
the Congo
A. M. E. Abdalla
Department of Biochemistry, College of Applied Science, University of Bahri, Khartoum, Sudan

# The Author(s), under exclusive license to Springer Nature Singapore Pte 101
Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_5
102 B. M. Bakadia et al.

oropharyngeal, and skin cancers. We also provided the current knowledge of

microbiota-based therapeutics and management of different types of cancers
through different therapies.

Keywords

Microbiota · Cancer biomarkers · Gastrointestinal microbiome · Skin

microbiome · Cancer management

5.1 Introduction: Microbiome as Cancer Biomarkers

Microorganisms are generally perceived to be harmful to health because these are all
wrongly assimilated to pathogens (Yang and Yu 2021); however, not all
microorganisms are harmful and some are very useful for humans and perform
different important roles in the body. Microbiome is the collection of all
microorganisms, including bacteria, fungi, and viruses, living on the surface and
inside the body (Temraz et al. 2019). These microorganisms reside in different parts
of the body such as digestive tract, mouth, skin, and other parts. In humans, most of
bacterial communities live in the intestine: the intestinal microbiome is made up of
100,000 billion microorganisms (Temraz et al. 2019). The intestinal microbiota
maintains a strong mutualistic relationship with the host, which depending on the
situation, will be qualified as “eubiosis” or “dysbiosis” (Song et al. 2020; Yang and
Yu 2021). This amazing relationship plays an important role in the regulation of
metabolism such as the chemical composition of brain, provide protection against
pathogens, and regulate the immune system (Sánchez-Alcoholado et al. 2020). It
seems that the intestinal microbiota is one of the major players involved in carcino-
genesis (Yang and Yu 2021). This hypothesis seems to be confirmed over different
studies. The result is dysbiosis of the intestinal microbiota, with abnormal over-
representation of commensal bacterial species (Brüssow 2020). The most relevant
are Bacteroides fragilis, Fusobacterium nucleatum, and Escherichia coli
(Wieczorska et al. 2020). According to published studies, the gut microbiome
plays an important role in modulating the mechanisms of response and resistance
to cancer immunotherapies. Changes in the immune system brought about by the gut
microbiome can be exploited to identify precise targets and bioactive compounds
which could increase the response rates to cancer immunotherapies (Sánchez-
Alcoholado et al. 2020; Wieczorska et al. 2020). The most frequently diagnosed
cancers in the world are those of lung, breast, and colorectal cancer, while most
frequent cancer-causing deaths are due to cancer of lungs, liver, and stomach (Ferlay
et al. 2015). Colorectal cancer (CRC) has for several decades been an important
public health concern (Temraz et al. 2019). It is one of the most common cancers
worldwide, ranks third (10.2%) among different cancers with nearly 1.4 million new
cases diagnosed each year and mortality approaching 50% each year (Temraz et al.
2019). Occupying the third place of the most frequent tumors in men after prostate
and the second place in women after breast cancer, it is the second leading cause of
5 Microbiome as Cancer Biomarkers 103

cancer-caused deaths worldwide (Temraz et al. 2019). It will therefore be possible

that new biomarkers, resulting from the analysis of the microbiome, are emerging as
well as therapeutic or preventive measures aimed at modulating this microbiota, the
final objective of which will be to improve the care of cancer patients. One of the
most promising areas of research is the development of “onco-microbiotics”, or
adjuvant probiotics of the anti-tumor response. The probiotics constitute a new
preventive or therapeutic strategy for patients at risk for CRC. Awareness remains
a redouble weapon for the prevention of CRC, because a particular diet and lifestyle
modification could prevent risk of developing CRC and at the same time reducing
recurrence in people with CRC. Gastric cancer kills a million people worldwide
every year (Ferreira et al. 2018). Another study evaluated the intestinal microbiota
by 16S rRNA gene sequencing among 116 gastric cancer and 88 healthy controls
from China. The intestinal microbiota was categorized by enhancement of
Escherichia, Lactobacillus, and Klebsiella (Qi et al. 2019). A summary of human
microbiome associated with different types of cancers is given in Table 5.1.
This chapter provides a comprehensive overview of microbiome in different parts
of human body and their role as cancer biomarkers. It specifically discusses the
microbiome of intestine, mouth, lung, vagina, gut, uterus, and skin and their role in
detecting different types of cancers such as colorectal, pancreatic, liver, breast, lung,
cervical, oral and oropharyngeal, and skin. Each section in the chapter provides
specific examples of microbiota and their role in detection of specific cancer type.
Moreover, we provided information on the management of different types of cancers
through different types of therapies.

5.1.1 Intestinal Microbiome: Biomarkers of Colorectal Cancer

5.1.1.1 Colorectal Cancer

Colon, also called the large intestine, is the terminal part of the digestive tract. It
measures on average in adults 1.5 m in length and 8 cm in diameter and divided into
four consecutive segments: the right colon or ascending colon, the colon transverse,
the left colon or descending colon, and the sigmoid colon. The big intestine ends in
the rectum. Colon cancer and rectal cancer are commonly referred to as colorectal
cancer (CRC) (Raptis et al. 2015; Yang and Yu 2021). The colon and rectum contain
different types of cells which can each cause a specific form of cancer. In most cases,
CRCs develop from glands called Lieberkühn that line the inside of the colon and
rectum walls. CRC is a malignant tumor in the lining of colon or rectum (Yang and
Yu 2021). It affects all anatomical segments of large intestine, such as cecum,
ascending colon, transverse colon, descending colon, sigmoid colon, and rectum;
however, it does not affect anal canal cancer, which is a separate entity (Raptis et al.
2015).
CRC develops from the cells (healthy mucous membranes) along the lining of
internal colon and rectum due to the accumulation of mutations and modifications
epigenetics (Sobhani et al. 2019). In over 80% of cases, it comes from a benign
tumor, called adenomatous polyp, which grows slowly and eventually becomes
Table 5.1 Human microbiome associated with different types of cancers
104

Control Microbial change in Microbiome

Cancer type Population individuals Patients patients (biomarkers) Sample evaluation References
Colorectal Chinese 54 healthy 74 CRC patients F. nucleatum Feces Shotgun Yu et al.
cancer (CRC) Danish Controls Parvimonas micra sequencing (2017)
Chinese 25 healthy 30 CRC patients Fusobacteria Intestinal lavage 16S rRNA Shen et al.
Controls Proteobacteria fluid gene (2020)
sequencing
Japanese 127 healthy 54 CRC (stage F. nucleatum Feces Shotgun Yachida et al.
controls III/IV) Atopobium parvulum sequencing (2019)
Actinomyces
odontolyticus
Italian, 52 healthy 61 CRC patients Panel of 16 bacterial Feces Shotgun Thomas et al.
German controls markers sequencing (2019)
and
Japanese
Chinese 92 healthy 74 CRC Panel of 22 viral Feces Shotgun Nakatsu et al.
controls markers sequencing (2018)
Chinese 204 healthy 184 CRC Panel of 14 fungal Feces Shotgun Coker et al.
controls markers sequencing (2019)
Japanese 60 healthy 158 CRC F. nucleatum Feces Digital PCR Suehiro et al.
controls (2018)
Chinese 931 healthy 1024 CRC F. nucleatum, Feces qPCR Liang et al.
controls Clostridium (2017), Wong
Bacteroides clarus, et al. (2017),
Bifidobacte, Rium, Xie et al.
Faecalibacterium (2017), Guo
Prausnitzii et al. (2018)
Swedish 66 healthy 39 CRC patients F. nucleatum Feces qPCR Eklöf et al.
controls E. coli pks+ (2017)
B. M. Bakadia et al.
 5

American 343 healthy 251 CRC patients Fusobacterium Feces 16S rRNA Zackular et al.
controls Enterobacteriaceae, gene (2014), Baxter
Lachnospiraceae, sequencing et al.
Porphyromonas, (2016a, b)
Porphyromonadaceae,
Bacteroides
American 23 healthy 21 CRC patients Proteobacteria Feces 16S rRNA Shen et al.
controls Bacteroidetes gene (2010)
sequencing
Pancreatic American – Patients with Proteobacteria , Pancreatic tissue 16S rRNA Pushalkar
cancer (PC) PDAC Actinobacteria, gene et al. (2018)
Microbiome as Cancer Biomarkers

Fusobacteria, sequencing
Verrumicrobia
– Mice with PDAC Helicobacteraceae , Feces qPCR Pushalkar
Bacteroidales, et al. (2018)
Mogibacteriacae
Chinese 25 healthy 30 pancreatic head Haemophilus , Tongues coat 16S rRNA Lu et al.
controls carcinoma Porphyromonas, samples gene (2019)
Leptotrichia, sequencing
Fusobacterium
Chinese Ten healthy Ten PC patients Fusobacterium , Saliva 16S rRNA Sun et al.
controls and 17 pancreatic Periodonticum gene (2020)
disease Neisseria mucosa sequencing
American 371 healthy 361 PAC patients Aggregatibacter , Oral wash 16S rRNA Fan et al.
controls Actinomycetemcomi, samples gene (2018)
Tans sequencing
Porphyromona
gingivalis
Cervical Costarican – 273 women with Gardnerella Cervical Illumina Usyk et al.
cancer HR-HPV MiSeq (2020)
105

platform
(continued)
Table 5.1 (continued)
106

Control Microbial change in Microbiome

Cancer type Population individuals Patients patients (biomarkers) Sample evaluation References
Chinese 131 Women 59 patients with Prevotella bivia , Posterior vaginal 16S rRNA Chao et al.
without HPV cervical persistent Enterococcus durans, fornix samples gene (2020)
Infection HPV infection, Porphyromonas sequencing
139 patients with Uenonis
incidence HPV Lactobacillus iners ,
infection Prevotella disiens
Breast cancer American 48 control 48 breast cancer Clostridiaceae , Feces Illumina Goedert et al.
patients patients Faecalibacterium, sequencing (2015)
Ruminococcaceae and 16S
Dorea rRNA gene
Lachnospiraceae sequencing
Lung cancer Chinese 65 healthy 42 lung cancer 13 biomarkers based Feces 16S rRNA Zheng et al.
controls patients OTU gene (2020)
sequencing
Korean – 20 lung cancer Veillonella Bronchoalveolar 16S rRNA Lee et al.
patients Megasphaera fluid gene (2016)
sequencing
Chinese 18 healthy 24 lung cancer Streptoccocus Paired human 16S rRNA Liu et al.
controls patients Neisseria lung cancer and gene (2018)
tumor tissues sequencing
Gastric cancer Portuguese – H. pylori Gastric biopsies 16S rRNA Ferreira et al.
proteobacteria , gene (2018)
Lactobacillus sequencing
Chinese 81 healthy 116 gastric cancer Lactobacillus , Feces 16S rRNA Qi et al. (2019)
controls Escherichia gene
Klebsiella sequencing
B. M. Bakadia et al.
 5

Oral and American 242 healthy 121 patients with Streptococcus, Oral rinse 16S rRNA Börnigen et al.
oropharyngeal controls oral pharyn geal Actinomyces , samples gene (2017)
cancer carcinomas Corynebacterium sequencing
Endometrial American 75 patients 66 patients with Porphyromas somerae Hysterectomy qPCR Walsh et al.
cancer with EC EC and seven with tissue samples (2019)
atypical
Hyperplasie
American – 17 patients EC, Atopobium Fallopian, MiSeq of 16S Walther-
four patients with Vaginae ovarian, rDNA António et al.
hyperplasia and Porphyromonas peritoneal, and (2016)
ten with benign urine samples
uterine
Microbiome as Cancer Biomarkers

Prostate cancer American – 129 prostate Streptococcus Urine pellet 16S rDNA Shrestha et al.
cancer patients anginosus, samples Illumina (2018)
Anaerococcus sequencing
lactolyticus and 16S
Anaerococcus rRNA
obesiensis, sequencing
Actinobaculum schaalii
Varibaculum
cambriensem
Propionimicrobium
lymphophilum
Bladder cancer Chinese 26 non-patient 29 patients with Actinomyces europaeus Urinary samples 16S rRNA Bi et al. (2019)
bladder cancer bladder cancer gene
sequencing
and qPCR
Chinese 19 62 males patients Micrococcus , Mid-stream urine 16S rRNA Zeng et al.
non-neoplastic with bladder Brachybacterium sample gene (2020a)
controls cancer sequencing,
107

Illumina
MiSeq
sequencing
(continued)
Table 5.1 (continued)
108

Control Microbial change in Microbiome

Cancer type Population individuals Patients patients (biomarkers) Sample evaluation References
Chinese 18 62 males patients Sphingobacterium , Mid-stream urine 16S rRNA Wu et al.
non-neoplastic with bladder Acinetobacter samples gene (2018)
controls cancer Anaerococcus sequencing
Roseomonas
Serratia
Proteus
American 11 healthy 12 males patients Fusobacterium Urine samples 16S rRNA Bučević
controls with bladder gene Popović et al.
cancer sequencing (2018)
MiSeq.
sequencing
Hepatocellular Chinese 56 healthy 30 early HCC, 30 microbial markers Feces MiSeq. Ren et al.
carcinoma controls 45 advanced HCC including Phylum sequencing (2019)
(HCC) Actinobacteria,
Gemmiger,
Parabacteroides
Argentinian 25 individuals 25 HCC patients Erysipelotrichaceae , Fecal stool 16S rRNA Piñero et al.
without HCC leuconostocaceae , gene (2019)
Fusobacterium, sequencing
Odoribacter and MiSeq.
Dorea sequencing
Israeli 25 healthy 30 HCC-cirrhosis Clostridium Fecal stool 16S rRNA Lapidot et al.
individuals Patients, Alphaproteobacteria gene (2020)
38 cirrhotic sequencing
patients without
HCC
B. M. Bakadia et al.
 5

Chinese 100 healthy 113 HBV-related Clostridium XIVa Feces 16S rRNA Huang et al.
controls HCC patients Bacteroides, gene (2020)
Lachnospiracea sequencing
incertae sedis
Italian 20 healthy 21 NAFLD-related Ruminococcaceae , Feces 16S rRNA Ponziani et al.
controls cirrhosis and HCC Bacteroides gene (2019)
and 20 NAFL sequencing
D-related cirrhosis
without HCC
Chinese 15 healthy 21 CHB, 25 LC21 Bacteroidetes Feces 16S rDNA Zeng et al.
controls HCC patients Firmicutes gene (2020b)
Microbiome as Cancer Biomarkers

amplification
and
sequencing
PDAC pancreatic ductal adenocarcinoma, PC pancreatic cancer, PAC pancreatic adenocarcinoma, HPV human papillomavirus, HR high-risk, EC endometrial
cancer, NAFLD nonalcoholic fatty liver disease, CHB chronic hepatitis B, LC liver cirrhosis
109
110 B. M. Bakadia et al.

cancerous (Sobhani et al. 2019). This process, called the adenoma-cancer sequence,
is quite long and can extend over more than 10 years (Sobhani et al. 2019). Usually
10% of cases of CRC are hereditary while 90% are sporadic (i.e., without a family
history). Genetic inheritance or mutation can induce CRC such as syndrome Lynch
or hereditary nonpolyposis colon cancer (HNPCC) and polyposis familial adenoma-
tous (PFA) (Jasperson et al. 2010). Other pathologies such as diseases inflammatory
bowel disease (including Crohn’s disease and colitis ulcerative) can also progress to
CRC (Mattar et al. 2011). Similarly, the environmental factors, as well as risk factors
such as smoking, alcoholism, sedentary lifestyle, physical inactivity, overweight,
obesity, low consumption diet in fiber, excessive red meat or processed meats, would
play a role important in the development of CRC (Song et al. 2020). There is a well-
established link between inflammation and the development of CRC (Kang and
Martin 2017).
CRC is a cancer with a good prognosis when diagnosed at an early stage: 5-year
relative survival is 91% for localized stages and 70% for local stages with loco-
regional invasion (Young et al. 2014). In contrast, the 5-year survival is approxi-
mately 11% in metastatic situations which represent approximately 25% of patients
at time of diagnosis (Young et al. 2014). CRC death rate has fallen over the past
20 years thanks to the progress made in terms of care, i.e., early diagnosis and
improvement of therapeutic modalities (Zavoral 2014; Ogunwobi et al. 2020).

5.1.1.2 Relationship Between Intestinal Microbiota and Colorectal

Cancer
In recent years, researchers have conducted a series of studies aiming to investigate
the involvement of intestinal microbiota in the development of CRC. The CRC
patients exhibit a significant increase in the density of B. fragilis, E. coli,
F. nucleatum, Enterococus faecalis, Peptostreptococus, Streptococcus gallolyticus,
Shigella, and Enterococcaceae or Campylobacter, and a decrease in density of
Bifidobacterium, Blautia, Faecalibacterium, Roseburia, and Clostridium
(Sánchez-Alcoholado et al. 2020). Some of these microbiota are discussed in the
following sections:

5.1.1.2.1 Bacteroides fragilis

Bacteroides are part of the germs of the intestinal microbiota. These are rightly found
in 109 germs per gram of stool. Bacteroides of the fragilis group are Gram-negative,
non-sporulating, and rod-shaped bacilli belonging to the kingdom Bacteria, phylum
Bacteroidetes, class and order Bacteroidales, family Bacteroidaceae, and genus
Bacteroides (Dahmus et al. 2018). Bacteroidaceae family constitutes a very impor-
tant family of anaerobic bacteria, in particular at the level of the intestinal tract.
These bacteria have in common the characteristic of not using oxygen as an electron
acceptor, their growth can even be inhibited in the presence of oxygen. This
indicates that Bacteroides of fragilis group do not develop contact with air, but
rather develop a capsule and have the particularity of fermenting glucose. A strong
growth of B. fragilis is observed in the presence of bile (Dahmus et al. 2018). It is the
most frequently isolated anaerobic bacterial species from humans during soft tissue
5 Microbiome as Cancer Biomarkers 111

infections. Bacteroides are easily recognized by action favoring that the bile exerts
on their growth. People, who consume large quantities of meat have higher
concentrations of Bacteroides in their intestine (Madigan et al. 1997). B. fragilis is
the most frequent opportunistic pathogen among species in the group B. fragilis (other
species include B. thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus,
B. uniformis, and B. acidifaciens) (Könönen et al. 2015). There are two types of
B. fragilis: enterotoxigenic B. fragilis (ETBF) and non-toxinogenic B. fragilis
(NTBF). The spread in the bloodstream “bacteremia” is more common with
B. fragilis than with any other anaerobic bacteria. Infections are usually endogenous,
caused by the gut’s own flora of the patient (Könönen et al. 2015). These infections
give rise to various manifestations, such as pleuro-pulmonary, peritoneal, gyneco-
logical, parietal, or septicaemic, and abscesses intra-abdominals. Studies have
shown that ETBF is also a causative potential for acute diarrhea in children and
adults. Bacteroides produce beta-lactamases which inactivate penicillins and
cephalosporins with the exception of cephamycins. ETBF strains were first isolated
from individuals with diarrhea in an uncontrolled study in 1987 (Myers et al. 1987).
Due to their delicacy, they are difficult to isolate and are often overlooked. Their
isolation requires proper methods of collection, transport, and culture of specimens.
Treatment is complicated by three factors: slow growth, increasing resistance to
antimicrobial agents, and the synergistic poly-microbial infection. Many cultures of
Bacteroides strains form colonies with brown or black pigmentation on blood agar
due to esculin hydrolysis (Balows et al. 2013).
The pathogenicity of B. fragilis is linked to its carbohydrate capsule, proteins of
outer membrane, and production of specific enzymes, including recently recognized
enterotoxin called fragilysin or bft (Sánchez-Alcoholado et al. 2020). This 20 kDa
zinc-metalloprotease toxin is produced only by certain sources of B. fragilis, and it
has three subtypes: bft-1, bft-2, and bft-3. The strains which produce this toxin are
known to be ETBF. An increased prevalence of ETBF has been reported in patients
with CRC compared to controls (Boleij et al. 2015; Sánchez-Alcoholado et al. 2020).
In addition, the presence of ETBF is associated with the stage of the disease. This
bacterium is indeed found more frequently in tumor and healthy tissue of patients at
late stages than early stages of the disease (Boleij et al. 2015). Several mechanisms
of action of fragilysin have been described and may explain, at least in part, the
carcinogenic potential of ETBFs (Dahmus et al. 2018; Niederreiter et al. 2018). The
binding of fragilysin to a receptor, yet to be identified and expressed in level of
intestinal epithelial cells, induces the activation of MAPK signaling (mitogen-
activated protein kinases), NFκB (nuclear factor-kappa B), STAT3 (signal trans-
ducer and activator of transcription-3), and COX-2 (cyclooxygenase-2), and leads to
the expression and secretion of chemokines/cytokines such as TGFβ (transforming
growth factor beta), IL8 (interleukin-8), and PGE2 (prostaglandin-E2), as well as the
development of an immune response of type Th (T-helper). Fragilysin also leads to
the cleavage of ecadherin, inducing the release of βcatenin in the cytoplasm, which
can then pass into the nucleus and activate the transcription of genes involved in cell
proliferation such as the c-myc oncogene (Dahmus et al. 2018; Niederreiter et al.
2018). The cleavage of Ecadherin also leads to decrease in the barrier function of the
112 B. M. Bakadia et al.

colonic mucosa, which could contribute to exacerbation of inflammation (Dahmus

et al. 2018). Mutagenic properties are also attributed to ETBFs, as they are capable of
inducing the expression of spermine oxidase (SMO) which is responsible for
production of reactive oxygen species (ROS) and causing double-strand breaks in
DNA (Dahmus et al. 2018; Niederreiter et al. 2018). The ETBF strains, toxigenic
producers of fragilysin, are able to induce the inflammation and colonic tumor
formation in a genetic model of CRC (MinApc716 +/
mice which expressed a
mutant gene encoding an Apc protein truncated at the amino acid 716 position)
(Wu et al. 2009; Niederreiter et al. 2018). This induction results from activation, by
fragilysin, the STAT3 transcription factor (signal transducer and activator of
transcription-3) with the consequence of the establishment of colitis which is
characterized by a selective Th17-type immune response (T-helper lymphocyte
response-17), known to be associated with a poor prognosis in CRC (Niederreiter
et al. 2018). ETBFs stimulate cell proliferation and develop resistance to apoptosis.
Indeed, by binding to a receptor, yet to be identified, fragilysin induces the cleavage
of Acadherin, involved in both junction and adhesion complexes in cellular and
cytoplasmic sequestration of βcatenin (Wu et al. 2009; Niederreiter et al. 2018).
Released in the cytoplasm, βcatenin passes into the nucleus of cells and activates the
expression of genes like c-myc. Fragilysin could also participate in tumor develop-
ment by promoting the survival of tumor cells. Indeed, in vitro stimulation of cells by
this toxin induces an increased expression of the inhibitory protein cIAP2 (cellular
inhibitor of apoptosis protein-2) and apoptosis which depends on COX-2 cyclooxy-
genase (Kim et al. 2008a; Dahmus et al. 2018). Fragilysin could also actively
participate in causing genetic abnormalities of tumor cells. In fact, in vivo, this
toxin induces colonic epithelial at cell level and DNA damage, which could lead to
the accumulation of mutations (Goodwin et al. 2011).

5.1.1.2.2 Escherichia coli

E. coli is a commensal of human digestive tract and many warm-blooded animals
(mammals and birds). It represents most of the aerobic bacterial flora of intestine
(aerobic species dominant) where it is present at a rate of 108 germs per gram of stool
(total flora: 1011 to 1012 bacteria per gram). E. coli is a prokaryotic organism and
belongs to the kingdom Bacteria, phylum Proteobacteria, class Gamma
Proteobacteria, order Enterobacteriales, and family Enterobacteriaceae (Brüssow
2020).
E. coli being the binomial name belongs to the genus Escherichia. This large
family enterobacteriaceae enter the digestive tract during the first hours or days of
child birth and constitute the most dominant aerobic intestinal flora throughout an
individual’s life. E. coli is a radio-resistant Gram-negative bacillus, rod-shaped,
coccobacillary, or filamentous (in old stumps), and not sporulated. Its size varies
according to the environment, but remains at a fairly short size (2–3 μm) (Brüssow
2020). E. coli is arguably the most studied living organism to date. Owing to its easy
culturing and rapid growth (cell divides every 20 min at 37  C in a rich environment)
make it a study tool of choice. It is a bacterium which has no deaminase, which
5 Microbiome as Cancer Biomarkers 113

excludes it from the genera Proteus, Morganella, and Providencia (typically ex-tribe
of Proteae).
The pathogenicity of E. coli results from a multifactorial process. The modern
technologies of biochemistry, genetics, molecular biology, and cellular microbiol-
ogy have made it possible to identify and analyze the mechanisms involved in the
interaction of pathogenic E. coli with their host. We could thus establish that certain
“specialized” strains of E. coli are associated with various diarrhea, gastroenteritis,
urinary tract infections, meningitis, septicemia, and hemolytic uremic syndrome, as
well as extra-intestinal diseases both in humans and animals. Despite the diversity of
conditions caused by strains of pathogenic E. coli, all these strains use a classic
infection strategy, common to many other agents pathogens. We can distinguish it
from colibacilli, which is a normal host of intestine and does not cause any illness;
nevertheless, it possesses the pathogenic potential under certain circumstances
(opportunistic pathogens) (Brüssow 2020). When it penetrates through the ascend-
ing urethral (contiguity) into the urinary tree, it causes three quarters urinary tract and
swarming infections starting from the digestive system (cholecystitis suppurative,
peritonitis, and sepsis). In addition to these extra-intestinal pathovars (ExPEC), there
are several pathovars within E. coli, whose nomenclature is pathogenicity dependent
(Brüssow 2020).
Pathogenic E. coli could be a cofactor in the pathogenesis of CRC (Sánchez-
Alcoholado et al. 2020). In order to demonstrate the hypothesis that E. coli is
involved in the pathogenesis of CCR, researchers conducted a study. The purpose
of this study was to appear bacterial colonization of the colonic mucosa in patients
screened for a CRC in noncancerous controls (diverticulosis). Given that E. coli
represents the majority of cultivable aerobic bacteria in the colon and is positively
selected for chronic inflammation, the analysis was limited to E. coli. Two
parameters of E. coli were studied: E. coli associated with mucosa and internalized
in mucosa in patients with CRC, and in patients with receiving an operation due to
diverticulosis, an intestinal pathology not tumor (Brüssow 2020). The pathogenicity
of E. coli in CRC is linked to the genotoxins produced by it. The diversity of E. coli
is observed primarily by an organization of the species into several major phyloge-
netic groups (A, B1, B2, D, E, . . .) (Tenaillon et al. 2010). The strains of E. coli
which colonize patients with CRC mainly belong to the B2 and D phylogroups,
which group together mostly pathogenic E. coli (Tenaillon et al. 2010). These strains
of E. coli are mostly producers of genotoxins (cyclomodulins and colibactin), which
are capable of inducing DNA damage and thus disrupting the cell cycle of eukaryotic
cells. The scientific community has, for the moment, mainly focused on studying the
role of colibactin in CRC. A high prevalence of strains harboring the “pks” genomic
island is observed, which carries genes encoding polyketide synthases (PKS) and
peptide synthases non-ribosomal (PSNR) involved in the synthesis of this genotoxin
(Arthur et al. 2012). Bacteria producing colibactin can induce breaks in vitro and
in vivo double-stranded DNA, with the potential consequences of accumulating
chromosomal aberrations and an increased frequency of genetic mutations (Cuevas-
Ramos et al. 2010). Colibactin is an unstable compound that has not been purified to
date. However, an intermediary in its synthesis, although unable to break DNA, is
114 B. M. Bakadia et al.

able to alkylate DNA and induce strand bonds (Vizcaino and Crawford 2015;
Niederreiter et al. 2018). Cyclomodulin CNF (cytotoxic necrotizing factor) which
is a protein of approximately 115 kDa organized into three functional domains, was
initially described by the researcher Caprioli and his collaborators in 1983. These
proteins are grouped into three variants CNF-1, CNF-2, and CNF-3. The CNF-1 and
CNF-3 are encoded by chromosomal genes while CNF-2 is encoded by a plasmid
gene (Knust and Schmidt 2010; Niederreiter et al. 2018). EPEC strains have a type
III secretion system required for characteristic changes of attachment and effacement
that modify the cytoskeleton and apical surface of the host cells. The attachment
capacity and erase is encoded on an island of pathogenicity, known as the locus
enterocyte erase (LEE) that is made up of 41 open reading frames. The LEE encodes
different ESP genes (EPEC-secreted protein), thus the genes espA, espB, and espD
encode proteins secreted by this system which can form a translocation apparatus for
the delivery of effector molecules into the host cells. The EspF protein is also
encoded by the LEE via the system type III secretion (Niederreiter et al. 2018).
Colibactin-producing E. coli induce double-stranded DNA breaks, which is at the
origin of important chromosomal rearrangements, such as translocations and the
formation of circular chromosomes, leading to cell cycle arrest in G2 phase
(Niederreiter et al. 2018). In addition, cells infected with E. coli colibactin producers
can leave the cell cycle and enter senescence. This is accompanied by the secretion
of growth factors which support the tumor development. The CNF-1 toxin binds to
the tight junctions of host cells which internalize it via endocytosis. In the cytoplasm,
CNF-1 induces deamination of the glutamine residue of GTPases, enzymes bind and
hydrolyze the GTP (guanine triphosphate) of the Rho family, which leads the
appearance of actin stress fibers, DNA endo-reduplication, and inhibition of cytoki-
nesis inducing multinucleation. The activation of NFκB pathway (nuclear factor-
kappa B) by this toxin induces the activation of gene expression Bcl2 (B-cell
lymphoma 2) and Bcl-xl (B-cell lymphoma extra-large) inducing resistance to cell
apoptosis. EPEC strains are able to increase the frequency of spontaneous mutations
in the host cells, notably by altering the expression of mismatch repair (MMR) and
DNA repair proteins by inducting the stress oxidant and through the posttranscrip-
tional action of a secreted bacterial effector.

5.1.1.2.3 Fusobacterium
Fusobacterium is a filamentous, anaerobic genus of bacteria not forming spores,
narrow, with tapering ends, or pleomorphic. It is Gram-negative, identical to
Bacteroides (Madigan and Martinko 2005). This strain shows irregular coloring.
Fusobacterium belongs to the kingdom Bacteria, phylum Fusobacteria, class
Fusobacteria, order Fusobacteriales, and family Fusobacteriaceae (Madigan and
Martinko 2005). Fusobacterium has a potent lipopolysaccharide (LPS) which is an
essential component of the cell wall of Gram-negative bacteria—it is an endotoxin.
Species of the genus Fusobacterium are part of the normal flora of oropharynx,
digestive tract, and genital tract (Könönen et al. 2015). Infections can occur after
surgical or accidental trauma, edema, anoxia, tissue destruction, and animal bites
(Könönen et al. 2015). Fusobacterium is implicated in several pathologies in
5 Microbiome as Cancer Biomarkers 115

humans, especially periodontal disease, Lemierre syndrome, and skin ulcers tropical.
Different species are suspected in the development of CCR.
Several virulence factors of F. nucleatum show potential oncogenic activity.
F. nucleatum indeed produces a toxin which has immunosuppressive activity, FIP
or FIPA (fusobacterial immunosuppressive protein). It is able to inhibit the cell cycle
of T-lymphocytes during G1 phase, but its role in CRC remains to be defined
(Shenker and Datar 1995; Sánchez-Alcoholado et al. 2020). The FIP protein is a
cyclostatin which in its purified state has an apparent molecular mass of 90–100 kDa.
It is composed of two subunits of 44 and 48 kDa. The 44 kDa subunit has a FIPA
polypeptide, derived from the FipA gene, which is sufficient for mediation of the
immunosuppressive activities of the host protein complex (Demuth et al. 1996;
Sánchez-Alcoholado et al. 2020). F. nucleatum secretes an FadA adhesin
(Fusobacterium adhesin A) at its membrane which by binding to ecadherin
stimulates tumor growth. This causes the activation of WNT/βcatenin signaling
pathway that induces expression of genes involved in cell proliferation such as the
c-myc oncogenes and cyclin D1, and in inflammation such as the genes encoding
cytokines pro-inflammatory IL-6 and IL-8 (Rubinstein et al. 2013). The FadA
peptide expressed on the surface of F. nucleatum exists in two forms: intact
non-secreted pre-FadA consisting of 129 amino acid residues (aa) and the secreted
mature FadA (mFadA) consisting of 111 amino acids without the 18 amino acids
signal sequence, with the pre-FadA anchored in the membrane internal and mFadA
secreted outside of bacteria (Han et al. 2005). F. nucleatum is also able to inhibit the
antitumor activity of NK cells (natural killer) and T-lymphocytes which infiltrate
tumors by binding to its protein Fap2 (fusobacterial apoptosis protein 2) at the TIGIT
receptor (T-cell immunoglobulin and ITIM domain) which is expressed on the
surface of these immune cells (Gur et al. 2015). The Fap2 proteins are outer
membrane proteins, which vary in size between 200 and 400 kDa, classified as
type Va (T5SS) or auto-carriers (Desvaux et al. 2005). The gene fap2 codes for 3692
amino acids, resulting in a predictive molecular mass of 390 kDa (Gur et al. 2015).
Studies carried out in different countries including China, Denmark, France, and
Austria in order to evaluate the potential for diagnosing CRC from fecal
metagenomes on 74 patients with CRC found an associations between CRC with
several species such as F. nucleatum, Peptostreptococcus stomatis, Parvimonas
micra, and Solobacterium (Yu et al. 2017). Furthermore, 20 microbial gene markers
were identified which differentiated CRC and control microbiomes. These studies
highlighted the potential for using fecal metagenomic biomarkers for early diagnosis
of CRC (Yu et al. 2017). In another study comprising 30 CRC patients and
25 healthy individuals, the pathogenic microbiota was more abundant in intestinal
lavage fluid than in fecal samples revealing the association of the intestinal
microbiota and CRC (Shen et al. 2020). Moreover, in 54 CRC (stage III/IV)
Japanese patients with 127 healthy controls, a 55 bacterial markers were identified
by using shotgun sequencing. The shotgun sequencing was used to identify 16 bac-
terial markers in 61 CRC including 52 healthy controls Italian, German, and
Japanese patients. Furthermore, in Chinese discovery cohort: 111 CRC and
121 healthy controls, 14 fungal and 22 viral markers were identified by shotgun
116 B. M. Bakadia et al.

sequencing study platform. In 158 CRC Japanese and 60 healthy controls,

F. nucleatum was associated with CRC. In 1093 CRC Chinese patients,
F. nucleatum as a ratio to Bifidobacterium and Faecalibacterium prausnitzii, Clos-
tridium hathewayi, Bacteroides clarus, and Clostridium symbiosum were found as
best fecal markers for CRC. Additionally, in 39 Swedish CRC and 66 healthy
controls, F. nucleatum and E. coli pks+ were detected as bacterial biomarkers. In
251 American CRC patients, bacterial markers such as Fusobacterium,
Lachnospiraceae, Porphyromonas, Porphyromonadaceae, Bacteroides, and
Enterobacteriaceae were detected (Wong and Yu 2019).
In conclusion, CRC is a multifactorial disease dependent on environmental
factors, genetics, host, and microbiome (Fig. 5.1). The search for various promoters
(protagonists) must be wide. A link between chronic inflammation and the develop-
ment of CCR has already been established. The concept that there is a link between
the gut microbiota and CRC is recent. The gut microbiota is potentially involved in
the development of colorectal carcinoma by different mechanisms. The microbiota
and the immune response system, both innate and acquired, are involved in the
oncogenesis mechanism, in particular by their role in the initiation, regulation, and
maintenance of chronic inflammation. This inflammation, via its mediators, consid-
erably modifies the microbial composition, leading to dysbiosis (as opposed to
eubiosis). In response to these changes in their environment, microorganisms induce
complex changes in their transcriptional and metabolic responses and produce
metabolites, enzymes, and toxins (enterotoxins, cyclomodulins, FIP or FIPA)
which may influence the development of CRC. The microbiome must, therefore,
be considered as one of the important protagonists of the chain of events culminating
in cellular transformation malignant colonic epithelial cells. This section highlights a
pro-carcinogenic properties of bacteria. As this is a multifactorial disease, and the
development spans a long period (more than 10 years), the simple search for a
correlation between the bacterial species and CRC is too limiting. We should place
ourselves in the part of a complex microflora, including elements which can modify
the composition of the intestinal microbiota associated with CRC such as food. It
will also be necessary to understand the mechanisms of interactions between bacteria
which would make up the dysbiotic microbiota associated with CRC and the cell
carcinogenesis.

5.1.2 Intestinal Microbiome: Biomarkers of Pancreatic Cancer

Pancreatic cancer is the seventh leading cause of cancer death worldwide (Rawla
et al. 2019b). Its symptoms usually do not appear during early stage and the majority
of patients are already at an advanced stage before the cancer is diagnosed (Sun et al.
2020). The microbiome is known to influence tumor-initiating inflammation and
carcinogenesis. Thus the identification of microbiome biomarkers which could serve
as early diagnostic indicators could be very useful.
The microbiota of intestine and pancreas, both linked, are thought to play a major
role in the development of adenocarcinoma of pancreas, one of the most fatal cancers
 5
Microbiome as Cancer Biomarkers

Fig. 5.1 A summary of microbiome dysbiosis in CRC. (Figure reproduced from Saus et al. 2019)
117
118 B. M. Bakadia et al.

(Adamska et al. 2017; Wei et al. 2019). Pancreatic ductal adenocarcinoma (PDAC)
is the most common and serious pancreas cancer, with a 5-year survival rate of only
20% after surgery followed by chemotherapy (Adamska et al. 2017; Wei et al. 2019).
The gloomy statistic could be improved by taking the intestinal and pancreatic
microbiomes into account in the overall therapeutic strategy of PDAC, according
to the results of certain studies (Michaud and Izard 2014; Fan et al. 2018; Lu et al.
2019; Sun et al. 2020). According to these and some other studies, it appears that the
pancreatic bacterial population of patients with PDAC is richer in Proteobacteria. In
these subjects, the intestinal microbiota is also disturbed, with a more marked
presence of Actinobacteria, Fusobacteria, and Verrumicrobia compared to the
control stool samples (Pushalkar et al. 2018). These two microbiomes are also
linked: for instance, a study showed the possible translocation of bacteria from the
intestine to the pancreas, probably via the pancreatic duct that connects the two
organs. In mice with aggressive tumors, Helicobacteraceae, Bacteroidales, and
Mogibacteriacae were overrepresented in the gut microbiota, while Elizabethkingia,
Enterobacteriacae, and Mycoplasmatacae were found in mice whose tumors grew
more slowly (Pushalkar et al. 2018). The degree of intestinal dysbiosis could
therefore serve as a marker for the disease progression (Wei et al. 2019). A schematic
illustration of mechanism of microbiota affecting PDAC is shown in Fig. 5.2.
The pancreatic microbiome is found to be essential of the pathophysiological
process of the disease. A study reported that certain pancreatic bacteria, in particular
Bifidobacterium pseudolongum, inactivates the T-lymphocytes (by binding to
TLRs—Toll-like receptors), protecting tumors from any immune response. How-
ever, the administration of an antibiotic cocktail to the affected mice to eliminate
their pancreatic microbiota effectively slowed the progression of their tumors
(Pushalkar et al. 2018). According to the authors, the preventive antibiotic treatment
could be considered in certain high-risk patients with genetic susceptibilities or
already with advanced lesions, as well as the use of probiotics could correct a
possible intestinal dysbiosis. Another result concerning the curative aspect this
time showed that the suppression of pancreatic bacteria by the same antibiotic
therapy improved the effectiveness of checkpoint inhibitors. A recent form of
immunotherapy aimed at removing the immune brakes on cells tumors; however,
its effectiveness remains very limited in pancreatic cancer (Pushalkar et al. 2018).
From diagnosis to the treatment through prevention, the targeting bacteria in the
intestine and pancreas therefore seems most promising in the management of PDAC.
Although the changes in the intestinal microbiome have already been
characterized to be associated with pancreatic cancer, the abrupt microbiota
(microbiota dysbiosis) of the tongue layer as an indicator of the disease has not yet
been well-defined. Studies carried out in China reported that the disruption in the
microbial composition of the tongue layer could serve as a biomarker for early-stage
pancreatic cancer. Studies found that the tongue-layer microbiomes of patients with
early pancreatic stage were more diverse and composed of remarkably different
bacteria, with the tongue layers of healthy people (Michaud and Izard 2014; Lu et al.
2019). In another study, a total of 37 saliva samples were collected, including ten
from pancreatic cancer patients, 17 from mild pancreatic disease, and ten from
 5
Microbiome as Cancer Biomarkers

Fig. 5.2 A schematic illustration of mechanism of microbiota affecting PDAC. (Figure reproduced from Wei et al. 2019 distributed under the Creative
Commons Attribution (CC BY 4.0) license)
119
120 B. M. Bakadia et al.

healthy controls. A high concentration of Fusobacterium periodonticum and low

concentration of Neisseria mucosa were found in patients with pancreatic and
benign pancreatic cancer compared to the control subjects (Sun et al. 2020). These
results confirm the potential of N. mucosa and F. periodonticum as the diagnostic
biomarkers of pancreatic cancer. In addition, two prospective cohort studies selected
361 cases of pancreatic adenocarcinoma and 371 controls to study the association
between the oral microbiota and the risk of pancreatic cancer from oral lavage
samples. The Aggregatibacter actinomycetemcomitans and Porphyromonas
gingivalis were associated with higher risk of pancreatic cancer (Fan et al. 2018).
These studies provide helpful evidence that oral microbiome may play a function in
the etiology of pancreatic cancer.
If an association between oral microbiome and pancreatic cancer is established in
larger studies, this can possibly lead to the improvement of new microbiome-based
early preventive or diagnostic tools for the disease. Although more assenting studies
are needed, the study results add to the rising indication of an association between
oral microbiome disturbances and pancreatic cancer. An association between
microbiota dysbiosis of the tongue layer and pancreatic cancer could be linked to
the immune system. The pancreatic disease can initiate immune reactions that
promote the overgrowth of certain bacteria or vice versa. Research on the association
between the microbiome and pancreatic cancer opens up new opportunities to
develop biomarkers for identifying people at a high risk. Such studies could also
lay the foundations for the development of new treatment approaches related to
immunotherapies or even probiotics that could help prevent pancreatic cancer in
high-risk individuals.

5.1.3 Intestinal Microbiome: Biomarkers of Liver Cancer

The intestinal microbiota is in close contact with the digestive barrier and then with
the liver via the portal vein (Ma et al. 2018). Some advanced technologies of high
throughput sequencing and metagenomics have revealed imbalances in the intestinal
microbiome associated with cancers. Regarding liver disease, some recent studies
have proposed that the intestinal microbiome is involved in the development of
hepatocellular carcinoma (HCC) (Ma et al. 2018). The decrease in bacteria such as
Clostridium family in the intestinal tract promotes the accumulation of primary bile
acids (Ma et al. 2018), indicating a phenomenon which would participate in an
enhanced expression of anti-tumor NKT lymphocytes at the hepatic level (Ma et al.
2018). The LPS/TLR4 system (lipopolysaccharide/Toll-like 4 receptor) and more
generally, the intestinal bacterial motifs and cellular receptors (PRR type, pattern
recognition receptors), play a role in the hepato-carcinogenesis (Lv et al. 2017; Rao
et al. 2020). Among PRRs, the TLRs are crucial for establishing defense against
microorganisms (Bakadia et al. 2020). They are able to trigger the processes of
innate immunity and inflammation in response to dangerous signals of a prokaryotic
or eukaryotic nature (Bakadia et al. 2020). TLRs recognize PAMPs (pathogens-
associated molecular pattern) from microorganisms and DAMPs (damages-
5 Microbiome as Cancer Biomarkers 121

associated molecular patterns) from damaged tissues (Roh and Sohn 2018). After
interaction with PAMPs or DAMPs, the TLRs induce the secretion of IL-6 (interleu-
kin-6) and TNFα (tumor necrosis factor-α) as proinflammatory cytokines via an
intracellular molecular activation cascade involving, among others, Myd88 (myeloid
differentiation primary response 88) and IRAK (IL1-receptor-associated serine
kinase), which leads to the activation of transcription factors such as nuclear factor
κB (NFκB) and activator protein 1 (AP1), as well as the activation of factors
triggering the adaptive immune response (Jain et al. 2014; Bakadia et al. 2020).
HCC ranks fifth among cancers and is the third leading cause of cancer death
worldwide (Ren et al. 2019). HCCs is a genetically heterogeneous tumor that
develops in 75–85% of cases from cirrhosis, most often of alcoholic or viral origin.
The repetition of inflammatory episodes, the origin of which can be viral (hepatitis B
and C virus), alcoholic and metabolic, and the development of associated chronic
hepatopathies, such as steatohepatitis and cirrhosis, are major causes of hepatic
carcinogenesis (Lv et al. 2017; Rao et al. 2020). The successive cycles of aggression
of liver which precede the onset of HCC lead to real remodeling of the organ
(regenerative liver nodules). The latter is molecularly associated with profound
alterations in the signaling pathways regulating the cell proliferation, death, and
migration/invasion, and angiogenesis (Lv et al. 2017; Rao et al. 2020). It is also well-
known that cirrhosis is frequently accompanied by an alteration in the integrity of the
intestinal barrier associated with an increased susceptibility of cirrhotic patients to
bacterial infections originating in the digestive tract. The translocation of bacterial
products, such as lipopolysaccharide (LPS, component of the wall of Gram-negative
bacteria), by the portal venous system thus contributes to the perpetuation of
inflammatory lesions of the liver (Yu and Schwabe 2017). A study has shown that
the activation of LPS/TLR4 pathway promotes hepatocyte proliferation via the
secretion of TNFα and IL-6 by Kupffer cells (resident macrophages of liver), and
increases the resistance of hepatocytes to oxidative stress and apoptosis resulting in
the accelerated development of HCC in an experimental model chemo-induced by
diethylnitrosamine (Yu et al. 2010). In the experimental model used in this study, the
depletion of circulating LPS by antibiotic therapy or the interruption of the
LPS/TLR4 activation pathway through the inactivation of the gene encoding
TLR4 inhibited the initiation and progression of HCC (Yu et al. 2010). Another
study showed a potential upregulation of anti-tumor immunity via an action of the
gut microbiota. This study investigated the impact of the reduction of commensal
bacteria on the evolution of levels of primary and secondary bile acids, and therefore
of the immune response. Primary acids promote the accumulation of natural killer
T-lymphocytes (NKT), which produce anti-tumor interferons-gamma, while second-
ary acids have the opposite action (Ma et al. 2018). After administration of a cocktail
of antibiotics aimed at destroying the intestinal commensal bacteria in mice suffering
from primary or metastatic hepatic carcinoma, the researchers evaluated the effect on
the tumor growth kinetics, and concluded that the decrease in species belonging to
the genus Clostridium caused an increase in the number of NKT cells regardless of
the mouse genus, lineage, and tumor type. The correlation was confirmed after
colonization by a bacterium known to metabolize primary bile acids into secondary
122 B. M. Bakadia et al.

acids—Clostridium scindens—in mice with altered microbiota. This

renormalization caused a decrease in the number of NKT cells, and therefore lifted
the inhibition of tumor growth (Ma et al. 2018).
More studies (Piñero et al. 2019; Ponziani et al. 2019; Ren et al. 2019; Huang
et al. 2020; Lapidot et al. 2020; Zeng et al. 2020b) suggest a relationship between the
gut microbiome, their metabolites, and the hepatic immune response; a relationship
that might herald novel therapeutic methods in HCC, a main source of death in
oncology. Additionally, the results of some studies make it possible to stratify the
patients with HCC according to their metagenome, or even to identify particular
risky metagenomic profiles, which could then benefit from a treatment modulating
the gut microbiota of the antibiotic, prebiotic, or probiotic type.

5.1.4 Intestinal Microbiome: Biomarker of Breast Cancer

A relationship between intestinal dysbiosis and breast cancer has been suggested by
a number of clinical studies, where the researchers have emphasized the use of
antibiotics, which influence the intestinal microflora with an increased risk of breast
cancer (Velicer 2004; Friedman et al. 2006; Tamim et al. 2008). However, to date,
there is very little data available on the precise contribution of the gut microbiota in
the development of breast cancer. This section summarizes the experiments resulting
from animal models and clinical studies that have established the link between the
intestinal flora and breast cancer, as well as the hypotheses explaining this
relationship.
The intestinal microbiota has an essential functional role and interferes in partic-
ular with the exogenous and endogenous metabolites such as estrogen. Estrogens are
recognized as a causative factor in the etiology of breast cancer, and play an
important role in the initiation and promotion of breast tumor (Russo and Russo
2006; Yue et al. 2010). A high level of endogenous or circulating estrogen are
directly associated with an increased risk of breast cancer in postmenopausal women
(Lewis et al. 2005; Sampson et al. 2017). Estrogens and their metabolites undergo a
second metabolic step in liver, where these are conjugated. These are then excreted
in the intestinal tract or through the urinary tract, in conjugated forms. It has been
widely described that the human gut microbiota can contribute to the metabolism of
estrogen in humans. The authors in a study defined the term “estroboloma” as “the
set of enteric bacterial genes whose expression products are capable of metabolizing
estrogen” (Plottel and Blaser 2011). Among these metabolic reactions, the most
documented activities are the β-glucuronidase and β-gluconidase enzymatic
activities expressed by intestinal bacteria. As previously described, these enzymes
are known to participate in the deconjugation of xenobiotics, drugs, but also steroid
hormones such as estrogen. These can eliminate the glucuronic acid part (for
β-glucuronidase) or the glucose part (for β-gluconidase) from the conjugated
estrogens, and therefore promote the reabsorption of their free forms through the
enterohepatic circulation. Therefore, a bacterial community enriched with bacteria
possessing these enzymatic activities, including the β-glucuronidase activity, could
5 Microbiome as Cancer Biomarkers 123

lead to greater reabsorption of free estrogen, and thus influencing the development of
estrogen-dependent neoplasia (Kwa et al. 2016). Bacteria possessing
β-glucuronidase activity are distributed among the Enterobacteriaceae family
(in particular the E. coli), in two dominant clusters Clostridium leptum and Clostrid-
ium coccoides, such as the genera Roseburia, Faecalibacterium, Clostridium, and
Ruminococcus (Jefferson et al. 1986; Dabek et al. 2008). β-glucosidase activity is
represented by many members of Firmicutes, some species of Bifidobacterium, and
the species Bacteroides thetaiotaomicron (phylum Bacteroidetes) (Dabek et al.
2008). In 2012, Flores and co-workers demonstrated that the β-glucuronidase activ-
ity of intestinal microbiota is negatively correlated with the level of total estrogen
(conjugated and deconjugated) in fecal samples, in a study comprised of
29 individuals (seven postmenopausal women and 22 men). They also showed
that the richness and diversity of fecal bacteria positively influenced the excretion
of total estrogen in urine (Flores et al. 2012). In addition, according to a study in
60 postmenopausal women, the diversity of the gut microbiota was positively
associated with an increased ratio of estrogen metabolites to estrogen in urine
(Fuhrman et al. 2014). Elevated levels of urinary estrogen metabolites are associated
with the decreased plasma estrogen concentrations, and thus leading to a reduced
risk of hormone-dependent breast cancer (Moore et al. 2016). Studies, therefore,
suggest that the risk of developing breast cancer may be greater in women with a gut
microbiome favorable to estrogen deconjugation or with a low gut bacterial
diversity.

5.1.5 Intestinal and Lung Microbiome: Biomarkers of Lung Cancer

The Human Microbiome Project, launched in 2007 by the United States National
Institute of Health (NIH), studied various organs, including digestive tract, urogeni-
tal tract, skin, mouth, and nose (NIH HMP Working Group et al. 2009). When the
study of the microbiota took off, the lung was not viewed with interest. But the
vision of the pulmonary microbiota has changed recently, passing from that taught in
medicine (“A healthy lung is a sterile lung”), to that of an organ occupied by
microorganisms, most of which, in normal conditions, are good for its health
(Faner et al. 2017).
Lung cancer, often diagnosed at an advanced stage, is deadly. An earlier diagno-
sis would greatly improve care and the chances of survival. What if, as with many
diseases including other cancers, the intestinal dysbiosis is a sign of lung cancer (Liu
et al. 2020a). In a study, the intestinal microbiota of 42 patients with early stage lung
cancer of three different types (only three patients with metastases) as well as that of
65 healthy controls were analyzed through sequencing of 16S RNA. The intestinal
dysbiosis was observed in patients with lung cancer compared to the controls, which
showed increased presence of species belonging to the genera Ruminococcus and to
the families Lachnospiraceae and Enterobacteriaceae, among others. Thus, the
composition of the microbiota could change with the development of lung cancer.
Finally, the composition of the intestinal microbiota specifically indicated the stage
124 B. M. Bakadia et al.

Fig. 5.3 A classification of normal and dysbiotic microbiota in lungs and the microbiota axis
between lungs and gut/intestine. (Figure reproduced from Carbone et al. 2019 distributed under the
Creative Commons Attribution (CC BY 4.0) license)

of the tumor: certain bacteria were only present in patients with metastases (Zheng
et al. 2020). In order to provide a noninvasive diagnostic tool for early detection of
lung cancer, 13 biomarkers, based on operational taxonomic unit (OTU), have been
identified. Together, these can accurately predict (97.6%) the presence of lung
cancer. This model was confirmed in a second cohort (34 patients and 40 controls):
its predictive power remains high (76.4%), although lower than that of the initial
cohort. From this model, it was possible to construct a “patient discrimination
index”, easy to use in clinical practice (weighted score) to identify patients with
early lung cancer. Its predictive power in the initial cohort (92.4%) is also found to
be greater than that measured in the validation cohort (67.7%). Larger cohorts could
improve the model and its predictive power (Zheng et al. 2020).
The analysis of lung microbiota (Fig. 5.3) reveals characteristic signatures of
various respiratory symptoms, making it a diagnostic, prognostic, and a follow-up
tool, in step with the developing precision medicine (Mao et al. 2018; Xu et al.
2020b). In lungs, the airways are poorly described compared to other microbial
5 Microbiome as Cancer Biomarkers 125

deposits (Laguna et al. 2016). This is due to the difficulty of access by invasive
samples (bronchoscopy or bronchoalveolar lavage). The other difficulty comes from
the oropharyngeal contamination, which must be overcome to make the observation
reliable. Encountered by the increase in patients with chronic respiratory diseases
(more than 300 million people according to WHO), different research teams are
interested in studying the pulmonary microbiota, betting that it could be the key to
progress for diagnosis and cares (Sharifi et al. 2019). If the human intestine contains
around 100 trillion bacteria, the bacterial lung biomass is a million times smaller
(100,000 bacteria per cm3 of bronchoalveolar lavage) (Wouters 2019; Liu et al.
2020a). Despite the low density, this microbiota is distinguished by its exceptional
biodiversity (Xu et al. 2020b), due to different origins of bacteria. Lung is an ideal
playground (approximately 75–100 m2 of alveolar surface in adults) (Fröhlich et al.
2016) for bacteria, which mainly come from the oral sphere but also from the inhaled
air (100,000 microorganisms per liter of inhaled air) and the digestive tract
(by micro-aspiration) (Wouters 2019). The combined action of mucus and cilia of
the respiratory mucosa, to which are added the cough and the local antimicrobial
defenses, limit the pulmonary bacterial colonization, with a microbiota theater of an
incessant ballet where some bacteria settle and others leave by the muco-ciliary
escalator (Xu et al. 2020b; Liu et al. 2020a). Local lung conditions select the
microorganisms best suited to this ecosystem (Xu et al. 2020b; Liu et al. 2020a).
Colonization of airways begins early in life, influenced by the method of delivery
(Xu et al. 2020b). The maturation of this microbiota would be fundamental for lung
health, with three roles including barrier against pathogenic bacteria, education of
the local immune system, and modeling of the lung architecture. It is likely that other
roles will be identified, particularly in relation to the regulation of metabolic
pathways (Xu et al. 2020b). The pulmonary microbiota results from a dynamic
balance, whose disturbances accumulated since childhood could play a role in the
onset or aggravation of diseases such as asthma, chronic obstructive pulmonary
disease, or genetic diseases such as cystic fibrosis, which all result in a profound
change in the pulmonary microbiota and especially a decrease in commensal
anaerobes. Thus, for asthma, one hypothesis is that a modification of this microbiota
during infancy could modify the local inflammatory response, favoring its occur-
rence (Loverdos et al. 2019).
Some recent studies show that the gut microbiota may affect lung health through
cross communication with lungs (Xu et al. 2020b). This axis of communication is
carried out via the passage of microbial metabolites (short-chain fatty acids),
endotoxins, and cytokines in the bloodstream connecting the intestinal and pulmo-
nary epithelia (Mao et al. 2018; Xu et al. 2020b). The intestinal dysbiosis could thus
lead to acute or chronic lung diseases, such as asthma, tuberculosis, and lung cancer.
Conversely, some patients with lung disorders such as tuberculosis, asthma, and
chronic obstructive pulmonary disease (COPD) also have intestinal symptoms,
which clearly indicates interference between the intestine and the lungs (Mao et al.
2018; Xu et al. 2020b). Overall, intestinal homeostasis could affect pulmonary
homeostasis and vice versa.
126 B. M. Bakadia et al.

5.1.6 Intestinal and Gastric Microbiome: Biomarkers of Gastric

Cancer

Gastric cancer, the third leading cause of cancer death, has a poor prognosis because
it is often diagnosed at an advanced stage and therefore difficult to treat (Li et al.
2017; Gantuya et al. 2019; Ye et al. 2019). The highest death rates from stomach
cancer are seen in East Asia as well as in Eastern Europe and countries in Central and
South America (Wroblewski et al. 2010). The identification of obtaining a biomarker
capable of early detection of this cancer is essential to reduce the number of deaths
(Li et al. 2017). Helicobacter pylori is a pathogenic bacterium that colonizes the
stomachs of almost half of the world’s population (Gantuya et al. 2019). Its infection
is acquired during childhood and lasts for decades (Pacifico et al. 2010; Kotilea et al.
2018). It remains asymptomatic in most people, but in some cases the infection will
progress to gastric cancer (Pacifico et al. 2010; Kotilea et al. 2018). Today, it is
believed that H. pylori is responsible for around 90% of gastric cancers worldwide,
resulting in an estimated number of deaths of around 800,000 persons per year
(Moss 2017; Rawla and Barsouk 2019). The sequence of events triggered by the
bacterial infection that will ultimately lead to gastric cancer is starting to be
deciphered, with the instability of DNA of the infected cells at the heart of the
mechanism (Liu et al. 2020b). Indeed, previous studies have shown that H. pylori
causes DNA breaks and disrupts its DNA repair system by promoting the accumu-
lation of mutations that can target p53, a protein called the “guardian of the genome”
(Li et al. 2016b; Williams and Schumacher 2016). The p53 protein is essential for the
proper functioning of the cell, because it allows—in the event of significant damage
in the genome—to temporarily stop the cell cycle, time necessary to repair the DNA
(Williams and Schumacher 2016). An inactivation of p53 therefore promotes
genome instability and the transformation of a normal cell into a cancer cell
(Williams and Schumacher 2016). It is important to understand the cell transforma-
tion induced by H. pylori that promotes cancer development, in order to define a
marker of susceptibility. This would allow early management of patients and thus
could prevent the development of gastric cancer.
H. pylori infection of the stomach is acquired during childhood and lasts for
decades or even the entire life of the infected person (Gantuya et al. 2019). This
develops a strong local and humoral inflammatory response that gradually settles
into chronicity. In most people, chronic gastritis progresses without further conse-
quence and remains asymptomatic (Gantuya et al. 2019). A small proportion of
patients (about 10% of those infected) develops ulcer over time and in 1–3% of cases
stomach cancer; gastric cancer (adenocarcinoma), or gastric MALT lymphoma
(Rawla and Barsouk 2019). However, the data accumulated in recent years show
that these two clinical pictures are mutually exclusive, and that the evolution towards
one or the other of the pathologies is a function of the genetic predispositions of the
host of environmental factors (food in particular), and bacterial properties. The
evolution of H. pylori infection to ulcerative disease is associated with a predomi-
nantly antral gastritis (lower part of the stomach) and an acid hyper-secretion which
leads to colonization and inflammation of the duodenum, site of duodenal ulcer
5 Microbiome as Cancer Biomarkers 127

(which accounts for 95% of ulcer disease) (Stewart et al. 2020). The progression of
the infection to gastric atrophy and then gastric cancer is usually associated with
pangastritis (gastritis of the upper and lower parts of the stomach) (Raza and Bhatt
2020). It is generally observed in patients with acid hyposecretion and more particu-
larly affects populations over 50 years of age (Osefo et al. 2009). Thus, H. pylori has
the sad privilege of having been recognized as the first, and still today the only
bacteria directly involved in the genesis of cancer (Wroblewski et al. 2010). The
correlation between H. pylori infection and adenocarcinoma of the stomach is clearly
demonstrated. H. pylori causes chronic inflammation of the gastric mucosa (gastritis)
which can lead to a process of carcinogenesis. However, it appears that a number of
cancers are independent of H. pylori infection. These results were provided by the
work of Portuguese researchers, who studied and characterized the gastric bacterial
flora of 54 patients with stomach cancer before comparing it with that of 81 people
with chronic gastritis. The composition of the gastric microbiota of the two types of
patients is different. Patients with adenocarcinoma present with gastric dysbiosis
characterized by the decreased microbial diversity, enrichment in Proteobactria,
Lactobacillus, Clostridium, and Rhodococcus and a lower proportion of H. pylori.
This imbalance causes an increase in the enzymatic activity of nitrites and nitrate
reductases, which participate in the metabolism of nitrogen by producing, for
example, carcinogenic nitrogen compounds (Ferreira et al. 2018). This discovery
is the first evidence of a gastric dysbiosis (potentially genotoxic) associated with
stomach cancer different from that seen in chronic gastritis. It provides new elements
of the direct involvement of the gastric microbiota in the occurrence of stomach
cancer.

5.2 Oral Microbiome: Biomarker of Oral and Oropharyngeal

Cancers

The human oral microbiota is the set of microorganisms present in the mouth of
human beings (Aas et al. 2005). The oral microbiota can be normal or pathological.
It is still poorly understood because many oral bacteria cannot be cultivated
(Socransky et al. 1963). It is made up of several hundred to thousands of different
species of microorganisms (Schwiertz 2016), 700 species having been discovered by
screening (Paster et al. 2006). It is mainly its free (planktonic) forms that have been
studied, but it is mainly made up of colonial forms consisting of organized biofilms
(Schwiertz 2016). Each individual is home to 100–200 of these 700 species. The oral
microbiota, mainly made up of bacteria, has developed strategies of resistance and
perception of its environment allowing it to a large extent to evade the immune
system, or even to modify the host for the benefit of the microbe (for example dental
caries). Oral microbiota is more or less diversified according to age and individuals,
and it contributes to the smell of the breath and can have systemic effects on the
health of the whole organism and even on the intellectual capacities (Bik et al. 2010).
This microbiotic heritage is partly acquired from the mother and the father at birth or
in infancy (Alaluusua et al. 1991). It could be positively or negatively modified by
128 B. M. Bakadia et al.

brushing teeth, dental care (Anwar et al. 1992; Auschill et al. 2002), diet (sugar in
particular), and ingestion of antibiotic products (alcohol, natural antibiotics, certain
drugs, chlorinated water, etc.) with consequences that are still poorly understood
(Arweiler et al. 2014) which can contribute to the appearance of phenomena of
microbial resistance to antibiotics. A part of oral microbiota could be
transgenerational (Attar 2016). The oral environment (temperature, humidity, pH,
constant presence of saliva, and nutrients and mucous residues) selects adapted
populations (and sometimes pathogens) of microorganisms (Faran Ali and Tanwir
2012; Kilian et al. 2016).
In a healthy young or adult person consuming a healthy diet, the resident mouth
microbes adhere to the mucous membranes, teeth (including enamel) (Al-Ahmad
et al. 2009), some artificial implants (if present in mouth) (Fürst et al. 2007;
Al-Ahmad et al. 2010), and gums to resist leaching out by saliva, but are subse-
quently mostly destroyed by passage through the stomach (through the action of
secreted of hydrochloric acid) (Faran Ali and Tanwir 2012; Wang et al. 2014; Kilian
et al. 2016). The salivary flow (Watanabe and Dawes 1990) and locoregional
conditions inside the mouth vary (Cruchley et al. 1989), and these largely depend
on the time of day and personal habit, for example the individual sleeps with open or
closed mouth. From childhood to old age, the respective surfaces of different areas of
oral cavity (as well as their quality) including those of teeth evolve over the course of
life (Kerr et al. 1991) by interacting with the oral microbiota, according to factors
that science explore (Busscher and van der Mei 1997). Through the larynx, many of
these bacteria migrate to the respiratory tract where mucus is responsible for
repelling them. A part of this microbiota is involved or co-involved in the production
of factors (like γδ T or γδT17 cells) which promote autoimmune diseases such as
psoriasis, arthritis, as well as cancer of colon, lungs, and breast (Fleming et al. 2017).
Tobacco consumption and poor oral hygiene are the two major risk factors for
oral or throat cancer associated with the depletion of oral microbiota. What
differences does the latter then mark compared to that of a nonsmoker and concerned
about his hygiene? To answer this question, a German–American research team
analyzed the composition, diversity, and functions of microorganisms in the oral
cavity of 121 patients with oral-pharyngeal carcinomas and 242 healthy individuals
(Börnigen et al. 2017). The main risk factors for developing oral cancer (tobacco,
alcohol, oral hygiene, and papillomavirus infection) were assessed in 363 volunteer
participants. The microbial composition of the oral cavity was then detailed in each
individual in order to identify the main differences between the two populations.
While all of the cancer risk factors are not accompanied by significant changes in the
microbiota, smoking and poor dental health are a source of significant deterioration
of the oral ecosystem. This deterioration is accompanied in particular by a decrease
in microbial diversity and disruption of certain essential functions of the microbiota
(metabolism of sugars, transport of metals, organic compounds, etc.) (Börnigen et al.
2017). Above all, the study highlighted the major impact of total tooth loss. The
disappearance of a privileged “habitat” drastically reduced the microbial diversity
and their associated functions, which are essential for maintaining a healthy mouth.
Directly linked to the occurrence of oral and pharyngeal cancers, the absence of teeth
5 Microbiome as Cancer Biomarkers 129

could therefore also increase the risk by modifying the local microbiota. While there
is currently no certainty regarding this eventuality, the results of the study allow at
least to confirm that the presence of certain risk factors for oral cancer alters the
composition and functions of the oral microbiota (Börnigen et al. 2017).

5.3 Vaginal Microbiome: Biomarker of Cervical Cancer

Vaginal microbiome is comprised of a plethora of bacteria, with the most abundant

representation by Lactobacillus (Chee et al. 2020). Modern methods have been used
to characterize the healthy vaginal microbiome and to distinguish it between the
different healthy profiles that keep vaginal homeostasis in check (Chase et al. 2015).
It is well-established that a balanced microbiome in the vaginal region is pivotal in
avoiding infections of the genital tract; however, recent evidences show that it may
even influence the development of malignant changes in the cervix and other
constituents of the reproductive system (Chase et al. 2015; Champer et al. 2018).
Exploring the association between the vaginal microbiome and gynecological
malignancies is a developing and exciting area of research, as several studies that
have been published recently proposed that such a relationship may exist (Chase
et al. 2015; Champer et al. 2018). The general hypothesis is that the vaginal bacteria
play a major role in the tumor microenvironment.
Evidences for how the vaginal microbiome influence the human papillomavirus
(HPV)-induced cervical cancer is mounting (Ramchander and Crosbie 2018). The
HPV is the most common sexually transmitted infectious agent. While most infected
women clear it quickly, only a small fraction develops a persistent infection with a
high risk of developing precancerous lesions and then cervical cancer (Usyk et al.
2020). The cervico-vaginal microbiota (CVM) has also been shown to be involved in
the prevalence of the disease. However, its influence on the elimination of infection
or, conversely, on the progression to moderate to severe dysplasia is not yet known.
The presence of Gardnerella in the cervico-vaginal microbiota of women with a
high-risk oncogenic papillomavirus would be an indicator of an increase in microbial
diversity and a sign of progression to precancerous lesions (Usyk et al. 2020). A
study hypothesized that the vaginal dysbiosis promotes the progression of oncogenic
HPV infection to precancerous lesions. The study specified that biomarkers exist
within CVM and make it possible to identify patients at risk. In future, if other
studies confirm the central role of CVM in the progression of the disease, therapeutic
strategies could be considered to prevent the progression of the infection by
modulating CVM (Chao et al. 2020). Moreover, a study showed that the microbiome
in women with HPV demonstrates a greater diversity of substance from bacteria,
particularly Gardnerella vaginalis and Gasseri lactobacillus, which was
corroborated by another study which found higher diets of HPV in women with
generally lower levels of Lactobacillus and higher microbiome diversity
(Ramchander and Crosbie 2018). In addition, HPV elimination rates (and hence
the risk of malignant transformation) are also influenced by the vaginal microbiome
composition, and a bacterial genus that has repeatedly been linked to stagnant HPV
130 B. M. Bakadia et al.

clearance was Atopobium. Additionally, vaginal infection with Chlamydia

trachomatis appears to predispose women to HPV infection simply by altering the
vaginal microbiome (Mitra et al. 2016; Kyrgiou et al. 2017). Some studies have tried
to elucidate certain putative substance which alone can act. For example, Lactoba-
cillus crispatus has been associated with healthy women, while Lactobacillus iners
alone have been found in those with cervical cancer—or with HPV (particularly in
those patients with high grades of cervical intraepithelial neoplasia) (Mitra et al.
2016). A disruption of the vaginal microbiome may also be an indirect risk factor for
the development of endometrial and ovarian cancer (Kyrgiou et al. 2017). Recent
studies have shown that the ovaries, fallopian tubes, and uterus are characterized by
unique microbial profiles, and that differences in their composition may be related to
certain malignant conditions (Chase et al. 2015; Champer et al. 2018; Xu et al.
2020a).
In conclusion, the notion that the vaginal microbiome may retain a secret of
cervical carcinogenesis is intriguing, and may represent a comprehensive different
perspective on the optimal prevention and treatment of this frequent malignant
process. Nevertheless, supplementary studies which may support this association
are desirable, and thus there is simply not sufficient indication for the connection
between the vaginal microbiome and gynecological cancers.

5.4 Gut Microbiome: Markers for Modulation of Immune

System and Breast Cancer

It is widely accepted that inflammation is an essential part of pathogenesis and

progression of breast cancer. Neutrophil dysregulation is a hallmark of breast cancer
development, and a high ratio of neutrophils to lymphocytes predicts a poor clinical
prognosis in patients with breast cancer (Ethier et al. 2017). Moreover, neutrophils
and lymphocytes could be modulated by the intestinal microbiota and inflammation.
In genetically engineered female mice with a predilection for developing breast
cancer, studies have shown that orogastric infection with Helicobacter hepaticus
upregulates the inflammatory cells associated with tumor, including neutrophils. In
particular, systemic neutrophil depletion inhibits the formation of mammary tumors
induced by H. hepaticus. Furthermore, CD8 + T lymphocytes are recognized as the
most powerful immune cells, which are capable of eliminating foreign antigens and
mammary tumor cells (Gritzapis et al. 2008). Patients with a higher level of CD8 + T
cells infiltrating their breast tumors are associated with better survival (Wang et al.
2011). The gut microbiota plays an important role in conditioning systemic CD8 + T
cells to modulate other peripheral immune cells, such as plasmacytoid dendritic cells
and iNKT (invariant nature killer cells) (Wu and Wu 2012). In contrast to bacteria
with carcinogenic potential, there are also beneficial bacteria which can inhibit
carcinogenesis in extraintestinal tissues. The oral administration of probiotic bacte-
ria, such as Lactobacillus reuteri to mice has been reported to reduce the production
of systemic inflammatory cytokines and the accumulation of neutrophils, and to
activate CD4 + CD25 + lymphocytes, thereby reducing the risk of breast cancer in
5 Microbiome as Cancer Biomarkers 131

mice (Lakritz et al. 2015). These data demonstrate that host immune responses to
intestinal bacteria could cause or inhibit cancer progression in extraintestinal tissues,
such as mammary glands.
Recently, Goedert and co-workers showed that the gut microbiota of breast
cancer patients had less diversity compared to the controls. In addition, the patients
had higher levels of Clostridiaceae, Faecalibacterium, and Ruminococcaceae (phy-
lum Firmicutes) and lower levels of Dorea and Lachnospiraceae (phylum
Firmicutes) (Goedert et al. 2015). In order to validate the relationship between the
gut microbiota and breast cancer, some studies have been conducted to investigate
the changes in microbial composition in patients with breast cancer depending on
their pathological characteristics. At the same time, differences in the intestinal
microbial profile between the patients and healthy women were analyzed. First, a
significant reduction in gut microbiota diversity was found in breast cancer patients
compared to the healthy women, which was in agreement with the results presented
by Goedert and co-workers. It is accepted that a low bacterial richness and diversity
are found in subjects with more marked overall adiposity, insulin resistance, and
hyperlipidemia (Le Chatelier et al. 2013), which are associated with an increase in
the occurrence of breast cancer (Gunter et al. 2015). In addition, the diversity of gut
microbiota in postmenopausal women is positively associated with the increased
ratio of estrogen/estrogen metabolites in urine (Fuhrman et al. 2014). The elevated
levels of urinary estrogen metabolites are associated with a decreased plasma
estrogen concentrations and thus reducing the risk of hormone-dependent breast
cancer (Moore et al. 2016). In a study, it was observed that the abundance and
relative proportion of certain bacterial groups, such as C. coccoides cluster,
C. leptum cluster, F. prausnitzii, and Blautia sp. group were higher in patients
with severe clinical stage and a higher histopronostic grade. In agreement with
these results, a second study revealed that the gut microbiota of patients is enriched
in C. coccoides cluster, C. leptum cluster, F. prausnitzii, and Blautia sp. Thus most
of the bacteria expressing identified β-glucuronidase and β-glucosidase activities
belong to two clusters, C. leptum and C. coccoides, such as the genera Clostridium,
Faecalibacterium, Ruminococcus, and Roseburia (Dabek et al. 2008). The bacterial
enzymes can catalyze the hydrolysis of the conjugated form of estrogen, thereby
promoting the reabsorption of their free forms into the enterohepatic circulation.
Thus, the intestinal microbiota enriched in C. coccoides and C. leptum cluster could
contribute to the occurrence of breast cancer in these patients. The genus
Bifidobacterium has been recognized as one of the probiotics which exerts numerous
beneficial effects for the host (Hemarajata and Versalovic 2013). Kim et al. showed
that oral intake of the probiotic Bifidobacterium reduced β-glucuronidase and
β-glucosidase enzymatic activities in rats (Kim et al. 2008b). The probiotic
Bifidobacterium has also shown its beneficial effect on the immune response against
the growth of melanoma in several mouse models (Sivan et al. 2015). Concerning
the genus Coprococcus, a low abundance of these bacteria has been found in patients
with colorectal cancer in autistic children (Kang et al. 2013) and in subjects infected
with HIV (McHardy et al. 2013). In addition, the abundance of Coprococcus was
reduced in mice exposed to social disruption stress, and is negatively correlated with
132 B. M. Bakadia et al.

circulating levels of pro-inflammatory stressor-induced cytokines (Bailey et al.

2011). This indicates that some bacterial populations, such as the genera
Bifidobacterium and Coprococcus, may be beneficial in healthy women for the
primary prevention of breast cancer. In summary, these studies show that the
intestinal bacterial profiles differ between patients with breast cancer, depending
on their clinical situation, but also between patients and healthy women. Their
findings raise the question of whether gut bacteria could be used for interventions
aimed at preventing the onset of this disease. Future studies on large populations,
supplemented by the determination of the concentrations of estrogen and estrogen
metabolites in different matrices (serum, urine, feces), as well as analysis of diet
should be performed to clarify the relationship between gut microbiota and breast
cancer.
Breast cancer is considered to be one of the world’s top public health priorities
(Ghoncheh et al. 2016). The diagnosis of breast cancer is based on clinical examina-
tion, in combination with breast imaging, and confirmed by pathological evaluation
of the tumor (Li et al. 2016a). The management of breast cancer consists of excision
of the tumor, radiotherapy, or drug treatments (Moo et al. 2018). Differences in the
composition of the gut microbiota influence plasma estrogen levels. Thus, a high
diversity of the intestinal microbiota would reduce plasma estrogen levels and
consequently reduce the risk of developing hormone-dependent breast cancer. In
addition, it should be noted that the composition of gut microbiota is interconnected
with other preestablished risk factors for breast cancer such as obesity and eating
habits, alcohol, age, all leading to intestinal dysbiosis (Hullar et al. 2014; Hills et al.
2019; Sampsell et al. 2020). Thus, the study of the intestinal microbiota would make
it possible to assess the risk of the occurrence of breast cancer and would help to
develop strategies for preventing this type of cancer.

5.5 Uterine Microbiome: Biomarkers of Endometrial Cancer

Endometrial cancer—or cancer of the uterus (not to be confused with cervix

cancer)—is one of the most common gynecologic cancers after breast cancer, and
the most common of the female reproductive system (Kato et al. 2021). Indeed, the
endometrium is the covering inside the lining of the uterus, which is part of uterus
where the embryo develops during pregnancy (Kelleher et al. 2019). Diagnosed at an
early stage, the prognosis for the endometrial cancer is quite good (Matteson et al.
2018). Unfortunately, due to the lack of characteristic symptoms, the average age of
patients at diagnosis is 68 years (Matteson et al. 2018). Interest in the microbiome of
the genital tract has grown in recent years. Studies of vaginal and placental
microbiomes have shown the associations between these microbiomes and obstetric
outcomes and it is plausible that the uterine microbiome is also associated with these
findings (Molina et al. 2020). Since infection is a major cause of preterm birth, many
studies over a decade have focused on identifying microorganisms remaining in the
uterus and recording any association with pregnancy outcomes (Agrawal and Hirsch
2012). Current knowledge about the uterine microbiome has been greatly improved
5 Microbiome as Cancer Biomarkers 133

by the availability of molecular techniques in the field of microbiology, which has

led to the discovery of various bacterial taxa which had not been previously
described (Verstraelen et al. 2016). The vaginal microbiota, the microorganisms
inhabiting in the vagina, could facilitate screening for endometrial cancer. The
predominant taxa in endometrial cancer are Treponema, Porphyromonas,
Anaerotruncus, Bacteroides, Acinetobacter, Anaerostipes, Ruminococcus,
Comamonadaceae, Peptoniphilus, Escherichia, Cloacibacterium, Pseudomonas,
Arthrospira, Dialister, and Atopobium (Walther-António et al. 2016; Walsh et al.
2019; Winters et al. 2019).
Different studies identified a microbiome signature associated with endometrial
cancer, which is in part favored by postmenopause (Walther-António et al. 2016;
Walsh et al. 2019). These studies were aimed to understand how risk factors for
endometrial cancer alter the microbiome of the reproductive system and the risk of
endometrial cancer. The studies analyzed samples taken from the female reproduc-
tive system (vagina, cervix, ovaries, and fallopian tubes) from patients who had the
endometrial cancer or hyperplasia and compared them with samples taken from
patients with benign uterine conditions (Walther-António et al. 2016; Walsh et al.
2019). As reported, the high sequencing of next throughput DNA recovery was used
to identify microbiota present, which showed that several taxa were more abundant
in patients with endometrial cancer and hyperplasia with patients who have had mild
conditions (Walther-António et al. 2016; Walsh et al. 2019). In particular, with the
presence of the Atopobium vaginae and Porphyromonas somera, a high vaginal pH
was significantly associated with the presence of endometrial cancer. These
microbes have previously been shown to be associated with other pathologies and
their findings suggest a role for the microbiome in the cause and progression of this
cancer that needs more research (Walther-António et al. 2016; Walsh et al. 2019).
Another study comprising women suffering hysterectomy for endometrial tumors
and identified Pseudomonas, Comamonadaceae, Escherichia, Acinetobacter, and
Cloacibacterium as principal taxa (Winters et al. 2019).

5.6 Cutaneous Microbiome: Biomarkers of Skin Cancer

Although it has long been known that skin is colonized by commensal microbes,
their identification has traditionally been based on methods of culturing and counting
living colonies (Sender et al. 2016). This method of identifying bacterial species is
biased towards bacteria that can easily grow (Gill et al. 2006). The advent of
molecular techniques, which allows the sequencing of gene encoding 16S ribosomal
RNA specific for prokaryotic cells and sufficiently variable for phylogenetic analy-
sis, has revealed a greater diversity of species in the microbiota that lives with its host
(Grice et al. 2009). Indeed, more than 1000 bacterial species have been identified
which collectively constitute the human microbiota (Grice et al. 2009; Grice and
Segre 2011). While the classic skin microbiota includes bacteria such as Staphylo-
coccus epidermidis, Staphylococcus hominis (common commensal bacteria), Strep-
tococcus mitis, Propionibacterium acnes, Corynebacterium spp., Acinetobacter
134 B. M. Bakadia et al.

johnsoni (common commensal bacteria), and Staphylococcus warneri (infrequent

commensal bacteria), new techniques based on 16S ribosomal RNA have revealed
that skin contains more than 300 commensal bacterial subspecies (Grice et al. 2009;
Grice and Segre 2011). Another discovery made possible by the new identification
methods is the variability of the microbiota between different anatomical areas of
the skin: different skin areas harbor different bacterial communities. The level of
diversity depends on temperature, humidity, and lipid content of the skin (Nakatsuji
et al. 2013). Three main skin microenvironments can be defined: oily areas (face,
inside part of ears, back of scalp, upper torso, and back), wet areas (inside of nostrils,
armpits, between the fingers, cubital fossa, popliteal fossa, inguinal folds,
intergluteal fold, arch, and navel), and dry areas (arms, palms, and buttocks)
(Nakatsuji et al. 2013). By comparing the bacterial species found in these different
skin areas, it was shown that Propionibacteria predominate in fatty areas,
Corynebacteria and Staphylococci are abundant in wet areas, while a mixed popu-
lation is found in dry areas, among which β-proteobacteria are the most abundant
(Grice et al. 2009). Although the skin microbiota found in each skin area appears to
be stable over time for a given person, the relative frequencies of these bacterial
species can vary greatly between individuals (Grice et al. 2009). This inter-
individual variability of the skin microbiome raises many questions about its acqui-
sition (environmental, role of genetics) and about its role in the development of skin
diseases. The commensal bacteria that partly make up the skin microbiota are present
on the surface of the skin, in the epidermis, and also in deeper layers such as the
dermis. They protect the host against colonization by pathogenic bacteria by com-
peting for nutrients and secreting bacteriocins. They also induce the expression of
IL-17 by T-cells and antimicrobial peptides (AMP) by keratinocytes, leading to the
development of protective immunity against the risk of infection (Macpherson and
Harris 2004). The increase or reduction in bacterial diversity, called dysbiosis,
promotes the emergence of pathogenic bacteria and a disruption of immune
responses which can be the cause of the development of certain skin diseases such
as acne, atopic dermatitis, hidradenitis suppurativa, or maybe even psoriasis and
cancer (Seite and Bieber 2015).
There are two main types of skin cancer: carcinoma and melanoma. Carcinomas
are the most common skin cancers (Liu-Smith et al. 2017). They generally occur
after the age of 50, on exposed areas of the body (face, neck, shoulders, forearms,
and legs, etc.) (Liu-Smith et al. 2017). They are most often due to excessive and
chronic sun exposure. Carcinomas are easily curable in the majority of cases.
However, some of them, called “squamous cell carcinomas”, can lead to distant
lesions (metastases) if not removed in time (Liu-Smith et al. 2017). Cutaneous
melanoma is much rarer than carcinoma but it is the most serious of the skin cancers,
because of its “high metastatic potential”, that is to say of its ability to spread rapidly
to other parts of the body (Liu-Smith et al. 2017). A good balance of the skin
microbiome protects against skin cancer. Furthermore, a study showed that strains of
S. epidermidis, which are frequently present on normal skin, produce, alongside
antimicrobial peptides, a molecule (6-N-hydroxyaminopurine) (6-HAP) which has a
role in providing protection against cancerous growth (Nakatsuji et al. 2018). In
5 Microbiome as Cancer Biomarkers 135

culture, 6-HAP inhibits the proliferation of cancer cell lines, without affecting
normal keratinocytes. Injecting 6-HAP into the mice inhibits the growth of experi-
mental melanomas. In addition, mice whose skin is colonized by S. epidermidis
producing 6-HAP are more resistant to UV-induced carcinogenesis than those whose
skin is colonized with strains which do not produce 6-HAP (Nakatsuji et al. 2018).
This research confirms the importance of the composition of the skin microbiome in
the antitumor immune response. The anticancer activity of nucleotide bases such as
adenine analogue 6-HAP has long been known, and the digestive microbiome is also
known to play a role in controlling tumor growth (Nakatsuji et al. 2018). We can
expect new discoveries regarding the anticancer functions of bacteria in the
microbiome, and possibly the development of anticancer drugs derived from
metabolites of the bacteria.

5.7 Gastrointestinal and Urinary Microbiota: Biomarkers

of Prostate Cancer

The urinary microbiota of people with prostate cancer harbors pathogens which
could contribute to the onset and development of this cancer. This is the conclusion
of a US study, the most comprehensive to date, on the links between urinary
microbiota and prostate cancer. Recent work has shown that the bacterial infections
and chronic prostatic inflammation promote the development of cancer by
prompting the identification of species likely to be involved in the pathophysiology
of the disease. Thus, the existence of a urinary microbiota could promote repeated
exposure of the prostate to various opportunistic bacteria in the proximity of the
urethra. To explore this lead, researchers analyzed the composition of urinary
microbiota of 129 people with prostate cancer before a prostate biopsy. Among
them, 63 presented a benign tumor, 61 a malignant tumor, and five others an initially
benign tumor which progressed to cancer. The results indicate that the urinary
microbiota of all participants were substantially equivalent, composed of about
60 species dominated by the genus Corynebacterium, Staphylococcus, Streptococ-
cus, Lactobacillus, or Gardnerella. However, the presence of a group of bacteria
containing Streptococcus anginosus, Anaerococcus lactolyticus, Anaerococcus
obesiensis, Actinobaculum schaalii, Varibaculum cambriense, and
Propionimicrobium lymphophilum was more often associated with cancer (70.8%
of cases). However, all these species are involved in urogenital infections including
prostatitis. Researchers have also identified more pathogens such as Ureaplasma
parvum or Ureaplasma urealyticum in cancer and G. vaginalis in moderate to severe
chronic inflammation of the prostate. These data do not allow to conclude on the
specific role of certain bacteria in this cancer but shed light on the composition of the
urinary microbiota in patients with prostate cancer. They also suggest the presence of
pro-inflammatory and uropathogenic bacteria in patients with prostate cancer
(Shrestha et al. 2018).
Researchers have shown how the gut microbiota interacts with an oral drug used
in prostate cancer, indicating an important influence of certain bacteria in response to
136 B. M. Bakadia et al.

the treatment. Conventional therapies designed to deprive the body of androgens,

which are responsible for the growth of prostate cancer, are not always effective.
Abiraterone acetate is considered here, and unlike other treatments, it is taken orally.
As this reagent is poorly absorbed, a significant portion is excreted in the stool which
interact with the intestinal microbiota. Several studies have demonstrated the role of
the intestinal microbiota in the development and progression of certain cancers, as
well as in the effectiveness of treatments. However, knowledge of the involvement
of the gut microbiota in prostate cancer remains limited. Researchers therefore
sought to demonstrate how AA, which is very effective in this type of cancer
resistant to conventional therapies, impacts the intestinal microbiota, and whether
this could act on the response to treatment. To do this, they examined the composi-
tion of the gut microbiota by sequencing 16S rRNA from 68 patients with prostate
cancer and divided them into three groups: patients not receiving treatment (n ¼ 33),
patients receiving conventional therapy (n ¼ 21), and patients receiving conven-
tional therapy + AA (n ¼ 14). The androgen deprivation by conventional therapy
alone or in addition to AA led to a significant reduction in Corynebacterium,
pro-inflammatory bacteria that metabolize androgens like testosterone, compared
to the control group. The intake of AA induced a significant enrichment of
Akkermansia muciniphila, accompanied by an increase in the production of vitamin
K2, known for its antitumor properties. These results were confirmed in an intestinal
model, thus ruling out the possibility of immune involvement. These investigations
reveal that AA is metabolized by intestinal bacteria. The components resulting from
this degradation would have a selective impact on the intestinal microbiota
characterized by the growth of A. muciniphila. This species, known for its health
benefits and anti-inflammatory properties, is believed to play a key role in response
to treatment, according to the authors. Previous work had also demonstrated its
beneficial role in response to treatments of certain immunotherapies. This study
highlights the key role of the intestinal microbiota in response to an anticancer
treatment taken orally, through the mechanisms that still need to be clarified (Daisley
et al. 2020). Exploring the drug–microbiota interactions could further improve the
treatment outcomes for many diseases.

5.8 Urinary Microbiome: Biomarkers of Bladder Cancer

Urinary microbiota is located in the bladder. As a result, the urine produced by the
body is loaded with microorganisms (Andolfi et al. 2020). The number of urinary
bacteria remains heterogeneous from one individual to another, and thus this
microbiota is different (Andolfi et al. 2020). A major difference is observed
according to the sex: the microbiota of women and men are divergent (Ackerman
and Chai 2019; Andolfi et al. 2020). In women, the urinary microbiota is marked by
a certain proximity to the vaginal microbiota, largely dominated by the bacteria of
genus Lactobacillus, Gardnerella, and Streptococcus (Ackerman and Chai 2019;
Pohl et al. 2020). Thus the urinary microbiota and the vaginal microbiota have
several points in common, but remain very different from the intestinal microbiota
5 Microbiome as Cancer Biomarkers 137

(Ackerman and Chai 2019; Pohl et al. 2020). Most often, the urinary microbiota is
less important than the vaginal microbiota. A study has shown that the presence of
certain Lactobacillus could be associated with the urinary incontinence (Govender
et al. 2019). Likewise, another study linked the presence of two bacterial species
with overactive bladder problems (Siddiqui et al. 2014). In men, such links between
the urinary disorders and the makeup of the urobiome has not been found. The
presence of urinary microbiota would be closely related to the method of urine
collection (Hourigan et al. 2020). In men with benign prostatic hyperplasia, a study
found a possible link between the frequency of bacteria in urine and the severity of
urinary symptoms (Lepor 2004). While the urinary microbiota differs by gender,
several studies suggest that urobiome may play a role in the development of urinary
disorders (Aragón et al. 2018). The data indicate that the urinary microbiota evolves
with age: the diversity of this microbiota decreases with age (Liu et al. 2017).
Researchers have also looked at the urinary microbiota of patients with bladder
cancer. One such study showed that the urobiome of 29 patients with bladder cancer
contained more Actinomyces europaeus than the urobiome of 26 noncancer patients
(Bi et al. 2019). An another study showed that the urobiome of 62 males patients
with non-muscle invasive bladder cancer contains more bacteria, including Micro-
coccus and Brachybacterium after undergoing transurethral resection of bladder
tumor than the urobiome of 19 non-neoplastic controls (Zeng et al. 2020a). A
study including 31 male patients with bladder cancer showed increased strains of
Sphingobacterium, Acinetobacter, and Anaerococcus and decrease of Roseomonas,
Serratia, and Proteus, compared to the 18 non-neoplastic controls. The increase of
Bacteroides, Herbaspirillum, and Porphyrobacter was observed in cancer patients
with a high risk of recurrence suggesting that these bacteria could be considered as
biomarkers (Wu et al. 2018). Furthermore, a study comprised of 12 male patients
with bladder cancer exhibited more Fusobacterium than 11 healthy. Next, 42 bladder
cancer tissues selected as independent sample had 11 F. nucleatum sequences
detected by PCR (Bučević Popović et al. 2018). A summary of occurrence of
microbiota in healthy and non-muscle invasive bladder cancer is shown in Fig. 5.4.
Despite its recent discovery, the investigations into the urinary microbiota
revealed its importance in the development of various urinary disorders, but also
of certain pathologies such as benign prostatic hypertrophy or bladder cancer. Other
studies may confirm the importance of the urinary microbiota.

5.9 Conclusions

Different parts of digestive tract as well as other parts like mouth, skin, and others in
human contain a variety of microbial community. These microbial communities are
beneficial in performing several physiological functions such as digestion, homeo-
stasis, and metabolism in gut and perform protection functions. Most importantly,
these microbiota are serve as biomarkers for detection of different types of cancers.
These biomarkers predict the risk and prognosis. Among the different microbiota,
the intestinal microbiota represents a considerable biomass with many physiological
138 B. M. Bakadia et al.

Fig. 5.4 A summary of occurrence of microbiota in healthy and non-muscle invasive bladder
cancer. (Figure reproduced from Andolfi et al. 2020 distributed under the Creative Commons
Attribution (CC BY 4.0) license)

functions essential for the host, thus representing an extremely complex ecosystem.
Each individual has their own intestinal microbiota which is therefore unique in
terms of quality and quantity. The intestinal microbiota has local and systematic
effects on the development of different types of cancers.
The extensive research carried out on different microbiota has led to the emer-
gence of knowledge and establishing links between them, which could ultimately
lead to open up therapeutic options. For instance, a knowledge of the pulmonary
microbiota and its links with the intestinal microbiota opens up therapeutic potential
through the use of probiotics on different respiratory sites. Thus, the pulmonary and
digestive administration of a cocktail of Lactobacilli before infection with the
dreaded pyocyanic Bacillus seems protective, whether in cystic fibrosis or nosoco-
mial pneumonia. Moreover, deciphering the lung microbiota will also enable the
discovery of next-generation probiotics and tackle common diseases like asthma
from an entirely new perspective. Similarly, a deep understanding of the interaction
of immune cells with different microorganisms would clarify the difference between
the commensal and pathogenic bacteria which could influence the immunotherapeu-
tic treatment. It is highly desirable to develop novel probiotics which could be used
in combination with chemo- and immuno-therapies for treating different types of
cancers.
5 Microbiome as Cancer Biomarkers 139

Acknowledgment This work was supported by the National Natural Science Foundation of China
(21774039, 51973076), BRICS STI Framework Programme 3rd call 2019 (2018YFE0123700),
China Postdoctoral Science Foundation (2016M602291), and the Fundamental Research Funds for
Central Universities, Open Research Fund of State Key Laboratory of Polymer Physics and
Chemistry, and Changchun Institute of Applied Chemistry, Chinese Academy of Sciences.

Conflict of Interest All authors declare no competing financial conflict of interest associated with
the publication of this work.

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Predictive Biomarkers for Anticancer Drugs
6
Nosheen Fatima Rana and Tahreem Tanweer

Abstract

The adverse effects of anticancer drugs, the acquired drug resistance, and tumor
heterogeneity among cancer patients limit the effective clinical management of
advanced malignancies. To overcome these challenges, predictive biomarkers
emerged as an indispensable tool to aid medical oncologists in identifying cancer
patients who may respond to several anticancer therapies, hence increasing the
risk-to-benefit ratio. This chapter will offer a brief overview of predictive anti-
cancer biomarkers, their characteristics, and brief details about the tools and
techniques in practice for their identification. This chapter will also discuss the
validated and also commonly researched predictive anticancer biomarkers for
anticancer drugs against different cancer types. Finally, the challenges in identifi-
cation and commercialization of the predictive biomarkers for anticancer drugs
will be discussed.

Keywords
Anticancer drugs · Drug resistance · Predictive biomarkers · Techniques ·
Identification · Treatment

N. F. Rana (*) · T. Tanweer

Department of Biomedical Engineering and Sciences, School of Mechanical & Manufacturing
Engineering, National University of Sciences & Technology, Islamabad, Pakistan
e-mail: [email protected]

# The Author(s), under exclusive license to Springer Nature Singapore Pte 149
Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_6
150 N. F. Rana and T. Tanweer

6.1 Introduction

Cancer diagnosis and treatment are nowadays mainly dependent on morphological

features determined by histological assessments. While this approach results in a
confident diagnosis and prognosis in most cases, molecular characterization of the
tumor tissue can effectively determine tumor grade, it's aggressiveness and
also predicts possible outcomes for various existing medications. Therefore,
throughout the decision-making process, molecular characterization might be an
indispensable tool for clinicians (Buonaguro et al. 2014). A biomarker is an attribute
that can be objectively evaluated and used to define the normal or pathological
condition of an organism through studying biomolecules like protein, peptide, DNA,
RNA, as well as chemical modifications of these biomolecules (Institute of Medicine
2012). Yet, it should be noted that the term “biomarkers” has evolved in the last
decade. The World Health Organization (WHO) offered an extremely broad defini-
tion that “a biomarker is any substance or structure, that can be measured in the body
or its products can influence or predict the incidence of outcome or disease” (Lassere
2008; World Health Organization 2001). Based on the clinical value, the cancer
biomarker can measure the likelihood of developing cancer inside a certain tissue or
measure the chances of its advancement or probable response to therapy. Cancer
biomarkers are increasingly associated with the deregulation of several molecular
pathways and cancer pathogenesis to enable the use of several therapeutics or
intervention methods and supply important information in guiding clinical
decision-making. With the remarkable increase in the scope of omics examination
of clinical biospecimens following the traditional course of the biomarker deploy-
ment, the conceptual model of cancer biomarker discovery has also been
transformed (Institute of Medicine 2012).
Cancer biomarkers are categorized into many groups based on their application.
Predictive biomarkers are cancer biomarkers that predict response to certain treat-
ment modalities, like HER2 positivity in breast cancer, predicting response to the
trastuzumab. Likewise, in colorectal cancer, KRAS-activating mutations predicted
resistance to EGFR inhibitors like cetuximab (Goossens et al. 2015). Such predictive
biomarkers are quite often classified as biological markers that are assessed using
tissue or bodily fluids. They are identified by using different techniques, such as
through genomics and proteomics. They can also be identified through radiographic
approaches such as dynamic contrast-enhanced imaging to create imaging markers
(DCE-MRI) and standard Response Evaluation Criteria in Solid Tumors (RECIST).
They are routinely utilized during patient follow-up in multiple clinical trials or
routine practice (Mankoff et al. 2014; El Bairi et al. 2019). In evaluating the clinical
and substitute endpoints, such biomarkers offer great potential for assessing the
effectiveness and safety of therapies tried on cancer victims in a timely, efficient, as
well as pharmacoeconomic manner (El Bairi et al. 2019).
The earliest research on predictive biomarkers yielded significant results for
cutting-edge directed cancer therapies. Some of the validated predictive biomarkers
produced as a result include expression of PD-L1 to predict response to
pembrolizumab (Merck Sharp Dohme, Keytruda®), the status of BRAF to predict
6 Predictive Biomarkers for Anticancer Drugs 151

response to nivolumab (Bristol Myers Squibb, Opdivo®) as well as vemurafenib

(Hoffmann La Roche, Zelboraf®) and dabrafenib (Novartis Pharms Corp, Tafinlar®).
Other validated predictive biomarkers include RAS expression to predict response to
cetuximab (Erbitux®), panitumumab (Vectibix®, Amgen), and Imclone; PDGFR
status to predict response to imatinib (Novartis; Gleevec®), as well as BRCA
expression for PARP inhibitors. These predictive biomarkers for anticancer drugs
are currently widely used in the routine clinical practice of oncologists. Conse-
quently, logical use of such biomarkers is appropriate to realize targetable disease
features and has allowed the personalized medical care of cancer patients (El Bairi
et al. 2019).
This chapter will introduce predictive anticancer biomarkers and discuss tools
and techniques in practice for thier identification such as different clinical trial
designs, molecular imaging, and computational and statistical techniques. This
chapter will also discuss some validated and commonly researched predictive
anticancer biomarkers for anticancer drugs against different cancer types, e.g.,
circulating tumor cells (CTCs), mutations and polymorphisms, methylation, and
miRNA expression. Finally, the challenges in identifying and commercialization
of the predictive biomarkers for anticancer drugs will be discussed.

6.2 Introduction to Predictive Biomarkers

As the name indicates, the predictive biomarkers disclose details about the likelihood
of achieving a response to anticancer therapy. Therefore, they aid the oncologists in
therapeutic decision-making process (Nalejska et al. 2014). They could also give
information that can assist patients in evading the toxicity of conventional (systemic)
medications and determine their eligibility for targeted therapy. Most of the predic-
tive biomarkers have been discovered for prostate, lungs, and breast cancer. These
cancer types are presently the most prevalent cancer forms throughout the general
population (Nalejska et al. 2014).
Understanding a patient’s cancer molecular characteristics can lead to the
personalization of drug therapies with a better probability of effectiveness. Patients
with specific kinase domain mutations upon EGFR, for instance, may not give a
response to EGFR targeted therapy like erlotinib (Fig. 6.1a). This also has the added
benefit of limiting a patient’s exposure to toxic side effects from a drug from which
they may not even have benefitted (Goossens et al. 2015).
The Predictive markers were also reported for their ability to predict the
aggressiveness of a certain patient’s cancer, the anticipated course of the disease,
or even the prospect of response to the cancer therapy. Therapeutic predictive
markers also show the prospect of a patient’s response to a specific treatment, as
mentioned earlier. They help the patient and the oncologist decide a therapy against
cancer that is almost certain to be successful for that patient’s cancer type. Different
predictive biomarkers have dual capacities; they exhibit prognostic as well as
predictive characteristics. ER expression in breast cancer is one such example. It is
a prognostic as well as a predictive marker. Compared to non-ER-expressing
152 N. F. Rana and T. Tanweer

Fig. 6.1 (a) EGFR expression predicts response to erlotinib (b) ER expression predicts response to
endocrine therapy in breast cancer patients

cancers, cancer with ER expression is less aggressive and shows a better clinical
course.
Furthermore, an absence of ER expression envisages that breast cancer will not
respond to endocrine therapy (Fig. 6.1b). Recently, multigene testing like Oncotype
DX (Genomic Health Inc.) also aided in predicting tumor aggressiveness and
response to the therapy. These tests are frequently utilized to assess the necessity
for receiving aggressive treatment, for instance, adjuvant chemotherapy (Mankoff
et al. 2014).
Generally, the development of predictive cancer biomarkers follows a multistep
development process which includes (1) identification of biomarker, (2) development
of assay, (3) clinical application, (4) validation of the assay, (5) clinical testing
employing large prospective trial, and lastly, (6) approvals from the regulatory
authority and then integration into the clinical practice guidelines and marketing.
This sector has profited from some well-known international recommendations,
notably the REMARK guidelines, which were established to tackle the consistent
utilization of reporting in biomarker research studies (El Bairi et al. 2019; Sauerbrei
et al. 2018; Altman et al. 2012). Positive results from randomized controlled trials
led to introducing effective anticancer drugs into clinical practice. Therefore, the
identification of biomarkers has become a chief topic in cancer research since
biomarker research is expanding in tandem with the development of novel anticancer
treatments like targeted medicines, immune checkpoint inhibitors, and all those
6 Predictive Biomarkers for Anticancer Drugs 153

obtained from natural sources including plants and aquatic organisms (El Bairi et al.
2019).

6.3 Need for Predictive Biomarkers

Assistance and support required to develop predictive biomarkers mainly comes

from various sources, including regulatory, commercial, and clinical sides. These
considerations apply to several newly discovered cancer drugs which target specific
molecular abnormalities and are often successful against a fraction of the cancer
patients, often in the 10–20% range (Sawyers 2004). This suggests that around
$45 billion of the yearly expenditures on cancer treatments (approximated $60 billion
in 2010) will be spent on medicines with low benefits without patient selection. As a
result, there is a convincing reason for developing companion diagnostics that can
evaluate biomarkers, thereby identifying the responsive subset of patients. This
enhances therapy’s affordability, which works in parallel with improved clinical
utility and safer drugs. Several chemotherapy regimes may result in fatality rates
ranging from 0.5% to 2.0%, whereas 30–40% have grade 3/4 toxic effects, resulting
in a considerable morbidity burden, particularly if a considerable proportion of this
group does not benefit from the treatment. Targeted therapies could be hazardous in
the same way. Bevacizumab, for instance, has substantial adverse effects such as
gastrointestinal, cardiovascular, and renal damage (Badgwell et al. 2008; Segal and
Saltz 2009) The minimization of unnecessary treatment and undesirable effects
would be a key aspect of predictive biomarker-driven cancer treatment.
Due to evolving pharmacoeconomic environment and rising medication costs,
regulatory authorities have emphasized the necessity of predictive biomarkers and
the importance of predictive biomarker testing on patients’ and providers’ budgets.
Both the FDA and European Medicines Agency are urging drug manufacturers to
explore predictive biomarkers as the companion diagnosis, which would be widely
expected to become communal guidelines and general practices (Goodsaid and
Papaluca 2010). Generally, there is a rising demand for prediction tools to help
identify and treat individuals with responsive diseases (La Thangue and Kerr 2011).

6.4 Identification of Predictive Biomarkers

for Anticancer Drugs

The evidence for using predictive biomarkers as companion diagnostics to aid in the
clinical use of cancer therapies is growing. To date, only a few biomarkers have got
to the clinic and achieved the value of companion diagnostics. These biomarkers are
being revealed mostly by the retrospective assessment of clinical trial data and
random ad hoc genetic testing. Before drug development, historical information of
mutations and related molecular heterogeneity has been rarely used as an integrated
factor of the future clinical trial design. Nowadays, the issue that integrates existing
methodologies is to position predictive biomarkers in a methodical prospect-driven
154 N. F. Rana and T. Tanweer

approach, permitting drug development to advance in parallel with the allied bio-
marker, thus opening a new, better hypothesis-based method to develop personalized
cancer therapy (La Thangue and Kerr 2011).
For identifying predictive biomarkers, various high-throughput technologies in
use include DNA sequencing at large-scale, microarray-based transcript profiling,
proteomics, and single-nucleotide polymorphism (SNP) analysis (Nalejska et al.
2014; Schilsky 2010).
These approaches are responsible for generating correlative data, which is further
utilized to identify the genes and proteins associated with the cancer type or response
to the treatment. However, these approaches are very challenging to understand the
processes involved in cancer and the treatment response. A clinical study was
conducted to understand response to the treatment by a potential anticancer drug;
it showed a response rate of around 20% and improved survival of 3 months in an
unselected group of patients. However, the mean survival rate of the population
compared to this group (12.6 months vs. 12 months) could not reflect a significant
difference. Therefore it was not adequate to justify the clinical activity of this drug.
In another study, a population of cancer patients was evaluated for a predictive
biomarker. The stratified subset of the population, with 70% PPV for response and
followed by treatment with the new cancer drug, reported an average survival rate of
14.1 months. This improved survival rate was better when compared with the
unselected group, therefore requiring further clinical study (Sawyers 2004). As a
solution, research has concentrated on establishing platforms that enable the identi-
fication of functionally relevant biomarkers, which can then be justified in the new
drug’s mechanism of cancer cell killing and utilized to assist and enhance its clinical
development (La Thangue and Kerr 2011).

6.5 Tools and Techniques for Predictive Biomarker

Identification

6.5.1 Clinical Trial Designs and Analysis Techniques

During the last decade, a variety of biomarker-based design methods have been
proposed to study therapies in potentially heterogeneous patient subpopulations.
There are several levels to which these can be classified.
Clinical trials for selective therapies are broadly classified into different phases.
The first type is the phase 1 trial that involves simultaneous investigation of the
predictive biomarker and the treatment in two groups, i.e., the healthy and the tumor
group. Once the investigation is completed, the assay is validated, followed by
carefully selecting the appropriate biomarker positivity levels. The second type is
the phase 2 trials that involve searching and validating a marker-based population.
At this stage, the efficacy of the targeted therapy is more promising compared to the
phase 1 trial. In phase 3 trials, a typical population-based randomized treatment
comparison is carried out, based on the benefits achieved from the earlier phase
2 studies (Mandrekar and Sargent 2014; Renfro et al. 2016). The predictive marker-
6 Predictive Biomarkers for Anticancer Drugs 155

based trial designs are categorized as retrospective and prospective study designs. In
the retrospective trial design, the marker and the treatment outcome relationship are
assessed after the trial’s completion. Whereas, in future trial designs, the predictive
biomarkers are formally included in the design considerations. Future trial designs
are usually required to assess the clinical validity of the predictive biomarker.
Another classification of the predictive biomarker trial designs is strictly based on
statistical methodology, i.e., the frequentist or “classical” designs versus the Bayes-
ian designs. These designs have variations in the procedures for carrying out the
testing of hypothesis, decision-making, and the use of preceding (or historical)
knowledge (Renfro et al. 2016) (See Fig. 6.2).
For the last decade, substantial progress has been made in understanding the
molecular basis of cancer and clinical trial design methods to meet biomarker-based
goals. Future studies in all fields are needed to carry out fully customized cancer
management plans for standardized care. There is still a need for an adaptive design
paradigm within a clinical trial methodology that can prospectively identify both
combination or continuous biomarkers that predict treatment response and clinically
validate certain markers along with their thresholds or classifications, usually
achieved in different studies ad hoc basis. Additionally, when spontaneously contin-
uous or mixed biomarkers or signatures are used, then there is a necessity for more
robust methods and better techniques for the selection of biomarker threshold (i.e.,
cataloging the patients as a marker-“positive” versus marker-“negative”). As
advancements in clinical testing and trial methods are made, we must keep the
viewpoint of particular patients in mind. Participation in biomarker-based clinical
trials becomes a more rational and ethical choice (Meric-Bernstam et al. 2015). In
this age of personalized and selective medicine, rigorous review and implementation
of novel design techniques, both for early and final phase trials, would be needed to
ensure the suitable clinical validity of specific experiments and therapies (Renfro
et al. 2016).

6.5.2 In Situ Hybridization and Immunohisto Chemistry

Techniques

In Situ Hybridization (ISH) and Immunohisto chemistry (IHC) are morphology-

based techniques that directly explore the cancer cells (Schildhaus 2019).
IHC detects changes of protein expression qualitatively or as a semiquantitative
measurement. The reliability of IHC-based predictive biomarkers depends on
pre-analytical factors, selection of suitable antibodies, staining procedures, and an
assessment of staining. Specific reading and scoring approaches for different tumor
entities make evaluations complex, especially for evolving biomarkers in the context
of immuno-oncology treatment (Schildhaus 2019).
ISH assays are capable of detecting gene amplifications, large deletions, and gene
fusions. Definitions of amplifications are gene and entity-specific. Activating
rearrangements frequently involve genes encoding receptor tyrosine kinases,
which tyrosine kinase inhibitors can address (Schildhaus 2019).
 156

Fig. 6.2 Classification of clinical trial designs for identification of predictive biomarkers for anticancer drugs
N. F. Rana and T. Tanweer
6 Predictive Biomarkers for Anticancer Drugs 157

Fig. 6.3 Morphology based methods for identification of predictive biomarkers for anticancer
drugs (a) Immunohistochemistry (b) In situ hybridization

Most of the currently applied predictive biomarkers are based on either IHC or
ISH. The number of assays are steadily growing, great efforts are needed to achieve
and maintain the highest level of reliability. Future developments will introduce
multiplexing IHC and ISH (Schildhaus 2019) (Fig. 6.3).

6.5.3 PCR-Based Technologies and Multiplexed Gene Analysis

Polymerase chain reaction (PCR) has become an invaluable tool for assessing the
presence and type of nucleic acids in tissues and body fluids. It is the enzymatic
synthesis and amplification of particular DNA sequences in vitro. It can replicate a
single molecule of DNA or RNA into billions of duplicates in a matter of hours. This
enables mutation tracking for the management of any cancer, which is particularly
crucial in targeted therapies. Novel applications include analysis of blood for
circulating DNA for tumor-associated mutations. RNA analysis has been extensively
used for the quantification of gene expression. This forms the basis of multiple gene
expression assays, including multigene panels developed for prognostic and predic-
tive purposes (Gökmen-Polar 2019).
158 N. F. Rana and T. Tanweer

6.5.4 Microarray Technology

Microarrays allow for the simultaneous measurement of expression levels of multi-

ple genes inside a sample. The Microarrays have also been widely used in screening,
profiling, and quantifying genome-wide gene expression profiles. They are crucial in
identifying and also validating the predictive cancer biomarkers. In cancer
diagnostics as well as biomarker research, gene expression arrays, single nucleotide
polymorphism (SNP) arrays, as well as comparative genomic hybridization (CGH)
arrays are indeed the primary approaches for identifying gene expression patterns,
copy number variability, and sequence polymorphisms (Palanisamy 2019).

6.5.5 Massive Parallel Sequencing

Cancer research is advancing at an unprecedented rate, notably in identifying and

characterizing clinical features and biomarkers that would be used to predict treat-
ment response. The purpose of cancer therapy is to tailor the right treatment to the
appropriate patient. To that aim, some clinical studies are now utilizing patient
stratification by “Massive Parallel Sequencing” (MPS), widely known as “Next-
Generation Sequencing” (NGS), to detect clinically responsive targets in real-time.
The omics upheaval gives significant, meaningful insight further into origins,
processes of disorders, drug responses, roles of genetic and environmental factors in
cancer predisposition and developed resistance. It lays the groundwork for precision
medicine, which concentrates on factors unique to a particular patient to give
individualized care; details about patients’ genes, proteins, and environment are
utilized to prevent, diagnose, and customize medical care to an individual patient.
Omics data and NGS use are currently transforming the way of cancer patients’
treatment. Many different forms of cancer are presently being treated using targeted
medicines that take the benefit of the information about an individual’s unique
cancer cells.
We must also consider the multidimensional nature of cancer biomarkers while
developing predictive indicators. They will necessitate a systems biology approach
involving the integration of omics platform data in order to build innovative proba-
bilistic models for early diagnosis, prediction, prognosis, and prevention. They will
ultimately be based on existing tumor profiling, translating into optimal therapy that
will improve patients’ overall survival and quality of life (Abramovitz et al. 2019).

6.5.6 Flow Cytometry

Modern biology heavily relies on optics-based instruments to grasp the intrinsic

functional and structural features of cells and biomolecules. Flow cytometry itself is
such a technique that allows the biomarkers detected by imaging technology to be
measured quickly. Flow cytometry is distinguished by its ability to rapidly resolve,
count, and sort distinct populations of cells or organelles, dependent on their
6 Predictive Biomarkers for Anticancer Drugs 159

expression of markers. Laser flow cytometry is widely used in fundamental and

clinical testing, diagnostics, and disease control. Flow cytometry is used for a variety
of purposes, including immunophenotyping of surface and intracellular markers,
genome prediction, cell cycle analysis, antigen quantitation, membrane potential,
ploidy analysis, calcium influx, pH, chromosome analysis, fluorescent reporter
proteins, and so on (Tembhare et al. 2019). They have also been extensively used
to identify predictive cancer biomarkers (Tembhare et al. 2019).

6.5.7 Molecular Imaging

The PET imaging of an ER expression, which is most often conducted using the
18
F-fluoroestradiol, is an ultimate illustration of molecular imaging through a pre-
dictive biomarker. The absorption of 18F-fluoroestradiol has been shown to be
associated with tissue-based ER expression assays (Linden and Dehdashti 2013;
Mankoff et al. 2014). 18F-fluoroestradiol PET, including tissue-based ER assays,
predicts endocrine responsiveness of breast cancer, and notably, a lack of the
18
F-fluoroestradiol uptake positively predicted no response. Early researches on
other ER-expressed cancers, like endometrial cancer, suggests a possible predictive
role (Tsujikawa et al. 2009; Mankoff et al. 2014). There is currently an NCI-held
investigational new drug application (IND) for the 18F-fluoroestradiol to fund
clinical trials. Thanks to the sustenance of a National Cancer Institute (NCI) Cancer
Imaging Program, a limited prospective phase 2 trial has been completed success-
fully (Peterson et al. 2014; Mankoff et al. 2014). This approach can be useful in
clinical trials for the novel endocrine therapeutics in breast cancer, particularly in
helping the patients whose tumor expresses the therapeutic target (ER); nevertheless,
more rigorous, highly controlled, as well as properly powered experiments are
required to assess the negative predictive value of no 18F-fluoroestradiol uptake
versus the fine uptake thresholds. Multicenter trials together under NCI IND have
been planned to meet this need. Many other molecular imaging methods for
evaluating the territorial expression of cancer therapeutics’ targets, such as proges-
terone receptor, HER2, or epidermal growth factor receptor, are also emerging
(Kenny et al. 2011; Mankoff et al. 2014).

6.5.8 Digital and Computational Pathology

Digital pathology is also at the forefront of tissue analytics, offering a suite of new
advanced methods for biomarker testing, interpretation, detection, and translation.
For several years, the benefits of digital image analytics in tissue science have been
recognized. Recent developments in high-resolution whole slide scanning, image
analytics, web-enabled multisites collaboration, imaging information, and machine
learning, on the other hand, are now allowing researchers to boost quantitative
biomarkers’ development and transform the clinical translation and discovery of
160 N. F. Rana and T. Tanweer

lucrative diagnostics for accurate therapeutics for the first time (Hamilton et al.
2019).

6.5.9 Bioinformatics and Biostatistical Tools

Biomarkers are now an essential part of modern medicine. Biomarker identification,

clinical confirmation, and biomarker acceptance in clinical practice all present new
problems in bioinformatics and biostatistics. With the increasing availability of high-
throughput technologies, the medical research challenge is identifying individual
biomarkers or biomarker signatures that predict treatment or therapy outcomes.
Various bioinformatic and biostatistical tools and methods are used to study predic-
tive biomarker discovery and biomarkers testing in clinical trials. For reporting and
processing issues, using biomarkers in clinical routine (including the bioinformatics
and machine learning method) required omics data, clinical trials, and validation
(Perera-Bel et al. 2019).

6.6 Cancer Biomarkers for Predicting the Response Toward

the Treatment

6.6.1 Circulating Tumor Cells (CTCs)

Circulating cell-free DNA (cfDNA), which can be extracted from serum or plasma
through noninvasive techniques, has been identified as an appealing biomarker for
cancer patients to evaluate therapeutic response, identify drug tolerance, and predict
the clinical outcome (De Mattos-Arruda and Caldas 2016; Carpinetti et al. 2015).
Tumor cells have been shown in experiments to release genomic DNA into blood,
then circulating DNA may reflect tumor strain and biologic characteristics (Jiang
et al. 2015; Xia et al. 2015; Chen et al. 2020).
Elevated levels of cfDNA were found to be strongly associated with low survival
in Non-small cell lung cancer (NSCLC) patients. Furthermore, our findings suggest
that cfDNA could be a capable predictor of reaction to EGFR-TKIs in NSCLC
patients (Ai et al. 2016).
Circulating cell-free DNA (cfDNA) derived from blood serum or plasma by a
noninvasive technique has also been suggested as an alternative and appealing tool
for detecting early lung cancer, assisting in developing a tailored care plan, and
estimating patient treatment response (Carpinetti et al. 2015; De Mattos-Arruda and
Caldas 2016). cfDNA has shown to be feasible and predictive for cancer patients
(Newman et al. 2014; Zhou et al. 2012). Furthermore, cfDNA is more appropriate for
a general diagnostic test for cancer patients and even more relevant for clinicians in
monitoring cancer dynamics, unlike biopsy. In the case of NSCLC, several
experiments have looked into the success of cfDNA in cancer diagnosis and
prognosis (Wang et al. 2014; Zhang et al. 2013), and the findings differ for a variety
of reasons. Thus, a systematic meta-analysis was performed to analyze the
6 Predictive Biomarkers for Anticancer Drugs 161

diagnostic accuracy of cfDNA for cancer diagnosis and EGFR and KRAS mutation
and to govern the predictive role of cfDNA in NSCLC patients with a low prognosis
(Chen et al. 2020).
The CTC count analysis before, during, and then after treatments at various
periods allows for the estimation of treatment outcome. About the fact that the
number of isolated CTCs from the patient samples is very small, some researchers
have investigated new techniques for extremely efficient enrichment of CTCs
required to conduct the profiling of gene expression. The molecular characterization
of CTCs will aid in predicting response to therapy. Reduced mammaglobin
1 (MGB1) mRNA levels in CTCs obtained from the patients of breast cancer
(metastatic), for instance, may better predict response to therapy (Nalejska et al.
2014).

6.6.2 Mutations and Polymorphisms

Some of the early episodes in colorectal carcinogenesis involve the somatic

mutations inside the KRAS gene. In 1988, the first finding demonstrated a link
between KRAS mutations and this tumor type’s growth (Vogelstein et al. 1988).
Codons 12 and 13 are the most often mutated, whereas codons 61 as well as 146 are
the least often mutated (Loupakis et al. 2009). The status of mutations in codons
12 and 13 of the KRAS gene is a gold standard predictive biomarker for determining
whether patients with advanced CRC are eligible for targeted treatment with mono-
clonal antibodies targeted to the extracellular domain of the EGFR (Lewandowska
et al. 2012). EGFR promotes colorectal cancer (CRC) cells (through signaling
pathways including MAPK, JAK/STAT, pik3). The growth of tumor cells is reduced
due to therapy, while the rate of apoptosis increases. The absence of codon 12 and
13 mutations in the KRAS gene hence presents a significant predictive value.
However, it must be remembered that patients with high-level EGFR expression
without mutations in 12 and 13 codons of the KRAS gene may have a worse
response to therapy when the occurrence of somatic mutations in 61 or 146 codons
of KRAS genes or the somatic mutation of V600E in BRAF gene in their tumor cells
has been determined (Lewandowska et al. 2013). The occurrence of both V600E
mutations inside the BRAF gene as well as Codon 61 mutations inside the KRAS
gene, over 10% are determined by CRC mutation analyses (Lewandowska et al.
2013). Therefore, rapid molecular biology diagnostic assays for both the KRAS gene
(12, 13, and 61 codons) and BRAF gene (V600E) while using the very sensitive
PCR method is still appropriate, in particular when DNA is to be collected from
low-density body tissue with the limited detection for carcinoma pattern
(Lewandowska et al. 2013). Notwithstanding the alterations in the KRAS gene,
PIK3CA gene mutations and deletions inside the PTEN gene might also reduce the
response to monoclonal metastatic CRC antibody treatment (Sartore-Bianchi et al.
2009). The evaluation of mutations throughout the BRAF, PIK3, and PTEN genes
may also reflect targeted treatment in the future.
162 N. F. Rana and T. Tanweer

Thymidylate synthase (TS) is an important molecular target for several

chemotherapeutics, including 5-fluorouracil, since it is a crucial enzyme for
synthesizing the DNA (5-FU). Resistance to 5-FU is linked to TS overexpression.
In comparison to just two copies of the tandem repeats (TSER*2) in a TS promoter
region, the three copies of tandem repeats (TSER*3) presence promote higher TS
expression. Furthermore, individuals with the TSER*2/TSER*2 or TSER*2/
TSER*3 genotype had a greater response to 5-FU treatment. Compared to
individuals with TSER*3 homozygotes, these patients had a high OS (overall
survival) rate. It is imperative to notice that in patients having metastatic CRC, an
overexpression of TS (due to many gene copies) leads to failure of 5-FU treatment
and deteriorates the OS rates (mCRC) (Jiang et al. 2012).
The 5-FU catabolism is regulated by dihydropyrimidine dehydrogenase (DPD), a
pharmacogenetic biomarker validated by the FDA. 5-FU has significant toxicity in
individuals with DPD deficiencies, leading to death in some cases (Gross et al.
2008). The G>A transition at the donor splice site (IVS14 + 1G>A) (that conse-
quently leads to the skipping of exon 14) is the most important mutation resulting in
the reduction of enzymatic activity by the DPD protein (Raida et al. 2001). Patients
can be classified with a high-low risk of grade 3 or 4 toxicity, while 5-FU therapy is
based on genetic analysis of the DPD polymorphism (IVS14 + 1G>A). In other
investigations, individuals with grade 4 neutropenia were found to be homozygous
or heterozygous for an IVS14 + 1G>A gene in 50% of cases (Van Kuilenburg et al.
2002).
The investigation of tumor’s microsatellite instability (MSI) can also give prog-
nostic and predictive information. The findings of a single-factor study including
grade 2 and 3 CRC patients demonstrated that fluorouracil medication was more
beneficial in patients having stable microsatellites (MSS) or the low-frequency MSI
than in colorectal cancer patients having high-frequency MSI (MSI+). Later on,
fluorouracil did not alleviate cancer and even worsened the condition (Ribic et al.
2003).
The use of topoisomerase I inhibitor, i.e., camptothecin-11 (CPT-11), targets MSI
+ tumor cells with mutated genes that play a role in mismatch system repair (MMR),
has the opposite effects (Bras-Gonçalves et al. 2000). However, the MSI status’
prognostic significance is still under investigation, and currently, it is not widely
employed in cancer therapy.
The evaluation of mutations in an EGFR kinase domain may be employed as the
predictive biomarkers in NSCLC patients. Somatic mutations in exons 19 or 21 have
been associated with tumor susceptibility to TKIs (tyrosine kinase inhibitors) like
erlotinib and gefitinib medications. In the instance of deletion in exon 19, a median
survival rate was greater than in the case of a point mutation L858R in exon
21 (Riely et al. 2006).
In a Polish population, activating mutations of the EGFR genes were observed
in about 12% of adenocarcinoma patients during the assessment of deletions in
exon 19 (Krawczyk et al. 2012), and a substitution of L858R was observed in 13% of
adenocarcinoma patients while scrutinizing the 29 mutations in exon 18, 19,
20 and 21. TKI-susceptible NSCLC cells might develop resistance with time.
6 Predictive Biomarkers for Anticancer Drugs 163

Unfortunately, NSCLC cells susceptible to TKIs may acquire resistance over time.
Apart from alterations that make the tumor sensitive to TKIs, a somatic mutation
T790M in an EGFR kinase domain of exon 20 has been found in about half of all
patients of lung adenocarcinoma (Pao et al. 2005). At position 790, the substitution
of threonine for methionine enhances the affinity of ATP (adenosine triphosphate),
which is the fundamental mechanism of drug resistance development (Yun et al.
2008).
Furthermore, irrespective of EGFR gene’s T790M mutation, MET gene amplifi-
cation may be a significant source of drug resistance in 20% of cases. The stimula-
tion of the ERBB signaling pathway may result in acquired resistance. The H820 cell
line’s ERBB signaling is largely reliant on the activity of MET, according to studies
using XL880 molecules (MET kinase inhibitors) and small interfering RNA
(siRNA) that suppresses MET expression. The findings support the possibility of
low molecular weight inhibitors in lung adenocarcinoma patients resistant to EGFR
inhibitors yet have multiple copies of a MET gene (Bean et al. 2007). Soda et al.
discovered a minor inversion in the p region of chromosome 2 in NSCLC patients
for the first time in 2007, resulting in the fusion gene EML4-ALK. The inversion was
discovered in 6.7% of the patients in the study, which comprised 75 Japanese
subjects (Soda et al. 2007). A lower incidence rate of 4.9% was seen in research
with bigger groups (n ¼ 266) (Wong et al. 2009).
It’s important to notice that EML4-AKL mutation occurs in various histological
types of NSCLC in nonsmokers, and it’s not always associated with the mutations in
the KRAS and EGFR genes (Wong et al. 2009). Within that group of patients,
translocation is the most important factor in tumor development. Hundreds of
adenocarcinoma tumors appeared in both lungs after a few weeks of birth in
transgenic mice harboring the differentially expressed EML4-ALK fusion gene as
the model organism. This prompted the researchers to examine further the
oncogene’s dominant involvement in lung carcinogenesis (Soda et al. 2008). Fol-
lowing studies of MET and ALK inhibitors, the crizotinib was swiftly introduced in
a market (2011), while a predictive evaluation of a fusion gene EML4-ALK,
employing fluorescence in situ hybridization (FISH) for paraffin-embedded material
or the reverse transcriptase PCR (RT-PCR) for cytological content (Soda et al.
2012), is critical for determining the eligibility for the treatment of the patient by
this selective kinase inhibitor.
The presence of BCR-ABL (the crucial fusion gene for targeted treatment) in
chronic myeloid leukemia patients is another illustration of a fusion gene as a
predictive biomarker (CML). The newly formed chimeric protein, i.e., tyrosine
kinase BCR-ABL, is enabled by a translocation among the chromosomes 9 and
22 (Druker et al. 2006); however, for more than 10 years, the predictive analysis of
the presence of that protein has permitted the assessment of patients’ candidacy for
imatinib treatment.
The evaluation of somatic mutation in codon 600 of a BRAF gene in advanced
melanoma patients is another intriguing predictive biomarker. The only medicine
recognized by the FDA for this condition was dacarbazine (DTIC) for several years,
which had a very low response rate of about 10% (Chapman et al. 1999). The
164 N. F. Rana and T. Tanweer

discovery of the most prevalent mutation in a BRAF gene (V600E) and the inclusion
of vemurafenib (a powerful inhibitor) in treatment marked a significant milestone in
this type of cancer treatment. BRAF protein belongs to the raf/mil family of the
serine-threonine kinases that control the MAPk and ERK signaling pathways, which
regulate cell proliferation. Many tumor types have somatic mutations (missense)
within BRAF gene (V600E/K/D/R/M). Malignant melanoma, on the other hand, has
the greatest incidence rate (66%), with the V600E mutation accounting for the
majority of cases (c. 1799T>A) (Davies et al. 2002).
In untreated and inoperable individuals (having stage III or IV), melanoma
expresses the mutation V600E. The phase III clinical trial findings showed that
vemurafenib reduced the risk of mortality by 63% and the risk of tumorigenesis by
74% compared to dacarbazine. Patients treated with vemurafenib responded to
therapy in 48% of cases, compared to just 5% of patients treated with dacarbazine.
Patients with the V600K and V600D mutations were also included in the research
group. A significant number of patients (40%) having the mutation V600K well
responded to vemurafenib treatment (Chapman et al. 2011); therefore, it appears
critical to identify not only the prevalent mutation, V600E, but also much more
known activating mutations, using the real-time PCR or pyrosequencing (Spittle
et al. 2007).
Unfortunately, with CRC and NSCLC, developing melanomas resistance to the
abovementioned inhibitors is becoming manifest. The CRAF protein was more
active in drug-resistant clones produced from BRAF V600E M14 melanoma cell
lines treated with an inhibitor AZ628. The CRAF protein may obtain a higher
predictive value in evaluating the BRAF inhibitor therapeutic effectiveness follow-
ing these findings. The possible predictive value of geldanamycin (tumor cells with a
high degree of CRAF expression appeared vulnerable to this medicine) has also been
investigated (Montagut et al. 2008). Further molecular studies in personalized
medicines showed somatic point mutation in a MEK1 gene (in BRAF V600E
A375 melanoma cell lines), which contributes to the resistance toward the MEK
inhibitor (AZD6244) (Emery et al. 2009), also a somatic mutation in a KIT onco-
gene, which is found 21% in mucosal melanomas, 11% in acral melanomas, and
16.7% in melanomas linked with chronic damage induced by the sun. Furthermore,
mutations in the KIT gene have been found in imatinib-resistant melanomas (Curtin
et al. 2006), making the gene a prospective therapeutic target that has been
investigated for almost a decade.
Breast cancer is considered the most prevalent malignant tumor in women, and
researchers have been looking for biomarkers to predict its prognosis for more than a
decade. Somatic mutations and polymorphisms are both included in the list of
predictive biomarkers below.
Tamoxifen is known as a common drug in the treatment of breast cancer. The
CYP2D6*10/*10 (and CYP2D6*5/*10) polymorphism is linked to the endoxifen’s
lower concentration (tamoxifen’s active metabolite) and N-desmethyl tamoxifen’s
(NDM) higher concentration. This suggests that NDM accumulation in the plasma is
a straight result of damage to NDM metabolism into endoxifen (Lim et al. 2011).
The link between CYP2D6*10/*10 and low amounts of tamoxifen’s primary active
6 Predictive Biomarkers for Anticancer Drugs 165

metabolites (such as 4-hydroxytamoxifen and endoxifen), as well as the possibility

of a connection between these findings and a lower response to tamoxifen in the
treated female patients, has previously been discussed (Lim et al. 2007). This
hypothesis appears to be supported by certain research. For the first time, Xu et al.
(2008) looked and examined this association in a Chinese female population.
Compared to the wt C/C homozygotes’ control group, individuals who were homo-
zygous for the CYP2D6*10 variation T/T had a significantly lower concentration of
4-hydroxytamoxifen. Following that, the impact of this polymorphism on chemo-
therapy was investigated. Patients with the CYP2D6*10 T/T genotype exhibited
worse clinical outcomes than C/C and C/T genotypes, as predicted. Importantly,
there was no influence of this genetic change on survival among non-treated women
(Xu et al. 2008). In contrast, no statistically substantial correlations were seen among
the alleles CYP2D6*1, *4, *5, *9, *10, *41, and *UM and OS or BCSS (breast
cancer-specific survival) in 3155 patients medicated with tamoxifen and 3485
non-medicated patients over 7 years in a study that looked at the alleles
CYP2D6*1, *4, *5, *9, *10, and *41 (Abraham et al. 2010). A similar lack of
connection was seen in the female breast cancer patients from japan having tamoxi-
fen as adjuvant treatment and possessing the genotype CYP2D6*10 (Toyama et al.
2009).
Similarly, HER2 gene amplification analysis is the most common genetic test
performed in cancer diagnostics to determine patients’ eligibility for lapatinib
treatment or trastuzumab. Trastuzumab treatment, however, does not always provide
the anticipated response in patients. According to studies done with the NIH3T3
andMCF-7 tumor cell lines, the isoform HER16, which is associated with Src kinase,
improves its metastatic and carcinogenic potentials, may be linked to the aforemen-
tioned drug resistance. One of the recommended solutions in such conditions is to
utilize suitable inhibitors to segregate the HER and kinase pathways. In the defined
experiment, the dasatinib TKI use ensued the inhibition of the Src kinase (Mitra et al.
2009).

6.6.3 Methylation

The most well-studied epigenetic change in cancer is abnormal DNA methylation

(Baylin 2005). In eukaryotic cells, abnormal hypermethylation of promoters can
result in the silence of essential genes, such as tumor suppressor genes, and eventu-
ally illness. The reverse mechanism can also influence cancer growth.
Hypomethylation of normally methylated genes, such as oncogenes, can upregulate
the expression (Søes et al. 2014; Kamińska et al. 2019). DNA hypomethylation was
the first DNA methylation anomaly to be documented in human cancer (Feinberg
and Vogelstein 1983). Despite the original data published in 1983 and subsequent
work, epigenetics has received little attention in investigating the molecular
pathways that contribute to cancer (Curtin et al. 2011; Kamińska et al. 2019).
Instead, the loss of heterozygosity (LOH) and DNA mutation were emphasized;
Toyota et al. suggested the CpG Island Methylator Phenotype (CIMP) as another
166 N. F. Rana and T. Tanweer

cancer pathway in 1999. For the first time, they employed CIMP to characterize the
clinical and pathological aspects of colorectal cancer (CRC). The MINT31,
MINT27, MINT25, MINT17, MINT12, MINT2, MINT1, THBS1, MLH1, and
CDKN2A (p16) genes were observed methylated in tumor tissue in this pioneering
work (Weiss et al. 2017). We have now devised methylation in vitro diagnostic
(IVD) techniques for tissue and blood, over 20 years after the Toyota et al. (1999)
findings (Curtin et al. 2011). They’ve also been used successfully in the clinical
context for prognosis, prediction, and cancer screening (Weiss et al. 2017; Kamińska
et al. 2019).
A DNA mismatch repair gene i.e., MLH1, was one of the gene listed in the first
methylation mapping in 1999. Microsatellite instability is instigated by epigenetic
silencing of a MLH1 gene by hypermethylation of its promoter (MSI). MLH1
hypermethylation has been discovered in 13% of sporadic colorectal cancer, and a
BRAF c.1799T>A, p.Val600Glu mutation has been discovered often in the DNA of
tumor (Weisenberger et al. 2006). Lynch syndrome (one of the most frequent causes
of hereditary CRC) similarly causes MSI and loss of MLS1 (Rustgi 2007); however,
it is linked with the mutations in the MMR genes. Genetic evaluation of constitu-
tional mutations in the MMR genes is used to diagnose Lynch syndrome fully. The
two-level screening test is used to distinguish nonheritable CRC from Lynch syn-
drome. The first tier consists of MMR gene expression analysis and MSI testing.
When MMR expression is lost and MSI is positive, constitutional mutations in
MSH6, MLH1, MSH2, EPCAM, or PMS2 are studied. Alternatively, the BRAF
V600E mutation and the MLH1 methylation level can be determined. To confirm
Lynch syndrome, constitutional MLH1 epimutations testing is suggested (Giardiello
et al. 2014). Pérez-Carbonell et al. (2010) found that methylation examination of
MLH1 can enhance patient selection for the genetic testing of Lynch syndrome,
lowering the cost of discovering a mutation by nearly half. Methylation of MLH1
may be determined by the MS-MLPA (methylation-specific multiplex ligation-
dependent probe amplification) (Castillejo et al. 2015), and pyrosequencing (Newton
et al. 2014; Kamińska et al. 2019) in certain laboratories.
In glioblastoma, clinical studies have shown that O6-methylguanine-DNA
methyltransferase (O6-me–MGMT) is a helpful predictive and prognostic marker
(Wick et al. 2012; Cabrini et al. 2015). MGMT is one of the DNA repair genes that
help O6-meG eliminate cytotoxic and mutagenic alkyl groups. MGMT protects cells
from damage by preventing DNA alkylation, which causes mutations (Coulondre
and Miller 1977; Esteller et al. 1999). Temozolomide damages alkyl DNA, which
results in cell death. Because cancer cells’ DNA repair processes are disrupted, its
harmful impact is higher against rapidly proliferating cancer cells in comparison of
normal cells (Chakravarti et al. 2006). As a result, cells with hypermethylated
MGMT had a stronger response to temozolomide medication (Cabrini et al. 2015).
MGMT methylation has been observed in 40% of tumors in glioma and CRC and
25% of tumors in head and neck carcinoma, lymphomas, and non-small cell lung
carcinomas (NSCLCs). The assessment of MGMT methylation (which is a signifi-
cant step in the treatment algorithm and delivers valuable insights regarding the
temozolomide’s response) is one of the diagnostic recommendations for glioma. In
6 Predictive Biomarkers for Anticancer Drugs 167

addition, the methylation status of MGMT in conjunction with IDH1 mutations

serves as a predictive biomarker. The Glioma Patients having the IDH1 p.R132H
mutation and MGMT hypermethylation have a better prognosis (Roszkowski et al.
2016; Kamińska et al. 2019).
There are a variety of commercial tests accessible to determine the methylation
level of MGMT, including (1) methylation-specific polymerase chain reaction
(PCR): Predict the MDx Glioblastoma (MDx Health); (2) real-time PCR: MGMT
Methylation Detection Kit (EntroGen); (3) MS-MLPA: SALSA MS-MLPA probe
mix ME011 MMR genes (MRC-Holland); and (4) PY (Qiagen) (Kamińska et al.
2019).
The RB1 gene is mostly linked to retinoblastoma, instigated by losing the
functioning of RB1. An LOH or RB1 mutation is linked to the absence of expression
of this gene in retinoblastoma and other malignancies, like malignant neuroendo-
crine lung carcinoma and bladder carcinomas. Nonetheless, methylation of RB1 can
cause its expression to be silenced in some situations (Greger et al. 1989) It is
reported that, in addition to the LOH and mutations, RB1 methylation must be
evaluated for complete molecular diagnosis of retinoblastoma. According to Ohtani-
Fujita et al. (1997), RB1 gene hypermethylation is usually acquired and accounts for
around 9% of sporadic tumors. There are currently assays on the market for
determining the RB1 promoter methylation level, based on the MS-MLPA approach
(Livide et al. 2012; Kamińska et al. 2019).
GSTP1, RASSF1, and APC are tumor suppressor genes that are frequently
methylated in prostate cancers and are thus used as cancer biomarkers.
ConfirmMDx®, a commercially available test, uses a subset of these genes
(MDxHealth). Patients having the prostate biopsy with negative result might be
better stratified with this test. It makes use of the epigenetic fields effect, which is
predicated on the idea that normal cells around cancer foci might carry DNA
methylation alterations. Methylation Analysis to Locate Occult Cancer (MATLOC)
and Detection of Cancer Using Methylated Events in Negative Tissue (DOCU-
MENT) are two independent studies that confirmed ConfirmMDxpredictive®’s
value, with 68% sensitivity, 64%specificity, and 90% negative predictive value
(Stewart et al. 2013). Furthermore, it was shown that using the methylation-based
biomarkers RASSF1, APC and GSTP1 ensued in a decrease of up to 64% in the
number of unnecessary repeated biopsies (Stewart et al. 2013; Kamińska et al. 2019).

6.6.4 Gene and miRNA Expression

MicroRNAs (miRNAs) are 22-nucleotide noncoding RNA molecules that control

post transcriptional gene expression by the degradation or inhibition of their target
mRNA. Biogenesis of miRNAs is a dynamic process that starts in the nucleus and
moves to the cytoplasm (Kashyap et al. 2018). MiRNAs epigenetically control the
60% of mammalian genes which are involved in the diverse pathological and
physiological diseases (Kashyap et al. 2018). MiRNAs, which are considered as
cell-free miRNAs, may be found both within and outside of cells in body fluids like
168 N. F. Rana and T. Tanweer

the blood, saliva, serum, plasma, urine, and pancreatic juice (Kashyap et al. 2018;
Sohel 2016). According to reports, 10% of all known miRNAs may be detected in
bodily fluids, however, at varying quantities (Turchinovich et al. 2013). Serum/
plasma is the greatest option for the analysis of miRNA expression compared to
numerous other bodily fluids (Williams et al. 2013). The reason for this is that it is
easy to extract and contains a larger volume of miRNA, making it possible to
perform safe and exact global cell-free miRNA quantification (Max et al. 2018).
According to many studies, cancer patients had greater amounts of unique miRNAs
in their blood than healthy persons (Moldovan et al. 2014; Cortez et al. 2011;
Filipow and Laczmanski 2019; Kosaka et al. 2010; Hetta et al. 2019). Furthermore,
several studies have demonstrated the importance of miRNA (cell-free) in the
prognosis and diagnosis of cancer (Chonggao et al. 2018; Kodahl et al. 2014).
NSCLC was identified by scrutinizing the change in miRNA expression in the
blood of patient and was associated with prognosis and overall survival (Yu et al.
2016). Cell-free miRNAs with dysregulated expression can be utilized to identify the
CRC (colorectal cancer) from healthy controls, according to Zhang et al. (2013). The
miRNA levels in the plasma of ovarian and prostate cancer patients have been
evaluated and associated with disease progression (Kashyap et al. 2018). For
cervical cancer identification, the blood miRNAs screening may be a novel testing
technique in health care, as per a systemic study (Pardini et al. 2018). In vivo and
in vitro studies have demonstrated that cell-free miR-373 and 520c are the indicators
for metastasis in breast and prostate cancer (Eichelser et al. 2014; Huang et al. 2008).
Similarly, increased blood levels of miR-221 have been associated to tumor metas-
tasis in individuals with renal, lung, and pancreatic cancer (Li et al. 2018; Teixeira
et al. 2014). Several studies have also shown a link between cell-free miRNA
expression assessment and tumor progression, proliferation, and therapeutic
response in breast cancer (Li et al. 2015; Al-Khanbashi et al. 2016).

6.7 Challenges in Identification and Discovery of Predictive

Biomarkers

Identification of predictive biomarkers is a challenging process. One of the

challenges associated with identifying a predictive biomarker for immune check-
point blockage therapy is the lack of systematic technique for collecting tissue
before, during, and after the therapy. There is a lack of clinical models available
for the assessment of these biomarkers. One of the other issues with these markers is
that a single marker cannot be suitable for patients due to the complexity of the
immune system of every individual (Lei et al. 2021).
The reality is that developing new predictive biomarkers for personalized therapy
is a tedious and challenging process fraught with failure. This should be unsurprising
to any researcher since it reflects the complexities of pharmacological development
process. This is a method with a high failure rate and high cost. It necessitates a
multistep validation procedure during which a large supply of candidates comes, but
candidates are usually rejected at each level. Adopting comparable procedures and
6 Predictive Biomarkers for Anticancer Drugs 169

criteria to the one used in drug discovery will lead to a higher number of clinically
significant biomarkers. Biomarker development researchers must acquire knowledge
from the drug development process since it offers useful schema and aids in
preventing much more costly errors on the route to enhanced patient care (Hewitt
et al. 2007).

6.8 Conclusion

The modern age of precision cancer medicine has begun, with the groundbreaking
possibility of personalizing almost every new or current cancer drug. Predictive
response-specific biomarkers can now be identified and tested in the laboratory,
thanks to the incredibly efficient and reliable technologies that are currently avail-
able. Following that, the hypothesis would be tested in the scope of clinical disease.
Biomarkers discovered by robust experimental research and validated in targeted,
well-designed clinical trials would make for more successful clinical growth, with a
lower drug-attrition rate as a result. The benefits to cancer patients would be
immense due to the approval of much more effective and less toxic treatments. It
is now possible to associate biomarker expression with disease development, define
a biomarker “code,” and continuously customize therapy to optimize patient gain. In
effect, the long-desired dream of treating cancer as a chronic illness, with clinical
decisions driven by an insightful predictive biomarker “code,” has actually become a
possibility. A large-scale concerted campaign to provide biomarkers that advise on
drug responsiveness, which are then implemented in the cancer center as compre-
hensive companion diagnostics, provides us with a rare and unparalleled ability to
deliver customized cancer therapy on an ongoing and appropriate basis.

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Biomarkers in Cancer Survival and Drug
Resistance 7
Muhammad Ikram and Zia Uddin

Abstract

Cancer survival and resistance biomarkers are biological molecules which are
distributed in different tissues or fluids of the body, they can be considered as
indications of abnormal processes pointing toward cancer cell growth and drug
resistance. These biomarkers have attracted great attention in all aspects of
cancers like screening for primary cancer, patient assessment, estimation of
disease risk, differentiating benign tumors from malignant one, prediction, prog-
nosis determination in cancer-diagnosed individuals, and assessing the disease
status by either investigating the progression or unveiling drug response to
treatment. Currently, numerous biomarkers have been explored on the basis of
their applications and molecular changes, among them, drug resistance and
treatment response biomarkers which consist of predictive and prognostic
markers have important role in drugs selection and improving patient’s survival
rate. Among the various biomarkers, higher expression of specific microRNAs
(miRNAs) have been detected as potential lead biomarkers, which predicts the
survival rate in several types of human cancer and drug resistance. Tumors are
highly adaptable and the activation of survival signaling pathways and the
inactivation of downstream death signaling pathways can also lead to drug
resistance and cancer cell growth. Similarly, epigenetic changes and the effect
of cancer cells in microenvironment have also been recognized as vital players to
drug resistance. Moreover, failure of treatment strategies has been ascribed to the
presence of cancer stem cells, which are highly resistant to different treatment
approaches. Furthermore, the increasingly recognized molecular and genetic

M. Ikram · Z. Uddin (*)

Department of Pharmacy, COMSATS University Islamabad, Abbottabad, KP, Pakistan
e-mail: [email protected]; [email protected]

# The Author(s), under exclusive license to Springer Nature Singapore Pte 177
Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_7
178 M. Ikram and Z. Uddin

heterogeneity that is present in many tumors is another major problem that can
contribute substantially to resistance and cancer survival.

Keywords

Cancer · Cell survival · Biomarkers · Drug resistance · Prognosis · MicroRNAs

7.1 Introduction

A cancer biomarker is a chemical that can be used to detect the presence of cancers in
a person, these chemicals are produced by certain body reactions to the existence of
malignancy or other complications, which could help in different conditions such as
tumor prediction and identification, epidemiology, genetic, epigenetic, proteomic,
glycemic, and imaging. Such indicators should ideally be tested by not involving the
introduction of instruments in the body for obtaining body fluids such as urine and
blood (Calzone 2012; Herceg and Hainaut 2007; Mishra and Verma 2010). There are
numerous obstacles while taking biomarker analysis into the therapeutic setting, a
variety of indicators have been recognized, for example AFP (Hepatic cancer),
BCR-ABL (Chronic Cancer of Blood), BRCA1/BRCA2 (Breast/Ovarian Carci-
noma), BRAF V600E (Lymphoma/bowel Carcinoma), CA-125 (Ovarian Tumor),
CA19.9 (Cancer of Pancreas), CEA (Colon Cancer), EGFR (Non-small-cell lung
cancer), HER-2 (Breast Cancer), KIT (Stromal Cancer of Gastrointestinal tract),
PSA (Prostate-Specific Antigen) (Glandular Cancer), S100 (Carcinoma), and a
number of other genes have been identified and already been used in patient care
(Rhea and Molinaro 2011; Ludwig and Weinstein 2005). Similarly, mutant proteins
which have been derived from an existing tumor discovered by Selected Reaction
Monitoring (SRM) method is the topmost indicator of malignant tumors, hence most
of the malignancies been estimated as 40% of the total cases could be successfully
treated if early diagnosis is made (Wang et al. 2011).
Even though numerous effective biomarkers have been discovered so far, but the
advances in biogenetics and bioinformatics promises to unveil potential biomarkers
that can alter molecular biology’s application to human disease. Owing to the
specific link of genetic alteration in tumor cells, biomarkers are leading the way in
cancer research. As a result, DNA-based biomarkers are already being used in
everyday patient management which are highlighting the value of proper diagnostic
testing. Furthermore, cancer treatment has disclosed the disease complications,
which could be thoroughly investigated using proper biomarkers, disease categories,
disease development, and the multistep procedure of tumor therapy. Similarly, risk
evaluation, diagnosis for initial phase illness, identification and localization of
disease, disease classification and diagnosis, and screening for disease recurrence
for those who are no longer in treatment, these are all different scenarios for cancer
diagnosis based on cancer biomarkers (Arm 2017).
7 Biomarkers in Cancer Survival and Drug Resistance 179

7.2 Role and Uses of Biomarkers in Cancer Cell Survival

7.2.1 Risk Assessment

Cancer biomarkers, particularly those linked to hereditary abnormalities or epige-

netic modifications provide a quantifiable technique to predict when people are
prone to certain types of cancers. Predominant cancer biomarkers include mutations
in the nucleic acids KRAS, tumor protein TP53, epidermal growth factor receptor,
protein-tyrosine kinase, esophageal, hepatic, and pancreatic carcinoma. Moreover,
methylation of cancer inhibitor proteins such as p16, cyclin-dependent kinase
inhibitor 2B, p14ARF for brain tumor, hypermethylation of MYOD1, CDH1, and
CDH13 for ovarian cancer, hypermethylation of p16, p14, and RB1 for oral carci-
noma are examples of potentially predictive biomarkers (Verma and Manne 2006).

7.2.2 Diagnosis

Cancer biomarkers can also be used to narrow down cancer diagnosis process, this is
especially true when it comes to determining whether cancers are elementary or
secondary. To determine this differentiation, scientists can compare the nucleic acid
changes identified in cells from the main cancer location to those present in cells
from the metastatic tumor area. If the changes are the same, the metastatic cancer is
considered secondary; if the changes are not similar, then the cancer is considered a
separate elementary cancer (Lapin et al. 2018). Furthermore, after the death of tumor
cells several markers still present in the blood and other body fluids in such a way
that the patients have significant quantities of circulating tumor DNA (ctDNA).
These markers can be seen in body fluids like sputum, and blood of patients
(Li et al. 2019). In view of the great biological homogeneity of cancer, the prospect
of discovering a useful indicator for early-stage cancer detection has been underway
(Dragani et al. 2020).

7.2.3 Prognosis and Treatment Predictions

Apart from disease diagnosis, biomarkers are also used in tumor therapy and
investigation of different stages, which are performed after an individual has been
discovered with a tumor. Similarly, biomarkers are also used to determine the stages
of malignancy along with its possibility of reacting to a particular therapy. This is
partly related to the fact that cancers displaying specific biomarkers may respond to
treatments based on the presence or expression of that targeted biomarker. Higher
concentration of metallopeptidase inhibitor 1 (TIMP1) has been linked to more
combative forms of plasma cell malignancy which is a typical example of predictive
biomarkers (Terpos et al. 2010), breast cancer patients with higher expression of
estrogen receptors (ER) or progesterone receptors (PR) have a greater overall
survival rate (Harris et al. 2007). Particularly, amplification of the HER2/neu gene
180 M. Ikram and Z. Uddin

which indicates that breast cancer possibly reacts to Herceptin therapy and alteration
in exon 11 of the proto-oncogene c-KIT, which indicates that a gastrointestinal
stromal tumor (GIST) will possibly react to imatinib treatment and EGFR1 tyrosine
kinase zone mutations, this biomarker indicates that whether or not a patient’s
squamous cell carcinoma will react to gefitinib or erlotinib therapy (Herbst et al.
2005; Lynch et al. 2004).

7.2.4 Pharmacodynamics and Pharmacokinetics

Biomarkers which play their role in malignancy can also be used in diagnosis and to
figure out what kind of treatment is best for a specific individual who is suffering
from cancer (Sawyers 2008). Some patients alter the molecular arrangement of
medications due to variances in their genetic makeup, meanwhile reduced drug
metabolism can sometimes lead to harmful situations when large quantities of the
medication accumulate in the body. Meanwhile, screening of biomarkers can help
with drug-dose recommendations particularly in cancer therapies, such as enzyme
thiopurine methyltransferase is encoded by a gene thiopurine methyl-transferase
(TPMP) that alter metabolism of certain anticancer drugs and could be an important
biomarker in this area (Karas-Kuzelicki and Mlinaric-Rascan 2009). Hence, patients
with thiopurine methyl-transferase gene modification are incapable to digest higher
doses of mercaptopurine which is a medication used in the treatment of blood cancer,
the reduced metabolism of mentioned anticancer drug results in deadly reduction in
leukocytes count which leads to several complication. To address the affiliated side
effects, it is advised that individuals with thiopurine methyl-transferase gene modi-
fication should be given small doses of mercaptopurine (Relling et al. 1999).

7.2.5 Treatment Response Monitoring

Cancer biomarkers can also be used to track how effectively a treatment protocol is
working. In this regard, a lot of investigations have been done to know the potential
biomarkers having ability to significantly reduce patient care costs, as image-based
diagnostics like computed tomography scans and magnetic resonance imaging
which are currently used for observing cancer levels are quite expensive (Schneider
et al. 2012). Similarly, the protein biomarkers like S100-beta have received a lot of
attention as a biomarker for evaluating the response of melanotic carcinoma. Mela-
nin producing cells which are responsible for black color in our skin also produce
large levels of these S100-beta protein in such melanomas which are proportional to
the number of cancer cells. As a result, lowered amount of calcium-binding protein
(S100-beta) in the body fluid of such people is linked to good treatment response
(Harpio and Einarsson 2004). Similarly, apoptotic tumor cells can produce
biological compounds such as cytochrome c, nucleosomes, cleaved cytokeratin-18,
and E-cadherin which have been reported in additional laboratory studies. Apart
from these biomarkers other macromolecules have been detected which freely
7 Biomarkers in Cancer Survival and Drug Resistance 181

circulate in body fluids during cancer treatment, suggesting that they could be used
as clinical indicators to monitor the ongoing treatment (Relling et al. 1999).

7.2.6 Recurrence

Malignancy biomarkers may potentially aid in cancer recurrence prediction and

surveillance. To estimate the recurrence of breast cancer, a specific type of assay
such as Oncotype DX breast cancer assay is used to know about the recurrence of
such cancers. This test is usually recommended for the patient with initial-stage
(Stage I or II) invasive breast cancer who are taking hormones for their treatment.
The tumor cells are assessed using biopsy and a cluster of 21 genes are examined.
These test’s results are expressed as a recurrence score, which reflects the likelihood
of recurrence in next 10 years (Lamond et al. 2013; Biroschak et al. 2013).

7.2.7 Developing Drug Targets

Despite its widespread use in cancer therapy, the application of these biomarkers has
been extended to anticancer drug discovery and development. Researchers have
discovered the Philadelphia chromosome, which is genetic abnormality in chromo-
some 9 and 22, that is widely common in the chronic myelogenous leukemia
patients. Mutation in these chromosomes has led to the formation of BCR-ABL
gene, also known as fusion gene. For a long time, the BCR-ABL gene was merely
considered as a biomarker to classify various types of leukemia, meanwhile Imatinib
was developed as a potent medicine that efficiently blocked this protein and dramat-
ically reduced the generation of cells with the Philadelphia chromosome (Moen et al.
2007; Lemonick and Park 2001).

7.2.8 Surrogate Endpoints

Surrogate endpoints is another interesting area of biomarkers usage, where

biomarkers are utilized as substitute to replace the effects of medicine on over
whole cancer cells growth. Especially, using verified biomarkers would eliminate
the need for cancer biopsies and lengthy and costly clinical investigations to evaluate
whether a current medicine was effective. Currently, quality of treatment which
reflects efficacy of medicines is assessed by observing that whether the disease
progression slows down in humans which could also extend the life span. On the
other hand, potential biomarkers substitutes could unveil about the pharmacokinetic
and pharmacodynamic properties of the proposed drugs and save a lot of time,
energy, and money by eliminating unsuccessful drugs from the development before
clinical investigation (Cohen and Khuri 2003).
Circulating tumor cells (CTCs) and circulating miRNAs are the two promising
biomarkers that have attracted great interest as substitute markers (Lu et al. 2013;
182 M. Ikram and Z. Uddin

Balic et al. 2013; Madhavan et al. 2012; Redova et al. 2013). These biomarkers have
direct relationship with the number of cancer cells present in the blood, so in this way
they can serve as a substitute indicator for cancer growth and survival. Currently,
several obstacles still exist in the detection and monitoring of these circulating tumor
cells and miRNAs however, latest technologies and advanced research is needed that
could solve these issues (Joosse and Pantel 2013; Hou et al. 2013; Dhondt et al.
2019). All cancer biomarkers do not have to be cancer-specific, some of the
biomarkers detected in the blood could be used to assess abnormal proliferation of
cells, which are detected by performing blood tests. Having regular blood tests,
cancer biomarkers could be easily detected and diagnosed at early stages which
could be a nice strategy to properly treat the specific disease at early stage and stop its
spread at the right time.
Different types of cancers have nonspecific determinant in the form of neutrophil-
to-lymphocyte ratio. This ratio examines the functionality of important components
of immune system, both of these components; neutrophil and lymphocytes are
important players in inflammatory reactions, hence higher concentrations of these
markers have been reported in malignant cancers. Furthermore, the basic fibroblast
growth factor (bFGF) is a cell proliferation protein, this protein is very active in the
presence of cancerous cells which undergoes rapid multiplication which results in
rapid proliferation and cancer cells survival. Interestingly, recent studies have
reported that anti-bFGF antibodies are promising agents to treat different types of
rapidly growing cancers. In addition, cell proliferation and growth are also increased
by insulin-like growth factors (IGF-R), so it is predicted that they could have a
possible role in preventing apoptosis, or programmed cell death (Yu and Rohan
2000).

7.3 Drug Resistance

Cancer drug resistance is a condition in which a tumor becomes resistant to the

chemotherapeutics that further leads to cancer cells’ survival and ultimately death of
that individual. This resistance against the anticancer medicines comes from several
factors such as increased efflux of targeted medicines, genetic modification, and
other diverse cellular and biological processes (Wang et al. 2019). Drug resistance
refers to a medication’s reduced effectiveness in treating a disease or condition, this
word is used in the context of pathogens or tumors that have “acquired” resistance
(Alfarouk et al. 2015; Davies and Davies 2010). Because of heterogeneity,
malignancies tend to become increasingly heterogeneous throughout the time, the
tumor mass may have a variety of cells with different biomolecular impressions and
altered responses to the ongoing treatment. This heterogeneity could lead to an
uneven dispensation of genetically dissimilar cancer-cell subgroup across and inside
disease sites (spatial heterogeneity), and time-related fluctuations of genomic
makeup of cancer cells (temporal heterogeneity). Because tumor heterogeneity
fuels resistance against treatment, so a precise estimation of cancer heterogeneity
is critical for the evolution of successful treatment. Multiregional sequencing,
7 Biomarkers in Cancer Survival and Drug Resistance 183

single-cell sequencing, autopsy sample analysis, and liquid biopsy analysis are all
new technologies that have the ability to analyze the complex clonal architecture of
malignancies (Dagogo-Jack and Shaw 2018).

7.3.1 Types of Drug Resistance

7.3.1.1 Intrinsic and Acquired Drug Resistance

The drug resistance during cancer treatment can be classified as either intrinsic or
acquired one depending on when the concerned resistance develops. Particularly, the
intrinsic resistance is present even prior to medication therapy and has no relation-
ship with cancer therapy, whereas acquired resistance develops after treatment that
could be due to the drugs used during treatment, both types of resistances prevail in
50% patients each, who are suffering from different types of cancer resistance
(Lippert et al. 2008; Kelderman et al. 2014; Wang et al. 2019).

7.3.1.2 Intrinsic Resistance

Intrinsic resistance is defined as the inborn resistance that exists prior to the use of
anticancer medicines and the drugs have no role in imitation or development of
intrinsic resistance. This type of resistance causes decrease in the potency of the
targeted anticancer drugs, which could be either caused by: (1) preexisting genetic
mutations which can lead to decreased drug response such as in the case of triple
breast cancer resistance is developed against the targeted drugs, (2) tumor heteroge-
neity which include preexisting unresponsive subpopulations like cancer stem cells
are chosen based on drug therapy. Intrinsic resistance is caused by a variety of
factors: (1) preexisting or inherent genetic mutations in most of the cases causing
cancer cells to be less sensitive to both chemotherapy as well as target medicines,
e.g., triple negative breast cancer cells, (2) Heterogeneity of cancers which include
medication treatment option preexisting unresponsive subpopulations, such as can-
cer stem cells, resulting in decline in later stages of treatment strategy, (3) stimulation
of intrinsic mechanisms involved in environmental toxin defense (such as anticancer
drugs). Because of genetic mutations in genes involved in proliferation of cancer
cells and/or program cell death, in cancer cells intrinsic drug resistance may be
present before therapy. For example, HER2 overexpression has been linked to a
poorer result of cisplatin treatment in patients with gastric carcinoma (Wang et al.
2019; Huang et al. 2016). The increased expression of the HER2 gene results in
increased level of snail which is a transcription factor causing a morphological shift
similar to the epithelial-mesenchymal transmission (EMT), making neoplastic cells
to be more resistant. Moreover, individuals who were snail/HER2 double positive
had a poorer survival rate than those who were just single positive or double negative
genetically.
Snail and Slug transcriptional repressors were discovered to be involved in EMT
as well as resistance to apoptosis induced by a self-renewal mechanism and p53
(Kurrey et al. 2009; Wang et al. 2019). Cancer stem cells (CSCs) become much
resistant to chemotherapies as well to radiations as a result of the second and third
184 M. Ikram and Z. Uddin

activities. Resistant cells were also discovered to be more mesenchymal in nature

(Witta et al. 2006; Sayan et al. 2009; Wang et al. 2019). Intrinsic drug resistance is
linked to EMT and CSCs by these concurrent alterations.
Relapse after chemotherapy may also be caused by preexisting resistant
subpopulations in malignancies. The occurrence of intra-tumoral genetic variability
in primary cancers predates clinical intervention, according to a growing body of
research (Burrell et al. 2013; Turner and Reis-Filho 2012; Kreso and Dick 2014). As
majority of tumor cells are responsive to the medicine as a result patients would
initially respond to treatment. However, after pharmacological therapy, resistant
subclones would multiply and induce recurrence (Kuczynski et al. 2013; Greaves
and Maley 2012). Because the tumor shrinks first after treatment and the resistance
appears to be gained as a result of therapy, intrinsic drug resistance is sometimes
confused with acquired resistance. CSCs are a type of cancer cells that have the
ability to self-renew and differentiate, and they play a key role in tumor genesis and
progression (Frank et al. 2010). They’ve been linked to chemotherapeutic drug
resistance in a variety of cancers including glioblastoma, gastric carcinoma and
leukemia (Viale et al. 2009). To combat medication resistance, it may be necessary
to use a combination of therapies that target both CSCs as well as majority of tumor
cells. Intrinsic mechanism activation, which is utilized as a shield against environ-
mental contaminants, together with anticancer treatments, can diminish a drug’s
therapeutic effects. The ATP binding cassette or shortly ABC, transporter-mediated
drug efflux and the glutathione (GSH)/glutathione S-transferase system, which
operate to minimize cellular drug aggregation and detoxify cancer cells treated
with drugs are two examples of these defensive mechanisms (Gillet et al. 2012;
Traverso et al. 2013).

7.3.1.3 Acquired Resistance

Acquired resistance is defined as a steady decrease in a drug’s anticancer activity
after treatment, that can be caused by a variety of factors, including: (1) Activation of
a second proto-oncogene which transforms into a new emerging driver gene,
(2) Mutations in drug target or changes in its expression and (3) alterations in the
tumor microenvironment (TME) after treatment. When new mutations are generated
in target proteins or their level of expression changes, cancer cells can develop
resistance to targeted treatments. The mutation of threonine 315 to isoleucine
(T315I) in the BCR-ABL kinase domain is an excellent example of secondary
mutations within the target kinase. The BCR-ABL which is a target of imatinib, a
tyrosine kinase inhibitor (TKI), is routinely practiced in the treatment of chronic
myelogenous leukemia, however about 20–30% of individuals will develop post-
treatment resistance or recurrence (Quintás-Cardama et al. 2009). A mutation point
T315I in the tyrosine kinase protein fusion is one cause of resistance (Quintás-
Cardama et al. 2009; Jabbour et al. 2013; Kimura et al. 2014). The removal of
hydrogen bond required for imatinib to bind to the ATP-binding region of
BCR-ABL, occurs when threonine 315 is changed to isoleucine, resulting in dra-
matically reduced therapeutic efficacy.
7 Biomarkers in Cancer Survival and Drug Resistance 185

Drug resistance can also be achieved as a result of TME dynamic changes during
treatment. Crosstalk lies between tumor cells and their tumor microenvironment
throughout malignancy and the development of resistance. The interaction is aided
by exosomes generated by cancer and stromal cells. Researchers discovered that
cancer cells and tumor associated macrophages in the TME communicated through
exosomes are generated by cancer cells and carry specific miRNAs. NBL cells
secrete exosomic miR-21 in cisplatin-treated neuroblastoma (NBL) tumors, which
causes TAMs to create exosomic miR-155, which silences the TERF1 gene in NBL
cells. Reduced expression of the TERF1 protein, which inhibits telomerase resulting
in enhanced activity of telomerase and chemotherapeutic resistance. As a result, drug
resistance may be facilitated via exosomic miRNA exchange in TME between
stromal and tumor cells.
The resistance mechanisms (intrinsic and acquired) outlined above might coexist
throughout the growth and treatment of tumor. Acquired drug resistance can have
completely distinct mechanisms than intrinsic drug resistance. It could also be the
result of a selective growth of innate drug resistance. The extent of intrinsic drug
resistance defines a cancer cell’s sensitivity to a specific medication. To eliminate
any preexisting drug resistance, genomic as well as other biochemical investigations
ought to be undertaken prior to the creation of drug therapy plan. Following the
development of acquired drug resistance, therapeutic strategies must be changed
consequently. One important goal of medication therapy must be to successfully
inhibit or slow down the tumor growth while avoiding the development of acquired,
or at the very least unmanageable acquired drug resistance. Ideal pharmacological
therapeutic strategy must consider preventing or delaying acquired/adaptive drug
resistance (Wang et al. 2019; Holohan et al. 2013). Some cancers can quickly
become resistant to targeted treatment. For drug researchers, the goal is to predict
these consequences and reduce the hazards by selecting new drug targets for patients
who do not respond to targeted drug therapy.

7.3.2 Role of MiRNAs in CRC Drug Resistance Regulation

Reduced intracellular drug accumulation, enhanced DNA damage repair, reduction

in apoptosis, altered oncogenes expression and tumor suppressors, among other
factors, can all contribute to drug resistance. MiRNAs are closely implicated in
these processes and so affect the drug resistance developments, including resistance
to 5-FU, oxaliplatin, and EGFR-targeted treatment, the latter of which is extensively
researched. Autophagy is expected to increase chemoresistance by boosting cellular
energy generation, making it a key mechanism of cancer cell chemoresistance. By
assisting tumor cells in surviving under metabolic and therapeutic stress, autophagy
has emerged as one of the most chemotherapy resistant mechanism. In vitro and
in vivo, miR-22 suppressed autophagy and induced apoptosis, increasing the sus-
ceptibility of CRC cells to 5-FU therapy. B-cell translocation gene 1 (BTG1) has
been recognized as a novel target of miR-22, with the potential to reverse miR-22-
induced autophagy suppression. Thus, by BTG1 posttranscriptional suppression,
186 M. Ikram and Z. Uddin

miR-22 could serve as a key switch between autophagy and apoptosis to modulate
5-FU sensitivity (Zhang et al. 2015a). The stimulation of the PI3K/AKT pathway by
the miR-204/HMGA2 axis regulated tumor cell resistance to 5-FU in HCT-116 and
SW480 colon cancer cells. These findings suggest that the miR-204/HMGA2 axis is
involved in colon cancer cells’ resistance to 5-FU (Wu et al. 2016).

7.3.2.1 MicroRNAs as Drug Response Noninvasive Biomarkers in CRC

Circulating nucleic acids provide a noninvasive means of detecting cancer early,
assessing prognosis, and predicting medication response. In blood, circulating
miRNAs are stable and repeatable, making them interesting targets for research
(Chen et al. 2008; Mitchell et al. 2008). miRNAs have recently been reported in
human plasma or serum has recently been reported and has gained key attention for
biomarker development (Slaby et al. 2009). We have compiled a list of miRNA
biomarkers present in blood for CRC patients’ responses to 5FU/oxaliplatin chemo-
therapy and EGFR target specific treatment.

7.4 Biomarker’s Assessment Methods

Tumor biomarkers rapidly fluctuate during cancer growth or treatment, it is very

important to develop invasive methods to assess drug effects. Blood collection has
become a popular method for academics and physicians to measure surrogate
endpoints (Twomey et al. 2017). While genotyping cancers, RNA and miRNAs
can all be used, and they are being studied as prognostic biomarkers. The EGFR
Mutation Test v2 is utilized to detect EGFR exon 19 deletions or exon 21 L858R
replacements in NSCLC patients undergoing Tarceva medication (MacFarlane and
Murphy 2010; Ma et al. 2012). When circulating miRs are combined with proven
predictive biomarkers, real-time detection of emergent drug resistance is possible.
miR-210, miR-125a-5p and miR-125b, in breast cancer patients, have been found to
be associated with HER2 status that may be used as a less invasive predictive
biomarker for HER2 targeted therapy (Twomey et al. 2017).
Other comprehensive systems-level analytics, such as full-scale proteomics,
phenotypic alterations, spatio-temporal regulation of oncoproteins and the role of
TME and the immune system, should be included in biomarker research and
development. Monitoring PD-L1 levels in the tumor microenvironment could con-
tribute to the development of immune and target specific therapies and serve as a
secondary biomarker for emerging drug resistance (Twomey et al. 2017). Neither
PFS nor OS were linked with other biomarkers (ABCB1, ABCC1, p53, cyclin E, and
AKT2) (Kim et al. 2012). RAS, phosphatidylinositol-4,5-bisphosphate 3-kinase
catalytic subunit alpha (PIK3CA), and BRAF, have all acquired prominence as
chemotherapy predictors and have been used to establish criteria for combined
treatment with anti-epidermal growth factor receptor drugs (Therkildsen et al.
2014). Serum levels of carcinoembryonic antigen (CEA) tumor markers and carbo-
hydrate antigen 19-9 (CA19-9) are often used as predictors in colorectal cancer;
although, the status of serum CEA and CA19-9 concentrations in metastatic colon
7 Biomarkers in Cancer Survival and Drug Resistance 187

cancer (MCC) patients as helpful survival predictors is still uncertain. Circulating

tumor cells (CTCs) are also regarded as predictive predictor, however, due to the
high cost and difficult methodology, measuring CTC levels is not now widely
employed. Serum levels of the tumor markers CA19-9 and CEA are the most
broad and cheapest chemotherapy predictors (Therkildsen et al. 2014; Taniguchi
et al. 2015).
According to Adam et al., increased blood CA19-9 levels following chemother-
apy in patients with ineradicable recurring colon cancer were associated with shorter
recurrence-free and overall existence (Hashizume et al. 2019; Adam et al. 2004).
Sakamoto et al. found that high blood CA19-9 levels after chemotherapy reduced
recurrence-free and overall survival, while high serum CEA levels after chemother-
apy was a major recurrence risk factor (Sakamoto et al. 2015; Hashizume et al.
2019). In patients with colon cancer and unresectable liver metastases found that
serum CEA levels of 100 ng/mL and CA19-9 levels of 100 U/L before chemother-
apy were predictive markers for poor prognosis (Hashizume et al. 2019; Mitsuyama
et al. 2012). miRNAs are involved in a variety of biological processes in cancer,
including tumor initiation, tumor development, and drug resistance, and can operate
as oncogenes or tumor suppressors (Xu et al. 2015; Fang et al. 2015; Li et al. 2015;
Rossi et al. 2010; Zhang et al. 2007).
A vast number of miRNAs dysregulation have been found in CRCs, and few of
them have been connected to anticancer therapy response via drug transport, drug
metabolism, DNA damage response, or cell death regulation (Zheng et al. 2010).
Notably, a subclass of miRNAs could be used as noninvasive biomarkers in the
circulation as potential predictive indicators for medication response. Furthermore,
improved RNA technology ensures miRNA-based therapies to be a unique thera-
peutic option for treating CRC treatment resistance (Zheng et al. 2010).

7.5 Therapy-Related Biomarkers

Acquired/adaptive resistance to treatment, which develops after chemotherapeutic

drugs exposure and contributes to MDR and recurrence, is one of the most signifi-
cant constraints in children ALL (chALL) treatment. The discovery of biomarkers
linked to medication resistance could lead to new methods in targeted therapy and
better patient outcomes. This section briefly introduces therapy-related biomarkers
(Aberuyi et al. 2020). The poor treatment responses, EFS, and OS (218 BCP-ALL
and 47 T-ALL) are closely associated to TP53 tumor suppressor gene mutation/
deletions. Utilizing direct sequencing and multiplex ligation-dependent probe ampli-
fication in 23 matched samples, changes in the TP53 gene were found in relapsed
patients after treatment (Aberuyi et al. 2020). DNA studies of paired chALL samples
at relapse, diagnosis and complete remission (CR) found that several missense
mutations are more common in relapsed patients following treatment as compared
to those that are at diagnosis or complete remission. RGS12, LPHN1, PRMT2,
CAND1, USP7, NIPSNAP1, CBX3, TULP4, COBRA1, SCARF1, SDF2, FBXO3,
DPH5, NEGR1, SMEK2, NT5C2, MIER3, DOPEY1, NT5C2, and ZNF192 genes
188 M. Ikram and Z. Uddin

all have mutations. RNA sequencing of ten matched cases revealed that unique
somatic mutations in the NT5C2 gene are acquired after relapse but not at diagnosis.
The enzymatic activity of the protein cN-II, a 50 -nucleotidase enhanced with these
mutations and also caused resistance to nucleoside analogue treatments in an
additional 61 relapsed individuals, according to enzyme analyses (Aberuyi et al.
2020).
According to a study of microRNA expression on paired diagnosis/relapse
samples (11 B-ALL and six T-ALL patients), some of the miRNAs are expressed
distinctly at various disease stages. MicroRNAs like miR-23a, miR27a and
miR-223, are examples of miRNAs whose downregulation occurs at diagnosis,
recovered at complete remission, and again downregulated at relapse. On the other
hand, certain miRNAs, for example, miR-181a, miR-181b, miR-128b, and miR-708,
during diagnosis and relapse, show overexpression. These miRNAs play important
roles in leukemogenesis and medication resistance after treatment (Staal et al. 2010).
Methylation study of certain genes revealed hypermethylation of
O6-methylguanine-DNA methyltransferase (MGMT) and p16 in eight patients
having B-ALL and one with T-ALL, both at diagnosis and relapse. On the other
hand Retanoic acid receptor beta (RARB) was hypermethylated during the time of
relapse that is thought to be linked to chALL progression and should be followed
during the treatment (Aberuyi et al. 2020). When comparing matched diagnosis/
relapse childhood BCP-ALL samples, high-throughput analysis indicated more
common somatic deletions in the KZF1, VPREB1, NR3C1, and EBF1 genes in
the relapse stage than at the disease beginning. Furthermore, during relapse,
BCP-ALL had a considerably higher hypermethylated genome than T-ALL. Only
relapsed chALLs, not those at the outset of the disease, had promotor
hypermethylation, which resulted in downregulation of the tumor suppressor genes
CDKN2A, PTPRO, and CSMD1. Furthermore, other genes like FANCD2, CENPM,
OBSL1 and FOXM1 were exclusively expressed after relapse, not when the pediat-
ric BCP-ALL was diagnosed (Hogan et al. 2011).

7.5.1 VEGF/VEGFR-Targeted Therapy

Angiogenesis is a process that plays a key role in tumor development and metastasis,
making it a viable therapeutic option for cancer treatment (Warren et al. 1995). The
vascular endothelial growth factor (VEGF) is a key pro-angiogenic factor, and
inhibiting the VEGF pathway for cancer treatment, including mCRC, has been
extensively used in clinical practice (Zerbini et al. 2008; Zhang et al. 2015b). The
most widely used VEGF-A antibody is bevacizumab, a humanized monoclonal
antibody.
7 Biomarkers in Cancer Survival and Drug Resistance 189

7.5.2 Genetic Determinants as Susceptibility Biomarkers

The genes capable of identifying patients with high risk of specific cancer, represent
another set of biomarkers. A scientist demonstrated that nonsense vs. missense
Protein patched homolog 1 (PTCH1) alterations, correlated with specific protein
profiles particularly in nevoid basal-cell carcinomas syndrome (NBCCS), has been
designated in fibroblast conditioned media among patients with NBCCS, peculiarly
during overexpression of matrix metalloproteinases 1 (MMP). Genetic elements are
applicable for the progression of disease to cancer as well as for disease of patho-
genic origin (Buonaguro et al. 2014; Duffy 2004, 2001).

7.6 Guidelines for Tumor Biomarkers

7.6.1 Alpha-Feto Protein

A glycoprotein (70 kDa), homologous to albumin found in serum. Due to glycosyl-

ation up to varying levels this protein exhibits micro heterogeneity. AFP is more
fucosylated produced by malignancies than that produced by normal tissues.
Existing assay does not variate between different forms. Mainly limited to three
malignancies:

1. Hepatocellular carcinoma (HCC)

2. Germ cell tumors in testis, ovary and at some other sites
3. Hepatoblastoma (in children and extremely rare among adults) (Buonaguro et al.
2014; Duffy 2004, 2001).

7.6.2 Cancer Antigen 125 (CA125)

Mu16 is the protein detected by this antibody, physiological functions are not
established yet.

Benign conditions with high levels

• Endometriosis
• Acute pancreatitis
• Cirrhosis
• Inflammatory pelvic disease
• Ascites of nonmalignant type
• Peritonitis
190 M. Ikram and Z. Uddin

Malignancies with high levels

• Up to 85% of all ovarian epithelial cancer have high level of CA125 protein.

High levels in physiological states

• Pregnancy and menstruation (>100 kU/L usually) (Buonaguro et al. 2014; Duffy
2004, 2001)

7.6.3 Carcinoembryonic Antigen (CEA)

A nitrogenous substance called mucin reacting with monoclonal antibody such as

1116 NS 19-9. Biological function is considered as cell adherence. There is variation
in reference range; from 0 to 37 kU/L to 0–100 kU/L. In serum, half-life is approxi-
mately 1 day, but still variation from less than 1–3 days exist.

Benign situations with high levels

• Hepatocellular jaundice (cirrhosis)
• Acute and chronic pancreatitis
• Acute cholangitis

Malignant conditions with high levels

• Colorectal carcinomas (30% approximately)
• Gastric Carcinomas (50% approximately)
• Pancreatitis, acute and chronic both

It is thought to be helpful in the diagnosis of pancreatic carcinoma (Buonaguro

et al. 2014; Duffy 2004, 2001).

7.6.4 Human Chorionic Gonadotropin (hCG)

It is a Heterodimer, composed of two glycosylated alpha & beta subunits (chains).

Alpha subunit chain of HCG is nearly undistinguishable to alpha chain of follicle-
stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing
hormone (LH) but the beta subunit chain differs from consistent chains. Distinct
24 amino acid C-terminal extension is present. Hormone in serum is found in
multiple forms (1) Intact and integral two chain of peptide and (2) Free α and β
chains (3) Numerous degradation products like the β core fragment. Physiological
function: to maintain and stimulate progesterone production during pregnancy in the
corpus luteum. HCG Can be detected within 1 week after conception (Buonaguro
et al. 2014; Duffy 2004, 2001).
7 Biomarkers in Cancer Survival and Drug Resistance 191

7.6.5 Prostate Specific Antigen (PSA)

PSA is single chain glycoprotein of 237 amino acids with 28.4 kDa molecular
weight, containing chymotrypsin-like serine protease. PSA forms complexes with
α1-antichymotrypsin (PSA-ACT) (present in major quantity), α1-antitrypsin (found
in trace quantity), α2-macroglobulin (not detectable by existing immunoassays),
while the noncomplex free form of PSA has physiological function such as partially
participate in the liquefaction of seminal fluid coagulum thus promoting the release
and motility of spermatozoa (Buonaguro et al. 2014; Duffy 2004, 2001).

7.6.6 Estrogen and Progesterone receptors (ERs & PRs)

Both Estrogen receptors (ERs) and Progesterone receptors (PRs) level should be
measured in all malignant breast cancers, among the two forms of ERs the ERα has
more validated role, compared to ERβ. Similarly the PRs both forms dubbed PRA
and PRB, both are easily detectable by immunohistochemistry assays. (Buonaguro
et al. 2014; Duffy 2004, 2001).

7.6.7 Human Epididymal Secretory Protein 4 (HE4)

Mechanisms of oncogenesis or tumorigenesis could be explained by the productivity

and liberation of cancer biomarkers in cancer specific cells, blood or several body
fluids. During early stage of cancer development, metastases and progression release
of these molecular biomarkers get elevated. This increase of cancer biomarkers in
any kind of the biological fluid can be described via three key mechanisms.
The overexpression means profuse target protein expression or genes product
amplification, or augmentation of epigenetic changes, and upregulation of protein
human epididymal secretory protein 4 (HE4) are considered as key mechanisms. In
ovarian cancer, protein human epididymal secretory protein 4 (HE4) release as a
result of DNA methylation. HE4 biomarker of ovarian carcinoma shows
overexpressed mechanism and can be identified in serum. Clinical evaluation of
this biomarker shows that in case of endometrial, breast, and bronchial adenocarci-
noma it also get overexpression. The second mechanism for elevation of cancer
biomarkers could be normally applied on biological fluid serum, this includes hyper
secretion of cellular proteins in serum or membrane proteins shedding. The third
mechanism is angiogenesis and cell invasion, both play a critical role in cancer, each
process occurs with expression of prostate-specific antigen (PSA). Generally, it is
expressed by means of prostatic epithelium, however increased level of PSA takes
place because of distortions of basement membrane of prostatic cell due to lymph
angiogenesis and cell invasion.
Various cancer biomarkers like circulating protein targets for management of
cancer has revolutionized the new era mainly with the accessibility of auspicious
techniques that are used for the discovery of “omics” cancer biomarker in body
192 M. Ikram and Z. Uddin

Table 7.1 Representative biomarkers in cancer survival and drug resistance

Specificity (organ/
Biomarker cancer) type of
No. (cancerous) cancer Uses/Application
1 Prostate specific BPH/prostate Screening, diagnosis and monitoring
antigen
2 Carbohydrate Ovarian Detecting recurrence and monitoring
antigen 125 therapy, diagnosis, prognosis,
3 Carcinoembryonic Colorectal/hepatic Monitoring therapy, Prognosis, Detecting
antigen recurrence, Screening for hepatic
metastases
4 Carbohydrate Breast carcinoma Monitoring therapy
antigen 15.3
5 Progesterone and Breast carcinoma Stratification/select patients for endocrine
Estrogen receptors therapy
6 HER2 Breast carcinoma Monitoring trastuzumab therapy
7 Carbohydrate Breast carcinoma Monitoring
antigen 27.29
8 Human chorionic Testicular Monitoring therapy, Detecting recurrence,
gonadotropin β carcinoma Diagnosis, stages
9 Alfa-fetoprotein Hepatocellular Monitoring therapy, Detecting recurrence,
carcinoma Diagnosis
10 Calcitonin Thyroid carcinoma Diagnosis and monitoring therapy
11 Thyroglobulin Thyroid carcinoma Monitoring
12 CA 19-9 Pancreatic Monitoring therapy
carcinoma
13 Nuclear matrix Bladder carcinoma Monitoring and prognosis, Screening,
protein 22
14 Prostate cancer Prostate carcinoma Prognostic
antigen 3

fluids, which may be represented as novel markers. These are extremely sensitive
diagnostic techniques for the detection of early stages of cancer (Buonaguro et al.
2014; Duffy 2001) (Table 7.1).

7.7 MicroRNAs as Potential Biomarkers

The microRNAs (miRNAs) are potential biomarkers which are conserved micro
noncoding RNAs, which are also known as micromanagers of gene expression.
Polymorphisms in the miRNA (miR-polymorphisms) is an emerging area having
promising potential to be used in the diagnosis and prognosis of different types of
cancers. Recent advances in miRNA research have highlighted that deregulated
miRNA genes are involved in cancer cells survival, a number of polymorphisms
in different stages of pre-miRNA and miRNA binding sites have been reported to be
affiliated with different types of cancers. Moreover, it has been reported that miRNA
7 Biomarkers in Cancer Survival and Drug Resistance 193

Table 7.2 Resistance of 5-FU in CRC involving miRNAs, Upregulated: "; downregulated: #
miRNAs Expression in resistent cells Gene targets
miR-203 # TYMS
miR-218 # TYMS, BIRC5
miR-494 # DPYD
miR-27a, miR-27b # DPYD
miR-21 " MSH2
miR-10b " BIM
miR-23a " APAF-1, ABCF1
miR-425-5p " PDCD10
miR-139-5p # BCL2
miR-200c, miR-302, miR-369 " MRP8
miR-519c # ABCG2, HuR
miR-22 # BTG1
miR-204 # HMGA2

Table 7.3 Resistance of oxaliplatin in EGFR-targeted resistance CRC involving miRNAs

miRNAs Therapy Expression in resistant cells Gene targets
miR-7 EGFR-targeted # EGFR, RAF1
miR-20a Oxaliplatin " BNIP2
miR-133b EGFR-targeted # EGFR
miR-143 Oxaliplatin # IGF-1R
miR-153 Oxaliplatin " FOXO3a
miR-203 Oxaliplatin " ATM
miR-199a-5p, miR-375 EGFR-targeted " PHLPP1
miR-409-3p Oxaliplatin # Becline-1
miR-520g Oxaliplatin " P21

polymorphisms also affect the tumor characteristics in the microenvironment and

cancer patient’s survival. Hence, miRNAs is an important player in tumorigenesis
and oncology, particularly it has wide spread usage in miRNA microarrays which
has enabled the confirmation of several miRNAs as potential cancer biomarkers
(George and Mittal 2010) (Tables 7.2 and 7.3).

7.8 Additional Factors Contributing to Drug Resistance

and Cancer Survival

Numerous other factors, apart from the above mentioned biomarkers may also have
potential role to contribute toward drug resistance and cancer survival e.g.,
autophagy, MDR, and regulatory genes.
194 M. Ikram and Z. Uddin

7.8.1 Autophagy and Multidrug Resistance in Cancer

Drug resistance is developed after long-term cancer treatment, the kind of drug
resistance which is developed against multiple drugs being used against cancer is
known as multidrug resistance (MDR) that leads to tumor recurrence and cancer cell
survival. Therefore, suppression of MDR is an important approach to control cancer
cells survival and increase the cancer drugs effects. Similarly, autophagy, a self-
degradative strategy, occurs during the treatment of MDR cancer. Autophagy has
dual functions: it contributes in the development of MDR and provides shelter to the
cancer cells from chemotherapeutics, on the other hand it can also kill MDR cancer
cells in which apoptosis pathways are not properly working. Moreover, anticancer
drugs induced autophagy could also activate apoptosis signaling pathways in MDR
cells and hence reverse the MDR. Therefore, recently the autophagy-based research
has attracted great attention which could suppress MDR and its related
complications (Wang et al. 2019) (Table 7.4).

7.9 Challenges in Clinical Applications of Biomarkers

As biomarkers hold a crucial role at almost every level of disease, so it is important

that these shall be monitored very carefully in terms of their analytical validation and
clinical utility assessments prior to their use in daily clinical care. The assessment
and validation of a recent biomarker is almost equal to the development and design
of a new drug. Identification, authentication and launching of a novel biomarker is
just as tough as developing and approving of a fresh drug. Out of 3–50% biomarkers,
only 3–5% get access to clinical usage. Herein, we discuss some problems validation
and application of the biomarker for clinical trials. A lot of problems are faced in the
process of new drug development, including complication and biological difference
to tumor response therapy which involves a complex molecular pathway with
adaptive feedback and loop of cross talk. Besides the abovementioned problems,
different sorts of measuring errors may be observed during the process of validation.
The basic determinant which assures the efficacy of biomarkers analysis is the
standard protocols application for the collection of sample and also their storage
and processing. Another logistical component of the process of authentication of the
biomarker data is statistical evaluation which is a tough task regarding consistency in
data management, biostatistical, and bioinformatics methodologies. The statistical
approach should be able to notice by chance relationship from those coming from
true biological association. However, omic features (i.e., epigenetics, genomics,
proteomics, etc.) and/or environmental and routine factors, may play a role in drug
response. It is crucial to identify biomarkers by either with lifestyle or omic data
alone, e.g., age, diet, etc., which are studied in the personalized medicine in order to
get a famous phrase of pharmaceutical sciences of “right dose at the right time to the
right patient.” The identification of a valid biomarker’s cohort studies is performed
which involve collection of large number of samples.
7 Biomarkers in Cancer Survival and Drug Resistance 195

Table 7.4 Resistance mechanisms of various biomarkers

Therapy
(targeted) Type of cancer Targets Mechanism of resistance
Bevacizumab Colorectal cancer, VEGF Activation of alternative signaling
NSCLC, glioblastoma pathways (such as IGF1R, PDGFR,
and renal cell carcinoma FGFR or MET), Hypoxia-induced
autophagy
Induction of tumor dormancy or an
increase in the cancer stem cell niche
Bortezomib Multiple myeloma and Proteasome Mutation in the binding site of
mantle cell lymphoma bortezomib
Anti-apoptotic mechanism
Cetuximab Head and neck cancer EGFR KRAS mutation
and colorectal cancer EGFR-S492R mutation inhibits
cetuximab binding
Increased ERBB family signaling
Crizotinib NSCLC EML4- Secondary EML4-ALK mutations
ALK or rearrangement
COT-mediated MAPK reactivation
CD74-ROS1 rearrangement
Dasatinib ALL and CMIL BCR- T315 mutation in ABL1
ABL1
Gefitinib NSCLC EGFR EGFR kinase domain mutations (for
example, T790M)
Epithelial-mesenchymal transition,
Epigenetic mechanisms
Increased ERBB family signaling or
MET amplification
Imatinib CML, ALL and GIST BCR- Mutation of the target (for example,
ABL1, KIT T315 in ABL1, T670I in KIT and
and T674I in PDGFRα) Elevated MDR1
PDGFRa expression
Nilotinib CML BCR- BCR-ABL1 upregulation
ABL1 T315 mutation in ABL1
Trastuzumab ERBB2-positive breast ERBB2 PTEN loss, Truncation of ERBB2,
cancer Activating mutations of PIK3CA
Activation of alternative signaling
pathways (such as IGF1 and
ERBB3)
Vemurafenib Melanoma BRAF- Elevated BRAF-V600E expression
V600E Acquired mutation in KRAS, NRAS
or MEK1
Activation or EGFR, IGF1R and
PDGFR β pathways

In order to find effective and reliable biomarkers, cohort studies should be applied
assessing huge quantity of sample groups. At this point of view, the limited count of
children involving in research studies is the main challenge to identify novel
196 M. Ikram and Z. Uddin

biomarkers as there is painful or invasive operations which are involved for

gathering of data for sample collection. Age-matching control finding of samples
is another difficulty, which is crucial to observe values of normal reference. Besides,
a small number of participants with children malignancies are not ready to partici-
pate in the trail based interventions.

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Biomarkers in Tumor Recurrence
and Metastasis 8
Dilawar Khan and Mudassir Khan

Abstract

Biomolecules that are produced or secreted by tumor cells themselves or in

response to tumor by other cells of body are cancer biomarkers that further define
cancer at molecular level. Biomarkers can be specific cells, genes, products of
genes, hormones, molecules, enzymes found in blood, urine, or tissues. Every
cancer has its unique signatures in terms of biomolecules/cancer biomarkers
which give precise and identifiable characteristics to that cancer. Cancer
biomarkers hold specific biochemical and molecular characteristics with roles
in diagnosis, screening of clinical complications, and response to therapy or
treatment stratification for patients. Besides these roles, cancer biomarkers play
very essential role in metastasis and recurrence/relapse of cancer as well. The
focus of this chapter is to highlight cancer metastasis and recurrence with a
particular focus on the role of key biomarkers involved in metastasis and
recurrence.

Keywords
Biomarkers · Hormones · Metastasis · Recurrence · Diagnosis · Screening ·
Clinical complications

D. Khan (*) · M. Khan

Department of Healthcare Biotechnology, Atta-ur-Rahman School of Applied Biosciences
(ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan
e-mail: [email protected]

# The Author(s), under exclusive license to Springer Nature Singapore Pte 201
Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_8
202 D. Khan and M. Khan

8.1 Introduction

Cancer is the second leading cause of death and is a major public concern. In 2020,
approximately 10 million deaths were reported, 19.3 million new cases of cancer
were diagnosed according to GLOBOCAN (Sung et al. 2021), and this number
increases every year (Cho 2007). Cancer control and its timely diagnosis, treatment,
appropriate follow-up, and comprehensive use of predictable measures (cancer
biomarkers) are certainly effective tools that help to reduce the burden of cancer.
US Food and Drug Administration (FDA) defined biomarkers as “Any measurable
diagnostic indicator that is used to assess the risk or presence of diseases” or they can
be defined as “A characteristic that is objectively measured and evaluated as an
indicator of normal biological processes, pathogenic processes, or pharmacological
responses to therapeutic intervention”.
Cancer biomarkers are tools for cancer screening/early detection, diagnostic,
prognostic, or can also be used to predict patients overall outcome. Recent techno-
logical advances in DNA and RNA sequencing as well as proteomics have enabled
identification of predictive, diagnostic and prognostic biomarkers, also it improved
more precise clinical management of cancer patients (Ilyin et al. 2004; Ansari and
Andersson 2021). Even patients subpopulations can be identified by cancer
biomarkers who are most likely to react to specific treatment (Goossens et al. 2015).
Metastasis describes the spread of cancer cells to distant organs and surrounding
tissues from the primary tumor (Tarin 2011). During metastasis cascade, metabolism
is adapted either dynamically or selectively by metastasizing cells (Bergers and
Fendt 2021). Tumor cells interact with host cells and extracellular matrix during
metastasis (Valastyan and Weinberg 2011).Tissue destructive macrometastases dis-
rupt the function of organ and are also a major source of morbidity and lethality for
almost all solid tumor types (Gupta and Massagué 2006; Hanahan and Weinberg
2011; Dillekås et al. 2019). Variety of cellular processes are involved with different
molecular programs that regulate migration, survival, and proliferation of cancer
cells during metastasis (Stracke and Liotta 1992; Nguyen and Massagué 2007;
Lambert et al. 2017). Molecular changes such as proteolytic and adhesion enzyme
expression as well as cellular processes influenced usually by cytoskeleton dynamics
are the processes that are required by cells for migration (Tahtamouni et al. 2019). In
addition, cell lines grown in culture fail to recapitulate metastatic behavior in in vivo.
Although different cancers share the same process of metastatic progression, the
diversity of molecular programs across (and within) cancer types necessitate the
analysis of cancer-type-specific molecular and cellular programs that contribute to
metastasis (Ko et al. 2021).
The stroma that surrounds the tumor, as well as the immune system effectors,
hormones, blood platelets, and other humoral factors, make up the tumor microen-
vironment (Goubran et al. 2014).Tumor metastasis is also associated with changes in
tumor microenvironment. For example, immune response to cancer cells and surgi-
cal removal of the tumor tissue can stimulate tumor recurrence and growth of the
metastatic tumor in distant parts of the body (Nishikawa 2008).
8 Biomarkers in Tumor Recurrence and Metastasis 203

The primary tumor releases a significant number of disseminated tumor cells, but
only a small number of cells survive as viable seeds (Deyell et al. 2021). Primary
tumor cells metastasize before diagnosis of primary cancer and can remain for years
in patients after treatment. These cells have a special biology that enables them to
avoid immune surveillance, form tumors, and colonize metastatic
microenvironments (Ganesh and Massagué 2021). These dormant malignant cells
remain undetectable, asymptomatic, occult for long time and can awaken after
decades which leads to relapse/recurrence (Friberg and Nystrom 2015; Phan and
Croucher 2020). Tumor dormancy and distant metastatic relapse regulation are
influenced by a number of molecular factors (Werner et al. 2021). Dormant cells
have self-renewal capacity, potential to escape from different treatment options, and
detected more frequently in different types of cancer (Müller et al. 2005); prostate
cancer (Pound et al. 1999; Amling et al. 2000), B–cell lymphoma (Davis et al. 1998;
Press et al. 2001), breast cancer (Meng et al. 2004), and melanoma (Faries et al.
2013; Callaway and Briggs 1989). A subpopulation of primary tumor cells like
cancer stem cells (CSCs) have been discovered that play critical role in tumor
progression, proliferation, relapse (Castelli et al. 2021) and are resistant to current
therapeutics, can metastasize, escape immune system, may lead to dormancy, or may
be left after primary tumor excision in case of solid cancers. The role of CSCs in
cancer recurrence can be viewed from a variety of angles. The development of CSCs,
which retains a community of tumor cells where recurrence occurs, causes resistance
to chemotherapy and radiation (Dave et al. 2012). CSCs role in cancer recurrence
can be viewed from a number of perspectives. Resistant to chemotherapy and
radiation, cancer cell growth is the source of a collection of tumor cells in which
recurrence occurs. The development of cancer stem cell model usually helps to
explain many unresolved questions, including why destruction of non-CSCs may
involve little improvement in patient outcomes. The centrality of CSCs in cancer has
recently been discovered, providing a conceptual basis for researchers to overcome
ambiguous mechanisms in cancer pathophysiology and care also it provided new
insights and objectives to oncology (Chang 2016).
Tumor metastasis also occurs “silently” during the clinical treatment process,
according to a significant number of case studies. Successful cancer metastasis
inhibition is difficult due to the heterogeneity of the surface of metastatic cancer
cells (Hirota et al. 1996; Chen et al. 2019), for example,surface antigens are different
in metastasis of the same melanoma at different locations (Zeltina and Bowden 2017;
Staalsoe et al. 2004). The second reason is that most molecular chemodrugs are
ineffective against metastatic tumors like in metastatic prostate cancer, resistance
was observed against docetaxel–prednisone (Seruga et al. 2011).
Different therapies for different forms of cancer have been approved by the FDA.
Personalized medicines can be designed for the treatment of tumor metastasis, but
the problem is detection and identification of the specific biomarkers which is related
to therapeutic response of tumor. Metastasis-related biomarkers of cancer patients
can help identify spread, preferred metastatic sites, and probability of recurrence
(Brinton et al. 2012). Profile of biomarkers vary a lot with original cancer type,
metastatic site, and metastasis histotypes. In breast cancer metastasis of human,
204 D. Khan and M. Khan

established receptors like epidermal growth factor receptor 2 (EGFR2), progesterone

receptor (PR), and estrogen receptors (ERs) are essential biomarkers to predict
response to anti-HER2 and endocrine therapies in decision-making process of
clinical settings (Martinez-Perez et al. 2019). Different biological components linked
to tumor metastasis processes and signaling pathways are targeted by treatment
methods have also been explored by researchers that include PIK3CA mutation,
mTOR, PI3K, CDK4/6, BRCA1/2 germline mutation, ERBB2 mutation, positive
programmed death ligand-1 (PD-L1), a high level of tumor-infiltrating lymphocytes
(TILs), etc. (Dieci et al. 2020). To effectively diagnose and inhibit malignant tumor
metastasis is still a major challenge and hurdle in current clinical cancer treatment
(Obradovic et al. 2019; Lu and Kang 2019).
Metastasis and recurrence both are major problems in the treatment of cancer
(Kong et al. 2021). Treatment failure, as well as tumor recurrence or relapse, is
believed to be caused by inability of traditional therapies to completely kill all
infiltrative tumor cells. In a variety of tumors CSCs which is a small subset of cancer
cells, have been suggested and believed to be responsible for cancer genesis, tumor
formation, metastasis, drug resistance, recurrence, escape from immune system, and
ability to self-renew (Visvader and Lindeman 2008; Chen et al. 2013), and results in
tumor metastasis and recurrence after treatment (Lei et al. 2021). The role of CSCs in
cancer recurrence can be viewed from a variety of angles. Growth of CSCs is the
reason of accumulation of tumor cells which generally leads to recurrence; this is the
cause of resistance to chemotherapy and radiation (Dave et al. 2012). Existing
therapies failed to offer long-lasting therapeutic responses in patients (Mehlen
2006). Despite all advances in cancer treatment, the key target for any anti-cancer
strategy is tumor metastasis and recurrence (Hanahan and Weinberg 2011). A major
therapeutic goal is eradicating cancer cells, that initiate metastasis and contribute to
tumor growth, survival, and treatment resistance (Mimeault and Batra 2010).

8.2 Cancer Metastasis and Recurrence

After surgery of cancer patients, most common reasons of death includes metastasis
and recurrence. Recurrence can be distant, regional and local, different factors like
adjuvant therapy, surgical stress and trauma, and cancer malignancies determine the
incidence of recurrence, severity and type (Horowitz et al. 2015). Anesthetic
techniques can be a possible factor during surgery in contributing recurrence and
relapse of cancer. During cancer patient’s surgical treatment shorter recurrent free
life has been reported in patients who received inhalational anesthetics in compari-
son to intravenous anesthetic propofol (Wigmore et al. 2016). The ability to detect
disease early can have the greatest impact on mortality, because if any advanced
treatment is possible for the patient, mortality rate can be decreased. It is very critical
to prognosticate, diagnose the stage, and control treatment, as well as detect cancer
recurrence, the ability to diagnose the disease early could have the largest impact on
mortality. Improving detection methods for early-stage cancer in asymptomatic
population is a particular challenge at present. Cancer is a persistent disease, genetic
8 Biomarkers in Tumor Recurrence and Metastasis 205

reports show that many cancers last a decade or more before significant clinical signs
appear (Yachida and Iacobuzio-Donahue 2013; Brown and Palmer 2009).
The pattern of growth is a serious consequence of patient’s outcomes, almost
without exception, cancers that have been diagnosed earlier and have confined
organs are treated with acceptable surgical excision combined with a range of
adjuvant therapies, such as radiation or chemotherapy. Although metastasis is the
cause of the majority of cancer deaths, it is also the most mysterious aspect of disease
(Chaffer and Weinberg 2011).

8.2.1 Cancer Metastasis

When genetically labile cancer cells adapt to a microtissue environment far from the
underlying tumor, this is known as metastasis. This process includes cancer cells as
well as substances that are useful for stromal tumor properties and the recruitment of
traits that can regulate metastatic cell invasion (Gupta and Massagué 2006).

8.2.1.1 Mechanism of Metastatic Cascade

There are three major processes in the metastatic cascade:

8.2.1.1.1 Invasion
It occurs as tumor cells gain the ability to infiltrate neighboring tissues, passing
through the extracellular matrix, and basement membrane (Yousefi et al. 2021;
Reuten et al. 2021).

8.2.1.1.2 Intravasation
It involves the penetration of the lymphatic or vascular system by the motile tumor
cells (Zavyalova et al. 2019; Majidpoor and Mortezaee 2021).

8.2.1.1.3 Extravasation
It involves the journey of metastatic cancer cells through the circulatory system at a
secondary site to infiltrate the extracellular matrix and vascular basement membrane
(Miles et al. 2008) (Fig. 8.1).

8.2.1.2 Changes in Extracellular Matrix (ECM)

To become independent of the main tumor mass and invade the surrounding stroma,
metastatic cancer cells must undergo the loss of cell–cell adhesion and changes in
cell–matrix interactions (Poturnajova et al. 2021; Niland and Eble 2021). These
changes include the secretion of heparinases and matrix metalloproteinases for
basement membrane and ECM degradation, as well as suppression/expression of
proteins involved in motility and migration. Many metastatic biomarkers have been
associated with the dysregulation of the ECM to promote metastasis. Within the
clinic, the tight junction transmembrane protein Claudin-7 is utilized as a prognostic
biomarker in the analysis of invasive ductal breast cancer and is being explored as a
cancer biomarker to distinguish chromophobe renal cell carcinoma from other renal
206 D. Khan and M. Khan

Fig. 8.1 Cancer cell metastasis

tumor subtypes (Bernardi et al. 2012; Rashed et al. 2021). A change in extracellular
matrix metabolism may be involved in a variety of pathways and circulating TIMP-1
or MMP-9 levels could be excellent markers (Hansson et al. 2011).
Claudin-1 expression is used as a prognostic biomarker and potential drug target
for colorectal cancer and lung adenocarcinoma cases (Chao et al. 2009; Zuo et al.
2020). Vimentin and E-Cadherin are known as prognostic biomarkers for non-small-
cell lung cancer treated with erlotinib and colorectal cancer (Richardson et al. 2012;
Niknami et al. 2020).

8.2.1.3 Epithelial to Mesenchymal Transition

The epithelial to mesenchymal transition (EMT) is a multi-stage mechanism wherein
epithelial cells weaken their cell adhesion and polarity and become invasive meta-
static cells. Wnt, TGF, and Notch are among the signaling pathways and growth
factors that control this transition. These signaling pathways show crosstalk that
facilitates EMT and subsequent cell invasion also Notch signatures and pathway is
involved in metastasis and progression of cancer (Li et al. 2020a, b; Misiorek et al.
2021). Expression of vimentin, APC, and β-catenin may be markers of malignant
transformation (Galichon and Hertig 2011). A variety of additional factors have been
shown to contribute to EMT, such as hypoxia, metabolic stressors, matrix stiffness,
and epigenetic and post-translational modulators. The precise contribution of each of
these factors to EMT remains unknown and may vary according to each specific
cancer (Fares et al. 2020). Clinically, a variety of EMT biomarkers are used in the
diagnosis and prognosis of different cancer types. EMT is characterized by
downregulation of E-cadherin and a connection between E-cadherin is lost, EMT
has been established in a variety of cancer types (Martin et al. 2013; Nader et al.
2020). Overexpression of MUC1, MUC4, and MUC16 are all utilized in the
8 Biomarkers in Tumor Recurrence and Metastasis 207

diagnostic and prognostic assessment of cancers, including breast, pancreatic, acute

myeloid leukemia and ovarian cancer (Vergara et al. 2016; Abdelhady et al. 2020).

8.2.1.4 Angiogenesis and Lymphomagenesis

Local diffusion for removal of waste products from site of tumor and transfer of
nutrients will only be sufficient for very small tumors, so the tumor must initiate
angiogenesis otherwise it will fail to grow. Detached tumor cells may enter the
circulatory system via blood vessels in the tumor’s vicinity and spread to secondary
sites. Imbalances in angiogenic and lymphangiogenic processes are thus, frequently
involved in cancer and lend several biomarkers that indicate cancer progression and
metastasis (Kilvaer et al. 2015), other metastatic biomarkers are given in Table 8.1.
Vascular endothelial growth factor (VEGF) has been a key therapeutic target in
the development of anti-angiogenic therapies to treat a range of cancers.

Table 8.1 Biomarkers of cancer metastasis

Cancer type Biomarker References
Lung cancer CA-125 and CEA Clevers et al. (2021)
TRAcP-5b Yao et al. (2011)
Angiopoietin-2 Dong et al. (2018)
NTx Tamiya et al. (2013)
Breast cancer Oxidative stress-responsive kinase Li et al. (2020a, b)
1 (OSR1)
CAPG, GIPC1 Westbrook et al. (2016)
Itgbl1 Li et al. (2015a, b)
DOCK-4 Westbrook et al. (2019)
nPAK4 Li et al. (2019)
LPC1, PRDX4 Tiedemann et al. (2019)
PRL-PRLR Shemanko (2016),
Sutherland et al. (2016).
Osteopontin Shevde et al. (2010),
Rodrigues et al. (2007)
CTX Lipton et al. (2011)
P1NP Brown et al. (2018)
Adenocarcinoma of Nectin-2 and DDX3 Miao et al. (2013)
gallbladder
Colorectal cancer Circulating exosomal miRNAs Alves dos Santos et al.
(2021)
ECA39 Yoshikawa et al. (2006)
Prostate cancer P1NP Koopmans et al. (2007)
Pancreatic cancer Long-non-coding RNAs (MALAT-1) Cheng et al. (2018),
Huang et al. (2016)
Renal cell C-reactive protein (CRP) Casamassima et al.
carcinoma (2005)
Cervical cancer Metastasis-associated lung adenocarcinoma Guo et al. (2010)
transcript 1 (MALAT1)
208 D. Khan and M. Khan

Fig. 8.2 Mechanism of endothelial cells activation via tumor cell in metastasis. (1) The aggrega-
tion of platelet tumor cells is normally mediated by p-selectin and integrins via fibrinogen and fibrin.
(2) The aggregation of neutrophils, which encourage tumor cell proliferation, improves the survival
of circulating tumor cells. (3) P and E selectin mediate tumor cell contact with the endothelium, and
integrins facilitate tumor cell firm adhesion, as seen with VCAM-1 on tumor cells. (4) Monocytes
promote endothelial cell–tumor interactions and help to initiate tumor cell extravasation. (5) Specific
biomarkers in metastasis. Modified from Läubli and Borsig (2019).

Bevacizumab is a VEGF neutralizing antibody that has been approved for use in a
number of cancers, including non-squamous non-small-cell lung cancer and colo-
rectal cancer, as well as epithelial ovarian cancer (Sato and Itamochi 2012; Syed
2020). Lymphatic metastases are common, with a number of cancers first
metastasizing to regional lymph nodes. Biomarkers for lymph angiogenesis and
metastatic spread are being investigated, with VEGF-C and VEGF-D and VEGFR3
emerging as biomarkers for breast cancer, colorectal cancer, and non-small-cell lung
cancer (Al-Rawi and Jiang 2011; Albrecht and Christofori 2011; Huang et al. 2021)
(Fig. 8.2).

8.3 Biomarkers Related to Cancer Metastasis

The spread of cancer from the primary tumor to other tissues and organs is known as
metastasis. This mechanism is a key factor in determining cancer morbidity and a
defining feature of cancer malignancy (Gupta and Massagué 2006; Ganesh and
Massagué 2021). ETNK2 overexpression in the GC promotes hepatic metastasis,
likely by disrupting p53–Bcl-2-associated apoptosis. Expression of ETNK2 may be
used as a therapeutic target biomarker and to predict recurrence of hepatic cancer
(Miwa et al. 2021).
8 Biomarkers in Tumor Recurrence and Metastasis 209

The prediction of tumor metastatic potential is important and could allow for
personalized therapy at an early stage to better treat cancers (Parol et al. 2021; Singh
et al. 2021). Accurate biomarkers of metastasis thus, represent an enormous advance
in the potential cancer treatment. The use of these cancer biomarkers in the clinic
could help detect initial stages of metastasis, preferred sites of cancer metastasis, and
the probability of cancer recurrence (Pavlou et al. 2013; Lokshin et al. 2021), early
cancer detection using miRNA biomarkers and their use as new diagnostic tools
(Galvão-Lima et al. 2021).
To escape circulation and establish a metastatic tumor involves the synthesis of a
number of conditions and events, such as traversing the endothelial wall and trans
endothelial migration and angiogenesis. Consequently, many of the biomarkers of
extravasation reflect earlier metastatic processes, such as VEGF, chemokines and
cytokines, and ECM components such as heparinase and matrix metalloproteinases
(Martin et al. 2013; Winkler et al. 2020). Additionally, the tumor microenvironment
plays a key role in metastasis and offers a number of biomarkers for clinical utility.

8.3.1 Prostate Cancer

Prostate cancer is the most common cause of death in men all over the world
(Velonas et al. 2013). As current prognostication and detection methods are limited
in precision, diagnosis and treatment remain the most complex clinical challenges
(Bidarra et al. 2019). Therefore, clinically significant molecular biomarkers for
prostate cancer diagnosis are in fierce competition, serum measurements show that
ICTP and PINP are useful in the timely identification of skeletal metastases in
prostate cancer patients, also other various biomarkers have been reported for
metastasis (Koopmans et al. 2007).

8.3.2 Breast Cancer

Breast cancer is uncontrolled growth of breast cells. It has different types which
depend on the type of breast cells involved that turn to cancer (Aroef et al. 2020).
They include lobular breast cancer, inflammatory breast cancer, mucinous breast
cancer, mixed tumor breast cancer, and ductal breast cancer. Breast cancer is often
diagnosed in men also (Alimkhodjaeva et al. 2020). Overexpression of osteopontin
is related to cancer metastasis and has been found in melanoma, ovarian cancer,
stomach cancer, colorectal cancer, lung cancer, and breast cancer (Westbrook et al.
2016). The presence of epithelial cells in the bone marrow, circulating tumor cells,
HER2/ErbB2, PR, ER, and uPA/PAI1 are all potential prognostic biomarkers for
breast cancer metastasis (Van De Vijver et al. 2002; Lorusso and Rüegg 2012),
nucleobindin-2 (Zeng et al. 2017) and E-cadherin (Fry et al. 2013).
210 D. Khan and M. Khan

8.3.3 Lung Cancer

Lung cancer is the most common cancer and one of the leading causes of death
worldwide. Late diagnosis is a major hindrance in lung cancer prognosis (Hoseok
and Cho 2015). CircRNAs (circular RNAs) are continuously being shown to play a
key role in lung cancer cell invasion, migration, and proliferation (Di et al. 2019).
Lung cancer biomarkers, especially those that predict metastatic risk, are of great
importance. Just a few biomarkers, such as epigenetic changes and DNA mutations
can necessitate tissue from the risk site; others, including micro RNAs (miRNAs)
and serum proteins, are less invasive, but may not be lung-specific (Subramaniam
et al. 2013).

8.3.4 Colorectal Cancer

Colorectal cancer is a form of cancer that affects specifically the colon and rectum.
Colorectal cancer is reported as the second leading cause of death in the world
(Hampton et al. 2021; Bach et al. 2021). ECA39 is an influential biomarker in
colorectal cancer for distant metastasis (Yoshikawa et al. 2006).

8.4 Cancer Recurrence

After the recovery period, when cancer restores, it is known as recurrence or relapse.
Cancer recurrence occurs when some of your cancer cells persist despite your best
efforts to get rid of cancer, or because cancer cells have metastasized to your organs
at the time of resection or had circulated via vascular and lymphatic systems
(Kinoshita and Goto 2021).
These cells may be in the place where cancer started at first or can be in another
part of body, still it is named after the part of the body from which it originated.
These cancer cells may have been dormant for a long time, but eventually begin to
grow, leading to cancer recurrence. It can happen weeks, months, or even years after
the first or primary cancer treatment. Hematogenous and loco-regional recurrence
require careful monitoring (Sugiyama et al. 2012). Cancer recurrence can be detected
biochemically with an increased level of cancer biomarkers before any radiological
or clinical evidence is appeared. Cancer biomarker can be a threatening sign of
cancer relapse earlier by 3–12 months prior to diagnosis. Many cancer biomarkers
can be used for detection of cancer metastasis, recurrence, or monitoring therapy,
i.e., in prostate cancer PSA, Ovarian Cancer Antigen 125 (CA125), and in colorectal
cancer CEA (Bast et al. 2001).
8 Biomarkers in Tumor Recurrence and Metastasis 211

8.4.1 Mechanism of Cancer Recurrence

Cancer recurrence is one among the most commonly known cancer treatment failure.
This biological phenomenon can be caused by incomplete tumor eradication after
radiotherapy and chemotherapy. Tumors are heterogeneous and CSCs population
can be found in the tumor. Tumor cells are destroyed by radiotherapy and chemo-
therapy, but CSCs are resistant to these treatments and can live for years after being
exposed to them.Therapeutic resistance raises the risk of recurrence and tumor
re-formation also, meanwhile tumor cell metastasizes and spread into other body
tissues. Tumor relapse is much less likely when radiotherapeutic and chemothera-
peutic interventions are directed at CSCs (Esmatabadi et al. 2016; Spring et al. 2020)

8.5 Biomarkers Related to Cancer Recurrence

Individual’s survival can be improved with early diagnosis because survival is

inversely associated with the stage of the disease (Chang et al. 2011). Cancer
biomarkers are used to quantify tumor burden or calculate clinical benefit, harm,
lack of benefit or damage (Goossens et al. 2015; Levenson 2004).
Cancer biomarkers should detect cancer early or at a stage when there are no
symptoms, this will improve safety and reduce morbidity and complications of
cancer patients. To reduce the number of false positives, screening tests must be
highly specific. Ideal screening programs should be low-cost and noninvasive and
they must result in a clear reduction in mortality and morbidity, as well as an
improvement in survival rate. Generally, screening programs are aimed at cancers
that are highly prevalent, as well as further treatment and follow-up are necessary
(Duffy 2001), other biomarkers for recurrence are enlisted in Table 8.2. Limiting
factors for cancer are low specificity and diagnostic sensitivity of many of the
biomarkers which are in use currently that act as screening markers and then later
on in cancer treatment if it escalates. However, some biomarkers are being used as
screening biomarkers for hepatocellular cancer in high-risk individuals, such as AFP
(Xu et al. 2021), in screening of prostate cancer PSA (Clift et al. 2021), in ovarian
cancer CA125 (Zhang et al. 2021), in newborns for screening of neuroblastoma
vanillylmandelic acid (VMA) (Duffy 2015), and for colorectal cancers screening
fecal occult blood testing (FOBT) (Lee et al. 2011). FDA approved PSA as bio-
marker for screening of prostate cancer, however, on individuals with inflammatory
or benign conditions false positive elevated levels of PSA can be found like
prostatitis and benign prostatic hyperplasia (Catalona et al. 1991). The role of PSA
screening in lowering mortality is still a source of debate (Schroder et al. 2009;
Andriole et al. 2009).
212 D. Khan and M. Khan

Table 8.2 Cancer biomarkers in recurrence

Biomarker in cancer
Cancer type recurrence References
Papillary thyroid cancer MiR-9 and miR-21 Sondermann et al.
(2015)
Breast cancer Long non-coding RNAs Zhou et al. (2016)
(IncRNAs)
CA15-3 Keshaviah et al. (2007)
Ki-67 Nishimura et al. (2010)
ACLY Chen et al. (2020)
Colorectal cancer miRNA-29c Yang et al. (2013)
ExosomalmiRNA Matsumura et al.
(2015)
TMEM240 Chang et al. (2020)
miR-34a-5p Gao et al. (2015)
Carcinoembryonic Tan et al. (2009)
antigen (CEA)
miRNA-148a Tsai et al. (2013)
Lung Small cell lung cancer Long-non-coding RNA Ono et al. (2014)
cancer HOTAIR
Exosomal miRNAs Munagala et al. (2016)
.
Histone macroH2A Sporn et al. (2009)
Non-small cell or small cell miRNA-451a Kanaoka et al. (2018)
lung cancer miRNA-486 Li et al. 2015a, b
Collagen XXIII Spivey et al. (2010)
Adenocarcinoma CA 19-9 and CA 125 Isaksson et al. (2017)
.
Anterior gradient Hanada et al. (2012)
homolog 2 (AGR2)
Hepatocellular carcinoma Mortalin (HSPA9) Yi et al. (2008)
miRNAs Sugimachi et al. (2015)
NDRG1 Cheng et al. (2011)
Cytokeratin 19 (CK19) Uenishi et al. (2003)
Osteopontin (OPN) Pan et al. (2003)
mRNA
Alpha-fetoprotein (AFP) dos Santos Schraiber
et al. (2016)
MCM6 Liu et al. (2018)
DCP, GP73 Yamamoto et al. (2010)
Ovarian cancer circSETDB1 Wang et al. (2019)
Human epididymis Plotti et al. (2012)
protein 4 (HE4)
CA125 Murakami et al. (2006)
KLK6 and KLK13 White et al. (2009)
KLK7 Tamir et al. (2014)
Bladder cancer Nuclear matrix protein 22 Shariat et al. (2005)
(continued)
8 Biomarkers in Tumor Recurrence and Metastasis 213

Table 8.2 (continued)

Biomarker in cancer
Cancer type recurrence References
Cystatin B Feldman et al. (2009)
Cervical cancer SCC-Ag and hsCRP Hoogendam et al.
(2013)
Pancreatic cancer CD44 Hsu et al. (2018)
Gastric cancer miRNA27a Huang et al. (2014)
miRNA-203 Imaoka et al. (2016)
Acute myeloid leukemia miR-15a/16-1 Gao et al. (2011)
Acute lymphoblastic leukemia miR-135a Diaz-Beya et al. (2014)
miR-708 Han et al. (2011)
LINC00152 Bárcenas-López et al.
(2020)
Multiple myeloma Circulating exosomal Luo and Gui (2020)
circMYC
ITD+ leukemia IL-10, IL-1β, TNF- α Rickmann et al. (2013)

8.5.1 Breast Cancer

It is reported in a study that ACLY is an independent and potential biomarker for the
prediction of recurrence in breast cancer patients (Chen et al. 2020).
When a patient’s primary breast cancer relapses then immunohistochemistry is
often used to guide treatment adjustments. Confirmatory tissue sampling and detec-
tion of HER2, PR, or ER switches that change clinical care for one in every six
patients must be part of relapsed breast cancer management (Thompson et al. 2010)

8.5.2 Prostate Cancer

Despite early prostate cancer treatment, most men experience an increase in PSA.
Although some of those men may undergo a metastatic or local recurrence, requiring
further treatment, some will show no evidence of progression of the disease
(Nakagawa et al. 2008). Prostate cancer studies over the years developed metabolite
profiling to classify prognostic, diagnostic and predictive biomarkers (Kelly et al.
2016). Additional biomarkers of prostate cancer aggressiveness are being sought by
urologists. Higher risk of prostate cancer recurrence is linked with increased level of
insulin in serum (Lehrer et al. 2002).

8.5.3 Leukemia

The optimal cure for acute myeloid leukemia (AML), which is a heterogeneous
disorder and cancer of white blood cells of myeloid origin, depends on molecular
markers and cytogenetic risk factors. MicroRNAs (miRNAs) have been linked to a
214 D. Khan and M. Khan

greater risk of AML relapse in many studies, a study reported that low levels of
miR-135a and miR-409-3p being linked to a greater risk of AML patients relapse
(Diaz-Beya et al. 2014).
In the last decade, evidence of the role of long-non-coding RNAs (lncRNAs) in
leukemogenesis has emerged. These genes have been suggested as diagnostic and/or
prognostic biomarkers in children with acute lymphoblastic leukemia (ALL). In the
last decade, evidence of the role of long-non-coding RNAs (lncRNAs) in leukemo-
genesis has emerged.Early relapse and mortality in patients, LINC01013 and
LINC00152 were among the most differentially expressed genes (Bárcenas-López
et al. 2020).

8.6 Applications of Cancer Biomarkers in Most Common

Cancers

Cancer has been identified as leading cause of death for several years; in 2012, an
estimated 8.2 million cancer patients died around the world (Bray et al. 2012).
According to the GLOBOCAN worldwide, incidence of cancer and mortality rate
reported in 2015 by the International Agency for Research on Cancer (IARC), breast
cancer, liver cancer, and ovarian cancer is among the most prevalent cancers in
women worldwide (Ferlay et al. 2015). Cancer biomarkers can be used for the
assessment of cancer risk for screening in the asymptomatic community,
distinguishing benign from malignant tumors, prognosis, and following treatments
(Henry and Hayes 2012).

8.6.1 Breast Cancer

Breast cancer is the most common malignancy in women and the leading cause of
death worldwide; its incidence rate is rising at an alarming rate at the moment (Pisani
et al. 2002). Early intervention and proper case management is an important way to
control cancer. The most common clinical signs of breast cancer are nipple dis-
charge, lump in breast, and prominent change in skin or nipples. According to the
American Cancer Society’s screening recommendations, women above the age of
40 can get a mammogram and a clinical breast test once a year or every other year
(Sabatino et al. 2015). Cancer biomarkers role in breast cancer treatment is primarily
very helpful with prognosis, monitoring of treatment, and follow-up; in particular,
does not seem to be very useful for early detection. (Ludwig and Weinstein 2005).
European Society of Medical Oncology has proposed testing for ER and PR for
diagnosed breast cancers to predict response to advanced breast cancer cases (Mirza
et al. 2002; Duffy 2006) “HER-2” is an additional prognosis marker which is also
most effective for targeting early or metastatic breast cancer patients in Trastuzumab
therapy (Ross et al. 2003); or estimate resistance of tamoxifen treatment in early
stages of breast cancer (Duffy 2005). For the treatment of breast cancer, screening
scheme should be introduced and discovery of “BRCA1 or BRCA2 gene
8 Biomarkers in Tumor Recurrence and Metastasis 215

mutations”, 5% of cases of breast cancer. Because of high vulnerability to breast and

ovarian cancer, it should be advised to females for regular cancer screening to
observe BRCA1 or BRCA2 mutations (Vietri et al. 2012). Low levels of “urokinase
plasminogen activator (uPA)” and “plasminogen activator-1 (PAI-1)” have been
confirmed to link with a decreased risk of relapse of breast cancer (Harbeck et al.
2001). During therapy, serum biomarkers are used which normally aided in postop-
erative surveillance, and CBs included in that group include “CA15.3, CEA, and BR
27-29” (Molina et al. 1998) used for conjugation with radiological and therapeutic
evaluation instruments to track chemotherapy in advanced breast cancer cases. The
elevation of serum levels of markers may propose for recurrence or disease progres-
sion (Sparano 2006).

8.6.2 Prostate Cancer

Prostate cancer is the most common cancer in men and the leading cause of death
(Pisani et al. 2002). Strong evidence suggests that the “PSA test” transfigured
screening and detection of prostate cancer in earlier stages and also helps in lowering
prostate cancer mortality (Bjartell 2013). Many experiments later showed that using
“PSA subtractions and isoforms [-2] (proPSA)” improved the sensitivity of PSA as a
diagnostic marker significantly. Patients with PSA levels range from 4 to 10 g/L,
these fractions can aid in the differentiation of benign and malignant prostatic tumors
(Gao et al. 2003). “Human kallikrein type 2”, “prostate cancer antigen 3 (PSA3)”,
and “prostate stem cell antigen” are the biomarkers under investigation. The PCA3
urine assay has the potential to improve prostate cancer diagnostic accuracy (Haese
et al. 2005). Increased amounts of the protease family “members metalloproteinase
2 and 9” (MMP-2 and MMP-9) have been linked to prostate cancer diagnosis (Moses
et al. 1998). MMPs have been investigated as clinical screening biomarkers of
prostate cancer (Morgia et al. 2005).

8.6.3 Ovarian Cancer

Mostly patients are diagnosed late with epithelial ovarian cancer stages III and IV at
the time of diagnosis, ovarian cancer diagnostic biomarkers must be responsive and
specific (Coticchia et al. 2008). CA 125 is one of the most commonly used and well-
known CBs. CA125 has been used in combination with vaginal ultrasound as a
diagnostic biomarker for females having a family history of ovarian cancer (Duffy
et al. 2005). CA125 is often used as a screening biomarker, as its level drops after
beginning chemotherapy or surgery, which coincides with a positive reaction.
Follow-ups at frequent intervals should be strongly recommended 2–3 weeks before
starting clinical intervention (Morgan et al. 2008). Other biomarkers have been
studied extensively in the monitoring of ovarian cancer and its prognosis; however,
more research is required to validate their exact function. “Kallikreins, osteopontin,
Her-2/neu, tumor-associated inhibin, CEA, trypsin inhibitor, hCG, interleukin-6
216 D. Khan and M. Khan

(IL-6), prostasin, TPA, lysophosphatidic acid, and plasminogen activator inhibitor-1

are all included in this panel (PAI-1)” (Coppola et al. 2004).

8.7 Conclusion

Cancer biomarkers have a vital role in cancer for screening, risk assessment,
diagnosis, response to treatment, prognosis, and recurrence or relapse. It is important
for researchers and clinicians to have thorough understanding of clinical utility,
molecular aspects, and biomarker’s reliability to know in which state biomarkers are
important in clinical use for better evaluation and patient care. Biomarkers can
facilitate the diagnosis and therapy of patients and can have vital role in personalized
medicine. Despite many efforts to identify the risk factor, the possibility of recur-
rence is unlikely to be eliminated. However, there is an urgent need to develop or
identify biomarkers which show us patient-related factors and can be used to predict
cancer in advance. Cancer biomarkers can be used for novel targeted and
personalized treatments as it investigates patient therapeutic profile and
characteristics.

8.8 Future Perspectives

The drawbacks of the clinical use of these new markers, at the detection level, have
been reduced to the rapid use of markers for validation and clinical practice, owing to
the increased detection of potential biomarkers. To ensure cost-effectiveness, oncol-
ogy practice will be tracked for the next decades. Biomarkers that can diagnose
cancer, predict cancer outcomes, and affect treatment choices would be crucial in
evaluating the efficacy of clinical cancer treatment. It necessitates a method that is
simple, efficient, safe and dependable. No biomarker is likely to be accurate enough
to affect treatment decisions when it comes to predicting disease outcomes. As a
result, cancer diagnosis in the future can depend on small plaques of 6–10 markers
that provide an accurate molecular measure indicating the probability of metastatic
involvement and the need for rapid systemic care. Advanced technologies will aid in
the development of several new biomarkers for a specific disease at the same time.
Today, with microchip technology, 2D gels, and mass spectrometers are not readily
available in the clinical setting and require well-equipped laboratories and skilled
personnel. To advance biomarker detection in clinical practice, simple, fast, and
responsive microbe-based protein chips, unlabeled detection systems and antibody-
based chip systems have been developed. We are getting closer to a time when
biomarkers can be used to help track and control cancer diagnosis, progression and
care. A key aim for the future of cancer is to develop simple test kits that correctly
and efficiently diagnose cancer and can be used in clinics or on potential patients.
8 Biomarkers in Tumor Recurrence and Metastasis 217

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Biomarkers for Cancer Immunotherapy
9
Haseeb Ahsan, Salman Ul Islam, Muhammad Bilal Ahmed,
Young Sup Lee, Mughal Qayum, and Jong Kyung Sonn

Abstract

Management of advanced-stage cancers of various types has revolutionized with

the introduction of immune checkpoint inhibitors (ICIs) in 2010. However,
achievement of optimum benefit is limited to a small number of patients only.
Identification of these responsive patients prior to administration of ICIs gives
rise to the critical need for predictive biomarkers for immunotherapeutic agents.
In this chapter, we have focused on the current status of famous biomarkers along
with their predictive utility in various types of cancers. PD-L1, mutational
burden, TILs, neutrophil/lymphocyte ratio, LDH, miRNA, and microbiota have
been discussed in detail. Multiple studies exemplifying their usefulness have been

H. Ahsan
School of Life Sciences, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National
University, Daegu, Korea
Department of Pharmacy, Faculty of Life & Environmental Sciences, University of Peshawar,
Peshawar, Khyber Pakhtunkhwa, Pakistan
S. U. Islam
School of Life Sciences, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National
University, Daegu, Korea
Department of Pharmacy, Cecos University, Hayatabad, Peshawar, Pakistan
e-mail: [email protected]
M. B. Ahmed · Y. S. Lee · J. K. Sonn (*)
School of Life Sciences, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National
University, Daegu, Korea
e-mail: [email protected]
M. Qayum
Department of Pharmacy, Kohat University of Science & Technology, Kohat, Khyber
Pakhtunkhwa, Pakistan

# The Author(s), under exclusive license to Springer Nature Singapore Pte 229
Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_9
230 H. Ahsan et al.

presented. It is pertinent to mention that optimization of response biomarkers

precedes the optimization of ICIs. For this purpose, tumors and their microenvi-
ronment must be characterized for selection of specific biomarkers to predict ICI
response. Further studies and deeper insights are required to eliminate all the
drawbacks associated with the predictive precision of the biomarkers for the
successful design of individualized immunotherapy regimens.

Keywords
Biomarkers · PD-L1 · Lymphocytes · Tumor mutational burden · Microbiota ·
miRNA · Lactate dehydrogenase · Pembrolizumab

9.1 Introduction

Managing cancer via drugs or agents affecting immune system has changed the
landscape of anti-neoplastic therapies. Extensive research in the recent years has
made it clear that modulation of immune system is effective in the elimination of
distant metastatic tumors. Cancer immunotherapy has recently shown promising
outcomes with respect to their safety andeffectiveness profiles, making it a strong
candidate besides radiotherapy and chemotherapy (Callahan et al. 2016; Hanahan
and Weinberg 2011). Although much needs to be done pertaining to the assessment
and standardization of their manufacturing with respect to environment &
sustainability, current priority is given to the evaluation of pharmacological and
clinical benefits (Ahsan et al. 2020; Capela et al. 2019). Research in this field has
resulted in the introduction of more than 2000 immunotherapeutic agents for cancer
(Tang et al. 2018; Walk et al. 2020).
Induction of lymphocytes to generate vigorous immune response in the tumor
environment has proven to be effective in various cancer patients (Suzuki et al. 2017;
Sheikh et al. 2013; Torphy et al. 2018). A major disadvantage associated with
positive clinical outcome is that cancer immunotherapy has been found beneficial
in only 20% of cancer patients onassessment through clinical trials. Selectivity of
clinical response is the biggest disadvantage of immuno-oncological therapies.
Moreover, adverse drug reactions, wasted financial cost, burden on private, and
government drug procuring facilities are additional disadvantages. Other sets of
reasons for unsuccessful development of an ideal predictive biomarker for immuno-
therapy, include tumor heterogeneity, individualized host–immune responses, dis-
tinct molecular, pathological, and genetic changes in the tumors. Pre-assessment of
outcome is necessary to determine benefit-to-risk ratio. (McKean et al. 2020).
Selection of patients for a specific immunotherapy drug to achieve optimum
clinical care and targeted therapy at minimal cost necessitates the development and
use of efficacy biomarkers of cancer immunotherapy. Therefore, the prerequisite for
effective personalized cancer immunotherapy is the development of biomarkers that
act as response predictors of specific immunotherapies (Kourie and Klastersky
2016). These would also enable the clinicians to understand the complex mechanism
9 Biomarkers for Cancer Immunotherapy 231

of cancer immune cycle and the stages at which relevant biomarkers affect and
predict the outcome of immunotherapy (Schumacher et al. 2015). Personalized
analysis and interpretation of biomarker testing will eventually be required for
selection of individualized regimens (McKean et al. 2020). Herein, we discuss the
potential response biomarkers in cancer immunotherapy.

9.2 Programmed Death-Ligand 1 (PD-L1)

Use of this endogenous molecule as a biomarker was first determined during the
study of PD-1 inhibitors in 2010 (Yarchoan et al. 2019). This biomarker, present on
T cells and tumor cells, has become the most popular and widely used standard prior
to commencement of PD inhibitors as mode of cancer immunotherapy (Brahmer
et al. 2010; Patel and Kurzrock 2015). It is the ligand of PD-1, one of the important
receptors on T lymphocytes involved in controlling tumor-immune responses. The
ligand causes decreased proliferation of T cells and also helps in escape of tumor
from immune responsiveness. Antibody against the PD-1 receptor to inhibit its
activation via PD-L1 will result in making the tumor cells susceptible to intense
immune response (Fig. 9.1) (Shindo et al. 2015).
A study reported the results of earlier clinical trials with an anti-PD-1 antibody
against carcinoma of various organs such as kidneys, lungs, colon, and prostate. 36%
of patients expressing PD-L1 showed anti-neoplastic response to the administered
antibody, while patients not expressing PD-L1, did not show any anticancer effects
(Topalian et al. 2012). Another study reported the association of PD-L1 biomarker
with efficacy of pembrolizumab (anti-PD-1). The trial revealed that patients with
non-small-cell lung cancer (NSCLC) respond to the antibody when PD-L1 expres-
sion was high. Greater and better response was directly related to better disease
outcome and survival (Garon et al. 2015; Topalian et al. 2012). PD-L1 expression,
determined via tumor proportion score, showed that there is direct relationship of
PD-L1 with efficacy of pembrolizumab. Hence, PD-1 inhibitors can prove to be

Fig. 9.1 Mechanism of action of anti-PD-1 agents. TCR T-cell receptor; MHC II Major histocom-
patibility complex II; CD28 cluster of differentiation 28
232 H. Ahsan et al.

good candidates for cancer immunotherapy and its effectiveness can be determined
by prior determination of PD-L1 expression (Allgäuer et al. 2018; Balar et al. 2017;
Herbst et al. 2016; Reck et al. 2016). Correlation of PD-L1 expression with immu-
notherapy was also found in patients with Hodgkin’s lymphoma. A response of 87%
to immunotherapy was observed along with higher expression of biomarker in the
patients (Ansell et al. 2015). In addition to its expression in tumor cells, it has also
been reported that degree of PD-L1 expression on TILs is also a representation of
patient suitability to immune checkpoint inhibition therapy (Herbst et al. 2014; Zou
et al. 2016).
PD-L1 testing is carried out by immunohistochemical analysis. The ratio of
PD-L1+ tumor and immune cells to the total tumor population is measured. This
procedure is considered prerequisite before recommendation of anti-PD-1 agents for
the management of advanced head and neck cancer, esophageal cell carcinoma
(second-line treatment recommendation),and adenocarcinoma of stomach and
esophageal junction (as third-line therapy recommendation). Versatility of PD-L1
as response biomarker has been verified by many other studies showing improve-
ment in clinical severity and increase in overall survival of patients (McKean et al.
2020).

9.3 Tumor Mutational Burden, Mismatch Repair Deficiency,

and Neoantigens

Tumor mutation burden (TMB), defined as the total number of nonsynonymous

somatic mutations within protein encoding area of tumor cell genome (Allgäuer et al.
2018). These mutations in tumor genome express to produce “neoantigens” which
are specific to tumor cells only. These antigens are expressed on cell surface of the
tumor cells followed by their immune recognition by lymphocytes (Schumacher and
Schreiber 2015).
A total of 2–5 base pairs sequences occurring repeatedly between coding and
noncoding regions of gene bear the deficiency of DNA mismatch repair, making
them prone to develop instability, and mutations. The unstable microsatellites
phenotype has been associated with greater neoantigen production, making the
tumor more responsive to immunotherapy (Gupta and Heinen 2019).
It has been reported that greater mutational burden causes greater immune
responsiveness. Patients with melanoma when treated with ipilimumab showed
greater responsiveness, progress, and survival to therapy when TMB was higher
(Snyder et al. 2014). One study presented similar results in another group of
individuals to whom pembrolizumab was administered for NSCLC. Alexandrov
et al. have reported the results of their studies on TMB that tumors with abundant
mutational load (TMB) are more likely to respond to cancer immunotherapy,
because multiple mutations generate neoantigens causing activation of immune
system (Alexandrov et al. 2013).
With the innovation of liquid biopsy, estimation of TMB in circulating tumors
has been reformed. One research group studied the effect of atezolizumab in NSCLC
9 Biomarkers for Cancer Immunotherapy 233

patients. The intensity of response to drug was compared with proportion of TMB
estimated (by liquid biopsy) to find if the correlation exists between objective
response and TMB. Researchers found that patients with higher TMB showed
greater and better pharmacological response to immune checkpoint inhibitors
(ICIs) therapy than those with lower TMB (Gandara et al. 2018). In its continuation,
tumors with faulty and deficient DNA repair mechanisms producing neoantigens
respond strongly to immunotherapy than those tumors with efficient DNA repair
mechanisms and lesser neoantigens (Chang et al. 2018). Recently, a study reported
variable response of immunotherapy in cancer patients with high TMB. The
variability of results was attributed to the nature and significance of genomic
mutations and their effect in inducing the robust immune response against the
tumor cells. Authors also suggested immunoediting as one of the escape mechanisms
from host’s immune system. This might generate immune-resistant tumors against
which anti-PD-1 agents would not be optimally effective. In NSCLC, it has been
reported that greater expression of neoantigens in the initial stages of malignant
transformation makes the tumor more responsive to immunotherapy. Hence,
neoantigens are also considered predictive response biomarkers of cancer immuno-
therapy (Jamal-Hanjani et al. 2017; McGranahan et al. 2017). Hugo et al. 2016 have
also reported that BRCA2 mutations cause defects in DNA repair. Loss-of-function
mutations in this gene have, therefore, been associated with better response
outcomes when treated with anti-PD-1 drugs (Hugo et al. 2016; Pérez-Ruiz et al.
2020).
TMB has been positively correlated in many tumors as response biomarker for
ICIs. In one study, the ICI objective response rate in various types of tumors and its
subtypes was observed and correlated to TMB measurement (Steuer and
Ramalingam 2018). Analysis showed that TMB measurements were correctly
representing the response to ICIs (direct proportionality) in all tumor types.
Desmoplastic melanoma is one of the tumors having highest TMB. Likewise, this
cancer type also shows very good response to ICIs (Boussemart et al. 2019). Similar
trend of TMB was reported with improved overall survival with ICI therapy in
151 patients having various types of cancer in advanced stage. TMB was assayed by
next-generation sequencing (Goodman et al. 2017).
In a recently published study by Valero et al., it was demonstrated that the relation
between TMB and patient survival is significant from treatment perspective. They
concluded that higher TMB will result in greater improvement and survival from
cancer only when treated with immune checkpoint inhibitors (Valero et al. 2021).

9.3.1 Concerns About TMB

Despite substantial advantages of TMB, various concerns need to be addressed to

generalize its use as preferable response biomarker. Of prime importance is the
absence of standard assay method for TMB determination. Multiple tests based on
different principles and criteria are being used. One study has demonstrated that
TMB is a better predictive biomarker in younger age cancer patients (Wu et al.
234 H. Ahsan et al.

2020). Additionally, various cutoff values have been suggested for TMB which
makes it more difficult to assent on a single universally accepted value (McKean
et al. 2020).

9.4 Tumor-Infiltrating Lymphocytes (TILs)

Human immune system responds to cancerous cells via various lymphocytes, such
as cytotoxic T cells and natural killer (NK) cells. The number of these cells in the
tumor microenvironment is directly related to overall survival rate and response to
ICIs. All such cell types inside the tumors are classified as tumor-infiltrating
lymphocytes (TILs)(Uryvaev et al. 2018). These lymphocytes are present in tumor
environment after infiltration from systemic circulation under the influence of
immune system. They have been studied for their predictive and prognostic potential
of immunotherapy in various types of cancers (Rosenberg et al. 1988). Likewise,
they have shown to be good efficacy predictors of ICIs.
Tumor with robust immunogenicity involves infiltration of CD8+ and CD4+ T
cells in tumor microenvironment. Such tumors are termed as immune-inflamed
tumors (Chen and Mellman 2017). Tumor assessment for immune infiltrates is
carried out by immunohistochemical staining (Clark Jr et al. 1989; Elder et al.
1985; Hendry et al. 2017).
Immune-inflamed tumors have been associated with better response and overall
survival (OS) with ICIs, suggesting lymphocyte infiltration as predictive biomarker
directly related to the ICI therapy in various advanced cancers (Herbst et al. 2014;
McKean et al. 2020; Tumeh et al. 2014).
In the tumor environment, activated T cells release cytokines such as interferon-
gamma (IFN- γ) that causes upregulation of PD-L1 receptors (Ikeda et al. 2002). It
has also been suggested that defective IFN- γsignaling causes reduction in response
to ICI therapy (Garcia-Diaz et al. 2017; Stühler et al. 2019). This biomarker has
recently gained importance to help recruit patients with greatest response rates for a
specific anti-PD-1 therapy. In breast cancer patients, stromal TILs density (deter-
mined by H&E staining) have also positively correlated to response of immunother-
apy (Gonzalez-Ericsson et al. 2020; Hendry et al. 2017). Several studies have
demonstrated the correlation of CD8/CD4 ratio of TILs as efficacy predictor of
anti-PD-1 regimen (Bald et al. 2014; Garon et al. 2015; Ribas et al. 2015; Robert
et al. 2015). One study was designed to determine the role of CD4+ and CD8+ TIL
as response biomarkers of anti-PD-1 therapy in NSCLC (26 patients) and malignant
melanoma (30 patients). The researchers found interesting association of CD8+/
CD4+ ratio to treatment response. In both cases, their results demonstrated that the
TIL ratio greater than 2 produces better response to anti-PD-1 treatment. Other
studies conducted in smaller number of NSCLC patients were also suggestive of
better response to anti-PD-1 agents due to combined increased in PD-L1 expression
and TILs (Herbst et al. 2014; Herbst et al. 2016; Reck et al. 2016; Uryvaev et al.
2018). One study reported that cytotoxic T cell density in microenvironment of
metastatic melanoma is proportional to the overall response rate of specific
9 Biomarkers for Cancer Immunotherapy 235

immunotherapy drugs (Tumeh et al. 2014). Another study reported a strong

proportionality of CTL (expressing CTLA-4) density as response predictor of
immunotherapy (Daud et al. 2016; Loo et al. 2017). Similarly, increased TIL density
in bladder carcinoma were indicators of high response rate and improved survival
with anti-cancer immunotherapy agents (Sharma et al. 2007).

9.4.1 Effect of Chemokines on TILs

Th1 chemokines have been found in tumor microenvironment and have been found
to affect Tcell density in tumor. Therefore, these chemical cytokines have indirect
effect on the patient response to immunotherapy by affecting lymphocyte concen-
tration in tumor (Galon et al. 2006; Kryczek et al. 2009; Zhang et al. 2003).
Resistance to immune responses by tumors is also believed to be produced via
regulation of chemokine expression and signaling mechanisms. This might also
reflect the failure of immunotherapy to manage these cancers. One such mechanism
has been reported in mouse melanoma model where β-catenin negatively regulates
CCL4. CCL4 is responsible for migration of dendritic cells that leads to T cell
activation. Tumor halts lymphocytes activation by negative regulation of
chemokines (Spranger et al. 2015). In other models of cancers (ovarian and
colon), negative regulation of CXL9 and CXL10 cause T cell inactivation and
reduce the lymphocyte migration, infiltration, and density. Epigenetic pathways
found to support tumor progression and reduce anti-tumor immune responses, by
affecting the expression of chemokines, including polycomb repressive complex
2 (PRC2) and DNA methylation (Nagarsheth et al. 2016; Peng et al. 2015).

9.5 Neutrophil–Lymphocyte Ratio (NLR)

Advancement in knowledge of various pathways and parameters involved in man-

aging cancer has led us to discover the potential of NLR as predictive biomarker of
cancer immunotherapy. Many studies have shown NLR to be a very useful parame-
ter for judging inflammatory status of tumor and its surroundings (Chua et al. 2011).
Basic advantages of this parameter, include its simplicity, ease to collect sample, and
conduct test (Park and Lopes 2019).
Effect of NLR on tumor microenvironment is mediated by the release of
cytokines which regulate downstream expression pathways of various genes that
lead can aggravate or alleviate the tumor microenvironment (Fig. 9.2). A study
revealed that good prognosis of cancer is linked to high lymphocyte and low
neutrophil counts. Patients treated with nivolumab for melanoma having high
absolute lymphocytic count (>1000 μL) and low absolute neutrophil count of
equal to or less than 4000 μL was positively associated with patient OS (Nakamura
et al. 2016). Higher lymphocyte count observed in peripheral blood sample has
shown positive correlation to immunotherapy with pembrolizumab and ipilimumab
(Martens et al. 2016; Park and Lopes 2019; Weide et al. 2016). This ratio of
 236

Fig. 9.2 Consequences of low lymphocyte count (high NLR) resulting in cytokine-mediated initiation of various pathways aggravating tumor severity
H. Ahsan et al.
9 Biomarkers for Cancer Immunotherapy 237

neutrophil-to-lymphocyte is an index of robust systemic immune inflammatory

response (Zahorec 2001). Higher ratio is indicative of feeble immune response to
tumors and hence, less responsive to cancer immunotherapy agents in advanced
stages (Bilen et al. 2019; Maymani et al. 2018; Shindo et al. 2017b). It has been
suggested that tumors with lymphocyte count less than 15% of normal limit should
not be dealt with immunotherapy because of lesser chance of any possible improve-
ment in cancerous condition or clinical manifestation of the disease. Furthermore, it
has also been reported that NLR greater than 3 is suggestive of poor prognosis in
patients with colorectal cancer (CRC) receiving vaccine (Hazama et al. 2014).
Another related ratio is called derived NLR (dNLR) which includes monocytes
and granulocyte count while predicting the responsiveness of a certain tumor to
immunotherapy (Proctor et al. 2012). A relatively large number of patients with
melanoma treated with ipilimumab for the same were checked for dNLR. It was
observed that lower ratio (less than 3) correlated with better therapeutic response and
survival rates. Higher ratio corresponded with increased risk of fatality and disease
progression (Ferrucci et al. 2016). These results show that dNLR can be used as
predictive and prognostic agent in cancer patients to check suitability of immuno-
therapeutic agents.

9.6 Lactate Dehydrogenase (LDH)

LDH is the last enzyme in carbohydrate metabolism. It is part of glycolysis where it

is responsible for interconversion of lactic acid and pyruvic acid (Talaiezadeh et al.
2015). Due to Warburg phenomenon, LDH is increased in cancer patients
representing greater utilization of glycolysis (Wong et al. 2013). It has inverse
relationship with the patient’s prognosis. In a study on 230 patients with melanoma
who were treated with an anti-PD-1 agent ipilimumab, the overall survival of
patients was higher (10 months) when baseline LDH values were less whereas, the
overall survival was 2.9 months when LDH value was two-fold higher than upper
limit of normal value (Kelderman et al. 2014). Other studies exploring the correla-
tion between LDH and melanoma patient survival reported similar results with
pembrolizumab and nivolumab used as anti-PD-1 agents (Diem et al. 2016; Weide
et al. 2016; Wagner et al. 2018). Other studies were also conducted to evaluate LDH
levels with death rate in lung cancer patients. The studies concluded that higher LDH
levels are associated with higher death rate. Moreover, administration of immune
checkpoint inhibitors did not improve the survival of patients with elevated LDH
concentration (Inomata et al. 2020; Ichiki et al. 2019; Oya et al. 2017). Similar
results were shown in patients with esophageal cancer and high LDH, representing
reduced improvement in clinical symptoms when treated with camrelizumab
(Simeone et al. 2014).
238 H. Ahsan et al.

9.7 MicroRNA (miRNA)

miRNA comprises 18–25 base pairs that are of noncoding nature. They affect gene
expression post-transcriptional level. Although miRNA does not translate into a
protein, they regulate the transcription of many mRNAs (Shindo et al. 2017a;
Zaharie et al. 2015). Alterations in the expression of miRNA change the behavior
of tumor. These changes are sometimes favorable making the tumor more responsive
to anti-cancer regimen, while in other cases tumor becomes more resistant to
anti-cancer therapies. Hence, it is reported to be a prognostic and predictive bio-
marker for various types of tumors (Iorio et al. 2005; Nagao et al. 2012; Schetter
et al. 2008).
In advanced CRC patients, better OS was observed in those having lower
expression of miRNA-6826 and miRNA-6875 in systemic circulation (Kijima
et al. 2017). This indicates that miRNAs are involved in strengthening tumor
resistance to immune system. Hence, downregulation of these miRNA can increase
the vulnerability of tumors to immune system and agents potentiating immune
system (Halvorsen et al. 2018). Similarly, seven circulating miRNAs (215-5p,
411-3p, 493-5p, 494-3p, 495-3p, 548j-5p, and -93-3p) were also found to be
associated with better survival and response rates to nivolumab when treated for
NSCLC (Halvorsen et al. 2018). Therefore, miRNAs have versatile functions, some
being tumor suppressors while others acting as tumor inducers (Li et al. 2016).
MRX34, a liposomal miRNA-34a was the first miRNA developed to act against
cancer (Beg et al. 2017). In murine model, when administered as adjuvant to
radiotherapy, miRNA-34a inhibited PD-L1 expression and enhanced TILs (Cortez
et al. 2016). In NSCLC, miRNA-200 is an inhibitor of PD-L1 expression. It
increases lymphocyte infiltration, in tumor microenvironment, from systemic circu-
lation. Moreover, it has also been shown to retard metastatic potential of tumor
(Romano and Kwong 2018).

9.8 Microbiome

Intestinal flora affects personal health and host’s immune system (Picardo et al.
2019; Wang and Jia 2016). Intestinal microflora and cancer coincide in newer but
unexplored avenues of cancer biology. Complete elucidation of their relationship
and interdependence has not been done so far. Anti-cancer treatment modality such
as cyclophosphamide (Viaud et al. 2013) and PD-L1 inhibitors (Sivan et al. 2015)
affect the intestinal microbiota. Abundance of Akkermansia muciniphila in patients’
stools has been associated with better overall response rates to anti-PD-1 therapy in
renal cell carcinoma and NSCLC (Routy et al. 2018). It has also been reported that
bacterial transplant into murine cancer model improved the response rate to anti-PD-
1 therapy. The effect was thought to be mediated by IL-2 causing migration and
recruitment of CCR9+, CXCR3+, and CD4+ T cells to generate robust immune
response against tumor. Similarly, Bacteroides fragilis has been correlated with the
efficacy of CTLA-4 inhibitors (Vétizou et al. 2015). Therefore, cancer patients
 9
Biomarkers for Cancer Immunotherapy

Fig. 9.3 Effects of short-chain fatty acids produced by gut microbiota on the lymphocyte differentiation and tumor microenvironment
239
240 H. Ahsan et al.

receiving any antibiotic regimen which can harm intestinal microflora can indirectly
lead to less responsiveness of main anti-cancer therapy. The outcome worsens when
the antibiotic has broad or extended anti-microbial spectrum (Bunney et al. 2017;
Pérez-Ruiz et al. 2020).
Fusobacterium nucleatum has been associated with upregulation of oncogenic
genes, thereby helping in CRC development (Abed et al. 2016; Rubinstein et al.
2013; Tahara et al. 2014; Yamaoka et al. 2018). A study in cancer patients of
Japanese origin revealed that the F. nucleatum is associated with poor clinical
outcomes and less responsiveness to anti-cancer therapies (Abed et al. 2016).
Another study revealed that skin cancer patients who showed optimum response to
anti-PD-1 agents had abundant number of bacteria of family Ruminococcaceae
(Gopalakrishnan et al. 2018).
Metabolic compounds produced by gut microbiota have been considered as
response predictors of cancer immunotherapeutic agents (Fig. 9.3). Short-chain
fatty acids produced as bacterial metabolites, from carbohydrate fibers, induce
lymphocyte differentiation. They have also been reported to promote effector T
cell activity. Quantification of these fecal/microbial metabolic products can be linked
with magnitude of response to immune checkpoint inhibitors (Malczewski et al.
2020).

9.9 Conclusion

Profiling of multiple response biomarkers is in dire need of successful anti-

neoplastic immunotherapies. Methods for assay of the biomarkers need to be
improved with greater accuracy and better sensitivity. Continuous investment of
efforts is required to develop methodologies that minimize variations in assay to
avoid false-positive/-negative outcomes. Success of cancer immunotherapy in
improving quality of life lies in the development of accurate response biomarkers
for patient selection.

Acknowledgements This work was supported by the National Research Foundation of Korea
(NRF) funded by Korean Government (MSIT) under the grant number NRF-2019R1A2C1003003.

Conflict of Interest The authors declare no conflict of interest.

9 Biomarkers for Cancer Immunotherapy 241

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Role of Biomarkers in Personalized
Medicine 10
Salman Ul Islam, Muhammad Bilal Ahmed, Haseeb Ahsan,
and Young Sup Lee

Abstract

Biomarkers are a key tool in medicine, especially in the domain of personalized

medicine. They are valuable for the early detection, prognosis, and diagnosis of
disease as well as for the prediction of treatment response. They enable us to
select appropriate individuals for treatment with personalized medicine and
provide the right medication to the right patient. At present, the development of
individually targeted patient therapy remains the key objective of the medical
world. The achievement of this goal needs advances in biomarker discovery and
the development of therapeutic strategies that can be optimized for individual
drug and dose selection. This chapter discusses strategies for the use of
biomarkers and their impact on drug development. Further, it highlights the
establishment of enabling technologies involved in pursuing the goal of
personalized medicine. It is important that regulatory agencies, clinicians, and
scientists establish collaborations to address the challenges surrounding this field.

S. U. Islam
School of Life Sciences, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National
University, Daegu, Korea
Department of Pharmacy, Cecos University, Hayatabad, Peshawar, Pakistan
M. B. Ahmed · Y. S. Lee (*)
School of Life Sciences, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National
University, Daegu, Korea
e-mail: [email protected]
H. Ahsan
School of Life Sciences, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National
University, Daegu, Korea
Department of Pharmacy, Faculty of Life and Environmental Sciences, University of Peshawar,
Peshawar, Khyber Pakhtunkhwa, Pakistan

# The Author(s), under exclusive license to Springer Nature Singapore Pte 249
Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_10
250 S. U. Islam et al.

These challenges include enhancing approaches for the development of

biomarkers, minimizing the cost of drug development, and delving into the
contribution of next-generation sequencing tests in drug development.

Keywords

Biomarkers · Personalized medicine · Cancer · Screening · Diagnosis ·

Prognostication

10.1 Introduction

The Food and Drug Administration (FDA) has defined a biomarker as “a character-
istic that is objectively measured and evaluated as an indicator of normal biological
and pathogenic processes, or pharmacologic responses to a therapeutic intervention”
(Food 2014). A biomarker is simply an indicator of an alteration in normal physiol-
ogy. A biomarker can be any distinct alteration of DNA, RNA, or protein. Among
other applications, biomarkers are used as tools for the early detection of cancer and
the development of individualized treatment (Patel 2014; Ogunwobi et al. 2020;
Pellino et al. 2018).
Biomarkers can be divided into various groups based on their biology, measure-
ment, and purpose (Grecchi et al. 2012). Several categories of biomarkers defined by
the FDA (Group 2016) and the European Medicines Agency (Barcikowska 2018)
have been reviewed in an article by Karen D Davis and coworkers (Davis et al.
2020). With respect to biology, molecular, physiological, or morphological
characteristics can be used as biomarkers. Currently, scientists working with trans-
lational and personalized medicine prefer molecular markers. However, physiologi-
cal and morphological markers still play important roles in clinical assessment
(Banin Hirata et al. 2014). The generation of objective measurements is a crucial
characteristic of a biomarker, so that assay results are obtained with little or no
dependence on the subjective decisions of the observer. Biomarker tests can produce
quantitative, semiquantitative, or qualitative results. Biomarkers can be further
subgrouped into drug response or diagnostic markers. Several other types can be
defined based upon their specific applications, such as disease monitoring and
surveillance, prognosis, diagnosis, safety/toxicology assessment, pharmacodynamic
analyses, and stratification (Landeck et al. 2016).

10.2 Discovery and Validation of Biomarkers

The process of biomarker development is a systemized and directed task, starting

from recognition of the need for a biomarker followed by candidate biomarker
discovery, initial identification, and preliminary proof-of-concept investigations.
During this process, the degree of validation evidence supporting the use of the
biomarker exhibits the prime importance, which reaches to the highest level when-
ever intended purpose of the biomarker enters clinical practice (Food and Drug
10 Role of Biomarkers in Personalized Medicine 251

Fig. 10.1 Key steps for identifying and developing biomarkers for clinical use

Administration 2018) (Fig. 10.1). The biomarker development process may also
include investigations concerned with the verification of reliability and accuracy of
the detection method. Moreover, this process also encompasses the analysis of the
connection between the biomarkers and the clinical outcomes.
Various levels of validation are required after the biomarker identification.
Regarding analytical validation, it involves testing of the performance of the assay
or detection technology in a way that is feasible for the purpose of the biomarker.
Precision, dynamic range, and sensitivity of the detection method remain the note-
worthy variables assessed during the analytical validation process. The clinical
validation step is based on the assessment of the specificity and sensitivity of the
biomarkers for identifying, measuring, or predicting the clinical outcome. Of note,
specificity is linked with the rate of true negative findings, whereas sensitivity means
the rate of true positive findings. In the biomarker validation process, the degree of
evidence, required to provide the necessary confidence, is dependent upon context of
use. The required degree of validation evidence is going to be increased, requiring
further multisite validation data, as the context of use moves from research use to
accepted utility in clinical trials/practice.

10.3 Biomarker’s Role in Early Detection and Diagnosis

Identification of disease-based biomarkers is a crucial step of research supporting

diagnosis and predicting prognosis in almost all types of human disorders.
Biomarkers help establish guidelines for screening, response to treatment, and
monitoring of disease progression. In the following sections, we use examples of
certain critical biomarkers identified through various molecular biological
252 S. U. Islam et al.

techniques for diffuse large B-cell lymphoma (DLBCL), which provide insights into
disease mechanisms and pathogenesis.

10.3.1 B-Cell Lymphoma 2

B-cell lymphoma 2 (BCL-2), an oncogenic biomarker, is located on the mitochon-

drial outer membrane (Tilly et al. 2015). BCL-2 induces cell survival and inhibits
apoptosis. BAX and BAK, pro-apoptotic proteins belonging to BCL-2 family,
stimulate the release of cytochrome c from mitochondria, trigger the apoptotic
signaling cascade, and are blocked by BCL-2 itself (Siddiqui et al. 2015). Consider-
able research has shown that BCL-2 chromosomal translocation t(14;18) occurs in
DLBCL, resulting in elevation of BCL-2 levels as well as BCL-2-mediated resis-
tance to the apoptotic cascade (Tilly et al. 2015; Akyurek et al. 2012) (Fig. 10.2). The
presence of the BCL-2 chromosomal translocation t(14;18) has been observed in
20–30% of DLBCL cases, and is often associated with GCB-DLBCL-like variants
(Akkaya et al. 2016). When this translocation is present, the cells become
immortalized because of an elevated expression of BCL-2. High BCL-2 expression
in DLBCL results in poor prognosis and shortened life span (Adams et al. 2019;
Kawamoto et al. 2016). The inclusion of rituximab to standard chemotherapy helps
to overcome the impact of BCL-2 on adverse prognosis (Chiappella et al. 2017; Frei
et al. 2013). The prognosis remains consistently poor in “double hit lymphoma”
(DHL), in which a BCL-2 translocation goes along with a translocation of MYC [t
(8;14) for MYC, and t(14;18) for BCL-2] (Kawamoto et al. 2016). Patients
representing lymphoma cells coexpressing BCL-2 and MYC showed a good
response to ABT-737 (specific inhibitor of BCL-2), suggesting a key role for
BCL-2 in DHL (Mason et al. 2008; Li et al. 2019).

10.3.2 B-Cell Lymphoma 6

Chromosomal translocations and mutations result in B-cell lymphoma 6 (BCL-6)

being deregulated. Mice, which were engineered to constitutively express BCL-6,
developed DLBCL in germinal center (GC) B cells (Baron et al. 2004; Cattoretti
et al. 2005). Due to mutations in the BCL-6 locus, BCL-6 appears to be constitu-
tively active in individuals with active B-cell lymphomas (Ye 2000; Cerchietti et al.
2010). Aberrant blockage of the BCL-6 repressive function causes genetic instabil-
ity, which ultimately leads to neoplastic transformation (Aquino et al. 2014; Shustik
et al. 2010). BCL-6 has also been shown to autoregulate its own transcription, and
indirectly increases the expression of several genes, which then induce GC reactions
(Basso et al. 2012). B lymphocyte-induced maturation protein 1 (BLIMP1)
displaying a zinc finger domain (PRDM1)/B participates in the terminal differentia-
tion of GC B cells to plasma cells, and is one of the protein directly regulated by
BCL-6 (Alkodsi et al. 2019; Pasqualucci et al. 2006). PRDM1 appears to specifically
inactivate ABC-DLBCL. The deregulation of BCL-6 and inactivation of PRDM1/
 10
Role of Biomarkers in Personalized Medicine

Fig. 10.2 DLBCL can develop from diverse oncogenetic alterations in B cells. Somatic hypermutations, gene amplification, and translocation of genetic
material are the main oncogenic pathways involved in the development of DLBCL. The two important subtypes of DLBCL, the germinal center, and the
activated type, have been described
253
254 S. U. Islam et al.

BLIMP1 indicates the existence of alternative pathogenetic pathways causing inhi-

bition of postGC differentiation; subsequently promoting lymphomagenesis
(Pasqualucci et al. 2006; Vrzalikova et al. 2011; Wagner et al. 2011). The BCL-6
translocation and hypermutation at chromosome 3q27 with t(3;7) (q27;p12) was
shown in 30–35% cases of DLBCL (Shustik et al. 2010), and frequent somatic
mutations also occur in this chromosomal region. BCL-6 rearrangement is linked
with poor outcomes in patients receiving rituximab, cyclophosphamide, vincristine,
doxorubicin, and prednisone (R-CHOP) (Shustik et al. 2010). Barrans and
colleagues’ study found that individuals with poor prognosis had BCL-6
rearrangements as well as MYC translocation and BCL-2 deregulation. This study
demonstrates that the rearrangement of BCL-6 rarely appears as a sole genetic
disorder in DLBCL (Barrans et al. 2010).

10.3.3 Nuclear Factor Kappa-B

Nuclear factor kappa-B (NF-κB) is one of the family of inducible transcription

factors that is responsible for regulating multiple genes involved in a range of
immune and inflammatory responses. NF-κB can modulate biological processes,
such as stress responses, inflammation, B cell development, and lymphoid organo-
genesis (Hayden and Ghosh 2011). The activation of NF-κB is essential for growth
and survival of various types of cancer cells. Lymphoid malignancies evade apopto-
sis by the constitutive activation of NF-κB signaling (Park and Hong 2016; Hoesel
and Schmid 2013). Both the canonical and alternative NF-κB pathways get activated
in DLBCL (Compagno et al. 2009; Davis et al. 2010; Nagel et al. 2014; Zhang et al.
2015). Activated B-cell (ABC) DLBCLs show classical NF-κB activation, as they
have the potential for rapid phosphorylation and show frequent nuclear translocation
of p50/p65 heterodimers, while showing minor nuclear translocation of p50/c-rel
heterodimers (Davis et al. 2001). RelA/p65 and p50 are the major subunits of NF-κB
participating in the classical NF-κB pathway, and nuclear translocation of RelA/p65
is significantly linked with poor survival in individuals with early stage DLBCL
(Zhang et al. 2016). Multiple receptors, including CD40, BCR, and B-cell-activating
factor, stimulate the NF-kB pathway in B cells (Hoesel and Schmid 2013; Ying et al.
2013; Young et al. 2015). The NF-κB activation is believed to be a hallmark of
ABC–DLBCL (Camicia et al. 2015). Compared to GCB–DLBCL, a greater number
of NF-κB-regulated genes appear in ABC–DLBCL. Hence, ABC-DLBCL lines are
highly sensitive to the blockage of NF-κB. Mutations in CARD11 (a part of the
CBM), stimulate the activity of NF-κB in ABC–DLBCL (Jiang and Lin 2012;
Zachos et al. 2005).

10.3.4 MYC

MYC is a key regulator of cell proliferation and metabolism. Many oncogenic

pathways stimulate MYC leading to malignant transformation (Miller et al. 2012).
10 Role of Biomarkers in Personalized Medicine 255

The recombination of MYC with other genes has been observed in 3–16% of
DLBCL cases (Akyurek et al. 2012; Montero et al. 2018). The frequently occurring
t(8;14) (q24;q32) translocation involves MYC rearrangement in GCB–DLBCL,
resulting in its upregulation (Akyurek et al. 2012; Kawamoto et al. 2016; Akkaya
et al. 2016). In DLBCL, the fusion of MYC and Ig is known to result in the
upregulation of MYC expression. A meta-analysis revealed that rituximab treatment
did not overcome the consequences of MYC translocations (Zhou et al. 2014). In
DLBCL patients who received R-CHOP therapy, MYC served as a prognostic
factor, although these findings need further investigation (Akyurek et al. 2012).
The presence of an n-MYC rearrangement in DLBCL patients is often linked with
poor outcome (Chastain and Duncavage 2015; Logothetis 2014).

10.4 Role of Biomarkers in the Early Detection of Colorectal

Cancer (CRC)

In order to enhance survival outcomes in individuals with asymptomatic CRC, early

diagnosis is crucial. The sensitivity of CRC detection utilizing current FIT testing
(100 ng/mL) was 73.8% compared with 92.3% for a stool-based DNA test (Stiell
et al. 2003). The sensitivity of FIT testing for analyzing advanced precancerous
lesions remained at 23.8% compared with 42.4% with stool DNA assays (Stiell et al.
2003). These parameters indicate the shortcomings of current diagnostic testing and
indicate the difficulty of establishing reliable markers for the early detection of CRC.
Ongoing noninvasive screening of stools is not sufficiently efficient and sensitive for
detecting precancerous lesions with any confidence and may miss notable numbers
of early CRC cases. It is, therefore, necessary to maintain a low threshold at which
patients undergo the more invasive colonoscopy, and to use novel, advanced tools
for identifying early CRC.
Prognostic biomarkers, including early recurrence and mortality rates, can be
used to predict the progression of CRC (Patel et al. 2019; Pellino et al. 2018). A good
example of the use of prognostic biomarkers is the use of KRAS, a member of the
RAS proto-oncogene family of GTPases. Mutations in KRAS lead to an increased
risk of recurrent metastatic CRC (Tsuchida et al. 2016; Margonis et al. 2015; Tie
et al. 2011). Mutations in the BRAF are linked with decreased survival,
encompassing progression-free survival, and up to 50% worse overall survival
compared to wildtype BRAF (Guo et al. 2015; Venderbosch et al. 2014; Yokota
et al. 2011) (Fig. 10.3). In the novel field of radiogenomics, prognostic sensitivity
can be increased by the use of a combination of radiological and genetic features,
which attain a higher sensitivity than can be achieved by either of these modalities in
isolation (Badic et al. 2019; Horvat et al. 2019). A high-molecular-weight glycopro-
tein, carcinoembryonic antigen (CEA), has been successfully used as a biomarker in
the detection of early recurrence in postoperative patients, although it exhibited
despite low specificity and sensitivity (Chao and Gibbs 2009; Koulis et al. 2020).
Investigators are hopeful that prognostic markers will change the thresholds at which
256 S. U. Islam et al.

Fig. 10.3 Intracellular signals for CRC manifestation via EGFR. Activation of EGFR results in a
change from GDP- to-GTP form of the KRAS, leading to increased concentrations of BRAF to the
plasma membrane. BRAF activation leads to the stimulation of MAPK signaling pathway, which
subsequently regulates proteins involved in angiogenesis, proliferation, and metastasis

individuals are given more potent therapy, provide further insights into recurrent
disease, and improve the chances of early detection and intervention.
Predictive biomarkers can be used to tailor individual therapies according to
molecular subtype. Mutations in KRAS are linked with poor responses to therapy
with anti-EGFR receptor agents, including panitumumab and cetuximab (Karapetis
10 Role of Biomarkers in Personalized Medicine 257

et al. 2008; Amado et al. 2008). Compared to the 4% decrease in KRAS mutants, a
16% increase in overall response rate was observed in patients with KRAS wildtype
who received cetuximab and FOLFIRI. A topoisomerase inhibitor, irinotecan,
utilized as part of a FOLFIRI strategy, is metabolized by diphosphate-
glucuronosyltransferase 1A (UGT1A). Homozygosity for the UGT1A1*28 allele
is linked with dose-dependent toxicity compared to the UGT1A1*1 genotype
(Palomaki et al. 2009). Dihydropyrimidine dehydrogenase (DPD) metabolizes
more than 80% of 5-FU (Koulis et al. 2020). DPYD*13 and DYPD*2A variants,
however, contribute to increased toxicity of the treatment, and there is evidence that
a reduction in 5-FU dose by 25–50% reduces its toxicity (Amstutz et al. 2018).
These strategies may improve response to treatment, and decrease the toxicity
resulting from ineffective interventions. They can also assist in making the adjust-
ment of drug doses, to produce maximum benefit from a specific regimen. Although
several biomarkers are currently under investigation, there is a clear need for more,
and more effective, biomarkers. To date, only NRAS, KRAS, MSI, and BRAF status
are recommended by national guidelines, for use in following CRC therapy response
and predicting outcomes (Shinagawa et al. 2018).

10.5 Potential Biomarkers in Skin Cancer

Biomarkers have been extensively studied, and their use is well established, in skin
cancer. Prognostic biomarkers are the most important type of biomarker in skin
cancer. Tumor thickness is believed to be one of the most important and oldest
prognostic biomarkers in skin cancer. The expression nuclear cell proliferation factor
Ki-67 is another important example of a biomarker being used clinically (Gimotty
et al. 2005). In ulcerated melanomas, there is close correlation between survival and
CD2 count and number of tumor-infiltrating lymphocytes (de Moll et al. 2015). The
presence of tumor marker protein S100 beta in blood is utilized to assess disease
progression in skin cancer (Forschner et al. 2010). By the use of highly sensitive
assays, KIT D816V can be detected in peripheral blood leucocytes from most
patients with systemic mastocytosis, and is considered as a major step in early
diagnosis of the disease (Arock et al. 2015). Active nuclear I kappa-B kinase is
correlated with the risk of metastasis of cutaneous squamous cell carcinoma (Toll
et al. 2015).

10.6 Biomarkers for Asthma

Asthma is a highly heterogeneous disease with several underlying mechanisms;

different subsets or clinical phenotypes respond differently to standard therapy
(Seys et al. 2019; Kuruvilla et al. 2019). Biomarkers have been validated for Type
2 asthma (Diamant et al. 2019). Sputum eosinophils or blood eosinophil counts,
FeNO, and serum specific IgE have all been identified as important clinically
applicable biomarkers (Alving et al. 2020; Diamant et al. 2019). The biomarkers
258 S. U. Islam et al.

reflect different features of Type 2 inflammatory signaling, although some

overlapping of Type 2 biomarkers can occur within individuals (Diamant et al.
2019). These biomarkers, along with specific clinical characteristics, have led to
current guidelines using algorithms adapted to their use, which are hoped to be of
value in predicting responses to therapies, and can be utilized to monitor subsequent
therapeutic responses (Holguin et al. 2020; Agache et al. 2021). There are some
confounders of the existing biomarkers. Fractional exhaled nitrous oxide (FeNO) has
been shown to be correlated with dietary nitrate intake, smoking, virus infections,
and bronchoconstriction, whereas systemic corticosteroids and parasites have been
reported to be the most common culprits for circadian variation in blood eosinophils
(Diamant et al. 2010). Oxidative stress is caused by an excess of reactive oxygen and
nitrogen species. Investigators have reported multiple direct or indirect markers of
oxidative stress, including glutathione disulfide, malondialdehyde, bromotyrosine,
thiobarbituric acid, and isoprostane in plasma, urine, BAL fluids, and sputum of
individuals with asthma. The levels of these markers were linked with the severity
and clinical output of the disease (Comhair et al. 2000; Comhair and Erzurum 2002).
The collection of exhaled breath condensate is another noninvasive analytical
approach, which allows direct measurements of H2O2, pH changes, and numerous
indirect by-products of oxidation, such as ethane and 8-isoprostane (Aldakheel et al.
2016; Thomas et al. 2013). The detection of high levels of urinary bromotyrosine
represents another important noninvasive biomarker of oxidative stress for clinical
use in patients with asthma (McDowell and Heaney 2020; Sze et al. 2020).

10.7 Significance of Biomarker Strategies in Drug Development

The significance of personalized strategies has been tested in phase I, II, and III
clinical trials. A meta-analysis of phase I trials published over a 3-year period
included 13,203 patients. It was found that, compared to approaches that did not
utilize a biomarker, the biomarker-based cancer therapeutic strategies produced a
longer median progression-free survival (PFS) time, and an improved response rate
(Schwaederle et al. 2016). Phase II clinical trials were also reviewed in a meta-
analysis of single-agent studies published over a 3-year period. Here also, the
biomarker-based approach gave a higher median response rate, longer PFS, and
better overall survival. Nonpersonalized targeted approaches had poorer outcomes
than personalized, targeted strategies. The personalized strategies proved to be safer,
and resulted in a lower treatment-induced death rate (Schwaederle et al. 2015)
(Fig. 10.4). These investigations suggest that personalized therapy produces better
outcomes, and may improve the effectiveness of cancer therapies during all phases
of drug development (Schwaederle et al. 2015, 2016). The clinical utility of
personalized medicine has, therefore, been established, at least for some biomarkers,
but the cost/benefit ratio of targeted therapy is still a subject of debate (Aitken et al.
2018). Higher treatment costs may be ascribable to a longer treatment time due to
enhanced survival instead of higher monthly drug costs (Chawla et al. 2018). The
financial return from newly launched personalized drugs comes at a higher initial
10 Role of Biomarkers in Personalized Medicine 259

Fig. 10.4 General idea of personalized medicine. Biological variability can result in different
outcomes (beneficial or harmful) for a population of patients (upper panel). Prediction of biomarker-
based treatment response helps select appropriate patients for treatment and avoids the high risk of
adverse drug incidents (lower panel)

investment, and use is prolonged due to better efficacies. It is expected that advanced
technologies like artificial intelligence will influence cancer treatment and costs in
the future (Mak and Pichika 2019).

10.8 Personalized Medicine in Conventional Therapeutic

Approaches

New advances in the understanding of the underlying mechanisms of cancer have

opened a new horizon of personalized medicine. One example is that of Bacillus
Calmette–Guérin (BCG) vaccine therapy in non-muscle-invasive bladder cancer
(NMIBC). During the year 1976, Morales and colleagues introduced the idea of
utilizing BCG as a therapeutic and preventive approach in NMIBC (Moss and
Kadmon 1991). BCG antigens provoke an immune response which attacks tumor
cells, resulting in an anti-neoplastic effect when instilled during therapy (Saad et al.
2017). However, BCG treatment failed in many bladder cancer patients, and nearly
40% of them experienced recurrence (Alhunaidi and Zlotta 2019; Zlotta et al. 2009;
Ślusarczyk et al. 2019; Lima et al. 2013). The ability to identify patients unlikely to
respond to treatment would save time and hence, avoid progression of disease. Many
investigations have been carried out to identify biomarkers which could be used to
predict patient response to BCG. Good biomarkers would help physicians to effec-
tively select candidates, and put poor responders on an alternative therapeutic
strategy (Kamat et al. 2016). Patients with mutations in the AT-rich interaction
domain 1A (ARID1A) were highly prone to recurrence of NMIBC after BCG
therapy. This study suggested that the screening of BCG candidates could give
260 S. U. Islam et al.

useful insights into patient prognoses (Pietzak et al. 2017). However, further
investigations are needed to demonstrate the functionality of ARID1A as a reliable
biomarker for BCG therapy. Researchers have also struggled to establish the predic-
tive value of the tumor suppressor protein p53, for response of BCG in bladder
cancer (PAGES et al. 1998; Berggren et al. 2001). p53 mutation was not found for
predicting clinical response, but was utilized to predict cancer prognosis (Du et al.
2016; Malats et al. 2005). Cell adhesion molecules like sialyl-Tn (STn) and sialyl-6-
T (s6T), which play roles in cell–cell adhesion and immune responses, were also
included in trials for BCG response (Pinho et al. 2007). STn, alone or in combination
with s6T, appeared to be linked with lower recurrence rates after BCG instillation,
although the underlying mechanism remains poorly understood (Lima et al. 2013;
Severino et al. 2017). Researchers also studied ezrin, a cell adhesion molecule,
during BCG response, and found that the loss of ezrin was correlated with reduced
survival (Palou et al. 2009; Andersson et al. 2014). During an investigation into
BCG nonresponders versus responders, Kates et al. found that programmed death
ligand-1 (PD-L1) appeared in nearly 25% and 4% of BCG nonresponders and
responders, respectively. This study suggested that PD-L1 can be involved in the
NIMBC-induced resistance to BCG therapy (Kates et al. 2020). As recent
investigations lack standardization regarding response measurement criteria, study
validation techniques, and cutoff points, further intensive and qualitative
investigations are required to find a single biomarker which could be used to predict
patients response to BCG therapy (Kamat et al. 2018).

10.9 Personalized Medicine in Novel Therapeutic Strategies

The existence of fibroblast growth factor receptor (FGFR3) mutations, fusions, and
amplifications have been found in numerous tumors, including bladder cancer
(Nogova et al. 2017). It has been shown that FGFR3 appears in bladder cancer
preferentially in low-grade NMIBC, which indicates that FGFR3 may serve as a
crucial marker for disease severity and management (Akanksha and Sandhya 2019).
Researchers have utilized different techniques, such as the development of mono-
clonal antibodies and selective tyrosine kinase inhibitors, to interfere with FGFR3
signaling (Paul and Mukhopadhyay 2004; Qing et al. 2009). B701, a fully
humanized immunoglobulin, resulted in significantly improved survival when
included to novel PD-1 inhibitors or traditional chemotherapeutic agents (Holash
et al. 2016). MFGR1877S, an antibody targeting the FGFR3 receptor, and
LY3076226, a FGFR3 antibody conjugated to a cytotoxic drug (DM4), have
shown promising results, and are currently in Phase I clinical trials (Qing et al.
2009; Surguladze et al. 2019). Investigators have also attempted to influence FGFR3
signaling at a more distal point utilizing tyrosine kinase inhibitors (TKIs).
Pazopanib, a potent TKI, has shown partial responses in 7 out of 21 patients in a
Phase II clinical testing (Necchi et al. 2012). During Phase II clinical trials,
pazopanib monotherapy in individuals with advanced urothelial cancer (UC),
showed partial response in seven patients and stable disease in 14 out of 41 patients
10 Role of Biomarkers in Personalized Medicine 261

(Necchi et al. 2012). Although pazopanib showed encouraging results, two other
TKIs, brivanib, and dovitinib, failed to show a strong response (Milowsky et al.
2014; Hahn et al. 2017; Ratain et al. 2011). It has been noted that drug molecules
showing a more specific effect on the tyrosine kinase domain of FGFR produce more
optimistic results. For example, erdafitinib, a small molecule inhibitor of FGFR
approved for treating advanced or metastatic UC and marketed under the name
Balversa, which harbors FGFR2/3 alterations, gave a response rate of up to 40%,
although 37% of the responses were partial. However, the response rate was almost
60% among patients who previously received immunotherapy (Loriot et al. 2019). A
few other TKIs, like infigratinib, AZD4547, and pemigatinib, are currently under
trial (Marandino et al. 2019; Jones et al. 2016; Merz et al. 2021).
Boosting host immunity by blocking inhibitory receptors is another strategy
extensively used in bladder cancer (Khalil et al. 2016). The primary signal for the
stimulation of T cell is the recognition of antigens by the T-cell receptors (TCRs)
presented by APCs via MHC. A second signal for T cell activation involves the
binding of T cell CD28 with CD80/86 on APCs. The two most common immuno-
modulatory molecules, CTLA-4 and PD-L1, inhibit this interaction. CTLA-4 plays
its inhibitory role in blocking the secondary signal by competing for CD80 and
CD86 binding (Collins et al. 2002; Parry et al. 2005). PD-L1 blocks downstream
TCR signaling and results in the inhibition of T cell responses (Sage et al. 2018).
Strategies which block the inhibitory effects of these molecules would allow the
immune system to attack the tumor more aggressively.

10.10 Bioengineering and Personalized Medicine

The concept of medicine is diverging from the “one size fits all” mentality. It usually
occurs that patients having same disease respond differently to drugs. Therefore,
now is the time to deeply understand this response and provide patients with
individual treatment. Biomaterial engineers, specifically, can play a crucial role in
making personalized medication a reality. Biomaterials can present different effects
on cell growth and survival, and it is highly recommended that they should be
screened via high-throughput approaches for a given application. For example, a
dextran–dendrimer composite was shown to work as an adhesive differently in colon
cancer than in colitis, which involves the same organ with a different environment
(Artzi et al. 2009; Oliva et al. 2015). These studies suggest that the use of
biomaterials cannot be generalized, and they must be designed according to the
organ environment. The appropriateness of biomaterials for certain organs, tissues,
or cells can be determined using a combination of small and large animal models
(Vegas et al. 2016a, b; Lind et al. 2017). To avoid the use of living models, different
extracellular matrix formulations can also be utilized to observe the effects of
biomaterials on cell differentiation, proliferation, and apoptosis (Beachley et al.
2015). Optimal biomaterial formulations produced via novel engineering platforms
can enhance personalized biocompatibility and therapeutic outcomes.
262 S. U. Islam et al.

There is an emerging idea of “organ-on-a-chip platforms” for individualized

drug-screening investigations. Scientists have developed a microfluidics-based
model of human intestine, in which they recreated the complex gut microenviron-
ment. This model paved the way for monitoring the interactions between the
immune cells, gut microbiome, and bacteria. It also opened the way for the observa-
tion of the pharmacokinetics, absorption, and metabolism of drugs (Bein et al. 2018;
Prantil-Baun et al. 2018). Drug pharmacokinetics, absorption, and metabolism
potentials may also be used for designing personalized therapeutic strategies. Accu-
rate and timely detection of treatment response is needed for accurate personalized
treatment; the latter includes parameters, such as appropriate drug selection and
dosing regimens. The commonly used techniques for acquiring these parameters,
include urine and serum analyses, or imaging modalities, such as X-rays, MRI, CT,
and ultrasound, which could be narrow in terms of testing frequency. It has been
demonstrated that wearables and other novel technologies can help in overcoming
the problem of infrequent measurements, which would thereby improve the design
of personalized therapeutic strategies (Blicharz et al. 2018). Over time, new
breakthroughs in personalized medicine have been introduced, encompassing the
application of nondrug-based strategies like digital therapies, to cope with conditions
such as cognitive impairment, mental health, and substance abuse (Kee et al. 2019;
Davis et al. 2018; Cho and Lee 2019) (Fig. 10.5).
A common feature of these strategies is their ability to utilize only a subject’s own
data to direct only their own care. This approach has been exemplified regarding
artificial intelligence-driven drug dosing and engineered cell therapy. Another
advantage in the connection of personalized medicine and engineering is the parallel
adjustment of intervention and diagnosis for ongoing therapy optimization.

10.11 Personalized Cell Therapy and Drug Delivery

A major advancement in personalized cancer treatment is the approval of chimeric

antigen receptor T-cell (CAR-T) immunotherapy. Tisagenlecleucel (Kymriah,
Novartis) was the first approved CAR-T therapy, which is being used for treating
patients with acute lymphoblastic leukemia. During this treatment, T cells are
removed from patient, reprogrammed, and expanded in a processing facility and
are finally introduced to the patient (Prasad 2018). Axicabtagene ciloleucel
(Yescarta, KITE Pharma/Gilead Sciences) has recently been approved for the treat-
ment of aggressive non-Hodgkin’s lymphomas (Roberts et al. 2018; Mullard 2017).
Scientists worldwide are working to broaden the indications that are managed
utilizing CAR-T. The approval of CAR-T remains an ideal shift for the FDA towards
efficacious and safe living cell therapies. It has been demonstrated recently that
nonviral approaches, like sleeping beauty transposition, can improve the scalability
of CAR-T for broader deployment (Monjezi et al. 2017). This technique is based on
the use of simple DNA minicircles for inserting CAR genes, which effectively
reduces the risk of genotoxicity and mutagenesis associated with viral modalities
(Fig. 10.6). It also reduces the cost of CAR-T engineering and minimizes the
 10
Role of Biomarkers in Personalized Medicine

Fig. 10.5 From genome-guided medicine to CRISPR, multiple advanced and highly accurate technological platforms bridge engineering and personalized
medicine together, improving clinical outcomes. Moreover, striking improvement in personalized medicine approaches may be realized by linking wearable
technologies, artificial intelligence, and other engineering platforms
263
264 S. U. Islam et al.

Fig. 10.6 How does engineered T cell therapy work? Enough blood is obtained from patients to
collect T cells from it. T cells are purified and modified by viral vector transfection to express
specific CARs/TCRs on their surface. Following amplification and quality control, engineered
CAR-containing T cells are infused into the patient body to improve antitumor ability

regulatory hurdles faced. “Off-the-shelf” cell therapy, which does not need autolo-
gous T cells, is used by advanced engineering approaches for improving CAR-T
manufacturing (Sadelain et al. 2017; Cooper et al. 2018; Ruella and Kenderian
2017). Zinc-finger nuclease technology, a novel tool composed of engineered
DNA-binding proteins, facilitating targeted editing of the genome by creating
double-strand breaks in DNA at user-specified locations, is being utilized for
modifying both allogeneic and autologous cell therapies. This technology is
expected to broaden the chances of off-the-shelf CAR-T manufacturing, and signifi-
cantly decrease the treatment duration (Dolgin 2018).
It is now possible to reprogram induced pluripotent stem cells, obtained from a
patient, to a desired cell like a brain cell, a specialized kidney cell, or a beta cell,
which can be introduced into the body and will carry out their specific functions
(Vegas et al. 2016b; Peruzzotti-Jametti et al. 2018; Ma et al. 2018). Mitochondrial
replacement therapy (MRT) is an additional example of cell therapy implemented in
the United Kingdom. Mutations in mitochondrial DNA (mtDNA), referred to as
mitochondrial disease, can also be maternally transferred to the offspring, and leads
to severe disorders, such as deafness, epilepsy, optic neuropathy, and diabetes
10 Role of Biomarkers in Personalized Medicine 265

mellitus. In MRT, the healthy nucleus from a maternal egg with malfunctioning
mitochondria is transferred to a healthy egg, including the donor mitochondria,
without a nucleus, which can result in a fertilized egg containing nuclear DNA
from two parents and mtDNA from a donor, thus eliminating the genetic disorder in
children (Saxena et al. 2018).
Another particularly relevant area is the cellular engineering. Scientists have
designed synthetic cells for the early detection of malignancy and diabetes (Danino
et al. 2015; Courbet et al. 2015). With the application of synthetic cells and
biosensors, scientists become able to detect early disease markers and deliver
therapeutic entities to improve symptoms. This is a major goal of personalized
medicine, wherein the cell is an autonomous therapy and sensor, delivering thera-
peutic entities without physician or patient intervention. During an investigation,
chronic and acute psoriasis was observed by a population of synthetic cells
implanted in mice, an approach which provided a new opportunity for personalized
medicine (Schukur et al. 2015).
Delivering therapy in a personalized fashion is also a promising approach.
Personalized biomaterial-mediated controlled release or 3D printing technologies
are being introduced to serve as cornerstones for improving drug delivery. A study
reported the tailored release profiles from 3D-printed tablets, enabling the customi-
zation of the temporal administration of the drug (Sun and Soh 2015). A technique
termed stamped assembly of polymer layers (SEAL) has been successfully utilized
to produce drug-loaded 3D microstructures with temporal drug release control
(McHugh et al. 2017). Specific responses of individuals to combination therapy
are often monitored by unique dosing profiles. Hence, these microstructures and
tablets can be a feasible drug delivery platform for personalized medication.

10.12 Concluding Remarks and Future Directions

Many drugs show efficacy only in a subgroup of patients (Laserna-Mendieta et al.

2020; Wang et al. 2021). Therefore, the “hit-or-miss” utilization of these drugs is
costly, ineffective, and puts patients at risk. Drug development is expensive and the
number of FDA-approved drugs per billion US dollars of spending decreases by half
every 9 years. The cost of launching a new drug exceeds one billion euro, and this
exorbitant price raises concerns (Scannell et al. 2012). The era of personalized and
precision medicine is expected to solve this problem. In this era, patients will no
longer be restricted to dose escalation-defined administration protocols and target-
based drug selection.
High failure rates are a major cause of the high research as well as development
costs involved in discovery of drugs. Less than 1% of drug development projects
launched result in the approval of a new drug. It has frequently been observed that,
after several years of significant investments, drugs fail late in the clinical trials. It is
alarming that the traditionally low success rates for new drug development projects
in Phase II clinical trials decreased even further from 28% to 18%. In the past few
years, insufficient efficacy has remained the most frequent reason for failure
266 S. U. Islam et al.

(Arrowsmith 2011). Personalized medicine can help to address this challenge.

Smaller sized clinical investigations conducted using biomarker-based stratification
can show better results. It is recommended, even in clinical trials lacking a stratifi-
cation strategy, to include biomarker candidates. It would also be supportive to
acquire patients’ informed consent, to enable retrospective assays conducted later. If
retrospective assays produce favorable outcomes in biomarker candidates, these
findings need to be verified in another prospective clinical study. Although such a
protocol may increase the cost of the initial study, it could rescue a project. To
support findings related to dosage, pharmacodynamic biomarkers are also
recommended to be added more rigorously in clinical studies. This approach will
have the benefit of increasing tolerance and establishing recommended dosage.
Moreover, this approach could provide important experience in case of study failure.
In this case, the pharmacodynamic biomarker represents a full-target engagement,
showing that the target is not relevant to the disorder. When a biomarker engages the
target insufficiently, it can indicate that the compound, rather than the efficacy of the
molecular target, is the cause of the problem.
Scientists are of the opinion that future medical products can be introduced as a
“double pack”: the drug molecule and the diagnostic assay to identify the feasibility
of the patient for this approach. This approach requires the creation of consortia, for
closer collaboration between the pharmaceutical and the diagnostic industry (Salter
and Holland 2014). Various models are in use for various categories of biomarkers,
and many more are needed for different stages of the drug discovery and develop-
ment process (Asadullah et al. 2015; Lessl et al. 2011; Dorsch et al. 2015). Studies
have shown major inconsistencies between the number of biomarkers identified and
the number reported (150,000) and the few (100) entered in clinic trials (Poste
2011). The reproducibility of publications is also an important issue (Prinz et al.
2011). High transparency and more coordinated efforts are needed for biomarker
discovery, development, and validation, involving collaborations between academia
and the diagnostics and pharmaceutical industry, since this process requires signifi-
cant resources and complementary skills (Landis et al. 2012; Asadullah et al. 2015).
The Biomarkers Consortium (URL), the predictive safety testing consortium
(URL), and the Coalition Against Major Diseases (URL) are noteworthy examples
of successful consortia in the biomarker area, and it is expected that the number of
collaborations would increase in future (Wholley 2014; Stephenson and Sauer
2014).
Scientists are expecting changes in the number of biomarkers tested. Currently,
only one biomarker is used to guide a treatment protocol, whereas future molecular
diagnostics may result in the simultaneous comprehensive profiling of several
markers. This approach reflects a movement from the use of a single marker to a
signature, which would allow us to choose the most suitable and potent therapeutic
combination for each patient. Although in the discovery of biomarkers, panels of
markers are frequently already measured, much remains to be done in validating
candidates’ biomarkers. Further improvements in precision and personalized medi-
cine in the present population are required for a successful transfer of validated
biomarkers and personalized medicine platforms into the clinical setting. Several
10 Role of Biomarkers in Personalized Medicine 267

technology-linked validation challenges associated with ethics, healthcare econom-

ics, and data privacy need to be addressed (Reddy et al. 2020; Cohen 2019; Dinh-Le
et al. 2019; Lee et al. 2019). Biomedical engineering, which is currently playing a
key role in breakthroughs, is expected to eventually improve the human condition in
an individualized fashion.

Author Contributions Y.S.L.: conceptualization, supervision; S.U.I., and H.A.: writing—origi-

nal draft; M.B.A.: designed all the figures. All authors have read and agreed to the published version
of the chapter.

Funding This research was funded by a National Research Foundation of Korea (NRF) grant from
the Korean government (MSIT) (NRF-2019R1A2C1003003).

Conflicts of Interest The authors declare no conflict of interest.

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Development of Novel Cancer Biomarkers
for Diagnosis and Prognosis 11
Kholood Abid Janjua, Raheem Shahzad, and Adeeb Shehzad

Abstract

In the past few decades, extensive research has been carried out to find novel
biomarkers for various cancers. Cancer biomarkers play an important role in the
screening, diagnosis, and posttreatment prognosis of patients in all the stages of
the disease. The utilization of biomarkers is multifaceted; from screening healthy
patients for risk assessment to timely diagnosis, accurate staging, patient stratifi-
cation into risk groups, determining prognosis, and continued surveillance.
Biomarkers play an indispensable role in both diagnosing and managing cancer
treatment. Recent advancements in the field of precision medicine have led to the
discovery of novel diagnostic and prognostic cancer biomarkers. Different
protein-based, RNA, DNA, miRNA, and SNP-based biomarkers have been
identified and developed as accurate, noninvasive, and cost-effective alternative
towards managing different neoplastic diseases. The rapid progression in the field
of diagnostic and prognostic biomarkers has led to the development of compan-
ion diagnostics and targeted therapies for the treatment of cancer patients, which
has resulted in improved diagnosis and preventing unnecessary chemotherapy
along with the associated toxicities.

Keywords

Biomarkers · Noninvasive · Prognosis · Risk · Diagnosis · Cancer

K. A. Janjua · A. Shehzad (*)

Department of Biomedical Engineering and Sciences, School of Mechanical and Manufacturing
Engineering (SMME), National University of Sciences and Technology, Islamabad, Pakistan
e-mail: [email protected]
R. Shahzad
Department of Horticulture, University of Haripur, Haripur, Pakistan

# The Author(s), under exclusive license to Springer Nature Singapore Pte 277
Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_11
278 K. A. Janjua et al.

11.1 Introduction

In the past few decades, extensive research has been carried out to find novel
biomarkers for various cancers. Cancer biomarkers play an important role in the
screening, diagnosis, and posttreatment prognosis of patients in all stages of the
disease (Sanhueza and Kohli 2018). The utilization of biomarkers is multifaceted;
from screening healthy patients for risk assessment to timely diagnosis, accurate
staging, patient stratification into risk groups, determining prognosis and continued
surveillance (Fig. 11.1).
Researchers are keen to develop cancer biomarkers that can be quantified easily.
There has been a global interest in developing specific and highly sensitive markers
which may provide reliable and reproducible results. This can be achieved by the
development of novel noninvasive and cost-effective approaches. Different protein-
based, RNA-, DNA-, miRNA-, and SNPs-based biomarkers are developed and
identified as a diagnostic and prognostic marker by utilizing biological samples of
prostate cancer patients such as urine and prostate tissue, non-neoplastic tissue,
blood, etc. (Fig. 11.2). Some of these biomarkers are approved by the FDA, while
others are still under investigation (Sanhueza and Kohli 2018). In this chapter, novel
biomarkers for various cancer types such as breast cancer, ovarian cancer, prostate
cancer, lung cancer, leukemia, lymphoma, etc., are discussed in detail.

Fig. 11.1 Uses of the cancer biomarkers

11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 279

Fig. 11.2 Development of cancer biomarkers

11.2 Novel Biomarkers for Diagnosis and Prognosis of Prostate

Cancer

Prostate cancer is one of the most common types of cancer in males. It often
progresses slowly and stays limited to the prostate gland. Some kinds of prostate
cancer are slow-growing and treatable, while others are advanced and can be fatal.
An early diagnosis of prostate cancer in a patient increases the chances of successful
treatment (Litwin and Tan 2017).

11.2.1 Serum Prostate-Specific Antigen (PSA)

Serum prostate-specific antigen (PSA), the serine protease member of the kallikrein
family, is currently widely used as a prostate cancer biomarker (Tchetgen and
Oesterling 1997). It has been broadly used as a marker in almost all prostate cancer
stages, from initial screening to prognosis after receiving the therapy. It has also been
used in clinical practice to monitor cancer recurrence and evaluate drug efficacy
(Sanhueza and Kohli 2018; Tchetgen and Oesterling 1997). Unfortunately, the use
of PSA is limited due to its poor specificity. It is not able to differentiate the indolent
tumor from aggressive tumors. Therefore, it is now considered a suboptimal tool for
screening, diagnostic, and prognostic purposes (Tchetgen and Oesterling 1997).
Research has been carried out to improve the screening, diagnostic, and prognostic
capacity of the PSA by modifying the PSA testing. It has been reported that a group
of multiple kallikrein biomarkers such as total PSA, intact-PSA, free PSA, and Hk2
improves the diagnostic capacity of PSA as a prostate cancer biomarkers (Lazzeri
et al. 2013; Darson et al. 1999; Benchikh et al. 2010). Research has now been
focused on developing novel biomarkers that belong to different classes such as
DNA-based, RNA-based, cancer cell type, SNPs, proteins, etc. Some of the recently
developed biomarkers for prostate cancer are discussed here.
280 K. A. Janjua et al.

11.2.2 Field DNA Methylation

An important biomarker for prostate cancer is field DNA methylation, that is

observed around the prostate cancer foci. It can be considered a cell senescence
marker (Desotelle et al. 2013). Recently, a DNA field methylation-based novel assay
was developed, which was named ProCUrE. It demonstrated enhanced prostate
cancer diagnosis and patient identification in patients with clinically significant
disease. This assay was based on two novel biomarkers, i.e., HOXD3 and GSTP1
(Zhao et al. 2018).

11.2.3 mtDNA Deletion

mtDNA deletions is another field-effect-based biomarker observed around the pros-

tate cancer foci and can be an important diagnostic marker for prostate cancer.
mtDNA deletions are made from the damage of the genomic segment that affects
multiple genes and therefore becomes the cause of cancer in humans (Robinson et al.
2010). A 3.4 kb mtDNA deletion extracted from blood plasma has been reported to
predict prostate cancer accurately. It has been reported to correlate well with the
image-guided biopsy outcome in men’s first biopsy setting in the PSA’ grey zone. It
is considered a novel biomarker that provides novel information to evaluate risk
(Creed et al. 2017).

11.2.4 Alpha-Methylacyl Coenzyme a Racemase (AMACR)

Alpha-methylacyl coenzyme A racemase (AMACR) DNA is another DNA-based

biomarker that codes for mitochondrial and peroxisome enzymes. It is a prostate
tissue-based biomarker and was reported for its diagnostic capacity. However, it has
not been approved by FDA yet (Sanhueza and Kohli 2018). The Germline BRCA2
mutations are another set of novel prostate cancer biomarkers found in both organ-
confined and advanced prostate cancer patients (Eeles and Raghallaigh 2018). CpGs
are novel DNA-based biomarkers for prostate cancer reported to discriminate the
patients based on the metastatic lethal stage. CpGs were reported to be present on
five genes, i.e., FHAD1, KLHL8, ALKBH5, PI15, ATP11A (Zhao et al. 2016).

11.2.5 Single Nucleotide Polymorphisms (SNPs)

Single nucleotide polymorphisms (SNPs)-based biomarkers are also investigated as

an addition to the group of DNA-based biomarkers. They consist of a set of
biomarkers that are not very effective for diagnosis and prognostic purposes but
can effectively inform about the risk of developing prostate cancer (Sanhueza and
Kohli 2018). Recently, ancestry-informative markers (AIMs) based on SNPs were
investigated that provided information about the risk factors contributing to prostate
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 281

cancer. These SNPs can detect allelic differences between different populations and
are reported to classify genetic drivers of developing prostate cancer accurately.
However, they have the limitation not to provide how much genetic heterogeneity is
present between the populations (Mitchell and Williams 2019). If AIMs are
incorporated in the risk prediction models, they can effectively improve prostate
cancer detection and risk stratification in different populations (Mitchell and
Williams 2019).

11.2.6 ERG

The novel RNA-based prostate cancer biomarkers are also an important area of
cancer biomarker discovery. ERG is an important RNA-biomarker, with the most
frequently found molecular aberrations in prostate cancer. However, it has the
limitation of low sensitivity (Sanhueza and Kohli 2018).

11.2.7 PCA3

Another important RNA-based biomarker that is PCA3 has been reported to predict
pathologically insignificant prostate cancer. This noncoding RNA was reported for
its diagnostic capacity due to its high specificity (Sanhueza and Kohli 2018). It is not
expressed in the normal prostate tissue; however, it is highly expressed in prostate
cancer (Sanhueza and Kohli 2018). The urinary PC3-A levels are reported to be
elevated in case of elevated PSA levels in patients who were previously found
negative for prostate cancer. Its sensitivity is 69% compared to the 27% sensitivity
observed for PSA. There is no cutoff value reported for the urinary PCA-3 levels
(Sanhueza and Kohli 2018). However, it cannot predict aggressive prostate cancer
(Sanhueza and Kohli 2018). Besides having a diagnostic capacity, urinary levels of
post-DRE PCA3 were found to have a high probability of finding GS levels 7 and
higher prostate tumor stage in surgical pathology (Sanhueza and Kohli 2018). These
two biomarkers were recently combined for the detection of prostate cancer. They
were found to preserve 95% sensitivity for detecting aggressive prostate cancer.
Furthermore, it also improved the specificity from 18% to 39% (Sanda et al. 2017).

11.2.8 SAP30L-AS1 and SChLAP1

SAP30L-AS1 and SChLAP1 are another set of diagnostic markers that were recently
reported to distinguish prostate cancer. These two lncRNAs, when combined sepa-
rately with PSA, developed a moderate discriminating ability (Wang et al. 2018a, b).
282 K. A. Janjua et al.

11.2.9 Multiple Truncated AR Variants (AR-Vs)

Multiple truncated AR variants (AR-Vs) are RNA-based prognostic biomarkers that

were reported for their predictive capacity. They were expressed in castrate-resistant
prostate cancers (CRPC) (Cao et al. 2016). It has been reported that AR-V7 can be
used as a biomarker to look for the response rate in patients receiving therapy. They
are detected in tumor cells of patients in which the Androgen-depriving therapies
such as abiraterone and androgen-blocking agents seem to be futile. These patients
had a 0% response rate and shorter survival times (Sun and Abdollah 2015). Other
novel AR-Vs (T878A or L702H) were observed to be related to worse progression-
free and overall survival (O’Reilly 2019). In chemotherapy-naïve patients, AR-Vs
amplifications are found to be associated with lower response rates to treatment with
abiraterone and enzalutamide.

11.2.10 miRNAs

miRNAs-based biomarkers have been developed to monitor tumor growth, disease

progression, and metastasis. They can monitor cancer cell activities and predict
prostate cancer progression (Munteanu et al. 2020). They are found to be circulating
in urine, serum, and plasma.
Several downregulated miRNAs were observed in the urine samples of prostate
cancer patients. A novel diagnostic model consisting of three-miRNA that is
(miR-222-3p*miR-24-3p/miR-30c-5p) was reported as a diagnostic marker that
distinguishes BPH from prostate cancer patients (Fredsøe et al. 2018). Recently, a
study identified a set of seven mRNAs (miR-451a, miR-148a, miR-144-3p,
miR-3195, miR-512-5p) with prostate cancer predictive ability (Chaudhry et al.
2020). The list of miRNAs-based biomarkers for prostate cancer is long. A meta-
analysis that included 37 miRNAs reported overexpression of 15 while under the
suppression of 22 miRNAs in prostate cancer (Pashaei et al. 2017). Among them,
only the following microRNAs were correlated with prostate cancer: miR-1,
miR-133b, miR-449a, miR-137, miR-370, miR-221, miR-449b, miR-125a-5p,
miR-199a-3p, miR-301b, miR-340, miR-361, miR-363, miR-98. Among these,
MiR-1, miR-133B, miR-449B, and miR-221 exhibited significant utility in
predicting prostate cancer after having radical prostatectomy (Munteanu et al.
2020). Furthermore, another three-miRNA-based model (miR-125b-5p*let-7a-5p/
miR-151-5p) with prognostic ability was also recently developed to predict the time
for biochemical recurrence after receiving the radical prostatectomy (Fredsøe et al.
2018).

11.2.11 Circulating Tumor Cells (CTCs)

Circulating tumor cells (CTCs) shed from primary tumors and metastatic sites and
possess a half-life of around 1–2 h. Apart from the biomarkers mentioned above,
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 283

circulating tumor cells (CTCs) are FDA-cleared markers, showing increased CTC
counts in castration-resistant prostate cancer. They are associated with worse prog-
nosis in several phase III trials in CRPC patients (Kohli et al. 2017). They are
reported to have low sensitivity/yield in blood, notably in early cancer stages, and
limited capacity to monitor early-stage prostate cancer (Riaz et al. 2018; Ghosh et al.
2019).

11.2.12 Exosomes

Exosomes are extracellular vesicles bearing a diameter of 30–150 nm. They are
naturally produced from almost all mammalian cells by fusing multiple vesicular
bodies with the plasma membrane. Exosomes have recently gained growing atten-
tion due to their release from the outward budding plasma membrane (Kim et al.
2018). They also recently emerged as a potential source of noninvasive biomarkers
for prostate cancer. These are the nanovesicles that carry molecules from the cancer
cells and can therefore be detected in biofluids (Skotland et al. 2017). Flotillin 2 and
PARK7 is a set of exosomes that gave signals at specific thresholds in combination,
and they gave 68% sensitivity and 93% specificity (Wang et al. 2017c).
Exosomal biomarkers have been reported to have the ability to detect prostate
cancer and distinguish between indolent and malignant tumors with higher PPV.
Recurrence and treatment responses were also observed to be predicted through
these biomarkers (Wang et al. 2017a, b, c; Arancio et al. 2017; Ghosh et al. 2019).

11.3 Novel Biomarkers for Diagnosis and Prognosis of Ovarian

Cancer

Ovarian carcinomas-related morbidity and mortality ranks considerably higher than

other gynecological malignancies. Its early detection is difficult due to the lack of
precise and accurate screening methods and absence of physical symptoms.

11.3.1 CA 125

The classic “gold standard” tumor biomarker in ovarian cancer is CA 125. It is a

glycoprotein with a high molecular weight and has a sensitivity of 50–60% with an
overall specificity of 90% in postmenopausal women diagnosed with early-stage
ovarian cancer. The expression of CA 125 is enhanced above normal level in about
90% of patients diagnosed with epithelial cell ovarian cancer (Rein et al. 2011;
Delaney et al. 2020; Colaković et al. 2000). CA 125 is the only biomarker currently
used widely in cancer therapy (Rein et al. 2011). CA 125 can be used as a potential
biomarker for the early detection of ovarian cancer, as suggested by the literature
(McIntosh et al. 2004). Moreover, CA 125 is also helpful in determining chemother-
apy responses, differentiating malignant pelvic tumors and benign tumors, and
284 K. A. Janjua et al.

detecting recurrence. A decrease in CA 125 is considered a favorable sign during

chemotherapy, and its level is also important in assessing disease stabilization (Bast
et al. 1998, 2005; Guppy and Rustin 2002).

11.3.2 Circulating Fetal Protein Alpha-Fetoprotein (RECAF)

A study conducted by Tcherkassova et al. reported novel RECAF (Receptor for

circulating Fetal Protein Alpha-Fetoprotein), an oncofetal antigen biomarker in
conjunction with CA 125 for the early detection of ovarian cancer among healthy
women. It was noticed that the addition of RECAF to CA 125 increased the
sensitivity of detecting ovarian cancer to about 83% as compared to when CA
125 is used alone (Chu and Rubin 2006). Thus, due to the relatively low sensitivity
of CA 125 when used alone, adding it with different biomarkers can create a panel of
multiple biomarkers with increased efficacy (Jacobs et al. 2011; Menon et al.
2009a, b).

11.3.3 Human Epididymis Protein (HE4)

Human epididymis protein (HE4), having a molecular weight of 25 kDa, coded by

the gene “WFD2,” is a protein that belongs to a family of “four-disulfide core” that
consists of various groups of small proteins which are heat-stable and acid-resistant
made up of different functional groups. After being produced by the epithelial
ovarian cancer cells circulating in the blood, this protein can be detected using
enzyme immunoassay (Moore et al. 2012). The use of HE4 for diagnosing and
monitoring women with epithelial ovarian cancer was approved by the Food and
Drug Administration (FDA) in 2009. Scientists have reported the overexpression of
HE4 in Epithelial Ovarian Cancer (EOC) but not in other types of ovarian cancers
(Montagnana et al. 2011). Physicians always requested CA 125 combined with
identifying serum HE4 levels for the early detection of ovarian cancers as CA
125 is found to be elevated in various benign lesions and other diseases. Early
detection of ovarian cancers based on HE4 has an overall sensitivity of about 90%
and a specificity of 72.9%. However, the combined use of CA 125 and HE4 certainly
improves identifying different benign and malignant tumors (Moore et al. 2008). In
ovarian cancer, the overexpression of HE4 leads to the irritation of Human Epider-
mal Growth Factor Response (EGRF). It induces MAPK signaling, contributing to
inducing tumor cell growth, migration, and adhesion (Lu et al. 2012). The normal
reference range of serum HE4 is <140 pmol/L. HE4 levels are found to be raised in
conditions like pregnancy, aging, and post-menopausal women.
Food and Drug Authority (FDA) approved the use of ROMA (Risk of Malig-
nancy Algorithm) for measuring the levels of CA 125 and HE4 for diagnosing the
epithelial ovarian cancers in women with post-menopausal status and are presented
with pelvic masses. The sensitivity (90.7%) and specificity (93.1%) of this test are
higher than CA 125 alone. ROMA score of greater than and equal to 1.31 reflects a
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 285

high risk of ovarian cancer in premenopausal women. However, ovarian malignancy

in post-menopausal women is considered by a ROMA score of greater than and
equal to 2.71 (Moore et al. 2009; Bast et al. 1981).

11.3.4 Mesothelin

Mesothelin is a glycoprotein having a molecular weight of 40 kDa, which is

expressed on the surface of mesothelial cells. The serum and urine levels of
mesothelin are found elevated in some cancers, including mesothelial cell carci-
noma, ovarian cancer pancreatic cancer (Hassan et al. 2005; Hassan and Ho 2008).
Studies reported on ovarian cancer have demonstrated the interaction of CA
125 with mesothelin on the surface of cancer cells that mediate cell attachment
(Massova et al. 1998; Schorge et al. 2010). Overexpression of mesothelin in
mesothelial cell carcinoma and ovarian cancer triggers the MAPK, PI3K, and
NF-κB signaling pathways (Hilliard 2018). Sensitivity (60%) and specificity
(98%) of mesothelin alone are less as compared to when used in combination with
CA 125 (McIntosh et al. 2004). Urine assay of mesothelin is found to be more
effective as compared to serum assay. However, the amount of mesothelin in both
assays can be found by Enzyme-Linked Immunosorbent Assay (ELISA) (Badgwell
et al. 2007). Factors that affect mesothelin’s expression level include age, smoking,
and BMI (Lowe et al. 2008).

11.3.5 Kallikrein-Related Peptidases (KLKs)

Kallikrein-related peptidases (KLKs) are a group of serine proteases having a

molecular weight of 30 kDa with proteolytic activity. Fifteen related serine proteases
play various roles in the human body encoded by a cluster of genes on the 19q13
chromosome. Research on SKOV3 epithelial ovarian cancers indicated that some
kallikrein-related peptidases, including KLK4, KLK7, and KLK6, play certain vital
roles in ovarian cancers and are found to be overexpressed. The sensitivity of KLKs
in the early detection of ovarian cancers is low when used alone, but sensitivity
(72%) and specificity (90%) have been reported by the combined use of CA 125 and
KLK 42. ELISA measures kLKs serum levels, and levels of more than 4.4 mg/L
indicate patients with poor prognosis (El Sherbini et al. 2011).

11.3.6 Osteopontin

Osteopontin is associated with the invasion and metastasis of tumor cells. It is a

secreted, adhesive, extracellular glycoprotein synthesized by osteoblasts and vascu-
lar endothelial cells whose function is linked with immunity and bone remodeling.
Osteopontin has a sensitivity of 83.3% in detecting ovarian cancers. However, the
286 K. A. Janjua et al.

increased sensitivity is reported by the combined use of osteopontin with CA

125 (Nakae et al. 2006).

11.3.7 ApoA1

ApoA1 belongs to the family of highly dense lipoproteins. Reduced levels of ApoA1
are found to be reported in Ovarian Cancers. The mechanism through which the
levels of ApoA1 are reduced is unclear; however, this reduction is made in the
serum. Research conducted in the past reported that the reduction in the levels of
ApoA1 in ovarian cancers is linked with the destruction of cellular bio-membranes.
ApoA1s serve as a potential biomarker having a sensitivity of 93.9% and specificity
of 95% in detecting OvCa when used in combination with CA 125 (Gadomska et al.
2005; Su et al. 2010).

11.3.8 Vascular Cell Adhesion Molecules 1 (VCAM-1)

Vascular cell adhesion molecules 1 (VCAM-1) is a receptor located on the surface of

endothelial cells and mesothelial cells. It overexpresses in the mesothelium layer of
ovaries of women diagnosed with ovarian cancer. VCAM-1is responsible for the
stimulation of cancerous cells to move to the peritoneal cavity. It has a sensitivity of
86% in detecting early-stage OvCa and a sensitivity of 93%, and a specificity of 98%
in detecting the last stages of OvCa when combined with other biomarkers (Slack-
Davis et al. 2009; Yurkovetsky et al. 2010).
Different types of cancer, including ovarian cancer, are attributed to the mutations
in the tumor suppressor genes and genes responsible for the cell cycle, leading to
uncontrolled growth, survival, and tumor cells’ metastasis. Genetic biomarkers
including BRCA1, P53, and KRAS aid in early detection by identifying the
disease-subtype, stage, and prognosis and serve as a source of selecting effective
treatments and therapies. In ovarian cancer, genes that mutate normally include the
following:

11.3.9 BRCA1

BRCA1 is a gene located on chromosome 17q12-21, which has a vital role in family-
related ovarian cancer. The function of this gene is to help in genome repair.
Different studies reported the hyper-methylation of BRCA1 in ovarian cancers and
tumor cells. Owing to this hyper-methylation, a 12–16% reduction in expression
occurs, and this hyper-methylation is associated with diminished protein
concentrations of RNA and BRCA1, specifically in epithelial cells ovarian cancer.
The poor prognosis of OvCa is also linked with hyper-methylation (Miki et al. 1994;
Baldwin et al. 2000; Wilcox et al. 2005; Strathdee et al. 2001).
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 287

11.3.10 P53

P53 is a tumor suppressor gene that is involved in maintaining the cell cycle and
apoptosis. As reported by studies, 50% of ovarian cancers occur due to mutations in
P53. It serves as a potential genetic biomarker to identify the metastatic potential of
ovarian cancer and differentiate between epithelial cells, ovarian tumors, and other
types of ovarian cancers. In all stages of ovarian tumors, mutations of P53 are found
present (Milner et al. 1993).
KRAS is a GTPase belonging to the family of RAS protein. It is an early player in
various signaling pathways. In normal tissue signaling, KRAS serves a vital role;
however, a mutated version of the KRAS gene is a significant step in causing and
developing cancers (Tsuchida et al. 1982). About 25% of cancers are reported to
have mutated KRAS. In type I epithelial ovarian cancers, mutations are reported in
almost 40% of patients (Nodin et al. 2013).
EGFR, known as Endothelial Growth Factor Receptor, is a tyrosine kinase
receptor that plays an important role in normal cell function. Mutations in this
receptor are responsible for changing normal cells’ phenotype into tumor cells
(Huang and Harari 1999). In 70% of ovarian cancers, mutations in EGFR are
reported. Mutations in the receptor cause overexpression of endothelial growth
factors leading to the AKT signaling pathway. Aggressive forms of ovarian cancer’s
poor prognosis are attributed to the EGFR/AKT pathway’s overexpression (Siwak
et al. 2010; Zeineldin et al. 2010).

11.3.11 MicroRNAs

MicroRNAs (miRNAs) are made up of 21–24 nucleotides and are noncoding RNA
types. Its function is to act in the posttranscriptional regulation of gene expression
(Iorio et al. 2007). miRNAs that have been investigated to be used as a biomarker in
detecting ovarian cancer include miR-21, miR-141, miR-200a, miR-200c,
miR-200b, miR-203, miR-205, and miR-214 (Taylor and Gercel-Taylor 2008;
Szajnik et al. 2013).

11.4 Novel Biomarkers for Diagnosis and Prognosis of Lungs

Cancer

Lung cancer is among the top causes for cancer-related mortality in both men and
women in Western nations, accounting for 30% of cancer-related deaths in the
United States consistently (Granville and Dennis 2005).
The rate of spread of lung cancer is manifolds higher, as compared to that of
prostate cancer among men and about double of that in breast cancer among women.
Smoking has been attributed as one of the main causes, with the hazard in smokers
found to be ten times higher than in nonsmokers. Lung cancer is broadly categorized
as non-small cell lung cancer (NSCLC), making up around 85% of cases. Small cell
288 K. A. Janjua et al.

lung cancer (SCLC) contributes to 15% of cases and includes a few histological
sorts, like adenocarcinoma, large cell carcinoma, and squamous cell carcinoma
(Lehtiö and De Petris 2010).
Lung cancer is considered a heterogeneous disease involving subtypes with
distinct pathologic and clinical characteristics (Fujimoto and Wistuba 2014). It is
important to distinguish between these histologic subtypes of non-small cell lung
carcinoma (NSCLC), specifically adenocarcinoma, squamous cell carcinoma, and
large cell lung carcinoma to determine the best course of therapy and provide reliable
information regarding the disease prognosis (Kerr et al. 2014).
There have been significant breakthroughs in the recent years in developing
biomarkers for lung cancer that would allow the molecular categorization of differ-
ent cancer subtypes, leading to customization of anti-cancer therapy (Mok 2011).
The progression in molecular profiling and targeted treatment has also given rise
to a renewed interest in the characterization of NSCLC into major subtypes like
adenocarcinoma, squamous cell carcinoma, and large cell lung carcinoma (Travis
et al. 2015).
Lung cancers are typically analyzed and diagnosed by transthoracic core needle
biopsy and fine-needle aspiration (FNA), transbronchial needle aspiration,
endobronchial ultrasound-guided transbronchial needle aspiration, and endoscopic
ultrasound-guided FNA (Travis et al. 2013).
Proteomics may offer a significant advantage over genomics since protein
biomarkers offer more reliable information regarding a disease considering proteins,
and not transcripts, are the actual players (Stroncek et al. 2005).
Since a persistent dynamic inflammation state describes cancer, the cancer
microenvironment often contains infiltrating inflammatory cells and
pro-inflammatory cytokines. Acute phase reactant proteins (APRPs) are released in
response to the aggravated inflammation. The relationship between the altering
APRP levels and progression of neoplastic disease has been established previously,
but the latest proteomics studies showed that APRP adjustments are diverse in
specific tumor types (Pang et al. 2010). Hence, APRPs can likely be utilized
biomarkers for categorizing the different cancer types. Among APRPs, the hapto-
globin (Hp) β chain, serum amyloid A (SAA) (Sung et al. 2011), and apolipoprotein
A-1 (Apo A-1) (Maciel et al. 2005) proteins have demonstrated considerable poten-
tial as diagnostic markers for lung cancer.
A similar study has shown that the haptoglobin levels are three times higher in the
blood samples of lung cancer patients as compared to healthy controls. The Hp levels
also provide remarkable specificity when distinguishing between lung cancer
patients and those suffering from other respiratory diseases such as pulmonary
fibrosis, tuberculosis as well as bronchial asthma where the levels are similar to
those in normal population. Furthermore, the Hp levels in other types of cancers such
as breast cancer and hepatocellular carcinoma, are also found to be significantly
lower than that in lung cancer (Kang et al. 2011).
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 289

11.4.1 SAA Proteins

SAA proteins are apolipoproteins that are involved in various key processes like
cell–cell communication, cholesterol transport in the liver, induction of immune
cells to the site of inflammation, and the breakdown extracellular matrix (Uhlar and
Whitehead 1999).
The SAA1 and SAA2 of the SAA protein family have been recently investigated
as diagnostic markers for lung cancer. The expression levels of the proteins from
blood and tissue samples of lung cancer patients were analyzed using LCMS/MS,
ELISA, and immunohistochemistry examinations. It was revealed that the SAA1
and SAA2 levels in the samples from lung cancer patients were significantly higher
than the samples from healthy volunteers as well as from patients suffering from
other types of cancer (Sung et al. 2011).
In another study comparing the adenocarcinoma patients with healthy controls,
the upregulation of SAA1 and SAA2 in lung cancer was found to be associated with
reduced levels of Apo A-1, an APRP responsible for removal of endogenous
cholesterol from tissues (Maciel et al. 2005). In a more recent study, the proteome
of serum and pleural effusions was compared between NSCLC patients and
non-malignant lung disease using ‘two-dimensional difference gel electrophoresis’
(2D-DIGE). Considerably higher levels of biomarkers were found in pleural effusion
and serum of cancer patients, although the level in pleural effusion was higher than
that in serum (Rodríguez-Piñeiro et al. 2010).

11.4.2 Epidermal Growth Factor Receptor (EGFR)

The EGFR (Epidermal Growth Factor Receptor) is a tyrosine kinase receptor

belonging to the family of ERBB. At the short arm of chromosome no. 7, the gene
for EGFR is located at the twelfth position. Overexpression of EGFR is reported in
nearly 62% of NSCLCs associated with poor prognosis (Sharma et al. 2007). In the
US, around 10% of patients diagnosed with lung adenocarcinomas and 30–50% in
Asia are found to have developed cancer due to the mutations in the EGFR gene
(Sharma et al. 2007). Tyrosine Kinase Inhibitors (TKIs), including gefitinib and
afatinib, due to their high response rates of 55–78%, are established as standard
treatment for patients diagnosed with lung adenocarcinomas due to EGFR mutations
6. For identifying EGFR mutations, techniques like gene sequencing and polymerase
chain reaction (PCR) are used (Fujimoto and Wistuba 2014).

11.4.3 Anaplastic Lymphoma Kinase (ALK)

Anaplastic lymphoma kinase (ALK) is also a tyrosine kinase family belonging to the
insulin receptor superfamily. On the short arm of chromosome 2, the gene for ALK
is located at 23rd position (Zhao et al. 2015). Mutations of the ALK gene are
reported in a subset of NSCLC tumors which harbor a fusion of both ALK with
290 K. A. Janjua et al.

echinoderm microtubule-associated protein-like 4 (EML4) (Fujimoto and Wistuba

2014). 3.7–7% of NSCLCs have been associated with EML4-ALK fusion and are
found to be more prevalent in adenocarcinomas reported in young patients who have
never smoked. Patients with verified EML4-ALK fusion have reported high
response rates (57–74%) after treatment with ALK inhibitors such as crizotinib
(Solomon et al. 2014).

11.4.4 Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS)

KRAS is an oncogene located on the long arm of chromosome 12 at 12.1 positions

(McBride et al. 1983). It belongs to the RAS family of membrane-associated
G-proteins (Edkins et al. 2006). In 25–30% of patients with NSCLC, KRAS
mutations are reported specifically in adenocarcinomas of solid pattern, which are
found more in white people than Asians (Dogan et al. 2012). KRAS mutations lead
to unfavorable outcomes and are considered a negative predictor of chemotherapy’s
response (Ying et al. 2015; Macerelli et al. 2014). Additionally, it is also linked with
the development of the “second-time primary tumor”. It is also considered a predic-
tor of resistance in patients with NSCLC who are administered targeted EGFR-TKIs
therapy (Macerelli et al. 2014).

11.4.5 Receptor Tyrosine Kinase (ROS1)

Receptor tyrosine kinase (ROS1), a proto-oncogene, is a gene from the insulin

receptor family’s tyrosine kinase receptor (Bergethon et al. 2012). It is located on
the long arm of the 6th chromosome at position 22. ROS1 rearrangements are found
in 1–2% of patients with NSCLC (Yoshida et al. 2013). This rearrangement is
commonly reported in young females who have never smoked, with a diagnosis of
adenocarcinomas (Bergethon et al. 2012; Yoshida et al. 2013). Response rates of
80% are recorded in advance NSCLC patients with ROS1 mutations given crizotinib
treatment (Bergethon et al. 2012). Assessment of ROS1 rearrangement is an expen-
sive and laborious technique. Since this type of cancer is rare, screening by IHC is
considered a tool for identifying patients suitable for ROS1-targeted therapy
(Popescu et al. 1989).

11.4.6 The Human Epidermal Growth Factor Receptor 2 (HER2)

The human epidermal growth factor receptor 2 (HER2) gene is localized on chro-
mosome 17 at position 12 that encodes a transmembrane member of the tyrosine
kinase epidermal growth factor receptors, which are usually expressed less in all
epithelial cells in the tissues of the normal fetus and normal. These receptors are
essential in the proliferation and survival of cancer. Increased or over-expression of
HER2 mRNA is associated with the HER2 gene amplification, which is responsible
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 291

for triggering carcinogenesis by uncontrolled proliferation due to self-sufficiency in

growth signals, enhanced invasive and metastasis processes (Yarden and Pines
2012; Oh et al. 1999). Seven to 34.9% of NSCLCs have been reported to have
overexpression HER2 associated with a poor prognosis in patients with these tumors
(Ricciardi et al. 2014). Various studies reinforced this proposal of screening patients
with lung adenocarcinomas for HER2 mutations to select patients who could benefit
from targeted HER2 therapies such as afatinib and trastuzumab. The response rate in
NSCLC patients diagnosed with HER2 mutations is recorded as 50% (Mazières
et al. 2013).
RET proto-oncogene that encodes for a tyrosine kinase receptor is located on
chromosome 10 at position 11.2 51. Rearrangement of RET was initially recorded in
papillary thyroid carcinomas (Knowles et al. 2006). Around 1–2% of NSCLC are
found to have RET rearrangements which typically occurs in young patients who
have never smoked in adenocarcinomas with poorly differentiated solid
characteristics (Lipson et al. 2012). The standard assay used for diagnosing RET is
FISH. PCR is considered insufficient for identifying RET rearrangements’ various
isoforms (Lipson et al. 2012).

11.4.7 Phosphatidylinositol-4,5-Bisphosphate 3-Kinase, Catalytic

Subunit a (PIK3CA)

Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA)

codes 1 of 2 phosphoinositide 3-kinase (PI3 K) subunits (German et al. 2013) and
is a cancer gene. It is considered the most common mutated gene and KRAS in
human cancers (Samuels and Velculescu 2004). These mutations are found in 1–4%
of patients with NSCLC (Yamamoto et al. 2008; Fujimoto and Wistuba 2014).

11.4.8 The Neurotrophic Receptor Tyrosine Kinase 1 (NTRK1)

The neurotrophic receptor tyrosine kinase 1 (NTRK1) proto-oncogene, also known

as a tropomyosin-related kinase (TRK) A, belongs to tyrosine kinase. It is located on
1q21-22 chromosome 117. In lung cancer, rearrangement in the NTRK1 gene is
reported in 3% of patients diagnosed with adenocarcinomas that harbor NTRK2
fusions (Vaishnavi et al. 2013). Early phase 1 studies such as entrectinib and LOXO-
101 have shown good results in patients diagnosed with solid tumors with NTRL1
fusions (Passiglia et al. 2016).

11.4.9 The Fibroblast Growth Factor Receptor (FGFR)

The fibroblast growth factor receptor (FGFR) is a gene located on the eighth
chromosome at position 12 responsible for encoding TKR belonging to the FGFR
family (Jiang et al. 2015). In lung cancer, FGFR1 gene amplification is reported
292 K. A. Janjua et al.

higher in carcinomas of squamous cells (20%) as compared to adenocarcinomas

which is found amplificated in only 3% of the cases and is more commonly found in
current smokers as compared to the people who smoked and never smoked (Jiang
et al. 2015; Weiss et al. 2010).

11.4.10 The Discoidin Domain Receptor Tyrosine Kinase Two Genes

(DDR2)

The discoidin domain receptor tyrosine kinase two genes (DDR2) encodes for a
TRK receptor. It is located on the long arm of chromosome 1 at 23.3 positions. In
lung cancer, DDR2 is found to be reported in 3–4% of carcinomas in lung’s
squamous cells (Hammerman et al. 2011) as compared to the adenocarcinomas
(0.5) (Ding et al. 2008) and are only found to be present in smokers (An et al. 2012).

11.5 Novel Biomarkers for Diagnosis and Prognosis of Breast

Cancer

Biomarkers play an indispensable role in both diagnosing and managing cancer.

Over the last few decades, advancement in precision medicine has led to the
discovery of cancer biomarkers. This discovery and advancement in diagnostic
and prognostic biomarkers have led to the delivery of targeted therapies to the
patients, which has spared patients from getting overtreatment and has significantly
reduced the side effects of the diagnosis and treatment of cancer (Meehan et al.
2020).
In women, breast cancer is considered the most common cancer whose incidence
rates increase day by day (Jemal et al. 2010). Currently, biomarkers play a crucial
role in managing patients with breast cancer, specifically when deciding the nature of
systematic therapy administered to the patient. In 2005, guidelines were published
by the European Group on Tumor Markers (EGTM) on the use of biomarkers for the
treatment of breast cancer (Molina et al. 2005). However, thenceforth, many new
developments are made and reported, specifically tissue-based biomarkers.
The use of biomarkers in cancer is limited to providing additional information
about various clinical factors. Still, it is also linked to providing more treatments to
the patients with a better and controlled benefit–risk balance (Polley et al. 2013).
Analysis using biomarkers in breast cancer is being used as a routine practice.
Initially, it began with testing the expression of hormone receptors in guiding
therapy using tamoxifen. However, a revolution in the biomarker field started with
the succeeding inclusion of targeted therapies against HER2 (Human Epidermal
Growth Receptor 2) (Albanell et al. 2009).
Breast cancer accounts for about one-third cancer cases in women, and the overall
health burden worldwide due to breast cancer is reported as 10% (Jemal et al. 2010).
At the beginning of this century, with the introduction of mammographic screening
techniques, early detection of breast cancer has further led to identifying cases, thus
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 293

increasing the incidence rates. In developing countries, the incidence rates of breast
cancers are also increasing rapidly, which depicts that in the next decade, a major
disease burden in both developing and developed countries would be contributed by
breast cancer (Cedolini et al. 2014; Driul et al. 2013). Improvements and the use of
adjuvant chemotherapy and endocrine therapy have significantly reduced mortality
due to breast cancer to about 50%. However, increasing incidence rates of breast
cancer has led to the debates of patients with overtreatment, which has contributed to
the increase in family and social burden and has also resulted in causing unnecessary
side effects and harm to the patient (Esserman et al. 2013; Katz and Morrow 2013).
Thus, research is being conducted worldwide, which is focused on targeted drug
delivery with fewer side effects. In this regard, many new biomarkers have been
studied and introduced to serve both as an important prognostic tool that enables
determining whether the cancer is progressing slowly or aggressively and as a
therapeutic agent (Veer et al. 2002; Wang et al. 2005).
Breast cancers mainly consist of a heterogeneous group of tumors with an
extensive spectrum of morphologically and molecularly special subtypes, ensuing
in special biological behaviors, presentation, and prognosis. Along with the ailment
level and the affected person’s performance, recognizing the molecular pattern of
tumors is fundamental in identifying patients who will benefit from a certain and
more specific treatment type. In the standard care of all breast cancer patients
(primary, recurrent, and metastatic), the use of estrogen receptor (ER), the human
epidermal growth factor receptor (HER2), the progesterone receptor (PR), and the
Mib1/Ki-67 proliferation index are the molecular markers that are firmly established
and are considered the most important ones (Naoi and Noguchi 2016; Harris et al.
2016).
Human breast tumors rely on sex hormones for their growth as their origin is from
breast tissue, which is responsive to endogenous hormones (Zumoff et al. 1975). In
1896, it was noticed that bilateral oophorectomy could significantly reduce the
progression of breast cancer, especially in fertile age (Stockwell 1983). This has
led to the acceptance of endocrine therapy as a standard treatment for patients with
the barest cancer. However, the positive response rate was recorded in only one-third
of patients.
In the early 1960s, the existence of estrogen receptors was supposed due to the
concentration of radiolabeled estrogens in the target areas, which could be consid-
ered as a predictive factor in reporting positive, the responsive need of breast cancer
to oophorectomy (Jensen and Jordan 2003; McGuire 1975). About 60% Estrogen
Receptor (ER) positive tumors and 8% ER-negative tumors responded to the endo-
crine therapy. However, the small proportion of ER-negative patients who
responded to the endocrine therapy may be due to faulty receptor assay results.

11.5.1 Human Epidermal Growth Receptor (HER2)

This successful identification of the estrogen receptor has proven both a therapeutic
target for the treatment and prevention of breast cancer. It has also been recognized
294 K. A. Janjua et al.

as a selective molecular model for all succeeding efforts in designing targeted

oncological therapies. Thus, the estrogen and progesterone receptors, along with
the Human Epidermal Growth Receptor (HER2), represent the most fundamental
biomarkers in the standard care of all patients with breast cancer. Their assessment
was also critical in evaluating every diagnosed breast cancer.
Among women younger than 50 years of age, about two-third of invasive breast
cancers are expressed by hormone receptors, and around 80% of women are found to
be older than 50 years of age (Anderson et al. 2002). For the evaluation of breast
cancers, measuring and recognizing hormone receptors is a routine practice that
represents a potential predictive factor in determining the given hormone therapy’s
responsiveness. Increasing levels of both estrogen and progesterone receptors corre-
late with a better and more precise response, decrease chances of failure and long
survival rate (Buzdar et al. 2004; Ravdin et al. 1992).

11.5.2 ER Expression

The expression of hormone receptor also represents an important favorable factor

with prime prognostic properties, recognized as an important growth marker rather
than metastatic potential. Specifically, the prognosis of patients with ER+/PR+
tumors is better than patients with ER+/PR tumors, who have a better prognosis
than patients with ER/PR tumors (Bardou et al. 2003). Prognostic indicators,
including old age, a lower fraction of dividing cells, and low grading, lower genetic
mutation, are significantly associated with ER expression (Anderson et al. 2002;
Elledge et al. 1993; Wenger et al. 1993; Nadji et al. 2005).
The recurrence rates of breast cancer patients with ER-positive tumors can be
halved with adjuvant hormone therapy (Early Breast Cancer Trialists’ Collaborative
Group 2005). Moreover, due to its limited and reduced side effects, patients with
comorbidities and elderly patients can also be administered with high success rates.
In some patients with metastatic disease, its response can last for years. Compared to
the stage-I patients with ER positive tumors who receive no systematic therapy, the
recurrence rate in 5 years is 5–10% lower than those breast cancer patients who have
ER-negative tumors. On the contrary, ER-negative tumors respond better to cyto-
toxic chemotherapy than hormone therapy (Fisher et al. 1988). The human epider-
mal growth factor receptor 2 (HER2) gene is localized on chromosome 17 that
encodes a transmembrane member of the tyrosine kinase epidermal growth factor
receptors, which are usually expressed less in all epithelial cells in the tissues of the
normal fetus and normal. These receptors are essential in the proliferation and
survival of cancer. Increased or overexpression of HER2 mRNA is associated with
the HER2 gene amplification responsible for triggering carcinogenesis by uncon-
trolled proliferation due to self-sufficiency in growth signals, enhanced invasive and
metastasis processes (Yarden and Pines 2012; Oh et al. 1999). The HER2 gene
results are amplified in the certain breast, ovarian, bladder, endometrial (Saffari et al.
1995), salivary gland (Press et al. 1994) and gastric cancer (Park et al. 1989).
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 295

The most popular humanized monoclonal antibody against human endothelial

growth factor receptor (HER2), Trastuzumab, has significantly improved response
rates; time is taken for progression and survival when used alone or when added to
chemotherapy in both early-stage breast cancer patients and metastatic (Slamon et al.
2011). Among the other HER2-targeted medicines which include lapatinib (tyrosine
kinase inhibitor), pertuzumab (the monoclonal antibody), adotrastuzumab emtansine
T-DM1 (the antibody-drug conjugate) have improved outcomes in HER2-positive
metastatic breast cancers.

11.5.3 Mib1/Ki-67

Mib1/Ki-67 is a proliferation index used both as a prognostic and predictive marker,

though due to the lack of standardization, its widespread use is limited (Colozza et al.
2005; Harris et al. 2007). This proliferation biomarker is a significant predictor for
the responsiveness to both endocrine therapy and chemotherapy (Dowsett et al.
2017; Fasching et al. 2011). Moreover, the decrease in Mib1/Ki-67 in posttreatment
women given neoadjuvant therapies is a powerful, independent predictor depicting
better clinical outcomes (Ellis et al. 2011). Retinoic acid receptor α (RARA) is
considered a potential biomarker for tamoxifen resistance (Niederreither and Dollé
2008). About one-third of breast cancer patients with ER-alpha positive receptors
(ERα) experience relapse when treated with tamoxifen (Early Breast Cancer
Trialists’ Collaborative Group 1988). Anti-tumor properties of Retinoic Acid Recep-
tor α can be attributed to the interaction of the receptor with ERα receptors and their
combined binding sites (Hua et al. 2009). Cells resistant to tamoxifen were reported
to have high levels of RARA (Johansson et al. 2013). Breast cancer patients with
ERα positive tumors diagnosed with a high internal protein level of RARA
administered with tamoxifen displayed RFS (recurrence-free survival) compared to
patients diagnosed with low levels of RARA (Johansson et al. 2013).

11.5.4 Osteopontin

Osteopontin is associated with the invasion and metastasis of tumor cells. It is a

secreted, adhesion, extracellular protein (Senger et al. 1979; Brown et al. 1994). An
experiment was conducted by Pang et al. (2013), where he examined the clinical and
pathological effects of osteopontin, E-cadherin, and β-catenin adhesion molecules
and reported higher levels of all the above-mentioned adhesion molecules in patients
suffering from the breast when compared with the normal. Metastasis of lymph
nodes was associated with the expression of higher levels of osteopontin-c.
Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a
human multifunctional regulatory protein that is overexpressed in cancer cells
(Kuroki et al. 1992; Blumenthal et al. 2007). Its expression in atypical ductal
hyperplasia serves a potential role in the development of breast cancer. Breast
296 K. A. Janjua et al.

cancers that are invasive and treatment-resistant are also associated with CEACAM6
(Poola et al. 2006).

11.5.5 Phosphatidylinositol-4,5-Bisphosphate 3-Kinase, Catalytic

Subunit a (PIK3CA)

Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA)

codes 1 of 2 phosphoinositide 3-kinase (PI3 K) subunits (German et al. 2013) and
is a cancer gene. It is present in 20–40% of breast cancer patients (Young et al.
2015). Cizkova et al. (2013) has identified 17 tumors with PIK3CA mutations
among 80 breast cancer patients with positive HER2 treated and administered with
trastuzumab for 1 year. Breast cancer patients showing wild type.
PIK3CA showed an improved disease-free survival (DFS) as compared to the
patients possessing PIK3CA mutations. The prognosis of HER2 positive breast
cancer patients treated with trastuzumab was poor than for the patients possessing
wild-type PIK3CA. Therefore, patients with HER2-positive tumors are only
required to detect PIK3CA mutations.

11.5.6 Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has shown efficacy in protecting

cells from apoptosis. The first chemotherapy drug of choice for patients diagnosed
with lymph node metastasis is Paclitaxel (Hortobagyi and Holmes 1996; Olayide
et al. 2015). Currently, no biomarkers are available that could predict the suscepti-
bility to chemotherapy (Chromek et al. 2004). An epidemiolocal study conducted
previously has reported an association between TIMP-1 and responsiveness to
cyclophosphamide/methotrexate/5-fluorouracil and anthracycline-based chemother-
apy regimens. Higher levels reduced would be the responsiveness to various
therapies (Schrohl et al. 2006).

11.5.7 Ferritin Light Chain (FTL)

Ferritin light chain (FTL) is an iron-binding protein. In chordates, apoferritin has two
types, including light and heavy chain, which are assembled by 24 subunits. The
ratio between heavy chains and ferritin light chains depends on the type of tissue and
conditions (Arosio et al. 1976). The rise in ferritin protein from tissues affected with
cancer shows a correlation between these two. Ricolleau et al. (Zeidan and
Townsend 2008) calculated a ferritin light chain cut-off level in tumors as 2.4 by
studying the utilization of FTL (a prognostic marker) in breast cancer patients with a
positive lymph node metastasis. The metastasis-free survival rate was recorded low
in high FTL patients (Jézéquel et al. 2012).
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 297

11.5.8 Urokinase-Type Plasminogen Activator (uPA)

Urokinase-type plasminogen activator (uPA) is a tumor-associated proteolytic fac-

tor. In contrast, plasminogen activator type 1 inhibitor (PAI-1) plays a potential role
in invasion and metastasis of tumor 41, cell signaling and adhesion, migration, and
cell-proliferation (Duffy et al. 2014). C-reactive protein (CRP) is used as a marker of
acute inflammatory reactions and serves as an important prognostic marker in breast
cancer (Allin and Nordestgaard 2011; Pradhan et al. 2018). Chromosome 17 centro-
mere enumeration probe (Ch17CEP) is the second most dense chromosome that
codes for several genes, including BRCA1 and HER2, which play an important role
in breast cancer and repairing genes TP53, RAD51C, and RAD52B (Zody et al.
2006).

11.5.9 Soluble Human Epidermal Growth Factor Receptor 2 (sHER2)

Soluble human epidermal growth factor receptor 2 (sHER2) is a protein released

from the intracellular transmembrane and extracellular domains 51. sHER2 is
considered an important biomarker in breast cancer patients with HER2 positive at
all stages. 53. In early-stage breast cancer, HER2 positive patients sHER2 is found as
a suitable relapse prognostic biomarker and survival determination in relapse
patients (Moreno-Aspitia et al. 2013).

11.5.10 Mitotic Arrest Deficient Like 1 (MAD1L1)

Mitotic arrest deficient like 1 (MAD1L1) is considered a checkpoint gene linked

with chromosomal instability. Several different types of cancer, including colon and
lung cancer, have been reported with abnormalities in MAD1L1 (Foijer et al. 2008).
A study conducted has revealed that the expression of nuclear MAD1L1 seems to
have increased the treatment resistance and has affected the prognosis of breast
cancer, showing that breast cancer patients with positive MAD1L1 was not suscep-
tible to treatment with Taxol (Sun et al. 2013). Methylation of paired-like
homeodomain 2 (PITX2P2), including C-phosphate-G islands, is located within
the gene regulatory site and linked with gene expression suppression. The introduc-
tion of a methyl group (methylation) in the DNA—nucleotides of this gene is
commonly recorded as an early event succeeding the onset of cancer (Baylin and
Herman 2000; Jones 1996; Herman and Baylin 2003). Patterns of methylation are
specific to the tumor’s subtypes, including breast cancer (Frühwald 2003; Laird
2003; Absmaier et al. 2018). Data analyzed from several studies reported that
methylation of the DNA of PITX2 was associated with an increased risk of relapse
in metastasis of lymph nodes positive, hormone-receptor-positive breast cancer
patients taking adjuvant tamoxifen therapy (Qian et al. 2017; Bibikova et al. 2006).
298 K. A. Janjua et al.

11.6 Novel Biomarkers for Diagnosis and Prognosis

of Leukemia

Leukemia is a set of progressive hematological malignancies originating in stem

cells of bone marrow that contributes to a large proportion of cancer deaths. Novel
biomarkers have managed to revolutionize the clinician’s approach towards prog-
nostication and therapy.

11.6.1 CD123

A recent study revealed that the biomarker CD123, interleukin-3 receptor alpha
(IL-3Rα), has significant expression in Leukemic stem cells (LSCs), but not in the
normal hematopoietic stem cells (HSCs), and is associated with better treatment
response, as well as minimum residual disease detection (MRD) and prognosis.
Moreover, CD123 is a significant marker for detection and targeted therapy of LSCs
for relapsed and refractory leukemia. Anti-CD123 targeted therapies in preclinical
testing and clinical evaluation validate the effectiveness of anti-CD123 neutralizing
antibody drugs, CD3 and CD123 bi-specific antibodies, dual affinity retargeting
(DART), and anti-CD123 chimeric antigen receptor-modified T-cell (CAR-T) treat-
ment therapies in the process of development (Shi et al. 2019).

11.6.2 Tumor Necrosis Factor Receptor 2 (sTNFR2)

A research group from University of the Ryukyus (Nishihara, Japan) has success-
fully identified a novel biomarker for diagnosing acute T-cell leukemia (ATL). ATL
is an adult T-cell neoplasm that is linked to human T-cell leukemia virus type-1
(HTLV-1). Elevated levels of HTLV-1 carriers contribute to higher prevalence rates
of ATL in Japanese population. To identify a novel biomarker that helps in the early
detection of ATL, scientists identified a higher expression of soluble tumor necrosis
factor receptor 2 (sTNFR2). There is a need to study further the importance of high
STNFR2 levels in ATL onset prediction and examine the method by which tumor
necrosis factor 2 is cast off from the cell surface, as a possible therapeutic route
(Guerrero et al. 2020).

11.6.3 NPM1 and FLT3 Mutation

Novel biomarkers based on the nucleophosmin (NPM1) and FLT3 mutation help
decide individual treatment regimens for the patients of acute myeloid leukemia.
Similarly, BCR-ABL genetic relocation has worked as a novel biomarker for
diagnosis of chronic myelogenous leukemia (CML). Imatinib is a small molecule
inhibitor of ABL and has served as a modern era therapeutic regimen for CML,
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 299

effectively shifting the trend away from allogeneic stem cell transplant (Hsueh et al.
2013).

11.6.4 BCR-ABL Tyrosine Kinase Inhibitor (TKI)

Several proteins that act as promoters of cancers, can be utilized as biomarkers so

that effective targeted anti-cancer agents can be developed against them, such as
BCR-ABL tyrosine kinase inhibitor (TKI), imatinib. These TKIs have helped form
the groundwork for targeted oral therapy for various types of leukemia. Inhibitors of
BCR-ABL, FLT3, JAK2, Bruton tyrosine kinase have set a novel example of
success for leukemia treatment (Liu 2019).
Certain novel biomarkers, comprising of non-tyrosine kinase oncoproteins, have
been developed for targeted therapeutic action. For example, inhibitors of biomarker
BCL-2, isocitrate dehydrogenases (IDH1 and IDH2), PI3 kinase, BRAF, mTOR,
PARP, and CDK are currently under research to target many cancer types, including
leukemia (Liu 2019).
Gemtuzumab ozogamicin (GO) is an “antibody-drug conjugate” (ADC) targeting
the CD33 receptor that is commonly expressed on myeloid cells. GO has been
authorized for use in both newly diagnosed and refractory/relapsed (RR) acute
myeloid leukemia (AML). GO has been found effective when used alone and also
in combination with other chemotherapeutics. Several novel ADCs targeting CD33
are being investigated for clinical efficacy. Some of the more promising ones include
vadastuximab talirine (SGN-CD33A), IMGN779, and AVE9633 (huMy9–6-DM4)
(Liu 2019).
ADCs targeting the CD123, such as IMGN632 and SGN-CD123A, have also
undergone early phase trials. However, further development of SGN-123A was
halted due to safety concerns (Liu 2019).
BiTE and ADCs targeting CLL-1 are currently being investigated in preclinical or
early clinical trials for effectiveness against AML. CAR-T cells that target CLL-1,
are further tested in clinical trials for AML therapy (Liu 2019).

11.6.5 MicroRNAs

MicroRNAs (miRNAs) are small non-coding RNAs that block or degrade the target
mRNAs in the posttranscriptional phase. Several studies have demonstrated the role
of miRNA dysregulation in the initiation, invasion, proliferation, and metastasis of
cancer. It was also shown that secretory miRNA levels in the blood and other body
fluids have a significant correlation with cancer progression, therapeutic outcome,
and overall survival. The presence of miR92-a in the plasma of acute myeloid
leukemia and acute lymphoblastic leukemia patients may serve as a novel biomarker
for leukemia. In contrast, the miR-638 is present in healthy individuals and can be
used as a control during miRNA quantification. The ratio of miR-638 and miR-92a
in plasma is significantly associated with early detection of acute leukemias.
300 K. A. Janjua et al.

Similarly, miR-150, miR-195, miR-29, and miR-222 have been identified as poten-
tial biomarkers for CLL. Thus, secretory miRNAs hold a great potential as powerful
and noninvasive cancer biomarkers. Further research is required to verify different
approaches for the detection of miRNA detection in body fluids to aid early cancer
diagnosis and therapeutic efficacy prediction (Zhang et al. 2012).

11.6.6 Exosomes

Exosomes are extracellular vesicles (EVs) made up of characteristic lipid bilayer,

which may contain lipids, proteins, DNA, messenger RNA (mRNA), and
non-coding RNAs. Recent studies have indicated that the lipid membrane of
exosome acts as a protection for nucleic acids. Almost all types of cells in the
human body release exosomes into biological fluids. In cancer, the exosomes
derived from tumor cells contain miRNAs with deregulated expression that leads
to metastasis and treatment resistance. Owing to the extensive presence of exosomes
in various body fluids and the increased stability of miRNAs inside exosomes, the
exosomal miRNAs can serve as a new class of biomarkers for minimally invasive
and timely detection of cancer (Salehi and Sharifi 2018).

11.7 Novel Biomarkers for Diagnosis and Prognosis

of Lymphoma

Classical Hodgkin lymphoma (cHL) is one of the most common types of lymphoma
in the developed countries. Advancement in the therapeutic options for cHL have
resulted in improved outcomes leading to cure rates as high as 80%. Despite this
progress, a certain subset of patients still face unresolved clinical issues due to
relapsed or refractory disease and treatment-induced toxicity. The inclusion of
targeted drugs such as PD-1 blockers and the CD30 antibody-drug conjugate such
as brentuximab vedotin has expanded the available treatment options for cHL
patients. It has also highlighted the urgent need for identification of biomarkers
that will provide guidance for treatment selection, improve drug utilization, and
reduce drug-related toxicity. The characteristic biology of cHL is made up of tumor
microenvironment consisting of both tumor cells as well as non-malignant immune
cells. This environment provides a variety of potential biomarkers related directly to
the tumor cells, crosstalk in the tumor microenvironment or host immune response.
The following section discusses the use of prognostic models based on gene
expression and circulating tumor DNA as novel biomarkers for lymphoma (Guerrero
et al. 2020; Aoki and Steidl 2018).
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 301

11.7.1 Imaging-Based Biomarkers

The functional imaging assessment using ‘[18F]-fluoro-2-deoxy-D-glucose’ (FDG)

positron emission tomography (PET)/computed tomography (CT) has shown
promising results in evaluating the chemosensitivity and disease outcome in cHL
(Wiedmann et al. 1999; Gallamini et al. 2007).

11.7.2 Peripheral Blood-Based Biomarkers

Peripheral blood is an ideal, easy-to-access, and minimally invasive and biomarker

source. Various malignant cell-based, immune response-based markers, and
TME-based biomarkers, for instance, neutrophil-to-lymphocyte ratio, serum thy-
mus, lymphocyte-to-monocyte ratio, galectin 1, activation regulated chemokine
(TARC), micro-RNAs, serum CD30, and serum CD163, have been reported as a
potential tool as a prediction outcome using the peripheral blood that is taken at the
time of diagnosis (“pretreatment”) or during the treatment (“interim”) in cHL. (Jones
et al. 2013, 2014; Plattel et al. 2016). Captivatingly, serum TARC was reported to
correlate with the disease status and may be predictive for the PET/CT outcomes in
cHL, which indicates that the serum TARC has a strong potential for monitoring
tumor dynamics (Farina et al. 2014). These biomarkers reflect TME or the immune
system’s biology, for instance, the infiltrating lymphocytes and tumor-related mac-
rophage; though, more detailed understanding of the multifaceted ecosystem of
TME of the cHL-related lymphatic tissue may not be possible. Another significant
limitation of such biomarkers is the lack of sufficient data to determine the optimal
cutoff values for each marker (Farina et al. 2014).

11.7.3 Circulating Tumor DNA (ctDNA) Biomarkers

The circulating tumor DNA (ctDNA), collected from the peripheral blood, has been
studied extensively as a tool enabling the dynamic monitoring of tumor biology
(Kurtz et al. 2015; Roschewski et al. 2015). The ctDNA comprises DNA fragments
that are released from the apoptotic and necrotic cancer cells. These can be detected
and then quantified by the use of next-generation sequencing. The tumor-specific
immunoglobulin gene segments have also been identified in the peripheral blood of
cHL patients (Oki et al. 2015).
Even though the recent research in the field of cHL has established multiple
biomarker candidates, there is still a gap in identification of biomarkers that can
provide information regarding disease relapse. The existing candidates need
improvement in validity and reproducibility for use in patients with refractory or
relapsed disease.
302 K. A. Janjua et al.

11.7.4 Identification of Biomarkers Using Gene Expression Profiling

Gene expression profiling is a novel technique employing recent technological

advances to examine sample resources generated during routine diagnostic
procedures, such as formalin-fixed and paraffin-embedded biopsies. Due to the
absence of the HRS (Hodgkin Reed-Sternberg) cells in the whole-tissue biopsies
of cHL, analysis of the whole-tissue sections shows the structure of the tumor micro-
environment in the tissues with cHL involvement. For this reason, a growing number
of studies aimed at identifying biomarkers through gene expression profiling, are
focused on studying the TME.
One of these studies established the significant association between the macro-
phage count and clinical outcomes in treatment naïve cHL patients. These findings
were successfully extended by applying the NanoString platform in a follow-up
study. This resulted in development of a prognostic model to predict the overall
survival of cHL patients with advanced disease that received ABVD chemotherapy
regimen. The model involved 23 genes, including ones that reflected a macrophage
signature and TH1 response, cytotoxic T cells, cytokines, and natural killer cells. It
aimed to incorporate the intricate biology of the tumor–host interactions in the TME.
This model, however, failed at providing accurate prediction of patient outcomes in
the S0816 and RATHL trials, which can be linked to the response-adapted treatment
strategy used in these trials (Burton et al. 2017).

11.8 Novel Biomarkers for Diagnosis and Prognosis

of Adenocarcinoma of the Upper Digestive Tract

Adenocarcinoma involving the upper digestive tract (UDT), which includes the
gastro-esophageal junction (GEJ), distal esophagus, and stomach, is a serious
disease that impacts over 20,000 people every year (Mohammadi et al. 2021).
Since early detection and therapeutic selection are essential for disease management
and treatment, identifying effective biomarkers can significantly improve the prog-
nosis (Calanzani et al. 2021). The lack of predictive biomarkers, responsive to
conventional chemotherapy and radiotherapy, and selective therapy, is currently
one of the major challenges in the treatment. In the context of UDT adenocarcinoma,
several new trackable proteins have been investigated (Mohammadi et al. 2021).

11.8.1 Mammalian Target of Rapamycin (mTOR)

Multiple inputs involved in cellular growth stimulate the mammalian target of

rapamycin, that is a serine/threonine kinase. Phase II trials in severely pretreated
gastric cancer-focused at mTOR inhibition as a monotherapy. Both trials revealed
moderate tumor responses (Doi et al. 2010; Yoon et al. 2012a, b). Large levels of
phosphorylated S6 protein (the mTOR downstream target) were associated with a
better response to this therapy (Yoon et al. 2012a, b). GRANITE 1, the subsequent
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 303

phase III randomized trial, observed that mTOR inhibition individually promoted
progression-free survival but not overall survival (Kanagavel et al. 2015). This
approach can reliably be paired with conventional therapy but needs further
investigation.

11.8.2 c-Met

The c-Met gene, which is responsible for coding the hepatocyte growth factor
receptor, is another subject in UDT adenocarcinoma research. MET inhibition has
shown some persistent responses in patients with c-MET amplification. It is not
frequently expressed in UDT adenocarcinoma (Lennerz et al. 2011). However, in an
unselected gastric cancer population, Shah and colleagues observed no c-MET
repression effect as monotherapy (Shah et al. 2013). On the other hand, Cecchi
and colleagues reported that inhibiting c-MET in conjunction with chemotherapy
has a substantial advantage (Cecchi et al. 2012). This influence was most noticeable
in patients with c-MET stimulation, emphasizing the significance of proper patient
screening for investigational interventions.

11.8.3 Vascular Endothelial Growth Factor (VEGF)

One of the driving factors of tumor angiogenesis is VEGF-A. When VEGF binds to
several VEGF receptors (VEGFRs 1, 2, and 3), several signaling cascades associated
with angiogenesis are activated. Inhibition of VEGF signaling has been studied in
several solid tumors (Arnold et al. 2020). There is insufficient retrospective evidence
in UDT adenocarcinoma to indicate that VEGF overexpression is associated with
cisplatin sensitivity (Boku et al. 2007). Still, VEGF signaling has mainly been
studied in this disease as an underlying factor for intervention. VEGF targeting in
conjunction with standard treatment is reliable and effective in early phase II trials
(Chen et al. 2017; Shah et al. 2006). Despite these preliminary observations, the
AVAGAST trial showed that adding bevacizumab to therapy in the first-line setting
has almost no advantage (Sawaki et al. 2018). Higher serum levels of VEGF-A, and
low tumor neuropilin (a receptor that binds to VEGF-A), are correlated with
improved outcomes in a subset study of this trial, emphasizing patient significance
selection for this kind of trial (Butters et al. 2019). Keeping in view the discussed
conditions, VEGF could be the potent marker for targeting the UDT
adenocarcinoma.

11.8.4 Human Epidermal Growth Factor Receptor 2 (HER2)

and Epidermal Growth Factor Receptor (EGFR)

HER2 and EGFR are three-domain tyrosine kinases found at the cell membrane that
are closely related. Unlike EGFR, which has various recognized activating ligands
304 K. A. Janjua et al.

like EGF and TGF, HER2 has none. These two proteins can form homo- and
heterodimers, resulting in the activation of various signaling pathways. Several
agents that target EGFR and HER2 have been used in clinical trials. The most
glaring example is the addition of trastuzumab (a monoclonal antibody that inhibits
Her2 mediated signaling), which dramatically increased survival in HER2 (Sardesai
et al. 2020). Studies on HER2 overexpression in UDT adenocarcinoma are contra-
dictory. HER2 overexpression is associated with better survival in patients only
treated with surgery in one comprehensive study (Heidarpour et al. 2020). Other
reports have come to a different conclusion (Chan et al. 2012). HER2 heterogeneity
may be a factor, as patients with substantial heterogeneity in their HER2-positive
UDT adenocarcinoma have poorer outcomes (Yoon et al. 2012a, b). In patients
treated with adjuvant chemotherapy or chemoradiation, Her2 positivity does not
significantly alter response or outcome (Jácome et al. 2015). Since up to 20% of
patients with UDT adenocarcinoma overexpress Her2, targeting this receptor has
been studied and studied (Phillips et al. 2013).

11.8.5 Germline Alterations (Single Nucleotide Polymorphisms

(SNPs))

The need for tumor tissue makes it difficult to develop the potential predictive
markers of UDT adenocarcinoma responsiveness. Obtaining adequate
pre-therapeutic cancerous tissues for evaluation is difficult, time-consuming, and
tumor heterogeneity can skew the findings (Gerlinger et al. 2012). The expression of
several candidate biomarkers can be influenced by the medication, making post-
therapy markers unhelpful for evaluating response. As a result, germline genetic
alterations, i.e., single nucleotide polymorphisms (SNPs), have been evaluated as
potential predictive biomarkers for UDT adenocarcinoma. This method has the
advantages of being relatively noninvasive (blood specimen can be used) and cost-
effective. Several studies have focused on evaluating the association between thera-
peutic responses and SNPs in various genes (Khorasani et al. 2021; Takahashi et al.
2013; Yoon et al. 2011). SNPs in the kt/mammalian target of rapamycin (mTOR),
X-ray repair complementing protein 1 (XRCC1), epidermal growth factor receptor
(EGFR), p53, and cyclin D1 genes have all been related to therapeutic responses.
However, utilizing SNPs to determine therapeutic responsiveness has the limitation
of needing a greater patient cohort and validation sets to draw clinically useful
conclusions (Khorasani et al. 2021; Huang et al. 2017a, b; Stocker et al. 2009;
Takahashi et al. 2013).

11.8.6 Chemotherapy-Associated Metabolism Genes

5-Fluorouracil is a widely used chemotherapeutic drug in UDT adenocarcinoma

(5-FU). This uracil analog is rapidly transferred into cancer cells, where
dihydropyrimidine dehydrogenase converts it to uracil. It has been proposed that
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 305

evaluating these enzymes’ expression in tumors may provide information about the
5-FU response. In UDT adenocarcinoma treatment, i.e., 5-FU-based chemotherapy
alone or in conjunction with radiation, higher thymidylate synthase (TS) expression
has been associated with a poor therapeutic response and outcome (Harpole et al.
2001; Huang et al. 2017a, b; Smid et al. 2016). However, this observation is not
conclusive, as at least one study found no relation between TS levels and outcome
after treatment with a 5-FU. Dihydropyrimidine dehydrogenase (DPD),
methylenetetrahydrofolate reductase (MTHFR), thymidine phosphorylase (TP),
orotate phosphoribosyltransferase (OPRT) are all 5-FU-related enzymes that have
been studied in the framework of predicting responsiveness to 5-FU-containing
regimens (Miyazaki et al. 2010; Wang et al. 2016).

11.8.7 NF-kB

Chronic inflammatory mechanisms, regulated by NF-κB, are known to be involved

in invasive UDT adenocarcinoma progression (Yang et al. 2012). This protein is
overexpressed in UDT adenocarcinoma and associated with chemo- and
radioresistance in vitro (Li and Sethi 2010). Multiple studies have been done to
evaluate its involvement in predicting therapy response due to these findings. Izzo
and colleagues focused on pretreatment NF-κB expression in 43 patients with UDT
adenocarcinoma who were part of a prospective study (Gambhir et al. 2015). They
discovered that elevated NF- κB expression levels before treatment were linked to a
poor response to docetaxel-based chemoradiation and poor survival. Some reports
have corroborated this conclusion.
Furthermore, it has been shown that chemoradiation can activate NF-B, and this
effect is linked to poor survival (Izzo et al. 2009). Other studies also indicated that
chemoradiation-induced NF-B downregulation is linked to enhanced therapeutic
response (Duggan et al. 2018). This phenomenon tends to be specifically predictive
of chemoradiation response rather than generally prognostic. At least one group has
associated NF-B overexpression with a better prognosis in UDT adenocarcinoma
patients that were only treated with surgery (Huang et al. 2016a, b).

11.8.8 Excision Repair Cross-complementing 1 (ERCC1)

The development of cross-linked DNA adducts by cisplatin and other platinum

compounds is the primary process by which these substances exert their cytotoxic
effects. Nucleotide excision repair, a complex mechanism involving the identifica-
tion of impaired DNA strands, excision of the DNA-adduct, and “filling in” of the
excised strand, is thus used to repair these DNA lesions. ERCC1, required for the
impaired DNA strand’s cleavage, is a key protein in this process. Several studies
have investigated the relationship between low levels of ERCC1 expression and
responses to platinum-based chemotherapy. Decreased levels of ERCC expression
are related to higher response rates and longer survival. Pretreatment ERCC1
306 K. A. Janjua et al.

expression has been associated with responses to the platinum-based chemotherapy

and patient results in UDT adenocarcinoma in various retrospective and prospective
studies (Chen et al. 2013a, b; Fareed et al. 2010; Jinjia et al. 2019; Zhuo et al. 2020).
Furthermore, patients with elevated ERCC1 expression levels in their tumors
have poor responses to platinum-based chemoradiation (Warnecke-Eberz et al.
2004). This finding, however, is not universal, as two retrospective studies in UDT
adenocarcinoma found no relation between ERCC1 expression and responses to
platinum-based chemotherapy (Langer et al. 2005, 2010). As a result, ERCC1
expression is rarely used in clinical decision-making for the treatment of UDT.

11.8.9 ATP-Binding Cassette Transporters (ABC Transporters)

Efflux of the drug from the cell before reaching therapeutic concentration is one of
the highly investigated chemotherapeutic resistance methods. ABC transporters
execute this process as they move the drugs out of the cell in an ATP-dependent
manner (Ge et al. 2018). Chemotherapeutic resistance has been linked to several
proteins in the ABC superfamily. Overexpression of Multidrug Resistance Protein
1 ( p-glycoprotein, MDR-1) and multidrug resistance-associated protein-1 (MRP-1)
in UDT adenocarcinoma has been linked to poorer chemotherapy response (Langer
et al. 2010; Shi and Gao 2016). In UDT adenocarcinoma, MDR-1 overexpression is
attributed to a weak response to concurrent chemoradiation (Fujishima et al. 2017).
Also, chemotherapy treatment has been shown to suppress MRP-1 expression in
UDT adenocarcinoma (Blank et al. 2016). Thus, these proteins are the most valuable
predictive markers in reducing cancers cell resistance and combating the UDT
adenocarcinoma.

11.9 Novel Biomarkers for Pancreatic Disease Treatment

and Diagnosis

Pancreatic cancer is an incurable condition that often stays unmedicated. The

mortality-to-incidence rates for pancreatic cancer are the largest of any solid
tumor. Pancreatic cancer management includes a new, multidisciplinary approach
that combines fundamental clinical research in diagnosis, identification, and therapy
(Klein 2019). Multiple experiments and clinical studies have been conducted in
recent years to find potential biomarkers for various pancreatic cancer forms
(Khomiak et al. 2020). Serum and other body fluids, like urine and pancreatic
juice, are sources of less invasive biomarker screening sources (Sahni et al. 2020).
The majority of genetic abnormalities identified in pancreatic cancer are
deletions, duplications of various chromosomal loci, oncogene mutations and
tumor suppressor gene mutations/deletions. These genes include; KRAS, TP53,
BRCA2, MADH4/SMAD4/DPC4, and CDKN1A/p16 (Sahni et al. 2020).
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 307

11.9.1 Angiogenesis Factors

Antiangiogenic agents have been more commonly used in cancer therapy, but it is
essential to discover the candidate biomarkers to assess the resistance and response.
EGF (Epidermal Growth Factor), VEGF (Vascular Endothelial, Growth Factor),
heparanase, thrombospondin, cathepsins are the most important angiogenic factors
pancreatic cancer. Overexpression of EGF and its receptor EGFR has been
associated with tumor staging, but there is currently no firm proof of overall survival
(Heidemann et al. 2006). Another essential element that works on the angiogenesis
mechanism is VEGF. Pancreatic ductal adenocarcinoma (PDAC) is enhanced by
interacting with MMP-9, a cellular matrix remodeling component. In pancreatic
cancer, therapy targeting both MMP-9 and VEGF resulted in a substantial reduction
in PDAC progression and microvessel density than single target treatment (Pistol-
Tanase et al. 2008).
The level of thrombospondin (TSP-1) expression has been linked with the
progression of PDAC. TSP-1 protein is present in abundance in the stroma
surrounding tumor cells, and its expression is found to be inversely related to the
density of microvasculature (Tobita et al. 2002). The role of cathepsins in the
development and progression of pancreatic cancer is still controversial.
In PDAC, the proteins cathepsin l (CTSL) and cathepsin b (CTSB) is found to be
overexpressed. The role of cathepsins in local tumor invasion is inferred by an
association with perineural invasion and CTSB expression level (Niedergethmann
et al. 2000).

11.9.2 Gene Expression and Potential Factors

New potential factors implicated in pancreas oncogenesis are identified in studies

focused on gene expression levels quantification (Sahni et al. 2020). The STAT3
transcription factor is discovered to be activated constitutively in PDAC. It is a key
factor in the self-renewal of stem cells and cancer cell survival and inflammation.
The tyrosine phosphorylated STAT3 levels and the gp130 receptor are highly
associated. STAT3 activation in pancreatic cancer has also been related to an
upregulation of the IL6/LIF-gp130 pathway (Corcoran et al. 2011). According to
that study, STAT3 is necessary for the progression of precursor pancreatic lesions
such as acinar-to-ductal metaplasia (ADM) and PanIN, so gp130 and phospho-
STAT3 expression may be a potential biomarker.
Calcium-binding protein S100P also gained particular attention. This protein is
reported highly expressive in the pancreatic precursor lesions (PanIN 2 or PanIN 3)
and pancreatic tumor, thus making the way via quantification of its expression level
for earlier diagnosis (Prica et al. 2016). S100P mRNA expression was found higher
in the pancreatic juice of patients with pancreatic cancer and IPMN (Crnogorac-
Jurcevic et al. 2013). Three members of the S100A family (S100A2, S100A4, &
S100A6), on the other hand, have been associated with poor prognosis. Cell cycle
308 K. A. Janjua et al.

control and cell invasion are also controlled by members of the S100A family
(Tanase et al. 2010).

11.9.3 ZIP3 (Zinc/Iron-Regulated Transporter-Related Protein 3)

Zinc plays a key role in cellular processes such as cell division, immune response,
and free radical defense; zinc deficiency has been associated with a high risk of
cancer (Sapkota and Knoell 2018; Skrajnowska and Bobrowska-Korczak 2019).
In adenocarcinoma, Costello et al. discovered a significant deficiency of zinc in
the ductal and acinar epithelium compared to the normal epithelium. The reduction
in zinc quantity is a feature of both pancreatic cancer and precursor lesions. ZIP3
(basilar membrane zinc uptake transporter) gene expression is present in normal
ductal/acinar epithelium but absent in adenocarcinoma. The deficiency of zinc in
early and advanced malignancy is determined by lowered ZIP3 expression (Costello
et al. 2011).

11.9.4 Cancer Stem Cell Biomarkers

Several surface markers of pancreatic cancer cells with stem cell features have been
identified. The cell surface markers CD24, CD44, and ESA, have been used to
identify these cancer stem cells (Gzil et al. 2019). These human cells can self-renew
and produce distinct progeny when inserted into the pancreas of immunocompro-
mised mice, mimicking a tumor’s phenotype from which they were generated. A
highly tumorigenic subpopulation of cancer stem cells was isolated from PDAC
patients. These CD133+ cells were capable of inducing tumor development in
athymic mice. The presence of the CXCR4 receptor distinguishes this subpopulation
of circulating cancer stem cells, which is involved in tumor metastasis and conse-
quently could be good predictors in diagnosing and identifying the new ways of
treatment of pancreatic cancer (Zhu and Yuan 2015).

11.9.5 Saliva Biomarkers

Recent studies have proposed that saliva may quantify particular factors to distin-
guish between patients with pancreatic cancer and those with mild or chronic
pancreatitis. With a sensitivity of 90% and a precision of 95%, the Zhang et al.
group differentiate the pancreatic cancer patients from healthy control using
transcriptome profiles. The researchers discovered 12 mRNA biomarkers that are
specific to pancreatic cancer patients. MBD3L2, KRAS, STIM2, MBD3L2,
DMXL2ACRV1, CABLES1, DMD were found to be upregulated, while TK2,
GLTSCR2, CDKL3, DPM1, TK2, TPT1 were found to be downregulated (Zhang
et al. 2010).
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 309

Another group used mass spectrometry to determine metabolites in saliva and

came up with a similar finding. Based on a pancreatic cancer-specific characteristic,
cases of pancreatic cancer were successfully identified. Patients with breast or
pancreatic cancer had higher ornithine and putrescine levels, whereas patients with
oral cancer had somewhat higher levels. Pancreatic cancers have higher tryptophan
levels, while arginine levels are lower in many cancers, including breast, colonic,
and pancreatic cancers, which may be attributed to elevated arginine uptake by
tumor tissues with enhanced arginase activity (Sugimoto et al. 2010).

11.9.6 Pancreatic Juice Biomarkers

The evaluation of protein expression profiles in pancreatic juice samples taken from
a pancreatic duct can identify markers that could be used to differentiate between
benign and malignant pancreatic lesions and differentiate between various phases of
PDAC. Vareed et al. discovered that 56 proteins were observed to be escalated in the
pancreatic juice of PDAC patients compared to controls (Schneider et al. 1984).
Increased levels of many metabolic enzymes were also discovered in the PDAC-
associated secretory proteome study. Purine nucleoside phosphorylase (NP), an
enzyme involved in the purine salvage pathway that is active during inflammation
and neoplastic development, is the most important metabolic component (Bantia
et al. 2010). NP activity is elevated in cancer sera, and NP expression, in conjunction
with another component, adenosine deaminase (ADA), has been used to assess the
clinical severity of different types of cancers (Kutryb-Zajac et al. 2018).
The strong correlation between NP and inflammation prompted researchers to
consider this protein’s expression and activity in PDAC patients, especially those
with antecedent inflammatory conditions such as chronic pancreatitis and pancreatic
intraepithelial neoplasia (Rebours et al. 2010). As a result of this relationship,
measuring NP levels may be a valuable marker for monitoring the progression
of PDAC.

11.9.7 Plasma Biomarkers

New promising biomarkers for early detection of pancreatic cancer have been
published in recent papers (Balasenthil et al. 2017). Roberts et al. studied blood
samples from patients with pancreatic adenocarcinoma that was either locally
advanced or metastatic. The report identified one putative prognostic protein, i.e.,
alpha 1-antichymotrypsin (AACT), and two putative predictive proteins, comple-
ment factor H (CFH) and histidine-rich glycoprotein (HRG). Overall survival
(OS) was negatively associated with AACT, while CFH had no predictive signifi-
cance as a prognostic factor for OS (Longo et al. 2016). Thus, AACT may be a
reliable prognostic marker in patients with advanced-stage pancreatic carcinoma.
310 K. A. Janjua et al.

11.9.8 Metabolomic Biomarkers

Studying the metabolome is a new method for detecting cancer signatures. There are
currently not commonly used metabolomic markers in clinical practice for prognosis
analysis, diagnosis, or chemotherapy response estimation. However, several studies
(Di Gangi et al. 2016; Goldberg et al. 2019; Moore et al. 2019; Tumas et al. 2019)
have shown distinctions in metabolic profiles in PDAC patients, patients with some
other conditions, and healthy controls. Mayerle et al. aimed at the blood samples’
metabolic profile from 914 patients with CP, liver cirrhosis, PDAC, healthy and
non-pancreatic disease controls (Mayerle et al. 2018). The findings reveal a bio-
marker signature (nine metabolites plus CA 19-9) that effectively differentiated
PDAC from CP.
The metabolomic profiles of cancerous tissue from 25 patients who had
undergone curative resection and adjunctive gemcitabine-based therapy were
investigated in another study. Lactic acid levels were noted to be elevated in the
tumors of patients who had adverse health results after receiving gemcitabine.
Patients with lowered lactic acid levels and higher hENT1 protein expression had
a slightly longer survival time than the other groups (Phua et al. 2018). While further
standardization and validity of candidate metabolic signatures are needed,
metabolomics could be a successful method for developing novel diagnostic, prog-
nostic, or predictive markers for pancreas cancer.

11.10 Novel Biomarkers for Diagnosis and Prognosis

of Esophageal Cancer

Oesophageal cancer is a fatal disease with a poor prognosis and limited available
treatment options. Cancer Research UK has declared it as a “cancer of unmet need”
(Lavery and Turkington 2021). In developed countries, the 5-year survival rate for
esophageal cancer (EC) is less than 10%, with esophageal squamous cell carcinomas
(ESCC) accounting for more than 90% of cases. There are currently no appropriate
adjuvant diagnostic biomarkers for ESCC to help with dysplasia diagnosis (Ishiguro
et al. 2013; Taylor et al. 2013). Biomarker assessment may greatly strengthen the
diagnostic accuracy by reducing the subjectivity involved with histologic dysplasia
assessment (Couch et al. 2016). Identifying the suitable biomarkers for ESCC and
squamous cell dysplasia that would be suitable as adjunctive use for pathological
diagnosis and may provide a deep insight into molecular pathways leading to
carcinogenesis (Lavery and Turkington 2021).

11.10.1 Mutations and Polymorphisms

Gene-based biomarkers play a significant part in cancer care. Mutations in the TP53
gene are the most commonly diagnosed genetic alterations in EC (Hao et al. 2013). A
study demonstrated that the TP53 gene knockout rate is strongly associated with the
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 311

degree of differentiation and lymph node metastasis in EC (Niyaz et al. 2020). It may
be useful for determining the clinical condition of EC patients (HUILI Zheng et al.
2016).
Other significant genetic mutations that affect esophageal tumorigenesis are
single nucleotide polymorphisms (SNPs) (Wu et al. 2013). According to research
that links EC and genetic polymorphisms, polymorphisms can be potential markers
for evaluating the risk of EC (Li et al. 2014a, b). Renouf et al. found that esophageal
adenocarcinoma (EAC) patients with p53 Pro/Pro have a shorter survival time
(Renouf et al. 2013). Cescon et al. discovered that p53 Arg72Pro Pro/Pro is linked
to a twofold rise in EC patients’ mortality risk (Cescon et al. 2009). Several other
analyses found a link between TP53 Arg72 carriers and a lower EC risk (Jafrin et al.
2020).
CYP1A1 is another studied polymorphism (Zeng et al. 2015). Several researchers
investigated the relationship between the CYP1A1 polymorphism, and the EC risk
have shown inconsistent effects, which can be explained by ethnic and environmen-
tal factors (Gong et al. 2014; Shen et al. 2013; Zheng and Zhao 2015). In central
China, genetic polymorphisms in the CYP1A1 gene are related to a higher EC risk
(Yun et al. 2014). However, after controlling for contributing causes, a study
observed that no CYP1A1 genotypes and alleles are linked to the risk of EC (Shen
et al. 2013). More comprehensive studies with improved designs are required to
verify the relationship between CYP1A1 polymorphisms and EC vulnerability
(Zheng and Zhao 2015).

11.10.2 Genomic Instability

Both chromosome instability (CIN) and microsatellite instability (MSI) are

implicated in EC progression (Forghanifard et al. 2016). A small number of
researchers have reported its values in EC prognosis.
CIN, in a general context, is referred to as a range of chromosome changes.
Losses of chromosomes, i.e., 5q, 11p, 12q, and 17p, are common in EC and are
linked to the patient’s outcome. Certain essential genes are still involved in chromo-
some restructuring; these changes have been observed in certain genes like MYC,
FHIT, etc. (Paulson et al. 2009; Xing et al. 2009).
MSI is a valuable biomarker in EC because patients with a positive MSI have
specific features and prognosis. According to Matsumoto Y et al., esophageal
squamous cell carcinoma patients have a comparatively high MSI-L prevalence.
The degree of MSI-L is inversely related to the depth of invasion (Jiao et al. 2019).
Changes in these genes trigger a cascade of mutations, which eventually contribute
to the emergence of malignant phenotypes. This relation has been discovered in
genes like MLH1, MSH2, BAT25, and BAT26 (Dudley et al. 2016).
312 K. A. Janjua et al.

11.10.3 Proteomics Biomarkers

The serum contains a large number of peptides, some of which are promising
biomarkers for cancer management. An abnormal proteomics molecule levels are
associated with health status, thus helpful in the prognosis of EC (Amiri-Dashatan
et al. 2018).
Modern techniques used in proteomic studies have suggested several contender
biomarkers in EC tissues, such as HMGB3, ANGPTL2, LSD1, and EI24.
Tissue biomarkers’ detection via proteomics-based approaches accelerated the
assessment and management of EC. For instance, EI24 is identified as a potential
predictive biomarker by using iTRAQ and some other methods by a research group
for the prognosis and management of EC (Hong et al. 2015). Recently, in discover-
ing the new EC biomarkers, cell lines have also been used as valuable sources of
proteins (Jiménez et al. 2017). However, further investigation is required due to the
heterogeneity in various cell lines.

11.10.4 Epigenetic Biomarkers

LncRNAs have depicted a considerable potential as prognostic biomarkers in

EC. Some reports have indicated that EC has a lncRNA profile, with particular
upregulated or downregulated genes, and potentially valuable in the prognostication
approach (Sugihara et al. 2015; Yao et al. 2016). Furthermore, some of them can be
used to monitor EC, such as higher expression of the lncRNA SPRY4- IT1 in ESCC
patients is an independent prognostic factor (Xie et al. 2014). According to Li JY
et al., ESCC patients with higher lncRNA UCA1 expression have an advanced
clinical stage and a worse outcome than lower expression (Kang et al. 2018). Several
studies found that lncRNA HOTAIR is overexpressed in ESCC tissues compared to
normal controls and that elevated expression is linked to poor ESCC patients’
survival (Chen et al. 2013a, b; Ma et al. 2015). MALAT1 lncRNA expression is
also associated with the prognosis of EC patients (Huang et al. 2016a). lncRNAs like
lncRNA-uc002yug2, lncRNA LOC285194, lncRNA FOX CUT, lncRNA CASC9,
lncRNA ZEB1-AS1[156], and lncRNA CCAT2, have been linked to EC patients’
prognosis (Tang et al. 2015). According to Liu et al., an elevated level of lncRNA
BANCR in plasma is associated with shorter survival, indicating that it could be used
as a tumor biomarker for early detection of EC, a prospective prognostic biomarker,
and a possible therapeutic target for EC patients (Liu et al. 2016b).

11.10.5 miRNA

miRNA alterations have been discovered in EC tissues and cells. So, reshaping the
miRNA expression patterns can help to prevent cancers from developing malignant
phenotypes. Some of these miRNAs, such as miR-27a/b, miR-335, let-7c, miR-145,
miR-21, miR-133, miR-148, and others, can serve as biomarkers for predicting
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 313

prognosis (Hiyoshi and Watanabe 2015). Chen and colleagues reported the
MiR-133a/b as an independent prognostic factor for ESCC patients’ survival
(Chen et al. 2014).

11.10.6 DNA Methylation

DNA methylation is an epigenetic modification that occurs in CpG dinucleotides at

“carbon 5” of the cytosine ring (Irwin et al. 2019). Detection of methylation
biomarkers in body fluids like serum and plasma is the most significant aspect of
these markers. The p16 gene methylation is a typical biomarker found in body fluids.
P16’s methylation pattern can be observed in blood and used as a marker for early
disease detection and control (Abbaszadegan et al. 2008). Other serum markers, like
PTX3, and MGMT have recently been identified as valuable prognostic biomarkers
(Das et al. 2014; Wang et al. 2011). SOCS-1, HLA-I, CDH13, EPB41L3,
NMDAR2B, MTHFR677C > T, and other genes with altered methylation patterns
have also been identified as helpful prognostic markers (Yang et al. 2019).

11.10.7 Exosomes

Liquid biopsies are advantageous over traditional tissue biopsies regarding cancer
genotyping evaluation, thus making every tumor type distinct (Huang et al.
2017a, b). Liquid biopsy can also be a predictive biomarker for prognosis and
therapeutic response in patients with EC.
Exosomes are small particles (ranges from 30 to 120 nm) released from cells in
normal or abnormal conditions and play a significant role in intercellular interaction
(Golyan et al. 2020). Exosome formation is not only an essential regulatory process
during cancer progression, but it also plays a role in cancer diagnosis, treatment
evaluation, and prognosis interpretation (Li et al. 2017). Exosomes played an
important role in the growth of the EC (Liu et al. 2018). Studies have shown that
the number of exosomes can be used as an independent prognostic marker, such as
low level of exosomes demonstrates terrible effects in ESCC patients (Matsumoto
et al. 2016). In ESCC patients, the expression of exosomal miR-1246 and miR-21 is
linked to the tumor classification stage (Matsumoto et al. 2016). Exosome-associated
DNA may also be used to identify tumor-specific genetic variations or determine the
therapeutic response in esophageal adenocarcinoma (EAC) (Smith and Lam 2018).
Exosomes are likely to be novel biomarkers for EAC and ESCC, but the
observations mentioned above need to be validated in further animal models and
major cohort clinical trials.
314 K. A. Janjua et al.

11.10.8 Circulating Tumor Cells (CTCs)

Clinically, disseminated tumor cells (DTCs) and circulating tumor cells (CTCs) have
been linked to patient prognosis (Dasgupta et al. 2017). Wang and colleagues
observed that the occurrence of CTCs in blood samples is associated with poorer
disease-free survival (DFS) and progression-free survival (PFS) of EC patients as
compared to CTC-negative cases (Wang et al. 2017b). The CTCs marker appears to
be a promising therapeutic response marker, prognosis, recurrence risk, and treat-
ment decisions. However, as contrasted to data on biologic markers like squamous
cell carcinoma antigen (SCC) or arcinoembryonic antigen (CEA), there is also a lack
of supporting data to use CTCs systematically. Furthermore, there are certain
drawbacks associated with CTC identification approaches (Grover et al. 2014).

11.10.9 Circulating Tumor DNA (ctDNA)

Circulating tumor DNA (ctDNA) has recently been identified as another biomarker
with clinical implications (Gabriel and Bagaria 2018). Cellular apoptosis
(70–200 bp) and necrosis (200 bp–21 kbp) are the two major sources of ctDNA.
The tumor cells have many chromosomal alterations compared to the normal cells,
majorly depends on ctDNA and can be a novel biomarker (Leary et al. 2012). As a
result, ctDNA screening may be a promising and effective approach for early cancer
detection.

11.11 Novel Biomarkers for Diagnosis and Prognosis

of Colorectal Cancer

Colorectal cancer (CRC) is a common type of cancer, which poses a greater

morbidity and mortality rate worldwide (Rawla et al. 2019). Despite recent advances
in the development of screening programs and the management of colorectal cancer
patients, there are still many challenges to be addressed, ranging from prevention
and early detection to determine the prognosis. The development of personalized
medicine and increased survival rates could be encouraged by discovering new
biomarkers to assist CRC’s early detection or treatment.
An effective CRC biomarker must be easy to quantify, highly sensitive and
specific, reproducible and reliable. These priorities can ideally be accomplished
with a noninvasive and low-cost approach that uses readily available biological
samples like urine, breath, serum, or feces (Chand et al. 2018; Choi et al. 2017;
Loktionov 2020).
Colorectal cancer screening strategies lead to identifying and removing adeno-
matous polyps and other premalignant for decreasing CRC mortality (Alves Martins
et al. 2019).
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 315

11.11.1 Caudal Type-Homeobox 2

Caudal type-homeobox 2 (CDX2) encodes a homeobox protein majorly involved in

regulating cell division of the normal cell in the colon. CDX2 expression loss of
might results in colorectal cancer. It is also noted that immunohistochemical finding
of CDX2 protein expression could be useful in identifying the CRC. The study has
revealed a high level of CDX2 expression in CRC.
Detecting CRC by evaluating CDX2 expression is also a very responsive and
explicit method. CDX2 expression in CRC has been studied extensively, and its
specificity and sensitivity have been observed greater than 90% (Asgari-Karchekani
et al. 2020; Werling et al. 2003).

11.11.2 Cytokeratins (CKs)

Cytokeratins are keratin proteins present in an intracytoplasmic cytoskeleton of

epithelial tissue. Generally, to distinguish metastatic CRC from the other tumors,
tissues are stained for CK7 and CK20. CRC specimens stain positive for CK20 but
negative for CK7. Ten CK20 is only found in Merkel cells and gland cells of colonic
mucosa. CK7, on the other hand, is not found in the mucosa of the colon. CK7 is
found in the epithelia of the bladder, mesothelium, normal lung and female genital
tract (Kigasawa et al. 2017). For distinguishing the metastatic adenocarcinoma of
unidentified primary origin, CK staining patterns are one of the most effective
procedures. The CK7–/CK20+ pattern is a common method for diagnosing meta-
static CRC. 12 A CK7–/CK20+ pattern has been documented in 65–95% of CRC
cases (Bayrak et al. 2011).

11.11.3 Cadherin 17

Cadherins are cell-to-cell adhesion molecules that play a critical role in tissue
structure normally. Many human disorders, including carcinomas, are linked with
molecular abnormalities in cadherin expression (Lee et al. 2010). According to
studies, CDH17 is represented in 96–100% of primary CRC and 100% of metastatic
CRC. Some studies have shown that CDH17 is more specific and sensitive than
CDX2 for detecting CRC (Bian et al. 2017; Su et al. 2008).

11.11.4 Special AT-Rich Sequence Binding Protein 2 (SATB2)

Special AT-rich sequence binding protein 2 is a part of the region binding transcrip-
tion factors family. Even though the exact function of SATB2 in the GI tract is
unknown (Zhang et al. 2018). Magnusson et al. discovered that SATB2 is more
expressive in the epithelium of a lower GI tract, especially in the colon (Magnusson
et al. 2011). The researchers examined the SATB2 expression profile of 216 cancer
316 K. A. Janjua et al.

samples and discovered that the maximum CRC samples had elevated SATB2
expression. SATB2 was noted to be positive in 87.8% of CRC cases when used as
a single marker (Magnusson et al. 2011; Zhang et al. 2018). The addition of SATB2
to the standardized panels of CK7, CK20, and CDX2 has been studied extensively.
According to Dragomir et al., the addition of SATB2 to standardized panels did not
improve the specificity and sensitivity in diagnosing CRC (Dragomir et al. 2014).
But according to two recent studies, SATB2 has been found as a significant marker
for distinguishing the metastatic CRC from primary ovarian carcinomas (Moh et al.
2016).

11.11.5 GPA33

The GPA33 gene codes for the membranous protein A33, a membrane-bound
glycoprotein, have a homolog in an immunoglobulin superfamily (Opstelten et al.
2020). Although the role of A33 is unknown, experimental findings suggest that it
could be linked with immunological processes, proliferation, and colonic mucosal
repair (Pereira-Fantini et al. 2010). According to immunohistochemical studies, A33
express by epithelial cells in the colon and rectum. It is found expressed in 95% of
CRC cases in humans, notably in well-differentiated tumors, suggesting that it may
target CRC treatment (Murer et al. 2020). An immunohistological analysis compar-
ing A33 and CDX2 found that A33 has equal susceptibility to CDX2 but higher
specificity than CDX2 as a CRC immunomarker (Wong et al. 2017).

11.11.6 Telomerase

It is a ribonucleoprotein that regulates TTAGGG repeats to telomeres at the ends of

chromosomes for reverse transcription; telomerase utilizes intrinsic RNA as a
template (Jafri et al. 2016). Telomeres shorten with every cell division of normal
cells. When telomeres shorten to critical lengths, a signal of DNA damage is
triggered, resulting in replicative senescence (Victorelli and Passos 2017). By
upregulating telomerase, cancer cells suppress DNA damage-induced inhibitory
signaling pathways. It has been observed in 85–90% of all malignant tumors
(Shay and Bacchetti 1997). Telomerase has emerged as a recent diagnostic bio-
marker in CRC. According to some studies, it is expected to have 95% sensitivity
and specificity in CRC (Roig et al. 2009).

11.11.7 MicroRNA (miRNAs)

MicroRNAs are small non-coding RNAs with 18–25 base pairs that control gene
expression via binding to mRNA. By functioning as oncogenes, miRNAs are known
to be linked to various cancers, including CRC (Cui et al. 2019). MiRNAs have
extensive blood stability than mRNA because they are not affected by the
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 317

endogenous RNase. So, miRNAs are potential noninvasive cancer biomarkers.

Around 48 studies have tested their diagnostic potential for CRC (Condrat et al.
2020; Oh and Joo 2020).

11.11.8 Insulin-Like Growth Factors Binding Protein 2 (IGFBP-2)

Insulin-like growth factor ligands and IGF-1 receptors interact with a binding protein
called insulin-like growth factor binding protein 2 (Ding and Wu 2018). According
to many studies, elevated IGFBP-2 levels in serum are associated with colon cancer
as well as other tumors (el Atiq et al. 1994). High serum and plasma IGFBP-2 levels
could differentiate patients with colon polyps or CRC from healthier controls.
IGFBP-2’s sensitivity and specificity for early CRC and colon polyp detection are
ineffective, but combining it with other biomarkers such as CEA could enhance
sensitivity. Furthermore, high plasma IGFBP-2 levels are linked to larger tumor
sizes, suggesting that IGFBP-2 may be used as a diagnostic and prognostic bio-
marker for colorectal cancer (Renehan et al. 2000).

11.11.9 Long Non-coding RNAs (lncRNAs)

Long non-coding RNAs consist of more than 200 non-translatable nucleotides. More
than 150 human morbidities, including colon cancer and other cancers, are
associated with lncRNAs (Wang et al. 2019). Single or panel lncRNAs have been
used in 55 studies to determine their diagnostic potential for CRC (Oh and Joo
2020).
Hypoxia-inducible factor 1-alpha-antisense RNA 1 (HIF1A-AS1) level in serum
was substantially higher in 151 CRC patients than 160 healthy controls, indicating
that HIF1A-AS1 has a high diagnostic potential for CRC (Gong et al. 2017). A splice
variant of CRNDE-h (colorectal neoplasia differentially expressed) has been shown
to differentiate CRC patients from a healthy control (Liu et al. 2016a, b).
Other lncRNAs, such as ZNFX1 antisense RNA1 (ZFAS1), NEAT1, and
GAS560, have shown potential as diagnostic and prognostic biomarkers for CRC
(Fang et al. 2017; Peng et al. 2017).

11.11.10 Circulating Cell-Free DNA (cfDNA)

It is essentially a type of cell-free nucleic acid that enters the bloodstream after
necrosis or apoptosis. CfDNA releases from apoptotic cells in healthy individuals.
The DNA fragments are around 180 bp long, but cfDNA, on the other hand, is much
larger fragments present in tumor cells (Fernandez-Garcia et al. 2019). As a result,
quantitative study of circulating cfDNAs using the ratio of longer to shorter DNA
fragments or determining cfDNA integrity number while CRC diagnosis yielded
promising results. A systematic review and meta-analysis on circulating cfDNA as a
318 K. A. Janjua et al.

diagnostic marker for CRC were published in which the author scrutinized the
14 studies for a total of 1258 CRC patients with 803 positive controls. The sensitivity
and specificity of circulating cfDNA for CRC diagnosis were 73.5% and 91.8%,
respectively, indicating significant specificity for diagnosing the CRC (Wang et al.
2018a).

11.11.11 Microsatellite Instability

Microsatellites are 1–6 bp repeating DNA sequences present in both the genome’s
coding and non-coding regions. It is also linked with sporadic CRC (Sepulveda et al.
2017). Two mononucleotide repeats, i.e., BAT26 and BAT25 and three dinucleotide
repeats, i.e., D2S123, D5S346, and D17S250, are widely used microsatellite
markers (Salto-Tellez et al. 2005).

11.11.12 BRAF

RAS-RAF-MEK-ERK molecules play a major role mitogen-activated protein kinase

pathway. BRAF-activating mutations are found in about 14% of localized stage and
8% of advanced CRC cases (Caputo et al. 2019). The American Society of Clinical
Oncology released a recommendation for using BRAF as a molecular biomarker for
CRC in 2017. BRAF p.V600 mutations account for about 90% of BRAF mutations
and should be assessed for prognostic significance (Tiwari et al. 2016).

11.11.13 SMAD4

SMAD4 is mainly associated with cell cycle and cell migration (Zhao et al. 2018).
According to studies, SMAD4 mutations are present in 30–40% of CRC cases.
Voorneveld et al. did a meta-analysis to assess the prognostic importance of SMAD4
in patients with CRC (Voorneveld et al. 2015).

11.11.14 p53

Many studies have focused on p53 mutations and their prognostic importance in
CRC patients (Russo et al. 2005). When DNA is impaired, p53 induces cell cycle
arrest to restore the mutations; if it fails to restore, apoptosis is induced (Carethers
and Jung 2015). The findings, on the other hand, are contradictory. According to
some researches, p53 mutation/overexpression is linked to lower disease-free sur-
vival, relapse-free survival, and overall survival frequencies (Oh and Joo 2020).
According to other studies, there is a lack of proof that p53 has prognostic signifi-
cance for CRC (McGregor et al. 2015; Wang et al. 2017a, b, c).
11 Development of Novel Cancer Biomarkers for Diagnosis and Prognosis 319

11.11.15 Neutrophil-to-Lymphocyte Ratio

Lymphopenia is linked to a reduction in cell-mediated immunity, whereas

neutrophilia is linked to systemic inflammation (Ménétrier-Caux et al. 2019;
Soehnlein et al. 2017). Few systematic studies and meta-analyses have looked into
the role of NLR in CRC prognosis. According to a review, patients with higher NLR
had a significantly smaller overall survival and progression-free survival; following
a treatment. Patients with an NLR <5 before treatment have a higher chance of
having a 5-year overall survival and disease-free survival (Li et al. 2014a, b).

11.11.16 CEA Levels

The ASCO 2006 update the guidelines for the treatment of CRC patients supports
CEA as the only marker (“2006 Update of ASCO Recommendations for the Use of
Tumor Markers in Gastrointestinal Cancer,” 2006). Preoperative CEA levels have
been shown to have prognostic significance in several studies (Li Destri et al. 2015).
According to two large-scale case studies, preoperative CEA level is strongly
correlated with prognosis in CRC patients, metastasized to the liver (Fong et al.
1999; Nordlinger et al. 1996). One study focused on 2230 CRC patients and
discovered that CEA levels are strongly associated with patient outcomes (Park
et al. 1999).

11.12 Conclusion

The development of novel cancer biomarkers for diagnosis, prognosis, and drug
therapy response prediction has benefited greatly from a deeper unraveling of the
mutational landscapes in cancer initiation and progression. They have reformed the
approach to patient care in various cancer types. Despite considerable advances,
most cancer biomarkers for different types of cancers are still in the developmental
phase. Many of them are still not approved by the FDA. Future research is expected
to develop sensitive and specific diagnostic, prognostic, and predictive biomarkers
that are more clinically applicable and patient-acceptable, unlike conventional
biomarkers. There is a need to refine further their application for individualized
management of the prospective patients. Improved biomarkers should ultimately
lead to improvements in outcomes and more efficient, safe, cost-effective, and
evidence-based use of health resources.

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Nanotechnology for Cancer Biomarkers
12
Abdul Muhaymin, Uzma Azeem Awan, Adnan Haider,
and Muhammad Naeem

Abstract

Cancer biomarkers play an essential role in early diagnosis, progression,

prediction, and potential response of treatment in cancer patients. However,
the traditional cancer biomarkers detection techniques lack specificity and
selectivity with drawbacks, such as irreproducibility and overdiagnosis.
Nanomaterials, with their unique properties, provide efficient, reliable
biosensing methods with high sensitivity and selectivity. This chapter
represents an overview of the development of nanomaterials-based biosensing
techniques and the integration of these nanomaterials in other techniques, such
as mass spectrometry (MS), Raman spectroscopy, optical detection, electrical
and electrochemical detection, and lab-on-a-chip technology for detection of
cancer biomarkers.

Keywords
Nanomaterials · Cancer biomarkers · Nanobiosensors

A. Muhaymin
CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety & CAS Center for
Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing,
PR China
U. A. Awan · A. Haider · M. Naeem (*)
Department of Biological Sciences, National University of Medical Sciences, Rawalpindi, Punjab,
Pakistan
e-mail: [email protected]

# The Author(s), under exclusive license to Springer Nature Singapore Pte 345
Ltd. 2022
A. Shehzad (ed.), Cancer Biomarkers in Diagnosis and Therapeutics,
https://doi.org/10.1007/978-981-16-5759-7_12
346 A. Muhaymin et al.

12.1 Introduction

“A biomarker is any substance, structure, or process that can be measured in the

body or its products and influence or predict the incidence of outcome or disease”
(Lassere 2008). Therefore, a cancer biomarker is the one that can predict the
development and progression of cancer in a specific tissue or potential response to
cancer therapy (Micheel et al. 2012). Cancer biomarkers can be classified into
predictive biomarkers, prognostic biomarkers, and diagnostic biomarkers. A predic-
tive biomarker is the one that predicts drug response, e.g., response to trastuzumab in
breast cancer by measuring activation of HER2 protein (Slamon et al. 2001; Piccart-
Gebhart et al. 2005; Romond et al. 2005). A prognostic biomarker can predict future
development of tumor, e.g., 21-gene recurrence score that can predict survival and
recurrence in node-negative tamoxifen-treated breast cancer (Paik et al. 2004). A
diagnostic biomarker is used to identify the present condition of cancer, e.g., testing
for cancer DNA in the stools has recently been implemented for colorectal cancer
surveillance (Imperiale et al. 2014; Goossens et al. 2015).
Cancer biomarkers detection could have a significant contribution in the early
diagnosis of a tumor and hence, a great impact on the successful treatment of cancer
(Ye et al. 2018). However, traditional cancer screening methods, such as the pap
smear for cervical cancer, immunohistochemistry, conventional cytogenetics,
prostate-specific antigen (PSA) blood test for prostate cancer, and the fecal occult
blood (FOB) test are not significantly successful for the precise detection of early-
stage cancer biomarkers due to complication of overdiagnosis (Etzioni et al. 2002),
inconsistency (Cottet et al. 2006), and insufficient sensitivity/specificity of individ-
ual markers. The detection of cancer biomarkers has also been limited by several
other barriers, e.g., heterogeneity and low concentration of biomarkers in bodily
fluids, the low half-life of cancer biomarkers, and complications in the analysis (Hull
et al. 2014; Zhang et al. 2019). Thus, it is pivotal to develop novel technologies that
are sensitive and selective enough to detect cancer biomarkers (Zhang et al. 2013).
Therefore, efforts are focusing on the discovery of innovative, efficient, reliable, and
authenticated detection of biomarkers for early cancer diagnosis (Patel and Ahmed
2015; Chen et al. 2015). In this regard, by employing nanotechnology, the develop-
ment of nanosensor-based biomarkers amplifiers has improved assay sensitivities
and specificity (Jiang et al. 2015; Kwong et al. 2013). The history of biosensors in
different eras is illustrated in Fig. 12.1.
Nanotechnology defines as the formation and manipulation of materials at nano-
scale levels to create products that display unique properties. It has a multidisciplin-
ary approach that has the potential to emerge as one of the most promising fields in
cancer diagnosis and treatment (Sengupta et al. 2005). The application of
nanotechnology-based materials in cancer has exceptional potential for
revolutionizing cancer diagnostics and therapeutics (Misra et al. 2010). The
nanomaterials having at least one dimension in the nanoscale that have distinct
properties as compared to bulk counterparts (Kagan et al. 2016; Weiss 2010). It
provides a measurement of multiple targets simultaneously with high specificity and
selectivity and can improve specific targeting of biosensors by conjugation with
12 Nanotechnology for Cancer Biomarkers 347

Fig. 12.1 History of biosensors. (Source: Icons from www.flaticon.com)

nanoparticles/nanomaterials (Sharifi et al. 2019). In addition, the high surface area to

volume ratio of nanoparticles makes the detection of specific biomolecules a lot
simpler by making biosensors more sensitive (Doria et al. 2012). Polymer dots
(PDs), quantum dots (QDs), and gold nanoparticles (AuNPs) are the three most
common types of nanoparticle probes used in the detection of cancer biomarkers
(Harun et al. 2013).
This chapter focuses on the presentation of various nanomaterials-based
platforms for the biosensing and detection of cancer biomarkers. Further, how
nanotechnology is contributing to new methods in the screening of biomarkers in
cancers, with an emphasis on research works published during the recent past years.

12.2 Nanomaterials-Based Biosensing Platforms

Several nanomaterials-based biosensing platforms have been used in the detection of

cancer biomarkers. These platforms integrate several nanoscale components and
utilize both synthesis approaches of top-down and bottom-up technologies. In this
section, we will describe the most common nanomaterials platform developed for
biosensing cancer biomarkers.

12.2.1 Nanoparticles

Nanoparticles (NPs)based assays, sensors, and arrays have emerged for the detection
of cancer biomarkers (Malhotra et al. 2012; Zhang et al. 2013). During the past two
decades, NPs have been formulated to obtain desirable biosensing applications due
to their unique chemical and physical properties (Gopinath et al. 2015; Yin et al.
348 A. Muhaymin et al.

2015). The NPs provide the advantage of highly selective, rapid, and sensitive
cancer biomarker detection methods, enabling their application in the identification
of biomarkers even in trace amounts in bodily fluids, ranging from tears to urine.
Different types of NPs have been developed, such as lipid and hybrid NPs (Li et al.
2014), semiconductor (Lu et al. 2015), metal (Yoo and Yeo 2016), metal oxide (Qiao
et al. 2015), and polymer (Battistella and Klok 2017), for the application in cancer
biomarker detection. NPs can be employed in various sensing techniques depending
on their physicochemical properties. The development of nanosensor-based assays
has a major challenge of differentiation of ultra-low biomarkers concentration and
recognition in point-of-care (POC) devices (Gustafsson et al. 2010; Lewis et al.
2015).

12.2.2 Targeting Ligand–Conjugated NPs for Detection

of Biomarkers

Cancer biomarkers are detected in tissue samples, urine, and serum through a range
of targeting ligands, such as peptides and proteins (antibodies and their fragments),
nucleic acids (aptamers), small molecules, or others (vitamins or carbohydrates).
NPs conjugated with tumor-specific ligands having a high binding affinity to trace
cancer biomarkers. They can interact with a minimal amount of cancer biomarkers
and produce a detectable signal to measure them. Moreover, these NPs can also
facilitate the detection of biomarkers by enhancing their secretion from cancer cells
(Ye et al. 2018). Some of the examples are given below.

12.2.3 Protein-Conjugated NPs

Most of the protein-based targeting refers to antibodies and antibody fragments.

Proteins as a targeting ligand have been a major focus in the field of nanotechnology.
Likewise, the enzyme-linked immunosorbent assay (ELISA), immunoprecipitation,
immunoblotting, immunochromatography assays, and NP-based biomarker assays
are also based on specific antibody–antigen interactions. In one such design, an
antibody (Ab)-coated magnetic NP-based one-step assay was developed for the
detection of prostate cancer. These Ab-coated magnetic NPs can be directly injected
into blood plasma even in a very small amount. Prostate-specific antigen (PSA)
concentration is efficiently detected by NPs due to high rotation frequency and
biomarker-induced binding. In undiluted human blood plasma, the detection limit
for PSA measurement was found to be 400–500 fmolL 1 in a total assay time of
14 min, and an optically probed volume of only 1 nL (Ranzoni et al. 2012).
As one of the most common cancer biomarkers, the increased amount of
carcinoembryonic antigen (CEA)is associated with cancers of the liver, colon,
rectum, prostate, and ovary(Duffy 2001). An electrochemiluminescence (ECL)
sensor was developed through a multistep procedure to detect the threshold level
of CEA(Jie et al. 2011). The fabrication of the sensor first involves the coating of
12 Nanotechnology for Cancer Biomarkers 349

Fe3O4 NPs with CdSe-CdSNPs and then assembled it with gold NPs (AuNPs) on the
electrode. AuNPs enhance the ECL signal by accelerating the electron transfer in the
ECL reaction. Eventually, to remove unwanted nonspecific binding sites on
immunosensors, it was protected by addition of bovine serum albumin (BSA).
This sensor proves to be ideal for ECL immunosensing due to its display of stable
and intense ECL emissions in neutral solution (Myung et al. 2002).
Metal nanoisland is a new term that describes ultrafine particles consisting of a
few to several hundred metal atoms. Nickel nanoislands (NiNIs) are especially
interesting due to their selective attachment to proteins. A NiNI-based biosensors
technique has been described for recognition and label-free detection. Quartz crystal
microbalance (QCM) surface was deposited by 5 nm NiNIs conjugated to the
antibody fragments His-tagged (scFv)-F7N1N2 to detect biomarker GTPaseRhoA.
GTPase RhoA is a cancer biomarker, usually overexpresses in various kind of
tumors which has been chosen for this test. Results show that using NiNIs provide
the possibility of anchoring the cancer biomarker to sense it (Martínez-Rivas et al.
2010).

12.2.4 Aptamer-Conjugated NPs

Aptamers are single-stranded DNA (ssDNA) or RNA sequences that can be bind to
NPs as a ligand for targeting. Aptamers have low immunogenicity due to their low
molecular weight and high specificity for their targets, e.g., phospholipids, peptides,
ions, and bacteria even whole cells, etc. (Avci-Adali et al. 2011; Torres et al. 2017).
Prostate-specific membrane antigen (PSMA) is the most common targeted cancer
biomarker in this category. PSMA can be targeted by Cy5 incorporated- polymeric
NPs conjugated with A10 RNA aptamer. Cy5-polylactic acid (PLA)/aptamer-NPs
would not bind to PC3 cancer cell lines which do not show PSMA, while it would
bind to canine prostate adenocarcinoma cells and LNCaP cells, which express
PSMA. Cy5-PLA-NPs have shown excellent signals in the balb/c mice model, and
also show the feature of lowering the background signals in other organs (Tong et al.
2010).
In recent years, DNAzymes (oligonucleotides that show catalytic activity) have
attracted interest in the detection of cancer biomarkers. AuNPs conjugated with
DNAzymes have been developed for the detection of alpha-fetoprotein (AFP)
biomarker usually found in liver or germ cell cancer. An immunoassay strategy
was employed by sandwiching the two types of probes. One probe was AFP
monoclonal antibody conjugated magnetic microparticles and the other one was
AFP polyclonal antibody/double-stranded DNA functionalized AuNPs having one
complementary strand of peroxidase mimicking DNAzyme. Double-stranded DNA
was rehybridized after the formation of this sandwich complex. A chromogenic
reagent was added in the substrate solution that exhibited green color during the
DNAzyme catalysis as DNAzyme reacted with AFP. This immunoassay identified
AFP at a detection limit of 0.1ngmL-1 (Zhou et al. 2009).
350 A. Muhaymin et al.

In the bloodstream, circulating tumor DNA(ctDNA) fragments (approx. 100–200

base pairs) can be detected as cancer biomarkers (Schwaederle et al. 2017). These
ctDNA fragments can be released from benign tumors or metastatic cancer cells and
show specific genetic aberrations that can be used to predict cancer even before any
symptom occurs (Mehra et al. 2008; Tan et al. 2017). A single exon in the BRCA1
gene can be detected by a fluorescent probe made up of DNA silver nanocluster
(AgNC)(Hosseini et al. 2017). The detection limit was increased to 6.4  10-11 M
under optimized conditions. AgNC produced fluorescence as they recognized the
large deletion mutations in BRCA1. DNA-templated AgNC was hybridized to target
DNAs which enhance the fluorescence produced by AgNC with different intensities.
These differences were defined to identify BRCA1 depletion. The nanocluster
sensing system provides the advantage of high fluorescence as compared to other
sensing systems (Zhang et al. 2019).

12.2.5 Carbon Nanotubes (CNTs)

Carbon nanotubes (CNTs), also called buckytubes, are one of the most intensively
investigated nanomaterials (Balasubramanian and Burghard 2005). CNTs are
formed by rolling graphene sheets into cylindrical tubes that show a hollow structure
with a nanometer-scale diameter and comparatively long length. Their excellent
combination of mechanical, chemical, electrical, magnetic, and optical properties
provides a promising wide range of applications, include biosensing (Biju 2014; Le
Goff et al. 2011). Other compounds can be easily conjugated to the surface of CNTs
for functionalization (Arkan et al. 2015). Through functionalization, CNTs can
easily cross the biological barrier of a cell membrane that assists them to penetrate
individual cells (Pantarotto et al. 2004). A major interest has been developed for
intracellular biosensing application of CNTs due to the unique mechanism of
internalization and release of CNTs from the cells (Tîlmaciu and Morris 2015).
Several types of biomarkers, such as protein receptors, enzymes, and DNA
biomarkers have been detected by CNTs conjugates. These CNTs conjugates can
be subdivided into electrochemical-based CNTs biosensors, optical CNTs
biosensors, and immunosensors determined by their mechanism of action and target
identification. A major interest has been developed for the detection of cancer
biomarkers through CNTs by conjugation with targeting moieties, such as peptides,
proteins, aptamers, and enzymes.
Field emission transistor-(FET)based CNTs have been developed for the detec-
tion of PSA–ACT (prostate-specific antigen complex of protease inhibitor alpha
1-antichymotrypsin) in serum blood. In this case, these FET-based CNTs have been
modified with 1-pyrenebutanoic acid succinimidyl ester as the linker and
1-pyrenbutanol as the spacer. Antibodies for the attachment of PSA–ACT were
then immobilized on the surface of FET-based CNTs through linkers. Linkers to
spacer ratio as 1:3 on FET-based CNTs showed a detection limit of 1.0ngmL-1 of
PSA–ACT complex without any pretreatment (Kim et al. 2009b).
12 Nanotechnology for Cancer Biomarkers 351

The stacking of 20–30 nm carboxylated SWCNTs (single-walled carbon

nanotubes) in upright bundles termed as CNTs forests (Rusling et al. 2009). These
CNTs forest have shown a 4–ten-fold increase in sensitivity to detection of cancer
biomarkers as compared to bare pyrolytic graphite surface. For the detection of IL-6
in head and neck squamous cell carcinoma (HNSCC), an ultrasensitive CNTs forest
electrochemical immunosensor was developed. CNT forests were conjugated to Ab1
for IL-6 and then simultaneously combined with enzyme label HRP for a very low
detection limit of 30pgmL-1. The results have shown a 16-fold improvement over
ELISA. Ab2 attached to carboxylated CNTs with 106 HRP labels per 100-nm CNT
gave an ultralow DL of 0.5pgmL-1 for IL-6 in 10 μL of calf serum (Malhotra et al.
2010).

12.2.6 Quantum Dots (QDs)

QDs are zero-dimensional semiconductor nanocrystals in a size regime of 2–10 nm.

QDs are categorized into two subgroups based on their chemical composition, the
first category is made up of elements from group III (Boron, Aluminum, and
Gallium) to V (Nitrogen, Phosphorous, and Arsenic) of the periodic table, while
the second category, includes elements derived from subgroup II (Zinc and Cad-
mium) and the main group VI (Oxygen, Sulfur, and Selenium) of the periodic group
(Singh et al. 2018). QDs are characterized by a slow degradation, high molar
extinction coefficient as well as high quantum yield, and high-efficiency stokes
shifts (Medintz et al. 2005; Freeman and Willner 2012). They possess excellent
fluorescent properties along with low photobleaching. A small change in the size of
these nanocrystals can produce spans of colors ranging from large-sized QDs
production of red fluorescence to small-sized QDs production of blue fluorescence.
QDs emit a single wavelength when excited with an even broad wavelength range of
light that shows their property of narrow emission spectrum and broad excitation
range (Tan et al. 2011).
In recent years, QDs-based nanosensors have been studied for the detection of
cancer biomarkers. The detection of cancer protein biomarkers is usually performed
by sandwich-type assays. It consists of several components which include a capture
antibody along with a secondary antibody for attachment and a secondary capture
antibody (Chinen et al. 2015). This secondary antibody is stained or produces
fluorescence to be visualized. Two cancer biomarkers, neuron-specific enolase
(NSE) and carcinoembryonic antigen (CEA) have been detected using two
QD-conjugated antibodies sandwich-type assay with each QDs-Ab detection limit
of 1.0 ng/ml (Li et al. 2011a).
One of the biomarkers associated with cancer diagnosis is microRNA. miR-141, a
biomarker in prostate cancer, was detected by developing a two-step QDs sensing
system. The first step in developing a sensing system is to modify CdSe/ZnS QDs
with Forster resonance energy transfer (FRET) quencher-functionalized nucleic
acids, which are also conjugated with miR-141 recognition sequence and a telome-
rase primer sequence. The FRET quencher and CdSe/ZnS QDs are bonded together
352 A. Muhaymin et al.

through covalent bonding. A duplex-specific nuclease (DSN) was utilized to cleave

the duplex formed by the hybridization of miR-141 with the probe. This cleavage
results in the exposure of telomerase primer sequence and separation of the quencher
unit, thus activating the fluorescence of the QDs. The second step involved the
production of chemiluminescence with luminol/H2O2 along with stimulation of
primer unit by telomerase/dNTPs for elongation and final conjugation of hemin.
This sensing system was able to detect miR-141 in a serum sample and discriminated
prostate cancer carriers from healthy individuals (Jou et al. 2015).

12.3 Nanotechnology-Enhanced Detection of Cancer

Biomarkers

Several traditional biosensing techniques have been a part of biomarkers discovery

for laboratory and clinical applications. These techniques have some limitations in
detection due to biomarkers’ complexity, low molecular weight, low analyte
concentrations, etc. Nanotechnology provides a solution by enhancing the selectivity
and sensitivity of these assay techniques using nanomaterials as illustrated in
Fig. 12.2. This section will describe the nanomaterials-based enhancement of

Mass
Spectrometry

Microscopic Dichroic Cylindrical

objecve mirror lens

Laser
sample
lens
CCD
detector
Raman
Vibraonal
frequency
Spectroscopy
CCD
Posion on
spectrometer sample

Photodectectors Electronic box

Sensors

High Surface Light source

Lab-on-a-chip
Area to Volume
Lab-on-chip
Nanomaterials Rao
Analyte flow

Gold thin layer on

Glass slide
Fluorescence and
Luminescence
High Specificity Detecon
and Selecvity
hv hv hv

donor acceptor donor acceptor

FRET FRET Electrical and

Fluorescence
Fluorescence

intensity
intensity

Electrochemic
al Detecon
λ λ

Fig. 12.2 Enhancement of conventional techniques by nanomaterials. (Source: Icons and images
from hiq.linde-gas.com, stemm.info, www.flaticon.com) (Downes and Elfick 2010; Aristotelous
et al. 2015; Xu et al. 2013)
12 Nanotechnology for Cancer Biomarkers 353

conventional biosensing techniques, such as mass spectrometry (MS), Raman spec-

troscopy, optical detection, electrical and electrochemical detection, and lab-on-a-
chip technology.

12.3.1 Lab-on-a-Chip Technology

Lab-on-a-chip (LOC) technology defines microfluidic and nanofluidic devices that

are patterned on a silicon substrate. It has several components integrated, such as
sensors, actuators, microchannels, pumps, mechanical elements, reaction chambers,
and several other components of micro and nanoelectronics (Lin et al. 2009; Xu et al.
2012). Top-down approaches, such as photolithography or chemical etching
techniques are mostly used for developing these structures. Nanowires or
nanoelectrodes are also used in microfluidic channels in LOC technology for
biosensing in compact devices (Kim et al. 2009a; Tian et al. 2010). This miniaturi-
zation process has a lot of benefits which include lesser power consumption, a lower
limit of dead volume, lower consumption of reagents and samples, speedy analysis
within a short time, high reproducibility, and high throughput that enables simulta-
neous samples detection. A variety of nanomaterials, such as CNTs, QDs, and
polymeric and metallic nanoparticles have been incorporated in microfluidics for
biosensing (Ghazani et al. 2006; Zeineldin 2013).
Gastric cancer is the second leading cause of cancer-related mortalities worldwide
(Hsu et al. 2010). Early detection of biomarkers of gastric cancer can assist in
preventing this multistep malignancy. An efficient microfluidic system was devel-
oped for the identification of alpha 1-antitrypsin (A1AT), a secreted biomarker,
elevated in different types of malignancies including gastric cancer, and also
correlated with cancer staging. Fluorescently labeled polymer NPs were utilized in
the replacement of antibodies in the ELISA assay to amplify the optical detection
signals in the region of near-infrared. These nanotags have low tissue fluorescence
noise as a background. Finally, specific antibodies were attached to the nanotags for
the detection of A1AT. Results have shown that this new sensor system is feasible
for the detection of gastric cancer biomarkers as a real-time diagnostic kit (Khazanov
et al. 2012).

12.3.2 Mass Spectrometry

Traditional mass spectrophotometry (MS) usually faces challenges in the detection

of cancer biomarkers especially peptide cancer biomarkers due to their low molecu-
lar weight and their low concentration (Vita et al. 2005). There is also signal noise in
samples due to the abundance of high-molecular weight serum proteins. Recently,
nanomaterials have been applied to MS for the isolation and detection of cancer
biomarkers through several strategies (Qiao et al. 2015). NPs show ligand interaction
354 A. Muhaymin et al.

with biomarkers and tag them in bodily fluids (Zhang et al. 2016b) as well as some
nanomaterials also enhance signals in MS (Zhang et al. 2014). Matrix-assisted laser
desorption/ionization-time of flight MS (MALDI-TOF-MS) has been modified with
different nanomaterials for signal amplification in simultaneous multiple detections
of cancer biomarkers (Li et al. 2014).
AuNPs have been reported as a signal amplifier for PSA immunoassay in
inductively coupled plasma mass spectrometry (ICP–MS) by improving the size of
QD tags. Deposition of gold shell around QD tags has improved the sensitivity by
many folds, which enables immunoassay to detect very low PSA concentrations.
The results have shown that the detection limit of the new system has six-fold
improvement over conventional Mn: ZnS QD-labeled ICP–MS immunoassays
(26attograms over 0.02nanograms of PSA mL 1). Other than the AuNPs doped
ICP–MS high sensitivity, it can also exhibit a six times better broad dynamic range
to cover both PSA concentration in male and female sera. A side advantage of this
amplifier is that the biomarker of interest can be changed by altering the antibody,
since it is located on a secondary antibody (Garcia-Cortes et al. 2016).

12.3.3 Raman Spectroscopy

Conventional Raman spectroscopy lack detection of cancer biomarkers at very low

concentration due to the low signal intensity of Raman scattering from the probe
molecules (biological recognition interface and Raman reporter). Metal NPs such as
AuNPs can greatly enhance the intensity (108–1014 orders of magnitude) of the
Raman probe molecules due to the property of surface plasmons (Nie and Emory
1997).
Femto level detection for PSA was achieved by surface-enhanced Raman spec-
troscopy (SERS) with AuNPs (Grubisha et al. 2003). Due to the reason, that
response of different Raman reporter molecules is different on each wavelength,
multiplex detection was possible. For this procedure, different Raman reporter
molecules were attached to specific antibodies on the AuNP substrate body. The
most integral part of SERS is the attachment of AuNP nanotags for the detection of
cancer biomarkers. As compared to QDs, these SERS nanotags never show
photobleaching. Even at a low volume of 20 nL, these SERS nanotags were able
to detect two hepatocellular carcinoma biomarkers (AFP and alpha-1-antitrypsin)
simultaneously(Dinish et al. 2014a). The same SERS nanotags were also applied to
detect three intrinsic biomarkers—EGFR, CD44, and TGFβRII in a breast cancer
model (Dinish et al. 2014b). Early-stage T1 which is a biomarker of nasopharyngeal
cancer was also in the detection limit of AuNP based SERS tags (Lin et al. 2014;
Viswambari Devi et al. 2015). Multiplex detection of breast cancer biomarkers in a
homogeneous solution using SERS-based molecular sentinel (MS) technology was
demonstrated recently. Two MS nanoprobes, ERBB2-MS and KI67-MS, were
12 Nanotechnology for Cancer Biomarkers 355

designed to respectively target critical biomarkers for breast cancer, e.g., erbB-
2 gene and Ki-67 gene (Wang and Vo-Dinh 2009).

12.3.4 Fluorescence and Luminescence Detection

Nanomaterials can assist in effective fluorescence detection of cancer biomarkers

either it’s qualitative or quantitative. It can be attained in many ways; intrinsic
fluorescence production of nanomaterials (Ramesh et al. 2016; Qin et al. 2017),
trigging fluorescence by nanomaterials (Huang et al. 2016; Zhang et al. 2016a),
Förster resonance energy transfer (FRET) (Xu et al. 2016), metal-enhanced fluores-
cence (MEF) (Zhu et al. 2016), or immunochromatographic fluorescence (Shen et al.
2015).
In contrast to conventional ELISA tests, the detection of PSA cancer biomarker is
very quick in label-free optical biosensors. Surface plasmon resonance (SPR)
provides a way for label-free detection of biomolecular interactions. Utilizing SPR
and quartz crystal microbalance (QCM) a POC system for the detection of PSA–
ACT in human serum was reported. A detection limit of 0.29ngmL 1 for PSA–ACT
in 75% pure human serum was achieved by utilizing 40 nm AuNPs. The comparable
results were shown by both SPR sensor and QCM sensor which indicates that both
systems can be used for the detection of cancer biomarkers (Uludaǧ and Tothill
2010). For the detection of picograms level of PSA in serum samples, another
AuNPs fluorescence probe was developed in which AuNPs act as a quencher. The
probe (Ab2–RBITC–AuNPs) was composed of specific antibodies for detection, a
fluorescent dye, i.e., Rhodamine B isothiocyanate (RBITC) that acts as the donor
and AuNPs act as the acceptor or quencher. Due to NPs surface energy transfer
effect, the RBITC fluorescence is quenched by AuNPs nearby. As the cancer
biomarker, i.e., PSA attached to the mAbs, a sandwich is formed between Ab2–
RBITC–AuNP probe and PSA. Finally, cystamine is added to the complex to cause
displacement of RBITC from the surface of AuNPs to prevent quenching so that
fluorescence measurement would be possible (Liu et al. 2013).

12.3.5 Electrical and Electrochemical Detection

Electrical and electrochemical detection of cancer biomarkers is one of the most

common strategies for the development of modern biosensors fabricated through
top-down or bottom-up techniques. The top-down approach has some advantages
over the bottom-up approach regarding better integrated POC devices and higher
yields so it is more compatible with the standard of fabrication technologies. Some
reported biosensors with electrical cancer biomarkers detection are nanoporous
membrane (Blundell et al. 2016; Raza et al. 2018), label or label-free flexible
transistor-based sensor arrays(Shalev et al. 2013; Shehada et al. 2016),
356 A. Muhaymin et al.

electrochemical microfluidic chip(Xie et al. 2015), and electrochemical

microspheres on the electrode(Hu et al. 2012). An electrochemical detection (Kavosi
et al. 2015; Grinyte et al. 2016)is possible due to a change in conductance or
resistance which is recorded as a redox reaction occurs. Nanomaterials are enhancing
the promising properties of these electrical or electrochemical biosensors. Nanowires
are the most studied nanomaterials in this regard due to their good biocompatibility
and excellent electrical properties.
Cytokeratin-7 (CK-7), a discriminatory protein between benign and metastatic
adenocarcinoma is expressed in epithelial tissues as a cancer biomarker (Tot 2002).
Gold nanowires-(AuNWs) based electrochemical biosensor was developed for the
sensitive detection of CK-7. The immunosensor system was developed as a
sandwich-type assay in which Ab1 was attached to AuNWs and biotinylated Ab2
was attached to streptavidin-conjugated alkaline phosphatase (AP). Microelectrodes
detect the electrochemical change that occurs due to the enzymatic reaction between
AP and p-nitrophenyl phosphate which forms an electroactive p-nitrophenol. The
results were measured as anodic peak current which is directly proportional to AP
concentrations and in turn, could be correlated with the concentrations of CK-7 (Patil
et al. 2008) (Table 12.1).

12.4 Conclusion

Over the recent years, a lot of efforts have been made for the development of
nanomaterials (NMs)-based biosensors or probes for the detection of cancer
biomarkers. These NMs-based techniques have shown significant improvement
over traditional biosensing techniques with better selectivity and sensitivity. The
final goal of developing these techniques is to translate them into clinics and provide
benefits to the healthcare system. However, NMs-based techniques have issues with
their long-term efficacy, safety, and toxicity being evaluated. On the other hand, the
reproducibility of the results in different working conditions of a particular device is
a concern along with standardization of robust synthesis of whole NMs-based
techniques. Practically, NMs-based techniques should be cost-effective to reach
the market. Point-of-care (POC) devices with the integration of nanotechnology
will be more feasible in the foreseeable future for the detection of cancer biomarkers.
It can be expected that nanotechnology integrated POC devices will provide the best
performance and viability with the early detection of cancer biomarkers. Early
detection can be helpful to the healthcare system in terms of patients’ early treat-
ment, lower risk, lower mortality, and higher quality of life. Further development can
be done for the economic benefit of these techniques which will provide cheaper,
efficient methods with fewer side effects and relapse.
 12

Table 12.1 Common nanomaterials-based biosensors for detection of cancer biomarkers (Ye et al. 2018)
Nanoparticles Biomarker detection
Type of Dimensions Cancer Sensing
material Composition Morphology (nm) Biomarker type Source technique Ref.
Metal Au Nanoparticle 14 Sialic acid Breast, Liver Model cancer ICP-MS (Zhang
cells et al.
2016b)
Au/Mn: ZnS Nanoparticles with surface 100–150 PSA Prostate Human serum ICP-MS (Garcia-
deposition Cortes
et al. 2016)
Au Nanoparticle 40 fPSA, cPSA Prostate Spiked in LDI-TOF-MS (Yoo and
FBS solution Yeo 2016)
Au Nanoparticle 10–30 Diglyceride, Kidney RCC tissue SALDI-TOF-MS (Nizioł
octadecanamide et al. 2016)
Au Nanoparticle 60 Protein Breast Breast tissue SERS (Wang
Nanotechnology for Cancer Biomarkers

biomarkers et al. 2016)

(EGFR, HER2,
CD44, CD24)
Au Nanoparticle 100 Human IgG Prostate Human blood DLS (Zheng
et al. 2015)
Au Nanoparticle arrays 3–5 IL-6 Breast, Calf serum Electrochemical (Jensen
Cervical, measurement et al. 2011)
Oral,
Colorectal
Metal Oxide Fe3O4@SiO2- Core-shell nanoparticle 10 EVOM Breast Urine GC-qMS (Qiao et al.
C18 2015)
NDA-Fe3O4 Nanoparticle 10 PSA Prostate Human serum Electrochemical (Li et al.
measurement 2011b)
Semiconductor Silicon Nanowire-FET array 80 nm in CYFRA21-1, Lung, Human serum Electrical (Lu et al.
width, 25 μm PSA Prostate measurement 2015)
in length
Hybrid Porous-silicon Nanoparticle on porous <30 (AuNPs) Peptide Colorectal Human serum MALDI-TOF-M (Li et al.
gold template Fragments 2014)
357

(continued)
Table 12.1 (continued)
358

Nanoparticles Biomarker detection

Type of Dimensions Cancer Sensing
material Composition Morphology (nm) Biomarker type Source technique Ref.
Au-PDMS Nanoparticle on thin film 55 PDGF-BB Non-specific Model cancer Fluorescence (Zhu et al.
biomarker emission 2016)
NaYF4: Yb/Tm Nanoparticle 16 CEA Non-specific Model cancer Up conversion (Xu et al.
biomarker FRET 2016)
Ag@Pb Nanoparticle on MOF 20–50 PSA Prostate Model cancer Electrochemiluminescence (Ma et al.
(II)-β-CD (AuNPs) biomarker sensing 2016)
Au-RGO Nanoparticle on thin film 40–50 VOC Gastric Patient breath SERS (Chen et al.
(AuNPs) sample 2016)
Liposome Nanocapsule loaded with 100 (iron Matrix Colorectal Mouse urine Fluorescence (Schuerle
(DPPC,MSPC, magnetic nanoparticles and oxide 25 nm) metalloproteinase emission et al. 2016)
DPSE-PEG) peptides
Carbon CNT-FETs Single-walled nanotubes N/A PSA-ACT Prostate Human serum Electrical (Kim et al.
measurement 2009b)
CNT-FETs Single-walled nanotubes 5–6 OPN Nonspecific Model cancer Electrical measurement (Lerner
(diameter) biomarker et al. 2012)
CNTs Multiwalled nanotubes 20–35 AFP Liver, Human serum Chemiluminescence (Bi et al.
testicles, measurement 2009)
ovaries
CNTs Single-walled nanotubes 20–30 PSA Prostate Calf serum Electrical (Yu et al.
measurement 2006)
Metal sulfides/ Fe3O4@CdSe- Nanocrystals 537 nm CEA Colorectal Model cancer Electrochemiluminescence (Myung
selenides CDS: Au (bandgap) biomarker Sensing et al. 2002)
CdSe/ZnS Quantum dots 565–665 nm CEA, CA125, and Colorectal, Saliva, blood Fluorescent (Jokerst
(wavelength) Her-2/Neu Ovaries, measurement et al. 2009)
Breast
SiO2 QD tagged nanowires 200(diameter) IL-10 Lungs Model cancer Fluorescent (Sekhar
biomarker measurement et al. 2008)
A. Muhaymin et al.
12 Nanotechnology for Cancer Biomarkers 359

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