Clinical Trial Protocol

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VGH-TAYLOR: Comprehensive precision

medicine study protocol on the
heterogeneity of Taiwanese breast cancer
patients
Chun-Yu Liu1,2,3,4 , Chi-Cheng Huang3,5,6 , Yi-Fang Tsai3,4,6 , Ta-Chung Chao1,3,4 , Pei-Ju Lien3,7 ,
Yen-Shu Lin3,6 , Chin-Jung Feng3 , Ji-Lin Chen3 , Yen-Jen Chen3,4,6 , Jen-Hwey Chiu3,6,8 , Chih-Yi
Hsu4,9 & Ling-Ming Tseng*,3,4,6,10
1
Division of Medical Oncology, Department of Oncology, Taipei Veterans General Hospital, Taipei, Taiwan
2
Division of Transfusion Medicine, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
3
Comprehensive Breast Health Center, Taipei Veterans General Hospital, Taipei, Taiwan
4
School of Medicine, National Yang Ming Chiao Tung University, Hsinchu, Taiwan
5
Department of Public Health, College of Public Health, National Taiwan University, Taipei, Taiwan
6
Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan
7
Department of Nursing, Taipei Veterans General Hospital, Taipei, Taiwan
8
Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
9
Department of Pathology & Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
10
Division of Experimental Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan
*Author for correspondence: Tel.: +886 228 757 652; lmtseng@vghtpe.gov.tw

Heterogeneity in breast cancer leads to diverse morphological features and different clinical outcomes.
There are inherent differences in breast cancer between the populations in Asia and in western countries.
The use of immune-based treatment in breast cancer is currently in the developmental stage. The VGH-
TAYLOR study is designed to understand the genetic profiling of different subtypes of breast cancer in
Taiwan and define the molecular risk factors for breast cancer recurrence. The T-cell receptor repertoire
and the potential effects of immunotherapy in breast cancer subjects is evaluated. The favorable
biomarkers for early detection of tumor recurrence, diagnosis and prognosis may provide clues for the
selection of individualized treatment regimens and improvement in breast cancer therapy.

Lay abstract: We describe the rationale and design for the VGH-TAYLOR study, which includes Taiwanese
patients with breast cancer and with a wide spectrum of clinical scenarios covering different breast
cancer subtypes and clinical settings, such as the neoadjuvant, adjuvant and metastatic settings. The gene
expression profile and genetic mutations of breast cancer subjects with the primary and recurrent tumors
are compared. We also explore whether immune-related gene expression and diversity have any impact
on response to treatment and survival. This study aims to discover biomarkers of detection of cancer
relapse, diagnosis and prognosis that may enable personalized medicine and improvement in breast
cancer treatment.
Clinical trial registration: NCT04626440 (ClinicalTrials.gov).

First draft submitted: 28 January 2021; Accepted for publication: 5 August 2021; Published online:
19 October 2021

Keywords: biomarkers • breast cancer • next-generation sequencing • precision medicine • prognosis

Breast cancer harbors different histopathological and biological features exhibiting distinct clinical outcomes and
behaviors. To improve patient management and the overall survival of patients, studies of the early detection of
tumor recurrence, diagnosis, treatment and post-treatment care of patients with breast cancer are increasing [1]. In
Taiwan, breast cancer is the second leading cause of cancer-related death in women. The annual incidence of breast
cancer has continuously increased over the past 2 decades in Taiwan [2,3].

10.2217/fon-2021-0131 
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Clinical Trial Protocol Liu, Huang, Tsai et al.

The clinical practices for breast cancer diagnosis include molecular imaging and biochemical markers. Imaging
techniques, such as mammography, ultrasound and MRI, could be implemented for monitoring breast cancer
progression. CA15-3, carcinoembryonic antigen and circulating cytokeratins are the most commonly used serum
biomarkers [4,5].
After a confirmed diagnosis, clinicopathological characteristics are determined, including tumor–node–metastasis
(TNM) staging; histological grade; and the status of the estrogen receptor (ER), progesterone receptor (PR) and
HER2 as defined by immunohistochemistry (IHC). Several studies have used IHC-based molecular subtyping as
surrogate markers for the prediction of patient outcomes and complete response rates (pCR) [6]. Nevertheless, these
diagnostic and prognostic markers are associated with various limitations, including inadequate antibody specificity
and sensitivity and being somewhat costly [7].
Previous studies have suggested that multigene expression profiling is significantly associated with patient
outcomes. Multigene expression profiling serves as a predictive marker for the response to neoadjuvant chemotherapy
in patients with ER+ and HER2- breast cancers [8,9]. In addition, liquid biopsies, such as the analysis of circulating
cell-free DNA (cfDNA) and circulating tumor cells, could be used for monitoring breast cancer progression and
recurrence. Increasing evidence have suggested that cfDNA is more sensitive than CA15-3 and is tested for diagnosis
and prognostication of malignancies [10,11]. Detection of mutant cfDNA serves as an inherent biomarker for breast
cancer recurrence [12,13].

Introduction to the trial

The VGH-TAYLOR (Veterans General Hospital TAipei – Yung-Ling foundation sinO-canceR) study (ClinicalTr
ials.gov: NCT04626440) is a comprehensive precision medicine research project focusing on the heterogeneity of
Taiwanese breast cancer patients.

Background & rationale

For the purpose of early detection of tumor recurrence and to estimate the risk of breast cancer recurrence more
accurately, gene expression profiling tests have been developed for making treatment decisions [14]. A major caveat
of these gene expression profiling studies and outcome data is that the populations used for the development and
validation are restricted to patients from western countries, which certainly are not entirely reflective of breast
cancer patients in Asia [15–18]. In addition, current treatments are unlikely to cure advanced breast cancer; hence,
immunotherapy may be a therapeutic strategy for breast cancer [19]. Despite the promising results in other cancer
types, data from phase I/II clinical trials have shown disappointing response rates in patients with advanced triple
negative breast cancer (TNBC) receiving an immune checkpoint inhibitor [20,21].
Breast cancer has not only been a major public health problem but also a great socioeconomic impact, contributed
by relatively expansive new targeted therapies and anticancer therapies as well as considerable high recurrence rate,
leading to occupational incapacity and mortality [22–25]. There are limitations of the current tools for diagnosis and
prognosis thus establishing a database of Asian-based genetic profiles may improve breast cancer treatment. Accord-
ingly, this study aims to identify potential biomarkers for the early detection of tumor recurrence, prognosis and
diagnosis for breast cancer. In addition, to understand the potential effects of immunotherapy in breast cancer
subjects, the expression of immune-related genes and T-cell receptor (TCR) diversity will be determined. Our
approach employs a standardized next generation sequencing (NGS) panel across all patients rather than different
mutation-specific assays for each patient. This approach involved tracking a personalized mutational signature
derived from sequencing pretreatment plasma or tumor tissue but obviated the need for unique assays for each
patient.
Breast cancer therapy is unique in its multidisciplinary nature and frequently divided into neoadjuvant versus
adjuvant approaches for patients with operable breast cancers [4]. In daily practice, patients are informed of the choice
of neoadjuvant or adjuvant approaches according to multifactorial considerations (e.g., breast cancer molecular
subtypes, tumor staging, age, performance status and other patient-based factors) and can make final decision for
each approach. The current VGH-TAYLOR study protocol is a genomic biomarker study correlative to patients’
treatments and designed to grouping study subjects according to their treatment scenario.

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 VGH-TAYLOR: comprehensive study protocol on the heterogeneity of Taiwanese breast cancer patients Clinical Trial Protocol

Methods
Study design
The VGH-TAYLOR study includes a wide spectrum of clinical scenarios covering different breast cancer subtypes
and clinical settings such as the neoadjuvant, adjuvant and metastatic settings. By using several NGS-based platforms
(e.g., a multigene tumor panel, cfDNA, the expression of immune response genes and the TCR immune repertoire),
we will attempt to find biomarkers for Taiwanese patients with breast cancer.
This study was approved by the ethics committee of the Institutional Review Board ([IRB] approval number:
2018-09-007A) of Taipei Veterans General Hospital and was conducted in compliance with the Helsinki Dec-
laration. The principal investigator agrees to provide the IRB/IEC with all appropriate material, including the
informed consent document from participants.
This is a study consisting of 3 years of enrollment and approximately 4 years of follow-up after enrollment. The
overall study duration is approximately 7 years. It is planned to enroll approximately 2025 subjects over 3 years,
including 1875 subjects with breast cancer and 150 archival breast tumor formalin-fixed paraffin-embedded (FFPE)
samples from the established biobank (Biobank of Taipei Veterans General Hospital: https://wd.vghtpe.gov.tw/b
iobank/Index.action) and/or the paired blood samples (if available) from anonymized female subjects with a
confirmed diagnosis of primary invasive breast cancer or with recurrent breast tumors. The aims of this study will
be achieved by analyzing the genetic profiling from a large cohort of breast cancer subjects using the method of
NGS.

Study objectives
The objectives are the following: to conduct comprehensive genetic profiling of subjects with breast cancer; to
identify the differences in the genetic profiling of subjects with breast cancer recurrences, to establish the temporal
changes in the genetic profiling of breast cancer subjects using circulating tumor DNA (ctDNA), to identify
potential biomarkers for the early detection of breast cancer recurrence and predict patient outcomes and to
conduct genetic profiling of the immune system in different subtypes of breast cancer.

Subject grouping & assessment plan

Subjects from biobank and recruited patients will be enrolled in this study. Figure 1 illustrates the study flowchart.

• Archival samples from the biobank of Taipei Veterans General Hospital.

To establish the preliminary baseline genetic profiling of this study, archival FFPE and/or paired blood samples
(if blood sample are available) of anonymized female subjects with a confirmed diagnosis of primary invasive breast
cancer or with recurrent breast tumors will be assayed (Figure 1).

• Patient enrollment.

After enrollment, individual subject will be assigned into one of the four groups according to the medical
management received, the diagnostic stage of breast cancer or the clinical outcome of breast cancer at enrollment.
In addition, approximately 625 subjects will be enrolled each year to fulfill the goal of 500 evaluable subjects
for genetic profiling analysis, including 330 evaluable subjects in Group 1, 70 evaluable subjects in Group 2, 20
evaluable subjects in Group 3-1 and 80 evaluable subjects in Group 3-2 annually. FFPE tissues, fresh tissues and
blood samples will be collected to determine the genetic profiling of breast cancer. The study design of each group
is outlined in the following and Supplementary Figure 1.

1. Group 1 (surgery and adjuvant therapy setting): the evaluable subject number is 330 for each year, including 300
subjects who are planning to undergo surgery as their first-line treatment for breast cancer and 30 subjects with
a high risk of recurrence (i.e., with stage III breast cancer, TNBC, or both HER2+ and lymph node-positive
[LN+] breast cancer).
2. Group 2 (neoadjuvant therapy setting): the evaluable subject number is 70 for each year, of subjects who are
planning to receive neoadjuvant therapy as the first-line treatment for their breast cancer, including at least
20 subjects who may not achieve a pathological complete response (non-pCR) and may have a breast cancer
recurrence.

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Clinical Trial Protocol Liu, Huang, Tsai et al.

All subjects enrolled

(2025 subjects in 3 years)

625 subjects each year

(evaluable 500 each year)
Biobank
(150 samples)
Group 1 (evaluable Group 2 (evaluable Group 3 (evaluable
330 each year) 70 each year) 100 each year)

Planned subject number for

Samples each assay per each year
from Biobank
Assays Group 1 Group 2 Group 3-1 Group 3-2
TMO comprehensive assay 330 70
150 20 80 (10§)
(FFPE) (30†) (20‡)
TMO cfDNA assay (blood) – 300 70 20 80
TMO lRR assay
– 300 70 20 80
(fresh tissue)

TMO repertoire assay

– 300 70 20 80
(blood)
WGS (FFPE and blood) 150 30† 20‡ 20 –

Figure 1. Flowchart of the study. TMO comprehensive assay, Oncomine Comprehensive Assay v3; TMO cfDNA assay,
Oncomine Breast cfDNA Assay; TMO IRR assay, Oncomine Immune Response Research Assay; TMO repertoire assay,
Ion AmpliSeq Immune Repertoire Assay Plus-TCR beta.
† Subjects with breast cancer recurrence at screening.
‡ Subjects who do not achieve pathological complete response (non-pCR), have breast cancer recurrence, and with

paired tumor formalin-fixed paraffin-embedded tissues available.

§ Subjects who are currently receiving the first-line treatment for mBC and have PD within 3 months after initiating
the first-line treatment for mBC (subjects with rapid PD).
BC: Breast cancer; mBC: Metastatic breast cancer; PD: Progressive disease; TMO: Thermo Fisher Oncomine; WGS:
Whole-genome sequencing.

3. Group 3 (stage IV breast cancer): the evaluable subject number was 100 for each year, including 20 subjects in
Group 3-1 and 80 subjects in Group 3-2.
Group 3-1: subjects diagnosed with de novo and treatment naı̈ve stage IV breast cancer.
Group 3-2: subjects diagnosed with a stage IV breast cancer and with recurrence or stage IV subjects who had
received or are currently receiving treatments for breast cancer.

Tables 1–4 list all the scheduled visits and preplanned sample collection for each NGS testing. We designed this
treatment scenario-based protocol with the assumption that participants are fully intended to receive surgery (for
Group 1 and Group 2). However, participants are allowed to change their mind for any reasons. We preplanned
the collection of samples for each biomarker assays (visits) according to patients’ treatment journey (as listed in
Tables 1–4). Because of the preplanned nature of the study, there is some flexibility in protocol schedule based on
patients’ actual course. For subjects who did not receive surgery as planned for whatever the reasons, they can stay
on the study protocol as long as they do not fulfill the protocol-defined withdrawn criteria. For example, Group 2
participants would remain at visit 1 and would enter visit 2 only if they received surgery (Table 2). Alternatively,
these nonsurgery participants will end study whenever they have disease progressed.

Eligibility criteria
Inclusion criteria
• Archival samples from the biobank:

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Table 1. Schedule of assessments for Group 1 subjects.

Group 1† (surgery and adjuvant therapy setting)
Study activities Unscheduled Visit 1¶ Unscheduled Visit 2¶ Visits 3–8# Unscheduled
visit‡ visit¶ visit (confirmed diagnosis
Before surgery When adjuvant Every 6 months
of recurrence)
therapy completed after visit 2
Blood collection for WGS (leukocytes) X§
FFPE tissues collection for WGS X§ X§
FFPE tissues collection for TMO X X X‡‡
comprehensive assay
Blood collection for TMO repertoire assay X X X‡‡
Fresh tissues collection for TMO IRR assay X X‡‡
Blood collection for TMO circulating cell-free X X X†† X‡‡
DNA assay
† Subjects who are planning to receive surgery as the first-line treatment for breast cancer and followed by adjuvant therapy, or subjects with breast cancer recurrence at screening.
‡ This unscheduled visit could occur at screening and is only applicable for subjects with breast cancer recurrence at screening and enrolled in Group 1.
§ Only the paired FFPE tissues (primary and recurrent tumors) and blood samples collected from subjects with breast cancer recurrence at screening and enrolled in Group 1 will be used
for WGS analysis.
¶ These visits (visit 1, unscheduled visit, and visit 2) are only applicable for subjects who are planning to receive surgery as the first-line treatment for breast cancer and followed by
adjuvant therapy.
#
These visits (visits 3–8) are only applicable for subjects with high risk of recurrence (i.e., with stage III breast cancer, TNBC or both HER2+ and lymph node-positive breast cancer).
†† For visits 3–8, blood samples will be collected at approximately 6, 12, 18, 24, 30 and 36 months after the completion of adjuvant therapy, respectively. If subjects have breast cancer

recurrence within the 36-month period, the last time point for blood collection will be at the time when subjects are with confirmed diagnosis of recurrence.
‡‡ This sample collection will only occur when breast cancer recurrence is confirmed. The FFPE and fresh tumor tissues will be collected if available.

All scheduled visits for tissue and blood sample collection will follow the actual clinical practice. If the subject is not able to provide the tissue or blood sample at the specific time point
during the study period or the samples collected failed the quality check process, it will not be considered as a protocol deviation.
FFPE: Formalin-fixed paraffin-embedded; IRR: Immune response research; TMO: Thermo Fisher Oncomine; WGS: Whole-genome sequencing.

Table 2. Schedule of assessments for Group 2 subjects.

Group 2† (neoadjuvant therapy setting)
Study activities Visit 1 Visit 2 Visits 3–8/14¶ Unscheduled
visit (confirmed diagnosis
Before neoadjuvant When surgery is Every 3 or 6 months after
of recurrence)
therapy completed visit 2
Blood collection for WGS (leukocytes) X‡
FFPE tissues collection for WGS X‡ X‡ X‡
FFPE tissues collection for TMO comprehensive assay X X§ X††
Blood collection for TMO repertoire assay X X X††
Fresh tissues collection for TMO IRR assay X X X††
Blood collection for TMO circulating cell-free DNA X X X #
X††
assay
† Subjects who are planning to receive neoadjuvant therapy as the first-line treatment for breast cancer and followed by surgery.
‡ Only the paired FFPE tissues (primary and recurrent tumors) and blood samples collected from subjects who do not achieve pathological complete response (non-pCR) and with breast
cancer recurrence will be used for WGS analysis.
§ FFPE tissues will only be collected from non-pCR subjects at visit 2.
¶ For pCR subjects, visits 3–8 will be applied. For non-pCR subjects, visits 3 to 14 will be applied.
#
For pCR subjects, blood samples will be collected at approximately every 6 months after surgery (i.e., at 6, 12, 18, 24, 30 and 36 months). If subjects have breast cancer recurrence within
the 36-month period, the last time point for blood collection will be at the time when subjects with confirmed diagnosis of breast tumor recurrence.
For non-pCR subjects, blood samples will be collected at approximately every 3 months after surgery (i.e., at 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 36 months). If subjects have breast
cancer recurrence within the 36-month period, the last time point for blood collection will be at the time when subjects with confirmed diagnosis of breast tumor recurrence.
†† This sample collection will only occur when breast cancer recurrence is confirmed. The FFPE and fresh tumor tissues will be collected if available.

All scheduled visits for tissue and blood sample collection will follow the actual clinical practice. If the subject is not able to provide the tissue or blood sample at the specific time point
during the study period or the samples collected failed the quality check process, it will not be considered as a protocol deviation.
FFPE: Formalin-fixed paraffin-embedded; IRR: Immune response research; TMO: Thermo Fisher Oncomine; WGS: Whole-genome sequencing.

1. FFPE samples and the paired blood samples (if available) that were collected from female patients with a
confirmed diagnosis of primary invasive breast cancer or with a recurrent breast tumor.
• Enrolled subjects should meet all of the following criteria for enrollment:
1. Female subjects aged ≥20 years old.
2. Subjects with a confirmed diagnosis of primary invasive breast cancer who are planning to receive treatments
for breast cancer. However, subjects who have a breast cancer recurrence at screening or stage IV subjects who
had received or are currently receiving treatments for breast cancer can also be enrolled.

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Clinical Trial Protocol Liu, Huang, Tsai et al.

Table 3. Schedule of assessments for Group 3-1 subjects.

Group 3-1† (de novo and treatment-naive stage IV breast cancer)
Study activities Visit 1 Visit 2 Visits 3–14‡ Unscheduled visit
(confirmed diagnosis of
Before receiving the Achieve CR, PR or SD for at Every 3 months after visit
first PD)
first-line treatment for least 12 months after 2
mBC initiating first-line treatment
for mBC
Blood collection for WGS (Leukocytes) X
FFPE tissues collection for WGS X X¶
FFPE tissues collection for TMO comprehensive X X¶
assay
Blood collection for TMO repertoire assay X X X¶
Fresh tissues collection for TMO IRR assay X X¶
Blood collection for TMO circulating cell-free DNA X X X§ X¶
assay
† Subjects with de novo and treatment-naive stage IV breast cancer.
‡ These visits (visits 3–14) are only applicable for subjects who achieve CR, PR or SD for at least 12 months after initiating the first-line treatment for mBC.
§ For Visits 3 to 14, blood samples will be collected at approximately 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 36 months after subjects achieve CR, PR, or SD for at least 12 months,
respectively. If subjects have PD within the 36-month period, the last time point for blood collection will be at the time when subjects are with confirmed diagnosis of first PD.
¶ This sample collection will only occur when the first PD is confirmed. The FFPE and fresh tumor tissues will be collected if available.

All scheduled visits for tissue and blood sample collection will follow the actual clinical practice. If the subject is not able to provide the tissue or blood sample at the specific time point
during the study period or the samples collected failed the quality check process, it will not be considered as a protocol deviation.
CR: Complete response; FFPE: Formalin-fixed paraffin-embedded; IRR: Immune response research; MBC: Metastatic breast cancer; PD: Progressive disease; PR: Partial response; SD: Stable
disease; TMO: Thermo Fisher Oncomine; WGS: Whole-genome sequencing.

Table 4. Schedule of assessments for Group 3-2 subjects.

Group 3-2† (stage IV breast cancer subjects with recurrence or posttreatment stage IV breast cancer subjects)
Study activities Visit 1 Visit 2 Visits 3–14‡ Unscheduled visit (confirmed
diagnosis of first PD after
After enrollment/during Achieve CR, PR or SD for at Every 3 months after visit
enrollment)
current treatment least 12 months after 2
initiating the treatment for
mBC
FFPE tissues collection for Thermo Fisher X X ¶ ,#
Oncomine (TMO) comprehensive assay
Blood collection for TMO repertoire assay X X X¶
Fresh tissues collection for TMO Immune X X¶
Response Research (IRR) assay
Blood collection for TMO circulating cell-free X X X§ X¶
DNA assay
† Subjects with stage IV breast cancer and meet any of the following conditions: subjects with breast cancer recurrence or subjects who had received or are currently receiving treatments
for mBC.
‡ These visits (Visits 3–14) are only applicable for subjects who achieve CR, PR or SD for at least 12 months after initiating the treatment for mBC.
§ For visits 3–14, blood samples will be collected at approximately 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 36 months after subjects achieve CR, PR or SD for at least 12 months,
respectively. If subjects have PD within the 36-month period, the last time point for blood collection will be at the time when subjects are with confirmed diagnosis of first PD.
¶ This sample collection will only occur when the first PD is confirmed. The FFPE and fresh tumor tissues will be collected if available.
#
For subjects who are currently receiving the first-line treatment for mBC and with PD less than 3 months, FFPE tumor tissues will be collected at every subsequent time when PD is
confirmed until the last PD within the 3-year follow-up period.
All scheduled visits for tissue and blood sample collection will follow the actual clinical practice. If the subject is not able to provide the tissue or blood sample at the specific time point
during the study period or the samples collected failed the quality check process, it will not be considered as a protocol deviation.
CR: Complete response; FFPE: Formalin-fixed paraffin-embedded; IRR: Immune response Research; MBC: Metastatic breast cancer; PD: Progressive disease; PR: Partial response; SD: Stable
disease; TMO: Thermo Fisher Oncomine; WGS: Whole-genome sequencing.

3. Eastern Cooperative Oncology Group (ECOG) performance status ≤3.

4. Life expectancy ≥3 months.
5. Subject agrees to provide written informed consent.

Exclusion criteria
1. Subjects will be excluded if they had a primary cancer other than breast cancer within 5 years of screening for
inclusion.

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Withdrawal criteria
• Archival samples from the biobank:
1. The tumor content of the FFPE sample is lower than the specified percentage according to the standard of
the central laboratory.
2. The FFPE samples failed the DNA/RNA quality check. The criteria of the DNA/RNA quality check will
follow the standard of the central laboratory.
• Enrolled subjects will be withdrawn if one of the following conditions occurs:
1. Subject who withdraws consent.
2. Subject who refuses to provide specimens for evaluation after enrollment.
3. Subject for which all samples/specimens fail the DNA/RNA quality check. The criteria of the DNA/RNA
quality check will follow the standard of the central laboratory.
4. Subject who does not have sufficient FFPE samples, tissues or blood samples for genetic profiling analysis by
principal investigator’s discretion.
5. Subject who does not return to the clinical site for more than 6 months (based on their medical records) will
be considered as lost to follow-up. However, whether this subject should be withdrawn will be based on the
PI’s discretion.

Data collection & follow-up

The data collection period begins when the subjects enroll and ends at the last time point of data collection for
subjects in each study group. A case report form (CRF), together with an electronically recorded system (eCRF)
are generated to record all the necessary data. These CRF and guidance for eCRF operation have been sent for IRB
approval.
For Group 1: for subjects who are planning to undergo surgery as the first-line treatment for breast cancer
followed by adjuvant therapy, the last time point of the data collection will be approximately 3 years after the
completion of adjuvant therapy. The date of a confirmed diagnosis of breast cancer recurrence after the completion
of adjuvant therapy, or the date of death, whichever comes first, is also defined as the last time point of data
collection. For subjects with a breast cancer recurrence at screening, the data will be collected from the date of a
confirmed diagnosis of breast cancer to the date when the first breast cancer recurrence is confirmed. Group 2:
the final date of data collection is 3 years after surgery (mastectomy or breast-conserving surgery), the date of a
confirmed diagnosis of breast cancer recurrence after surgery or the date of death, whichever comes first. Group 3-1:
the final date of data collection is 3 years after subjects achieve complete response (CR), partial response (PR) or
stable disease (SD) for at least 12 months, the date of a confirmed diagnosis of the first PD after initiating first-line
treatment for metastatic breast cancer (mBC), or the date of death, whichever comes first. Group 3-2: for nonrapid
PD subjects, the last time point of data collection will be approximately 3 years after the subjects achieve a CR,
PR or SD for at least 12 months, the date of a confirmed diagnosis of the first PD after enrollment, or the date of
death, whichever comes first; or for rapid PD subjects, the last time point of data collection will be the date of the
confirmed diagnosis of rapid PD in the 3-year follow-up period or the date of death.
The clinical records including demographics, anthropometrics, ECOG score, menstruation status, family history
of cancer, medical history, details of the clinical characteristics of breast cancer at initial presentation, details of
surgical management for breast cancer (date and type of surgical management received for breast cancer – simple
mastectomy, modified radical mastectomy or breast-conserving surgery), details of medical management for breast
cancer (if any), details of first-line treatment for mBC (if any), response to treatment (for subjects in Groups 2,
3-1 and 3-2 only) and details of the clinical outcomes (events of disease progression, recurrence and deaths) of the
breast cancer will be recorded in the case report form.
During the follow-up, response to treatments (for example response to neoadjuvant chemotherapy or response to
first-line metastatic treatment) is assessed and defined by the Response Evaluation Criteria in Solid Tumors (RECIST,
version 1.1) [26,27]. Survival endpoints in this study protocol include disease-free survival (DFS), recurrence-free
survival (RFS), progression-free survival (PFS) and overall survival (OS) as defined in literature [28].

Study procedures (NGS tools)

FFPE tissues will be used for the Oncomine Comprehensive Assay v3 (a TMO comprehensive assay) and whole-
genome sequencing (WGS) analysis. The TMO comprehensive assay is an NGS-based targeted sequencing assay
that enables the detection of relevant single nucleotide variants (SNVs), gene copy number variations (CNVs),

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Clinical Trial Protocol Liu, Huang, Tsai et al.

gene fusions and indels from 161 unique genes that are designated to help inform drug discovery research and
clinical trial research programs [29,30]. Fresh tissues will be used for the Oncomine Immune Response Research
Assay (a TMO IRR assay). The TMO IRR assay is for research purpose in current study protocol, and it is also
a targeted sequencing gene expression assay that quantitative evaluates the expression of immune genes involved
in tumor-immune interactions, such as those associated with leukocyte subsets, antigen presentation and immune
checkpoint pathways. Blood samples will be used for the Oncomine Breast cfDNA Assay (a TMO cfDNA assay), the
Ion AmpliSeq Immune Repertoire Assay Plus-TCR beta (a TMO repertoire assay) and WGS analysis. The TMO
cfDNA assay is designated to detect several breast-cancer-associated biomarkers including AKT1, EGFR, ERBB2,
ERBB3, ESR1, FBXW7, KRAS, PIK3CA, SF3B1 and TP53 from cfDNA [31]. Whereas the TMO repertoire assay is
a mRNA-based sequencing for complete characterization of CDR1, CDR2 and CDR3 regions of TCR beta [32] and
is for research purpose in current study protocol. All of the assays will be conducted following the manufacturer’s
instructions (Thermo Fisher Scientific, MA, USA), and details of all assays are available at manufacturer’s website
(https://www.thermofisher.com/). The time schedules of the visits for the participants and the assessments for each
group are provided in Tables 1–4.

Data analysis
To determine the comprehensive genetic profiling of breast cancer, the frequency of gene mutations identified
by WGS and TMO comprehensive assay will be tabulated by the primary and recurrent tumors (if available) of
subjects in the Groups 1, 2, 3-1 and 3-2. To determine the differences of comprehensive genetic profiling for
recurrence in breast cancer subjects with the primary and recurrent tumors, the potential gene mutations and
types of mutation that associated with breast cancer recurrence will be identified by comparing the comprehensive
genetic profiling between the paired primary tumor and recurrent tumor of individual subject. To determine the
temporal changes in genetic profiling of breast cancer subjects using cfDNA, the frequency of gene mutations
identified by TMO cfDNA assay will be tabulated by different study groups at different time points (Tables 1–4,
Blood collection for TMO cfDNA assay). To determine the temporal changes in the gene expressions involved in
tumor–immune interactions in breast cancer subjects, the results of TMO IRR assay will be compared between the
paired primary tumor and recurrent tumor in subjects with BC recurrence. To determine the temporal changes in
the TCR diversity in breast cancer subjects, the genetic profiling of TCR will be determined using TMO repertoire
assay. The variable regions of TCR sequences and CDR3 will be tabulated by different study groups at different
time points.
SNVs (reported in the 1000 Genome Project with a minor allele frequency ≥1%) and copy number variants
of breast tumor tissues and cfDNA will be detected and analyzed. The potential gene mutations and types of
mutation that associated with breast cancer recurrence after neoadjuvant therapy will be identified by comparing
the comprehensive genetic profiling between the paired primary tumor and recurrent tumor of individual subject.
Survival endpoints including DFS, RFS, PFS and OS curves will be plotted for breast cancer patients with wild-type
or mutated gene. TCR repertoire diversity, including evenness, Shannon diversity, TCR convergence, CDR3 length
distribution and usage of TCR V(D)J gene segments, in pCR subjects versus non-pCR subjects will be calculated.
A receiver operating characteristic curve analysis is used for the prediction of pCR using these TCR features. To
determine the altered genes involved in tumor-immune interactions in pCR subjects versus non-pCR subjects, gene
with p-values ≤ 0.05 and fold change ≥1.5 will be selected for candidates. The hierarchical clustering analysis and
principal component analysis of genes will be performed. The significant impact of genomic and genetic alterations
on clinical efficacy and outcome will be evaluated.

Statistical analysis
In general, continuous variables will be summarized as the number of observations, mean, median, standard
deviation, minimum, maximum and 95% CIs. Categorical variables will be summarized as counts and percentages.
Logistic regression models will be used to determine the potential biomarkers for distinguishing the subjects. Chi-
square tests will be used to determine the differences between groups. Clinicopathological characteristics, including
cancer stage, histologic grade, lymphovascular invasion, gene expression and gene mutation, which are associated
with survival end points (DFS, RFS, PFS and OS) will be used as variables for the univariate and multivariate Cox
hazards model and logistic regression analysis. Survival curves of breast cancer subjects will be generated by the
Kaplan–Meier method and compared with the log-rank test. All statistical analysis will be performed using the SAS

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 VGH-TAYLOR: comprehensive study protocol on the heterogeneity of Taiwanese breast cancer patients Clinical Trial Protocol

statistical software system. Unless otherwise specified, all statistical assessments will be performed at the significance
level of 0.05.

Informed consent process & NGS results availability to participants

The archival delinkage samples from biobank by which the sample donors have given the informed consent at time
of donation will be applied from Biobank of Taipei Veterans General Hospital upon IRB approval. For subjects
to be enrolled in this study, the informed consent process will be applied. An IRB-approved informed consent
form explains how data safety is kept and all the potential implications of participating, including the possible
return of ‘incidental’ findings, in easy-to-understand language. In brief, NGS testing data will be interpreted by
investigators, and if necessary, a molecular tumor board to evaluate the benefits of returning NGS testing results
to participants and patients will be provided information with regard to use of these NGS data, in particular the
results of targeted sequencing from multigene panel and cfDNA. Those data that are for research purpose only,
such as results from immune response assay and immune repertoire, will not be made available to the participants.
All identifiable patient information will be delinked to guarantee confidentiality and security of the information in
the genetic material. Only in charging investigator will be able to discuss returning data with individual-responsible
participants.

Discussion
The current study has been designed to achieve the following outcomes: understanding the genetic profiles of
different subtypes of breast cancer in Taiwan, assessing the efficacy of different treatments for subjects with breast
cancer, defining the molecular risk factors and predicting the potential risk of breast cancer recurrence, assessing
the immune repertoire and the potential effects of immunotherapy in subjects with breast cancer and developing
new strategies for treating patients with TNBC or late stages of breast cancer.
A needle aspiration or solid biopsy followed by imaging techniques is required to confirm the results of mam-
mography and breast ultrasound. Because the incidence of breast cancer has continuously increased, these methods
for breast cancer diagnosis are insensitive and are no longer satisfying the medical demands. The available genomic
tests, such as Oncotype DX, MammaPrint, Prosigna and EndoPredict, may be used for identifying patients who
will benefit from adjuvant chemotherapy for ER+ and HER2- breast cancer [33]. Most of these products focus on
Western populations, and the performance of these genomic test panels in Asian populations remains controver-
sial [16]. In this regard, developing an Asian-based genetic profiling database is crucial for Asian populations with
breast cancer.
Liquid biopsies are less invasive to access and suitable for serial monitoring cancer progression compared to tissue
biopsies. In the bloodstream, the release of cfDNA results from apoptosis, necrosis and secretion by cells [34]. A
proof-of-concept analysis indicated that cfDNA carries tumor-specific alterations and there is a significant negative
correlation between ctDNA levels and overall survival in mBC [11]. Likewise, ctDNA harbors genetic and epigenetic
alterations of tumors, suggesting potential roles as cancer biomarkers. ESR1 and PIK3CA mutations in ctDNA may
be linked to drug resistance in hormone receptor-positive breast cancer [35,36]. The US FDA-approved a ctDNA
assay, the Cobas EGFR Mutation Test, for use as a companion diagnostic test for metastatic non-small-cell lung
cancer eligible for therapy with erlotinib [37].
Immunotherapy has already been used for treating several types of cancer, such as melanoma, lung cancer,
acute lymphoblastic leukemia and bladder cancer [38]. Immune checkpoint blockade by antibodies against PD-
1/PD-L1 and CTLA-4 exhibits long-lasting antitumor responses in multiple cancers [39]. Although breast cancer
is considered to be a poorly immunogenic tumor type, mounting evidence has suggested that the immune system
contributes to the prognosis and the chemotherapy response in breast cancer [40]. The tumor infiltrating lymphocytes
(TILs) have been reported to be positive prognostic markers, and they are predictive of a therapeutic benefit in
patients with breast cancer. High levels of PD-L1 expression has a positive correlation with the presence of TILs
in HER2-positive breast cancer and TNBC [41–43]. Recently, atezolizumab, a PD-L1 inhibitor, plus nab-paclitaxel,
has been approved for use in patients with PD-L1 positive TNBC [40].
TCR recognizes peptide-MHC epitopes, initiating adaptive immune responses. Activation of the responding T
cells results in clonal expansion and their progeny inherit an identical TCR sequence. The generation of a highly
diverse TCR repertoire results from random recombination of TCR V(D)J gene segments [44]. The development
of NGS-based TCR repertoire analysis provides a powerful tool to study the complexity of the adaptive immune
system and cellular immunology. Determination of the TCR repertoire diversity and complementarity-determining

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Clinical Trial Protocol Liu, Huang, Tsai et al.

regions can be employed to identify biomarkers of immune responses and immune-mediated adverse events [45].
Recent studies have provided evidence that TCR repertoire sequencing can serve as a biomarker of the immune
response in cancer patients receiving immunotherapy. TCR sequencing acts as a biomarker for TIL clonal expansion
after anti-CTLA-4 antibody ipilimumab therapy in breast cancer [46].

Conclusion
This study is designed as comprehensive precision medical research of Taiwanese patients with breast cancer.
The outcomes of this study will provide information about genetic profiles, the efficacy of different treatments
including immunotherapy, the risk factors of recurrence and the TCR repertoire in patients with breast cancer. The
identification of biomarkers for early detection of breast cancer recurrence and prognosis as well as the prediction
of responses to treatments may improve treatments for TNBC and advanced breast cancer.

Executive summary
• In Taiwan, breast cancer is the most common cancer and the leading cause of cancer death in women.
• Since the incidence of breast cancer has continuously increased, the clinical practices for breast cancer diagnosis
are insensitive and are no longer satisfying the medical demands.
Background and rationale
• For the purpose of early detection of tumor recurrence and to estimate the risk of breast cancer recurrence more
accurately, gene expression profiling tests have been developed for making treatment decisions.
• Our approach employed a standardized next generation sequencing panel which involved tracking a
personalized mutational signature derived from sequencing pretreatment plasma or tumor tissue.
Study design & eligibility criteria
• The VGH-TAYLOR study (ClinicalTrials.gov: NCT04626440) includes a wide spectrum of clinical scenarios covering
different breast cancer subtypes and clinical settings such as the neoadjuvant, adjuvant, and metastatic settings.
• It is planned to enroll approximately 2025 subjects over 3 years, including 1875 subjects with breast cancer and
150 archival breast tumor formalin-fixed paraffin-embedded samples from the established biobank.
• The study population will consist of the Taiwanese patients ≥20 years of age with primary invasive breast cancer
who are planning to receive treatments for breast cancer.
Outcome measures/end points
• The primary end points are information about genetic profiles, the efficacy of different treatments, the risk
factors of recurrence and the T-cell receptor repertoire in patients with breast cancer.
• The second end points are disease-free survival, recurrence-free survival, progression-free survival and overall
survival.
Conclusion
• The identification of biomarkers for early detection of breast cancer recurrence, prognosis and the prediction of
responses to treatments may improve treatments for advanced breast cancer.

Supplementary data
To view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/sup
pl/10.2217/fon-2021-0131

Author contributions
Conceptualization, methodology, investigation, coordination and conception: LM Tseng and CY Liu. Acquisition of data and revising
the work: CY Liu, CC Huang, YF Tsai, TC Chao, YS Lin, CJ Feng, YJ Chen, JH Chiu and CY Hsu. Drafting the article: CY Liu and
JL Chen. Study coordinator: PJ Lien. All authors approval of the version to be published and agreement to be accountable for
all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately
investigated and resolved.

Acknowledgments
The authors are grateful to the patients at Taipei Veterans General Hospital, who provided contributions to enable this research
project. Research was also supported by Biobank, Taipei Veterans General Hospital.

Financial & competing interests disclosure

This research is funded by Yong-Lin Healthcare Foundation (SINO-CANCER project) under the clinical study protocol no. QCR18002.
The funding body’s role was limited to funding with no influence on design, analysis and data interpretation and writing the

4066 Future Oncol. (2021) 17(31) future science group

 VGH-TAYLOR: comprehensive study protocol on the heterogeneity of Taiwanese breast cancer patients Clinical Trial Protocol

manuscript. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial
interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The whole study protocol was reviewed and approved by Institutional Review Board of Taipei Veterans General Hospital (2018-
09-007A). Tumors for immunohistochemical study were collected in accordance with the Declaration of Helsinki and informed
consents from sample donors were obtained at time of their donation.

Data availability
Taipei Veterans General Hospital retains the ownership of data, results, reports, findings, and discoveries related to this study.
Yong-Lin Healthcare Foundation has the priority authorization. The patient data is unavailable to the public. The findings will be
published in journals. The data might be available by requests which need to be approved by both institutions (Taipei Veterans
General Hospital and Yong-Lin Healthcare Foundation).

Open access
This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license,
visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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