biomedicines

Article
Gut Microbiota Signatures with Potential Clinical Usefulness in
Colorectal and Non-Small Cell Lung Cancers
Sofía Tesolato 1,2,† , Juan Vicente-Valor 1,2,† , Mateo Paz-Cabezas 2,3 , Dulcenombre Gómez-Garre 2,4,5,6 ,
Silvia Sánchez-González 2,4 , Adriana Ortega-Hernández 2,4 , Sofía de la Serna 2,7,8 ,
Inmaculada Domínguez-Serrano 2,7,8 , Jana Dziakova 2,7,8 , Daniel Rivera 2,7 , Jose-Ramón Jarabo 2,8,9 ,
Ana-María Gómez-Martínez 2,8,9 , Florentino Hernando 2,8,9 , Antonio Torres 2,7,9 and Pilar Iniesta 1,2, *

1 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Complutense University,

28040 Madrid, Spain; [email protected] (S.T.); [email protected] (J.V.-V.)
2 San Carlos Health Research Institute (IdISSC), 28040 Madrid, Spain; [email protected] (M.P.-C.);
[email protected] (D.G.-G.); [email protected] (S.S.-G.);
[email protected] (A.O.-H.); [email protected] (S.d.l.S.);
[email protected] (I.D.-S.); [email protected] (J.D.);
[email protected] (J.-R.J.); [email protected] (A.-M.G.-M.);
[email protected] (F.H.); [email protected] (A.T.)
3 Biomedical Research Networking Center in Cancer (CIBERONC), Carlos III Health Institute,
28029 Madrid, Spain
4 Cardiovascular Risk Group and Microbiota Laboratory, San Carlos Hospital, 28040 Madrid, Spain
5 Department of Physiology, Faculty of Medicine, Complutense University, 28040 Madrid, Spain
6 Biomedical Research Networking Center in Cardiovascular Diseases (CIBERCV), Carlos III Health Institute,
28029 Madrid, Spain
7 Digestive Surgery Service, San Carlos Hospital, 28040 Madrid, Spain
8 Department of Surgery, Faculty of Medicine, Complutense University, 28040 Madrid, Spain
9 Thoracic Surgery Service, San Carlos Hospital, 28040 Madrid, Spain
* Correspondence: [email protected]
Citation: Tesolato, S.; Vicente-Valor, J.; † These authors contributed equally to this work.
Paz-Cabezas, M.; Gómez-Garre, D.;
Sánchez-González, S.; Abstract: The application of bacterial metagenomic analysis as a biomarker for cancer detection is
Ortega-Hernández, A.; de la Serna, S.;
emerging. Our aim was to discover gut microbiota signatures with potential utility in the diagnosis of
Domínguez-Serrano, I.; Dziakova, J.;
colorectal cancer (CRC) and non-small cell lung cancer (NSCLC). A prospective study was performed
Rivera, D.; et al. Gut Microbiota
on a total of 77 fecal samples from CRC and NSCLC patients and controls. DNA from stool was
Signatures with Potential Clinical
Usefulness in Colorectal and
analyzed for bacterial genomic sequencing using the Ion Torrent™ technology. Bioinformatic analysis
Non-Small Cell Lung Cancers. was performed using the QIIME2 pipeline. We applied logistic regression to adjust for differences
Biomedicines 2024, 12, 703. attributable to sex, age, and body mass index, and the diagnostic accuracy of our gut signatures was
https://doi.org/10.3390/ compared with other previously published results. The feces of patients affected by different tumor
biomedicines12030703 types, such as CRC and NSCLC, showed a differential intestinal microbiota profile. After adjusting for
confounders, Parvimonas (OR = 53.3), Gemella (OR = 6.01), Eisenbergiella (OR = 5.35), Peptostreptococcus
Academic Editor: Ryota Niikura
(OR = 9.42), Lactobacillus (OR = 6.72), Salmonella (OR = 5.44), and Fusobacterium (OR = 78.9) remained
Received: 30 January 2024 significantly associated with the risk of CRC. Two genera from the Ruminococcaceae family, DTU089
Revised: 5 March 2024 (OR = 20.1) and an uncharacterized genus (OR = 160.1), were associated with the risk of NSCLC. Our
Accepted: 18 March 2024
two panels had better diagnostic capacity for CRC (AUC = 0.840) and NSLC (AUC = 0.747) compared
Published: 21 March 2024
to the application of two other published panels to our population. Thus, we propose a gut bacteria
panel for each cancer type and show its potential application in cancer diagnosis.

Copyright: © 2024 by the authors. Keywords: microbiota; biomarker; colorectal cancer; non-small cell lung cancer
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons 1. Introduction
Attribution (CC BY) license (https://
Non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) are among the most
creativecommons.org/licenses/by/
common cancers in terms of incidence and are leading causes of death from malignant
4.0/).

Biomedicines 2024, 12, 703. https://doi.org/10.3390/biomedicines12030703 https://www.mdpi.com/journal/biomedicines

Biomedicines 2024, 12, 703 2 of 19

diseases worldwide. In 2020, there were 1.8 million and 0.9 million deaths due to NSCLC
and CRC in the world, respectively [1,2]. Due to this high health burden, major efforts have
been invested in research on prevention, diagnosis, prognosis, and treatment.
Several factors may play a role in the genesis and development of the tumorigenesis
process. These are intrinsic factors related to individual characteristics, such as age and
random mutations, and extrinsic factors, which depend on environmental features, such as
pollutants, diet, and lifestyle [3]. The microbiota is one of the environmental factors that
significantly influence the onset of cancer [4]. The vast quantity of microorganisms harbored
by humans [5] can interact with host factors through metabolic, immunoinflammatory, and
toxic pathways, driving tumor formation. For example, bile acid synthesis by Clostridium
species has been shown to promote tumor growth [6], and short-chain fatty acids have
been proven to protect against CRC [7]. In a mouse model of lung adenocarcinoma and in
human samples, tumor tissue was enriched with γδ T cells in specific-pathogen-free mice
(SPF) but not in germ-free (GF) mice [8]. Fusobacterium nucleatum expresses a bacterial cell
surface toxin, FadA, an adhesion molecule that can activate β-catenin signaling by binding
to E-cadherin, thereby promoting carcinogenesis [9].
Several experiments have shown that fecal microbiota transplantation from cancer
patients to GF mice led to a greater number of tumoral lesions of larger size than in
SPF mice bearing commensal microbiota [10]. However, other studies have concluded
the opposite, where reduced commensal microbiota by antibiotics induced fewer tumor
lesions [8]. These data support the need to conduct well-characterized studies in order to
find generalizable biomarkers [11]. Additionally, some genera have been claimed to be
increased in cancer in some studies but decreased in others, e.g., elevated [12] or reduced
Pseudomonas [13] in tumor tissue from lung cancer patients. Therefore, human studies
remain relevant to shed light on the development of microbial biomarkers for the diagnosis
and prognosis of colorectal and lung cancer. The fecal microbiota has attracted the greatest
interest in terms of biomarker identification, although the local microbiota from tumor
and non-tumoral gut mucosa may also be of paramount relevance. Moreover, the gut
microbiota can influence tumorigenesis at a distant site, such as the lungs. This finding
has paved the way for the so-called gut–lung axis. According to this hypothesis, the gut
microbiota can affect lung homeostasis, leading to increased susceptibility or resistance
to lung diseases. Soluble microbial ingredients circulating via the gut–lung axis and
interaction with dendritic cells and lung group 2 innate lymphoid cells would stand for
the key interconnected means through which the gut microbiota participate in pulmonary
diseases [14]. Taken together, these data seem to suggest that gut dysbiosis might contribute
to CRC and NSCLC pathogenesis.
Recently, gut microbiome signatures have been developed across several cancer
types [15,16]. However, whether there is a specific microbiome signature for each type of
cancer, CRC or NSCLC, still remains under investigation. In CRC and NSCLC patients, a
central core of pathogenic bacteria has been shown to be enriched, but the reality is that
there has been a highly variable degree of species reported [15,17,18].
The main objective of this work was to compare gut microbiota profiles between
different groups of subjects: CRC and NSCLC patients and control subjects. Furthermore,
we intended to provide results to establish the relevance of the gut–lung axis. The analyses
were performed on feces since this type of sample can be obtained non-invasively.

2. Materials and Methods

2.1. Patients and Samples
A total of 77 fecal samples from 38 CRC patients, 19 NSCLC patients, and 20 controls
were collected and submitted to metagenomic analysis. Samples were obtained prospec-
tively between 2021 and 2023. All cases came from the San Carlos Hospital in Madrid
(Spain). Written approval to develop this study was obtained from the Clinical Research
Ethics Committee of the San Carlos Hospital in Madrid (C.I. 19/549-E_BC, 27/12/2019).
In addition, written informed consent was given by the subjects prior to investigation.
Biomedicines 2024, 12, 703 3 of 19

All participants were recruited subsequently and regardless of gender, age, or tumor
stage. Subject characteristics are depicted in Table 1. Patients were staged according to the
American Joint Committee on Cancer classification [19] and all subjects were categorized
according to their body mass index (BMI) values, following the criteria of the World Health
Organization (WHO).

Table 1. Characteristics of patients with CRC, NSCLC, and controls.

p Value p Value
CRC Group NSCLC Group Control Group
Variable (CRC vs. (NSCLC vs.
(N = 38) (N = 19) (N = 20)
Control) Control)
Age (Mean ± SD, years) 71.24 ± 12 72.79 ± 7.91 54.80 ± 14.97 <0.001 1, * <0.001 2, *
Gender, N (%) 0.016 3, * 0.265 3
Male 24 (63.2) 9 (47.4) 6 (30)
Female 14 (36.8) 10 (52.6) 14 (70)
BMI group, N (%) 0.004 3, * 0.008 3, *
Normal weight (BMI < 25 kg/m2 ) 8 (21.1) 6 (31.6) 3 (15.0)
Overweight (BMI ≥ 25 kg/m2 and <30 kg/m2 ) 20 (52.6) 9 (47.4) 3 (15.0)
Obesity (BMI ≥ 30 kg/m2 ) 10 (26.3) 4 (21.1) 14 (70.0)
Tumor location, N (%)
Right colon 20 (52.6)
Left colon 13 (34.2)
Rectum 5 (13.2)
TNM stage, N (%)
I–II 17 (44.7) 13 (68.4)
III–IV 21 (55.3) 6 (31.6)
CRC: colorectal cancer; NSCLC: non-small cell lung cancer; SD: standard deviation; BMI: body mass index;
1 Mann–Whitney U test; 2 Student’s t-test; 3 Chi-squared test; * Statistically significant for the comparison at
p value < 0.05.

Eligible controls were voluntary subjects without cancer and considering the following
exclusion criteria: previous history of inflammatory bowel disease; antibiotic treatment
in the month prior to obtaining the stool sample; previous intestinal resection surgery;
and oncological history, regardless of the time elapsed. In the cancer groups, eligible
cases were patients undergoing potentially curative surgery for CRC or NSCLC at the
San Carlos Hospital in Madrid. Cancer patients previously submitted to gastrointestinal
resection surgery, those affected by inflammatory diseases, and those who had received
antibiotic treatment one month before surgery were excluded. Moreover, chemo- and/or
radiotherapy prior to the surgery was also considered an exclusion criterion.
Before the surgery or any systemic treatment, fresh fecal samples were collected and
stored at −80 ◦ C.

2.2. DNA Preparation and Sequencing

DNA was isolated from fecal samples using the QIAamp® Fast DNA Stool Mini
Kit (Qiagen, Hilden, Germany). DNA concentration was quantified using the Invitro-
gen™ Qubit™ 3 Fluorometer with the dsDNA HS (high sensitivity) Assay (Thermo Fisher
Scientific, Waltham, MA, USA).
For microbiota analysis, amplification and sequencing of the 16S ribosomal RNA
(rRNA) bacterial gene were conducted. The Ion Torrent™ sequencing technology and
reagents from Life Technologies (a division of Thermo Fisher Scientific, Waltham, MA,
USA) were employed, following previously established protocols [20]. Briefly, the DNA
extracted from each sample (5 ng) underwent parallel polymerase chain reactions (PCRs).
According to the Ion 16S™ Metagenomics Kit protocol, two distinct primer sets were used to
amplify seven hypervariable regions of the gene (V2, V4, V8, and V3, V6–7, V9, respectively).
Subsequently, the resulting amplicons were quantified, pooled equimolarly for each sample,
and purified using Agencourt AMPure XP reagent beads (Beckman Coulter, Pasadena,
CA, USA). Barcoded libraries were generated using the Ion Plus Fragment Library Kit and
the Ion Xpress™ Barcode Adapters. After quantification using the Qubit™ 3 Fluorometer,
Biomedicines 2024, 12, 703 4 of 19

libraries were set to 22 pM, pooled, and subjected to emulsion PCR with the Ion OneTouch™
2 System and the Ion 520™ and 530™ Kit—OT2. Template-positive ion sphere particles
were collected, washed, enriched using the Ion OneTouch™ ES Instrument OT2, and finally
loaded onto an Ion 530™ Chip for sequencing with the Ion S5™ System.

2.3. Statistical Analysis

Bioinformatic analysis was conducted using the Quantitative Insight Into Microbial
Ecology 2 (QIIME2) pipeline [21]. Sequences were assigned to operational taxonomic units
(OTUs) at a 99% similarity threshold with SILVA 16S (v138). A minimum sampling depth
of 100k reads per sample was applied for quality control.
Alpha diversity metrics, including observed OTUs, Chao1 richness estimate, Shannon
diversity index, Pielou’s evenness index, and Simpson’s diversity index, were calculated
from rarefied OTU profiles. Parametric tests (t-test) or non-parametric tests (Kruskal–
Wallis test) were employed depending on data distribution for alpha diversity assessment.
For beta diversity analysis, permutation-based multivariate analysis of variance (PER-
MANOVA), analysis of similarities (ANOSIM), and analysis of multivariate homogeneity
of variances (PERMDISP) tests were conducted using the Jaccard and Bray–Curtis similarity
indexes. Principal coordinate analysis (PCoA) was used for visualization.
Linear discriminant analysis effect size (LEfSe) [22] was used to identify taxa with
significantly different relative abundances between groups. Taxa with a logarithmic LDA
score (log10) > 2 and p value < 0.05 were considered statistically significant. The relative
abundance values of taxa identified in the LEfSe analysis were further analyzed using the
Cutoff Finder application [23]. This facilitated the establishment of relative abundance
thresholds to distinguish between cancer patients and controls based on receiver operating
characteristic (ROC) curve analysis, considering optimal area under the curve (AUC),
sensitivity, and specificity values. Logistic regression was performed to adjust for potential
confounding variables such as gender, age, and BMI differences.
Diagnostic parameters (AUC of ROC curve, sensitivity, and specificity) were calculated
and compared between the identified fecal microbiota signatures and two large cancer
microbiome signatures [15,16]. Statistical comparisons of ROC curves were conducted
using the DeLong et al. method [24], with a significance threshold set at p value < 0.05.
Statistical analyses were performed using STATA IC16.1 (Stata-Corp LLC, College Station,
TX, USA) and IBM® SPSS® Statistics software package version 27 (IBM Inc., Armonk,
NY, USA).

3. Results
3.1. Comparison of Microbial Diversity between Feces from CRC Patients, NSCLC Patients, and
Control Group
Figures 1–3 show the box plots and p values for the comparison of the alpha diversity
metrics [Observed OTUs, (a); Chao1 index, (b); Shannon index, (c); Pielou’s evenness
index, (d); and Simpson index, (e)], performed between feces from CRC patients and
controls, NSCLC patients and controls, and CRC and NSCLC patients, respectively. When
comparing the alpha diversity of microbiota between fecal samples from CRC patients
and the ones from the control group, there were no significant differences. However, all
metrics were higher in the CRC feces (Figure 1). A similar tendency was observed when
the comparison was performed between feces from NSCLC patients and controls, with
differences bordering statistical significance for the observed OTUs and the Chao1 index
(Figure 2). Finally, the comparison of feces from the CRC patients and NSCLC patients
reported no significant differences in alpha diversity, nor a particular tendency between
both groups (Figure 3).
 the comparison was performed between feces from NSCLC patients and controls, w
differences bordering statistical significance for the observed OTUs and the Chao1 in
(Figure 2). Finally, the comparison of feces from the CRC patients and NSCLC patie
reported no significant differences in alpha diversity, nor a particular tendency betw
Biomedicines 2024, 12, 703
both groups (Figure 3). 5 of 19

Figure 1. Alpha diversity comparison between feces from colorectal cancer (CRC) patients and
Figure 1. Alpha diversity comparison between feces from colorectal cancer (CRC) patients and
controls. (a) Observed OTUs; (b) Chao1 index; (c) Shannon index; (d) Pielou’s evenness index;
trols. (a) Observed OTUs; (b) Chao1 index; (c) Shannon index; (d) Pielou’s evenness index; (e) Si
(e) Simpson index. Median values with interquartile range and p values are indicated.
son index. Median values with interquartile range and p values are indicated.
Biomedicines 2024, 12, x FOR PEER REVIEW 6 of 1
Biomedicines 2024, 12, 703 6 of 19

Figure 2. Alpha diversity comparison between feces from non-small cell lung cancer (NSCLC)
Figure 2. Alpha diversity comparison between feces from non-small cell lung cancer (NSCLC) pa
patients and controls. (a) Observed OTUs; (b) Chao1 index; (c) Shannon index; (d) Pielou’s evenness
tients and controls. (a) Observed OTUs; (b) Chao1 index; (c) Shannon index; (d) Pielou’s evennes
index; (e) Simpson index. Median values with interquartile range and p values are indicated.
index; (e) Simpson index. Median values with interquartile range and p values are indicated.

With respect to beta diversity, no significant differences between CRC or NSCLC pa

tients and controls were detected, although in the NSCLC vs. controls comparison differ
ences bordered statistical significance (in PERMDISP test, p value = 0.082 for the Jaccar
index). However, when feces from the CRC and NSCLC patients were compared, differ
ences in beta diversity became remarkable (in PERMANOVA test, p value = 0.010 for Bray
Curtis, and p value = 0.038 for the Jaccard index). Figure 4 shows bidimensional PCoA
plots and p values for Jaccard beta diversity index between the study populations: CRC
vs. control (Figure 4a), NSCLC vs. control (Figure 4b) and CRC vs. NSCLC (Figure 4c).
Biomedicines 2024, 12, x FOR PEER REVIEW 7 of 19
Biomedicines 2024, 12, 703 7 of 19

Figure 3. Alpha diversity comparison between feces from colorectal cancer (CRC) and non-small cell
Figure 3. Alpha diversity comparison between feces from colorectal cancer (CRC) and non-small
lung cancer (NSCLC) patients. (a) Observed OTUs; (b) Chao1 index; (c) Shannon index; (d) Pielou’s
cell lung cancer (NSCLC) patients. (a) Observed OTUs; (b) Chao1 index; (c) Shannon index; (d)
evenness index; (e) Simpson index. Median values with interquartile range and p values are indicated.
Pielou’s evenness index; (e) Simpson index. Median values with interquartile range and p values are
indicated.
With respect to beta diversity, no significant differences between CRC or NSCLC
patients and controls were detected, although in the NSCLC vs. controls comparison
3.2. Taxonomic
differences Comparison
bordered of Microbiota
statistical between
significance Feces from CRC
(in PERMDISP test, pPatients,
value = NSCLC
0.082 forPatients,
the
and Controls
Jaccard index). However, when feces from the CRC and NSCLC patients were compared,
differences in beta diversity became remarkable (in PERMANOVA test, p value = 0.010
Regarding phylum composition, all the groups shared Firmicutes, Bacteroidota, Prote-
for Bray–Curtis, and p value = 0.038 for the Jaccard index). Figure 4 shows bidimensional
obacteria,
PCoA plots Actinobacteriota
andand as dominant
p values for Jaccard taxa. index
beta diversity However,
betweenthethe
CRC and
study NSCLC groups
populations:
displayed a decrease
CRC vs. control in 4a),
(Figure phylum
NSCLC Bacteroidota
vs. control when
(Figurecompared
4b) and CRC tovs.
theNSCLC
control(Figure
group4c).
(CRC—
NSCLC—control: 42.3% vs. 42.9% vs. 46.3%).
Biomedicines 2024, 12, x FOR PEER REVIEW 8 of 19
Biomedicines 2024, 12, 703 8 of 19

Figure
Figure 4.
4. Principal
Principal coordinates analysis (PCoA)
coordinates analysis (PCoA) plots
plots based
basedon
onJaccard
Jaccardindex
indexfor
forfeces
fecesfrom
fromcolorectal
colorec-
tal cancer (CRC) patients, non-small cell lung cancer (NSCLC) patients and controls. (a) CRC vs.
cancer (CRC) patients, non-small cell lung cancer (NSCLC) patients and controls. (a) CRC vs. controls
controls (b) NSCLC vs. controls; (c) CRC vs. NSCLC.
(b) NSCLC vs. controls; (c) CRC vs. NSCLC.

We next focused
3.2. Taxonomic on the
Comparison oftaxonomic
Microbiota differences
between Feces between
from CRC fecal microbiota
Patients, NSCLCfrom patients
Patients,
with CRC, patients with NSCLC, and control subjects at genus level. Figure 5 shows both
and Controls
the Venn diagrams
Regarding and the
phylum LEfSe analyses
composition, at the
all the bacterial
groups genus
shared level for the
Firmicutes, comparisons
Bacteroidota, Pro-
between fecal
teobacteria, andsamples from CRC
Actinobacteriota aspatients
dominant vs. taxa.
controls (a), NSCLC
However, the CRCpatients
and vs. controls
NSCLC (b),
groups
and CRC patients
displayed vs. NSCLC
a decrease in phylum patients
Bacteroidota p values
(c). Thewhen corresponding
compared to the group
to the control bacterial gen-
(CRC—
era featured in the 42.3%
NSCLC—control: LEfSe vs.
analysis
42.9%arevs.reported
46.3%). in Table 2.
As
Weshown in Figure
next focused 5a, taxonomic
on the of the 1205differences
bacterial genera
between reported in the CRC
fecal microbiota vs. patients
from control
comparison, 875 were
with CRC, patients shared
with NSCLC, by both groups,subjects
and control 271 were only present
at genus in the 5CRC
level. Figure shows group,
both
and 59 were
the Venn only present
diagrams and theinLEfSe
the control
analysesgroup.
at theLEfSe analysis
bacterial genusat level
the bacterial genus level
for the comparisons
resulted
betweeninfecal17 genera
samples differentially increasedvs.
from CRC patients in controls
CRC feces,(a),12 belonging
NSCLC Firmicutes
to thevs.
patients controls
phylum (CAG-352,
(b), and CRC Lactobacillus,
patients vs. NSCLCRuminococcaceae,
patients (c). TheS5-A14a,
p values Peptostreptococcus,
corresponding to the Clostridium
bacterial
sensu
genera stricto 1, Eisenbergiella,
featured Gemella, are
in the LEfSe analysis Hydrogenoanaerobacterium,
reported in Table 2. Turicibacter, Parvimonas,
 Biomedicines 2024, 12, x FOR PEER REVIEW 11 of 19
Biomedicines 2024, 12, 703 9 of 19

Figure 5. Taxonomic comparison at the bacterial genus level between feces from colorectal cancer
(CRC) patients, non-small cell lung cancer (NSCLC) patients and controls (Venn diagrams and LEfSe
analysis). (a) CRC vs. controls; (b) NSCLC vs. controls; (c) CRC vs. NSCLC.
Biomedicines 2024, 12, 703 10 of 19

Table 2. Differentially increased bacterial genera in feces from colorectal cancer (CRC) patients,
non-small cell lung cancer (NSCLC) patients, and controls. (a) CRC vs. controls; (b) NSCLC vs.
controls; (c) CRC vs. NSCLC.

(a) (b)
Bacterial Genus p Values (LEfSe) Bacterial Genus p Values (LEfSe)
Control group
g__Sutterella 0.019
g__Blautia 0.005 Control group
g__Lachnoclostridium 0.044 g__Lachnoclostridium 0.031
g__Dielma 0.018 g__Megasphaera 0.040
g__Olsenella 0.024 g__Olsenella 0.020
CRC group NSCLC group
g__Campylobacter 0.004 g_Hungatella 0.009
g__Intestinimonas 0.049 g_Turicibacter 0.013
g__Parvimonas 0.004 g_Coprobacillus 0.007
g_Clostridium sensu
g__Turicibacter 0.006 0.015
stricto 3
g__Hydrogenoanaerobacterium 0.008 g_Intestinimonas 0.038
g__Gemella 0.016 g_Eisenbergiella 0.028
g__Eisenbergiella 0.008 g_Salmonella 0.015
g__Clostridium sensu
0.021 g_DTU089 0.034
stricto 1
g__Peptostreptococcus 0.006 g_Frisingicoccus 0.040
g__S5-A14a 0.023 g_Granulicatella 0.035
g__Ruminococcaceae 0.022 g_Incertae Sedis 0.010
g__Lactobacillus 0.024 g__Lactobacillus 0.017
g__Salmonella 0.011 g__Agathobacter 0.038
g__Eikenella 0.034
g__CAG-352 0.035
g__Fusobacterium 0.003
g__Escherichia-Shigella 0.018
(c)
Bacterial Genus p Values (LEfSe)
NSCLC group
g__Bifidobacterium 0.005
g__Agathobacter 0.004
g__Roseburia 0.050
g__Blautia 0.009
g__CAG-873 0.029
g__Incertae Sedis 0.039
g__Oscillibacter 0.005
g__Ammoniphilus 0.023
g__Pseudobutyrivibrio 0.005
CRC group
g__Campylobacter 0.030
g__Ruminococcaceae 0.042
g__Hydrogenoanaerobacterium 0.016
g__Sellimonas 0.021
g__Solobacterium 0.002
g__Sarcina 0.034
g__Parvimonas 0.005
g__Peptostreptococcus 0.003
g__Eikenella 0.033
g__CAG-352 0.037
g__Fusobacterium 0.004

As shown in Figure 5a, of the 1205 bacterial genera reported in the CRC vs. control
comparison, 875 were shared by both groups, 271 were only present in the CRC group,
and 59 were only present in the control group. LEfSe analysis at the bacterial genus
level resulted in 17 genera differentially increased in CRC feces, 12 belonging to the
Firmicutes phylum (CAG-352, Lactobacillus, Ruminococcaceae, S5-A14a, Peptostreptococcus,
Clostridium sensu stricto 1, Eisenbergiella, Gemella, Hydrogenoanaerobacterium, Turicibacter,
Parvimonas, and Intestinimonas), 3 to the Proteobacteria phylum (Escherichia-Shigella, Eikenella,
Biomedicines 2024, 12, 703 11 of 19

and Salmonella), 1 to the Fusobacteriota phylum (Fusobacterium), and 1 to the Campylobacterota

phylum (Campylobacter).
On the other hand, feces from the control subjects were enriched in five bacterial
genera with respect to CRC patients, three from the Firmicutes phylum (Blautia, Lachno-
clostridium, and Dielma), one from the Proteobacteria phylum (Sutterella), and one from the
Actinobacteriota phylum (Olsenella).
When fecal samples from NSCLC patients were compared to the ones from the control
group (Figure 5b), the Venn diagram reported 1085 genera, 812 in common between both
populations, 138 only present in the NSCLC population, and 135 only present in the
controls. LEfSe analysis indicated that 13 bacterial genera were significantly increased
in feces from NSCLC patients. Five of these genera were common to the CRC group
(Turicibacter, Intestinimonas, Eisenbergiella, and Lactobacillus, all belonging to the Firmicutes
phylum; and Salmonella, from the Proteobacteria phylum), whereas the other eight genera
were new for the NSCLC group (Agathobacter, Ruminococcaceae Incertae Sedis, Granulicatella,
Frisingicoccus, DTU089, Clostridium sensu stricto 3, Coprobacillus, and Hungatella, all of them
belonging to the Firmicutes phylum). Feces from controls were enriched in three genera
with respect to NSCLC patients: genera Lachnoclostridium (phylum Firmicutes) and Olsenella
(phylum Actinobacteriota), which were common to the CRC vs. control comparison, and
genus Megasphaera (Firmicutes phylum).
Finally, the comparison of fecal samples from CRC and NSCLC patients (Figure 5c)
reported 1017 bacterial genera, 804 of them shared between both cancer groups, 177 only
present in CRC, and 36 only present in NSCLC. Regarding LEfSe analysis, feces from
patients with CRC had 11 increased bacterial genera. Eight of these (Fusobacterium, from
phylum Fusobacteriota; CAG-352, Peptostreptococcus, Parvimonas, Hydrogenoanaerobacterium,
and Ruminococcaceae, all of them from phylum Firmicutes; Eikenella, from the phylum
Proteobacteria; and Campylobacter, from phylum Campylobacterota) were already found to
be increased in CRC patients with respect to the control group, whereas the other three
genera (Sarcina, Solobacterium, and Sellimonas, all from phylum Firmicutes) were new for this
comparison. Feces from NSCLC patients reported an increase in genera Agathobacter and
Ruminococcaceae Incertae Sedis (phylum Firmicutes), which were also increased with respect
to the control group, as well as in seven new bacterial genera (Bifidobacterium, phylum
Actinobacteriota; Roseburia, Blautia, Oscillibacter, Ammoniphilus, and Pseudobutyrivibrio, all of
them from phylum Firmicutes; and CAG-873, from phylum Bacteroidota).
Notably, when performing the taxonomic analysis between fecal samples from CRC
patients with respect to tumor location and TNM stage, some of these genera also appeared
to be increased in one of the groups. Particularly, genus Campylobacter was more abundant
in feces from stage I or stage II CRCs (p value = 0.027 in LEfSe analysis), whereas Ruminococ-
caceae was associated with stage III-IV CRCs (p value = 0.033 in LEfSe analysis). Regarding
primary tumor location, genus Hydrogenoanaerobacterium was increased in feces from rectal
cancers with respect to CRCs from other locations (p value = 0.027 in LEfSe analysis), and
both Hydrogenoanaerobacterium and Fusobacterium genera were more abundant in left colon
cancers with respect to right colon ones (LEfSe p value = 0.021 for Hydrogenoanaerobacterium
and p value = 0.048 for Fusobacterium).

3.3. Logistic Regression Analysis of Differentially Increased Bacterial Genera in Patients with CRC
or NSCLC
A logistic regression analysis was performed on the bacterial genera that were found
to be increased in either CRC or NSCLC patients with respect to the control group, in
order to evaluate their potential as biomarkers for cancer development. First, the relative
abundances of each taxon were categorized according to a threshold calculated by the
Cutoff Finder application. Next, a logistic regression analysis was performed to identify the
significant taxa after adjusting for sex, age, and BMI. The results from logistic regression
in CRC and NSCLC patients can be seen in Tables 3 and 4, respectively. As shown in
Table 3, a higher abundance of genera Parvimonas, Gemella, Eisenbergiella, Peptostreptococcus
Biomedicines 2024, 12, 703 12 of 19

Lactobacillus, Salmonella, and Fusobacterium was significantly associated with the presence
of CRC when compared to controls. Genera Campylobacter, Turicibacter, Clostridium sensu
stricto 1, Eikenella, and Escherichia_Shigella were also associated with CRC, although their
logistic regression bordered the statistical significance. On the other hand, increased levels
of genera DTU089 or Ruminococcaceae Incertae Sedis were associated with the presence of
NSCLC when compared to controls (Table 4), as well as genera Hungatella, Clostridium sensu
stricto 3, Salmonella, and Agathobacter, which bordered statistical significance.

Table 3. Logistic regression analysis of bacterial genera remarked as increased in colorectal cancer
(CRC) by LEfSe analysis, adjusting for sex, age, and body mass index (BMI).

CRC Bacterial Genera Unadjusted Adjusted OR

Group Unadjusted p Value Adjusted p Value
(Abundance Threshold) OR (95% CI) (95% CI)

Campylobacter Control reference - reference -

(0.003313) Cancer 6.13 (1.72–21.9) 0.005 * 4.44 (0.88–22.4) 0.071

Intestinimonas Control reference - reference -

(0.01827) Cancer 2.35 (0.78–7.11) 0.130 1.34 (0.28–6.32) 0.705

Parvimonas Control reference - reference -

(0.001859) Cancer 10.9 (2.69–44.1) 0.001 * 53.3 (3.26–870.5) 0.005 *

Turicibacter Control reference - reference -

(0.01017) Cancer 6.13 (1.72–21.9) 0.005 * 3.98 (0.83–19.0) 0.084

Hydrogenoanaerobacterium Control reference - reference -

(0.00367) Cancer 4.56 (1.44–14.5) 0.010 * 1.42 (0.295–6.82) 0.662

Gemella Control reference - reference -

(0.006006) Cancer 3.68 (1.18–11.5) 0.025 * 6.01 (1.20–30.0) 0.029 *

Eisenbergiella Control reference - reference -

(0.008012) Cancer 4.56 (1.44–14.5) 0.010 * 5.35 (1.08–26.5) 0.040 *

Clostridium sensu stricto 1 Control reference - reference -

(0.08304) Cancer 7.79 (1.95–31.2) 0.004 * 4.98 (0.93–26.6) 0.061

Peptostreptococcus Control reference - reference -

(0.0003669) Cancer 4.13 (1.24–13.7) 0.021 * 9.42 (1.38–64.2) 0.022 *

S5-A14a Control reference - reference -

(0.0003232) Cancer 6.55 (1.32–32.3) 0.021 * 2.54 (0.41–15.8) 0.318

Ruminococcaceae Control reference - reference -

(0.004992) Cancer 8.5 (2.19–33.0) 0.002 * 3.67 (0.62–21.7) 0.151

Lactobacillus Control reference - reference -

(0.04309) Cancer 5.67 (1.42–22.6) 0.014 * 6.72 (1.05–43.0) 0.044 *

Salmonella Control reference - reference -

(0.00081) Cancer 8.22 (2.40–28.2) 0.001 * 5.44 (1.02–28.8) 0.047 *

Eikenella Control reference - reference -

(0.0001615) Cancer 7.74 (0.92–65.1) 0.060 8.20 (0.73–92.1) 0.088

CAG-352 Control reference - reference -

(0.008875) Cancer 3.42 (1.10–10.7) 0.034 * 2.14 (0.47–9.61) 0.323

Fusobacterium Control reference - reference -

(0.01714) Cancer 11.2 (3.02–41.6) 0.000 * 78.9 (4.48–1389.0) 0.003 *

Escherichia_Shigella Control reference - reference -

(0.1569) Cancer 5.2 (1.61–16.7) 0.006 * 3.81 (0.80–18.2) 0.094
CI: confidence interval; OR: odds ratio; * Statistically significant for the comparison at p value < 0.05.
Biomedicines 2024, 12, 703 13 of 19

Table 4. Logistic regression analysis of bacterial genera remarked as increased in non-small cell lung
cancer (NSCLC) by LEfSe analysis, adjusting for sex, age, and body mass index (BMI).

NSCLC Bacterial Genera Unadjusted Adjusted OR

Group Unadjusted p Value Adjusted p Value
(Abundance Threshold) OR (95% CI) (95% CI)

Hungatella Control reference - reference -

(0.002103) Cancer 6.53 (1.61–26.5) 0.009 * 7.17 (0.92–56.0) 0.060

Turicibacter Control reference - reference -

(0.006865) Cancer 5.20 (1.32–20.5) 0.019 * 4.61 (0.73–29.1) 0.104

Coprobacillus Control reference - reference -

(0.0006056) Cancer 6.86 (1.63–28.9) 0.009 * 7.00 (0.59–82.9) 0.123

Clostridium sensu stricto 3 Control reference - reference -

(0.0004242) Cancer 5.50 (1.32–22.9) 0.019 * 6.11 (0.84–44.5) 0.074

Intestinimonas Control reference - reference -

(0.0208) Cancer 5.63 (1.36–23.3) 0.017 * 3.50 (0.45–27.3) 0.232

Eisenbergiella Control reference - reference -

(0.007163) Cancer 4.02 (1.06–15.3) 0.041 * 5.49 (0.69–43.9) 0.108

Salmonella Control reference - reference -

(0.0008436) Cancer 15.8 (2.80–89.0) 0.002 * 6.06 (0.73–50.4) 0.096

DTU089 Control reference - reference -

(0.007431) Cancer 5.50 (1.32–22.9) 0.019 * 20.1 (1.35–300.1) 0.029 *

Frisingicoccus Control reference - reference -

(0.0004159) Cancer 5.06 (1.30–19.7) 0.020 * 2.88 (0.49–17.1) 0.244

Granullicatella Control reference - reference -

(0.008393) Cancer 4.02 (1.06–15.3) 0.041 * 4.81 (0.63–36.6) 0.129
Control reference - reference -
Incertae Sedis
(0.1568) 160.1
Cancer 8.00 (1.74–36.7) 0.007 * 0.017 *
(2.44–10,506.36)

Lactobacillus Control reference - reference -

(0.03697) Cancer 4.13 (1.06–16.1) 0.041 * 2.07 (0.36–11.8) 0.414
Control reference - reference -
Agathobacter
(0.5153) 5.625
Cancer 0.017 * 6.94 (0.97–49.6) 0.053
(1.36–23.3)
CI: confidence interval; OR: odds ratio; * Statistically significant for the comparison at p value < 0.05.

3.4. Comparison of Fecal Microbiota Panels

Based on the results of the LEfSe analysis, we performed a comparison of the diagnos-
tic accuracy of a proposed bacterial panel for each cancer type (CRC or NSCLC), and the
signatures from two large, published cancer databases. The details of this comparison are
shown in Table 5. In both cases, the comparison of ROC curves yielded that our discovered
signatures significantly outperformed the diagnostic ability of the previously published
panels. The genera used for each panel can be found beneath Table 5. In the case of CRC,
the proposed panel, and the one with best performance, included the seven bacterial genera
increased in CRC feces with respect to control feces and of which the logistic regression
was found to be significant (i.e., Parvimonas, Gemella, Eisenbergiella, Peptostreptococcus, Lacto-
bacillus, Salmonella, and Fusobacterium). When applied to our population, this panel yielded
an AUC of 0.840, a sensibility of 78.9%, and a specificity of 80%. The same criterion was
applied for the NSCLC panel, comprised by two bacterial genera (DTU089 and Ruminococ-
Biomedicines 2024, 12, 703 14 of 19

caceae Incertae Sedis). This panel performed with an AUC of 0.747, 73.7% of sensibility,
and 75% of specificity, when applied to our population. ROC curves are displayed in
Figure 6, where Figure 6a,b correspond to the performance in our study population of
our proposed CRC and NSCLC panels, respectively, Figure 6c,d to the performance of the
Thomas et al. published signature [15], and Figure 6e,f to the performance of the Yang et al.
published signature [16].

Table 5. Comparison of the diagnostic performance between the new proposed signatures for
colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) and two previously published
signatures from large databases by test of equality of ROC areas for independent samples.

Panel Bacterial Taxa Included AUC (95% CI) Se (95% CI) Sp (95% CI) p Value
CRC (proposed) 7a 0.840 (0.720–0.923) 78.9 (63.7–88.9) 80.0 (58.4–91.9) -
Thomas et al. [15] 34 c 0.557 (0.420–0.687) 60.5 (44.7–74.4) 60.0 (38.7–78.1) 0.004 *
Yang et al. [16] 43 d 0.511 (0.376–0.644) 47.4 (32.5–62.7) 65.0 (43.3–81.9) 0.015 *
NSCLC (proposed) 2b 0.747 (0.583–0.872) 73.7 (51.2–88.2) 75.0 (53.1–88.8) -
Thomas et al. [15] 34 c 0.447 (0.288–0.615) 36.8 (19.1–59.0) 65.0 (43.3–81.3) 0.038 *
Yang et al. [16] 43 d 0.413 (0.258–0.582) 47.4 (27.3–68.3) 45.0 (25.8–65.8) 0.002 *
AUC: area under the curve; CI: confidence interval; Se: sensitivity; Sp: specificity; * Statistically significant
at p value < 0.05. a Proposed CRC panel: genera Eisenbergiella, Fusobacterium, Gemella, Lactobacillus, Parvi-
monas, Peptostreptococcus and Salmonella. b Proposed NSCLC panel: genera DTU089 and Ruminococcaceae Incertae
Sedis. c Signature by Thomas et al. [15]: Acidaminococcus, Actinomyces, Anaerotruncus, Bilophila, Butyricimonas,
Candidatus Schneewindia (CS. gallinarum), Campylobacter, Cloacibacillus, Dysosmobacter, Eggerthella, Eisenbergiella,
Enterocloster, Enterococcus, Erysipelatoclostridium, Escherichia-Shigella, Faecalicatena, Faecalitalea, Flavonifractor, Hafnia-
Obesumbacterium, Harryflintia, Holdemania, Hungatella, Hydrogeniiclostridium, Intestinimonas, Lachnoclostridium,
Lancefieldela, Lawsonibacter, Merdimonas, Pseudoflavonifractor, Pararuminococcus, Ruthenibacterium, Scardovia, Strep-
tococcus, and Veillonella; d Signature by Yang et al. [16]: Acidimicrobium, Acidiphilium, Actinomyces, Aeromonas,
Alcaligenes, Anaplasma, Bacteroides, Bifidobacterium, Bradyrhizobium, Campylobacter, Capnocytophaga, Cellulomonas,
Chamaesiphon, Eikenella, Flavobacterium, Fusobacterium, Gemella, Haemophilus, Halothiobacillus, Helicobacter, Hy-
phomicrobium, Leptotrichia, Methylophilus, Mycoplasma, Neisseria, Nostoc, Parvimonas, Pediococcus, Peptostreptococcus,
Porphyromonas, Prevotella, Prosthecobacter, Pseudomonas, Roseiflexaceae—uncultured, Roseiflexus, Rothia, Rubrivivax,
Selenomonas, Staphylococcus, Streptococcus, Treponema, Variovorax, and Veillonella.

Though the results shown are promising, an investigation in a larger cohort of patients
is needed. Based on our data, we could now hypothesize a prevalence exposure in the
control population of 45% for CRC (meaning that 45% of our control population had
overrepresentation of at least one “hazardous” genus from our CRC signature). In the
case of NSCLC, the prevalence exposure in our control population was 15% (meaning that
15% of our control population had overrepresentation of at least one “hazardous” genus
from our NSCLC signature). Additionally, considering a minimum OR of association of 2,
sample size for future studies considering CRC and NSCLC would be 266 (133 controls
and 133 CRC patients) and 416 (208 controls and 208 NSCLC patients), respectively.
 Campylobacter, Capnocytophaga, Cellulomonas, Chamaesiphon, Eikenella, Flavobacterium, Fusobacterium
Gemella, Haemophilus, Halothiobacillus, Helicobacter, Hyphomicrobium, Leptotrichia, Methylophilus, M
coplasma, Neisseria, Nostoc, Parvimonas, Pediococcus, Peptostreptococcus, Porphyromonas, Prevotella,
Prosthecobacter, Pseudomonas, Roseiflexaceae—uncultured, Roseiflexus, Rothia, Rubrivivax, Selenomona
Biomedicines 2024, 12, 703 Staphylococcus, Streptococcus, Treponema, Variovorax, and Veillonella. 15 of 19

Figure 6. Receiver operating characteristic (ROC) curves of diagnostic accuracy of fecal micro-
Figurebiota
6. Receiver operating characteristic (ROC) curves of diagnostic accuracy of fecal microbio
signatures applied to our study population. (a) Proposed colorectal cancer (CRC) signature;
signatures applied to our study
(b) proposed non-small population.
cell lung (a) Proposed
cancer (NSCLC) signature; colorectal cancer
(c,d) signature (CRC)
proposed bysignature;
Thomas (b) p
posedetnon-small cell lung
al. [15] applied cancer
to CRC (NSCLC)
and NSCLC, signature;
respectively; (c,d)
(e,f) signature
signature proposed
proposed byetThomas
by Yang al. [16] et al. [1
applied to CRC and NSCLC, respectively;
applied to CRC and NSCLC, respectively. (e,f) signature proposed by Yang et al. [16] applied
CRC and NSCLC, respectively.
4. Discussion
In this work, we compared the fecal microbiota from patients affected by CRC or
Though the results shown are promising, an investigation in a larger cohort of p
NSCLC with that from control subjects. These comparisons were based on the potential
tientsutility
is needed.
of fecalBased
samplesonasour data, we could
a non-invasive way to now hypothesize
detect a prevalence
pathologic changes exposure
in the gut
the control population
microbiota of 45%
and identify for
useful CRC (meaning
biomarkers that
for cancer 45% of The
diagnosis. our difference
control population
in age h
overrepresentation of at least one “hazardous” genus from our CRC signature). In the ca
between the control group and the groups of patients affected by cancer could be, at least
of NSCLC,
in part,the prevalenceofexposure
a consequence in our
the exclusion control
criteria population
applied wassubjects.
to the control 15% (meaning
It is not that 15
easy for an elderly patient not to meet any of the exclusion criteria that we established
of our(young
control population had overrepresentation of at least one “hazardous” genus fro
patients meet the necessary characteristics more easily). Additionally, in our case,
our NSCLC signature).
CRC is mainly Additionally,
a pathology considering
typically found a minimum
in older patients, OR of
which probably association
explains the of
age differences.
Biomedicines 2024, 12, 703 16 of 19

Some previous studies have reported a decrease in alpha diversity in cancer patients,
both at the tissue and the fecal level [25,26]. Controversially, our findings suggested a
tendency to higher alpha diversity metrics in both cancer groups with respect to the controls.
However, none of the differences observed was statistically significant, which is in line
with other research [27]. Importantly, the observed microbiome differences usually involve
relative quantitative differences in the abundance of specific taxa of bacteria [28].
A healthy gut microbiota is composed of a diverse group of commensal microorgan-
isms, most of them belonging to the bacterial phyla Firmicutes and Bacteroidota, which help
to maintain homeostasis by interacting with the host’s metabolism and immune system.
In this context, both the pathogen infection and the infectivity of resident pathobionts
are limited. Several diseases including different types of cancer have been linked to gut
dysbiosis, which involves a shift to a more pathogenic microbiota. Feces from CRC and
NSCLC patients included in the present study had a decrease in phylum Bacteroidota, when
compared to feces from controls. Other studies [29] have also highlighted the decrease in
this phylum in mucosal tissue as harmful.
At the genus level, our LEfSe analysis comparing feces from patients with CRC, pa-
tients with NSCLC, and controls revealed several bacterial genera which were enriched
in each of the study groups. Putting the comparisons together, feces from both CRC and
NSCLC patients shared five increased genera with respect to controls (Intestinimonas, Turi-
cibacter, Eisenbergiella, Lactobacillus, and Salmonella), thus making them potential biomark-
ers for cancer. Moreover, CRC feces included eight genera (Campylobacter, Parvimonas,
Hydrogenoanaerobacterium, Peptostreptococcus, Ruminococcaceae, Eikenella, CAG-352, and Fu-
sobacterium) which seem to constitute a more specific CRC profile, as they were found to
be differentially increased in this group of patients both when compared to controls and
to NSCLC patients. On the other hand, genus Agathobacter and the unidentified genus
Ruminococcaceae Incertae Sedis were increased in feces from NSCLC patients with respect to
both control and CRC patients, thus being more specific for the profile of this type of cancer.
After adjusting the genera reported by LEfSe analysis for variables that could have
influenced the composition of the microbiota, seven genera remained increased in CRC
patients (Parvimonas, Gemella, Eisenbergiella, Peptostreptococcus Lactobacillus, Salmonella, and
Fusobacterium), and two in NSCLC patients (DTU089 and Ruminococcaceae Incertae Sedis),
compared to controls. In CRC, an overrepresentation of Fusobacterium, Porphyromonas,
Parvimonas, Peptostreptococcus, and Gemella can be found as an indicator of microbial dys-
biosis [30]. Similarly, Salmonella [31] and Eisenbergiella [32] have also been linked to the risk
of CRC. The role of Lactobacillus in the risk of developing CRC is more controversial, as
the core scientific studies explore its benefits as a probiotic [33]. Some authors did not find
differences in the abundance of Lactobacillus between CRC and controls [34]. The increase in
Lactobacillus in our oncological population could be interpreted as reflecting a fight against
the tumor process, a dysbiosis inherent to the tumor process itself, or a reflection of an
incipient stage where the abundance of this particular bacterium has not been reduced. In
NSCLC, an uncharacterized member from Ruminococcaceae family [35] was also found to be
associated with NSCLC, as similarly were the uncharacterized genus Incertae Sedis and the
genus DTU089 in our study. Other bacteria genera that were close to statistical significance
in our regression studies had also already been related to cancer in previous studies. Of
the ones increased in CRC feces, Campylobacter spp., Escherichia spp., and Shigella spp.,
along with the already mentioned Salmonella spp., include pathogenic and toxin-producing
bacteria that have been related to the progression of cancer [36]. Turicibacter spp. and
Clostridium sensu stricto 1 spp. were found to be positively correlated with the expression
of several inflammatory cytokines as well as growth factor TGFb and transcription factor
STAT3 in a CRC mouse model [37]. Finally, Eikenella spp. was suggested as passenger
bacteria in CRC as it increased in tumors from CRC patients with respect to healthy control
mucosa [38]. In the case of the bacteria found in NSCLC feces, overrepresentation of
Agathobacter spp. and Clostridium spp. in feces from patients with this type of cancer had
been previously reported [39]. Hungatella spp. was increased in feces from NSCLC patients
Biomedicines 2024, 12, 703 17 of 19

with cachexia [40]. Finally, Salmonella spp. has been related to various types of cancer
including CRC, gallbladder cancer, and hepatobiliary carcinoma, [41], although its role in
lung cancer remains unclear. The mechanism by which the microbial taxa could be involved
in cancer development is still the subject of thorough investigation. Whether the main
actors are specific taxa or the dysbiosis as a whole is unknown; possibly, a combination of
mechanisms could provide an explanation to the role of microbiota in carcinogenesis. Both
types of imbalance would ultimately trigger epithelial–mesenchymal transition, inflam-
mation with increased reactive oxygen species and DNA damage, and suppression of the
immune response [42].
We then proposed two diagnostic bacterial panels for CRC and for NSCLC, based
on the logistic regression results. When we compared the diagnostic performance of
our augmented differential genera with that of previously published clusters, our panels
displayed significantly better accuracy in predicting cancer status. The diagnostic accuracy
of our proposed panels was considered acceptable (0.7 < AUC < 0.8) and very good
(0.8 < AUC < 0.9) [43]. The relevance of the published bacteria panels for being as less
applicable is uncertain for us, as their data come from a larger number of patients. It should
be noted that the signature from Yang et al. [16] was developed for cancer prognosis, not
with diagnostic purposes. However, both signatures previously published [15,16] showed
no discriminatory performance. These results may reflect some lack of external validation.
Therefore, the implementation of the microbiome features as a diagnostic cancer tool in
clinical practice may need further research. It may be that a signature panel for diagnostic
purposes cannot be applied with all the genera but only with a few of them. Moreover,
considering that there are 3.8 × 1013 bacteria [44] in the human body and that we harbor
over 10,000 species [45], this enormous biodiversity may defy the discovery of a universal
oncomicrobiome signature. We may have to develop specific signatures for each continent,
age range, cancer type, or even cancer stage.
As the main limitation of this work, we highlight the modest size of our population.

5. Conclusions
Our results indicate, as the main conclusion of the present study, that the feces of
patients affected by different tumor types, such as CRC and NSCLC, show a differential
intestinal microbiota profile. We propose a gut bacteria panel for each cancer type and
demonstrate its potential application in cancer diagnosis. Furthermore, we report for the
first time some bacteria associated with the cancer risk and perform a comparative analysis
with bacteria panels coming from previous meta-analysis studies.

Author Contributions: Conceptualization, S.T., J.V.-V., D.G.-G., A.O.-H., A.T. and P.I.; Formal analysis,
S.T., J.V.-V., M.P.-C., D.G.-G., A.O.-H., S.d.l.S., I.D.-S., J.D., D.R., J.-R.J. and P.I.; Funding acquisition,
A.T. and P.I.; Investigation, S.T., J.V.-V., M.P.-C., D.G.-G., S.S.-G., A.O.-H., S.d.l.S., I.D.-S., J.D., D.R.,
J.-R.J. and P.I.; Methodology, S.T., J.V.-V., M.P.-C., D.G.-G., A.O.-H., S.d.l.S., I.D.-S., J.D., D.R., J.-R.J.,
F.H., A.-M.G.-M., A.T. and P.I.; Project administration, A.T. and P.I.; Supervision, M.P.-C., D.G.-G.,
F.H., A.T. and P.I.; Validation, S.T., J.V.-V., M.P.-C., D.G.-G., S.S.-G., A.O.-H., S.d.l.S., I.D.-S., J.D., D.R.,
J.-R.J., F.H., A.-M.G.-M., A.T. and P.I.; Writing—original draft, S.T., J.V.-V. and P.I.; Writing—review
and editing, S.T., J.V.-V., A.T. and P.I. All authors have read and agreed to the published version of
the manuscript.
Funding: The present study was supported by grant PI19/00073 from the Carlos III Health Institute
(Ministerio de Economía y Competitividad), Spain and co-funded by the European Union through the
European Regional Development Fund (ERDF) ‘A way to make Europe’. Funders did not participate
in study design, data collection and analysis, decision to publish, nor preparation of the manuscript.
Institutional Review Board Statement: This study was conducted in accordance with the Declaration
of Helsinki, and approved by the Clinical Research Ethics Committee of the San Carlos Hospital (C.I.
19/549-E_BC, 27 December 2019).
Informed Consent Statement: Written informed consents were obtained from patients prior to
investigation assuring the confidentiality of their data.
Biomedicines 2024, 12, 703 18 of 19

Data Availability Statement: We have deposited the raw sequence data in a public repository:
Submission ID: SUB14179719. https://submit.ncbi.nlm.nih.gov/subs/sra/SUB14179719, accessed
on 18 March 2024.
Conflicts of Interest: The authors declare that there are no conflicts of interests.

References
1. Sharma, R. Mapping of global, regional and national incidence, mortality and mortality-to-incidence ratio of lung cancer in 2020
and 2050. Int. J. Clin. Oncol. 2022, 27, 665–675. [CrossRef] [PubMed]
2. Xi, Y.; Xu, P. Global colorectal cancer burden in 2020 and projections to 2040. Transl. Oncol. 2021, 14, 101174. [CrossRef] [PubMed]
3. Wu, S.; Hannun, Y. The importance of extrinsic factors in the development of cancers. Mol. Cell. Oncol. 2016, 3, e1143079.
[CrossRef]
4. El Tekle, G.; Garrett, W.S. Bacteria in cancer initiation, promotion and progression. Nat. Rev. Cancer 2023, 23, 600–618. [CrossRef]
[PubMed]
5. Gilbert, J.; Blaser, M.J.; Caporaso, J.G.; Jansson, J.; Lynch, S.V.; Knight, R. Current understanding of the human microbiome. Nat.
Med. 2018, 24, 392–400. [CrossRef]
6. Jia, W.; Xie, G.; Jia, W. Bile acid-microbiota crosstalk in gastrointestinal inflammation and carcinogenesis. Nat. Rev. Gastroenterol.
Hepatol. 2018, 15, 111–128. [CrossRef] [PubMed]
7. Hou, H.; Chen, D.; Zhang, K.; Zhang, W.; Liu, T.; Wang, S.; Dai, X.; Wang, B.; Zhong, W.; Cao, H. Gut microbiota-derived
short-chain fatty acids and colorectal cancer: Ready for clinical translation? Cancer Lett. 2022, 526, 225–235. [CrossRef] [PubMed]
8. Jin, C.; Lagoudas, G.K.; Zhao, C.; Bullman, S.; Bhutkar, A.; Hu, B.; Ameh, S.; Sandel, D.; Liang, X.S.; Mazzilli, S.; et al. Commensal
Microbiota Promote Lung Cancer Development via γδ T Cells. Cell 2019, 176, 998–1013.e16. [CrossRef]
9. Wang, N.; Fang, J.-Y. Fusobacterium nucleatum, a key pathogenic factor and microbial biomarker for colorectal cancer. Trends
Microbiol. 2023, 31, 159–172. [CrossRef]
10. Riquelme, E.; Zhang, Y.; Zhang, L.; Montiel, M.; Zoltan, M.; Dong, W.; Quesada, P.; Sahin, I.; Chandra, V.; San Lucas, A.; et al.
Tumor Microbiome Diversity and Composition Influence Pancreatic Cancer Outcomes. Cell 2019, 178, 795–806.e12. [CrossRef]
11. Manzoor, S.S.; Doedens, A.; Burns, M.B. The promise and challenge of cancer microbiome research. Genome Biol. 2020, 21, 131.
[CrossRef]
12. Dumont-Leblond, N.; Veillette, M.; Racine, C.; Joubert, P.; Duchaine, C. Non-small cell lung cancer microbiota characterization:
Prevalence of enteric and potentially pathogenic bacteria in cancer tissues. PLoS ONE 2021, 16, e0249832. [CrossRef] [PubMed]
13. Bello, S.; Vengoechea, J.J.; Ponce-Alonso, M.; Figueredo, A.L.; Mincholé, E.; Rezusta, A.; Gambó, P.; Pastor, J.M.; Galeano, J.; del
Campo, R. Core Microbiota in Central Lung Cancer with Streptococcal Enrichment as a Possible Diagnostic Marker. Arch. de
Bronconeumol. 2021, 57, 681–689. [CrossRef] [PubMed]
14. Liu, X.; Cheng, Y.; Zang, D.; Zhang, M.; Li, X.; Liu, D.; Gao, B.; Zhou, H.; Sun, J.; Han, X.; et al. The Role of Gut Microbiota in
Lung Cancer: From Carcinogenesis to Immunotherapy. Front. Oncol. 2021, 11, 720842. [CrossRef] [PubMed]
15. Thomas, A.M.; Fidelle, M.; Routy, B.; Kroemer, G.; Wargo, J.A.; Segata, N.; Zitvogel, L. Gut OncoMicrobiome Signatures (GOMS)
as next-generation biomarkers for cancer immunotherapy. Nat. Rev. Clin. Oncol. 2023, 20, 583–603. [CrossRef] [PubMed]
16. Yang, X.; An, H.; He, Y.; Fu, G.; Jiang, Z. Comprehensive analysis of microbiota signature across 32 cancer types. Front. Oncol.
2023, 13, 1127225. [CrossRef] [PubMed]
17. Wong, C.C.; Yu, J. Gut microbiota in colorectal cancer development and therapy. Nat. Rev. Clin. Oncol. 2023, 20, 429–452.
[CrossRef] [PubMed]
18. Birla, P.; Shaikh, F.Y. De-“bug”-ing the microbiome in lung cancer. Cancer Metastasis Rev. 2022, 41, 335–346. [CrossRef] [PubMed]
19. AJCC Cancer Staging Manual [Internet]. Available online: https://link.springer.com/book/9783319406176 (accessed on 19
December 2023).
20. Tesolato, S.; Ortega-Hernández, A.; Gómez-Garre, D.; Claver, P.; De Juan, C.; De la Serna, S.; Paz, M.; Domínguez-Serrano, I.;
Dziakova, J.; Rivera, D.; et al. Gut microbiota profiles in feces and paired tumor and non-tumor tissues from Colorectal Cancer
patients. Relationship to the Body Mass Index. PLoS ONE 2023, 18, e0292551. [CrossRef] [PubMed]
21. Bolyen, E.; Rideout, J.R.; Dillon, M.R.; Bokulich, N.A.; Abnet, C.C.; Al-Ghalith, G.A.; Alexander, H.; Alm, E.J.; Arumugam, M.;
Asnicar, F.; et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 2019,
37, 852–857. [CrossRef]
22. Segata, N.; Izard, J.; Waldron, L.; Gevers, D.; Miropolsky, L.; Garrett, W.S.; Huttenhower, C. Metagenomic biomarker discovery
and explanation. Genome Biol. 2011, 12, R60. [CrossRef] [PubMed]
23. Budczies, J.; Klauschen, F.; Sinn, B.V.; Győrffy, B.; Schmitt, W.D.; Darb-Esfahani, S.; Denkert, C. Cutoff Finder: A comprehensive
and straightforward Web application enabling rapid biomarker cutoff optimization. PLoS ONE 2012, 7, e51862. [CrossRef]
[PubMed]
24. DeLong, E.R.; DeLong, D.M.; Clarke-Pearson, D.L. Comparing the areas under two or more correlated receiver operating
characteristic curves: A nonparametric approach. Biometrics 1988, 44, 837–845. [CrossRef] [PubMed]
25. Ai, D.; Pan, H.; Li, X.; Gao, Y.; Liu, G.; Xia, L.C. Identifying Gut Microbiota Associated with Colorectal Cancer Using a Zero-Inflated
Lognormal Model. Front. Microbiol. 2019, 10, 826. [CrossRef] [PubMed]
Biomedicines 2024, 12, 703 19 of 19

26. Liu, W.; Zhang, R.; Shu, R.; Yu, J.; Li, H.; Long, H.; Jin, S.; Li, S.; Hu, Q.; Yao, F.; et al. Study of the Relationship between
Microbiome and Colorectal Cancer Susceptibility Using 16SrRNA Sequencing. Biomed. Res. Int. 2020, 2020, 7828392. [CrossRef]
[PubMed]
27. Zhuang, H.; Cheng, L.; Wang, Y.; Zhang, Y.-K.; Zhao, M.-F.; Liang, G.-D.; Zhang, M.-C.; Li, Y.-G.; Zhao, J.-B.; Gao, Y.-N.; et al.
Dysbiosis of the Gut Microbiome in Lung Cancer. Front. Cell Infect. Microbiol. 2019, 9, 112. [CrossRef] [PubMed]
28. Bhatt, A.P.; Redinbo, M.R.; Bultman, S.J. The Role of the Microbiome in Cancer Development and Therapy. CA Cancer J. Clin.
2017, 67, 326–344. [CrossRef] [PubMed]
29. Zhao, L.; Grimes, S.M.; Greer, S.U.; Kubit, M.; Lee, H.; Nadauld, L.D.; Ji, H.P. Characterization of the consensus mucosal
microbiome of colorectal cancer. NAR Cancer 2021, 3, zcab049. [CrossRef]
30. Cullin, N.; Azevedo Antunes, C.; Straussman, R.; Stein-Thoeringer, C.K.; Elinav, E. Microbiome and cancer. Cancer Cell 2021,
39, 1317–1341. [CrossRef]
31. van Elsland, D.M.; Duijster, J.W.; Zhang, J.; Stévenin, V.; Zhang, Y.; Zha, L.; Xia, Y.; Franz, E.; Sun, J.; Mughini-Gras, L.; et al.
Repetitive non-typhoidal Salmonella exposure is an environmental risk factor for colon cancer and tumor growth. Cell Rep. Med.
2022, 3, 100852. [CrossRef]
32. Qi, Z.; Zhibo, Z.; Jing, Z.; Zhanbo, Q.; Shugao, H.; Weili, J.; Jiang, L.; Shuwen, H. Prediction model of poorly differentiated
colorectal cancer (CRC) based on gut bacteria. BMC Microbiol. 2022, 22, 312. [CrossRef] [PubMed]
33. Ghorbani, E.; Avan, A.; Ryzhikov, M.; Ferns, G.; Khazaei, M.; Soleimanpour, S. Role of lactobacillus strains in the management of
colorectal cancer: An overview of recent advances. Nutrition 2022, 103–104, 111828. [CrossRef]
34. Sobhani, I.; Tap, J.; Roudot-Thoraval, F.; Roperch, J.P.; Letulle, S.; Langella, P.; Corthier, G.; Nhieu, J.T.V.; Furet, J.P. Microbial
Dysbiosis in Colorectal Cancer (CRC) Patients. PLoS ONE 2011, 6, e16393. [CrossRef]
35. Zheng, Y.; Fang, Z.; Xue, Y.; Zhang, J.; Zhu, J.; Gao, R.; Yao, S.; Ye, Y.; Wang, S.; Lin, C.; et al. Specific gut microbiome signature
predicts the early-stage lung cancer. Gut Microbes 2020, 11, 1030–1042. [CrossRef] [PubMed]
36. Yusuf, K.; Sampath, V.; Umar, S. Bacterial Infections and Cancer: Exploring This Association and Its Implications for Cancer
Patients. Int. J. Mol. Sci. 2023, 24, 3110. [CrossRef] [PubMed]
37. Yu, H.; Li, X.-X.; Han, X.; Chen, B.-X.; Zhang, X.-H.; Gao, S.; Xu, D.-Q.; Wang, Y.; Gao, Z.-K.; Yu, L.; et al. Fecal microbiota
transplantation inhibits colorectal cancer progression: Reversing intestinal microbial dysbiosis to enhance anti-cancer immune
responses. Front. Microbiol. 2023, 14, 1126808. [CrossRef]
38. Wang, Y.; Zhang, C.; Hou, S.; Wu, X.; Liu, J.; Wan, X. Analyses of Potential Driver and Passenger Bacteria in Human Colorectal
Cancer. Cancer Manag. Res. 2020, 12, 11553–11561. [CrossRef]
39. Ni, B.; Kong, X.; Yan, Y.; Fu, B.; Zhou, F.; Xu, S. Combined analysis of gut microbiome and serum metabolomics reveals novel
biomarkers in patients with early-stage non-small cell lung cancer. Front. Cell Infect. Microbiol. 2023, 13, 1091825. [CrossRef]
40. Hakozaki, T.; Nolin-Lapalme, A.; Kogawa, M.; Okuma, Y.; Nakamura, S.; Moreau-Amaru, D.; Tamura, T.; Hosomi, Y.; Takeyama,
H.; Richard, C.; et al. Cancer Cachexia among Patients with Advanced Non-Small-Cell Lung Cancer on Immunotherapy: An
Observational Study with Exploratory Gut Microbiota Analysis. Cancers 2022, 14, 5405. [CrossRef]
41. Maddi, A.; Sabharwal, A.; Violante, T.; Manuballa, S.; Genco, R.; Patnaik, S.; Yendamuri, S. The microbiome and lung cancer. J.
Thorac. Dis. 2019, 11, 280–291. [CrossRef]
42. Bagheri, Z.; Moeinzadeh, L.; Razmkhah, M. Roles of Microbiota in Cancer: From Tumor Development to Treatment. J. Oncol.
2022, 2022, 3845104. [CrossRef] [PubMed]
43. Hosmer, D.W.; Lemeshow, S. Area under the ROC curve. In Applied Logistic Regression, 2nd ed.; John Wiley & Sons: New York,
NY, USA, 2000; pp. 160–164.
44. Sender, R.; Fuchs, S.; Milo, R. Revised Estimates for the Number of Human and Bacteria Cells in the Body. PLoS Biol. 2016,
14, e1002533. [CrossRef] [PubMed]
45. NIH Human Microbiome Project—HMQCP. Available online: https://hmpdacc.org/HMQCP/ (accessed on 16 January 2024).

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.