Advertisement

The Role of Intestinal Microbiota in Metastatic Colorectal Cancer Patients Treated With Capecitabine

  • Author Footnotes
    & Shared first authorship
    Romy Aarnoutse
    Footnotes
    & Shared first authorship
    Affiliations
    GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands

    Department of Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands
    Search for articles by this author
  • Author Footnotes
    & Shared first authorship
    Janine Ziemons
    Footnotes
    & Shared first authorship
    Affiliations
    GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands

    Department of Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands
    Search for articles by this author
  • Judith de Vos-Geelen
    Affiliations
    GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands

    Department of Internal Medicine, Division of Medical Oncology, Maastricht University Medical Centre, Maastricht, The Netherlands
    Search for articles by this author
  • Liselot Valkenburg-van Iersel
    Affiliations
    GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands

    Department of Internal Medicine, Division of Medical Oncology, Maastricht University Medical Centre, Maastricht, The Netherlands
    Search for articles by this author
  • Aurelia C.L. Wildeboer
    Affiliations
    GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands

    Department of Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands
    Search for articles by this author
  • Anne Vievermans
    Affiliations
    GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands

    Department of Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands
    Search for articles by this author
  • Geert-Jan M. Creemers
    Affiliations
    Department of Medical Oncology, Catharina Hospital, Eindhoven, The Netherlands
    Search for articles by this author
  • Arnold Baars
    Affiliations
    Department of Medical Oncology, Hospital Gelderse Vallei, Ede, The Netherlands
    Search for articles by this author
  • Hanneke J.H.M.J. Vestjens
    Affiliations
    Department of Internal Medicine, VieCuri Medical Centre, Venlo, The Netherlands
    Search for articles by this author
  • Giang N. Le
    Affiliations
    Department of Medical Microbiology, Maastricht University Medical Centre, Maastricht, The Netherlands
    Search for articles by this author
  • David J.M. Barnett
    Affiliations
    Department of Medical Microbiology, Maastricht University Medical Centre, Maastricht, The Netherlands

    Maastricht Centre for Systems Biology (MaCSBio), Maastricht University, Maastricht, The Netherlands
    Search for articles by this author
  • Sander S. Rensen
    Affiliations
    Department of Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands

    NUTRIM - School of Nutrition and Translational research In Metabolism, Maastricht University, Maastricht, The Netherlands
    Search for articles by this author
  • Author Footnotes
    # Shared last authorship
    John Penders
    Footnotes
    # Shared last authorship
    Affiliations
    Department of Medical Microbiology, Maastricht University Medical Centre, Maastricht, The Netherlands

    NUTRIM - School of Nutrition and Translational research In Metabolism, Maastricht University, Maastricht, The Netherlands
    Search for articles by this author
  • Author Footnotes
    # Shared last authorship
    Marjolein L. Smidt
    Correspondence
    Corresponding author: Prof. dr. Marjolein L. Smidt, Department of Surgery, Maastricht University Medical Centre, P.O. P. Debyelaan 25, 6202 AZ Maastricht, The Netherlands
    Footnotes
    # Shared last authorship
    Affiliations
    GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands

    Department of Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands
    Search for articles by this author
  • Author Footnotes
    & Shared first authorship
    # Shared last authorship
Open AccessPublished:October 17, 2021DOI:https://doi.org/10.1016/j.clcc.2021.10.004

      Abstract

      Background

      Previous pre-clinical research has indicated that the intestinal microbiota can potentiate anti-tumour efficacy of capecitabine and that capecitabine treatment impacts intestinal microbiota composition and diversity. Using a longitudinal design, this study explores the associations between the intestinal microbiota and treatment response in patients with metastatic colorectal cancer (mCRC) during capecitabine treatment.

      Patients and Methods

      Patients with mCRC treated with capecitabine were prospectively enrolled in a multicentre cohort study. Patients collected a faecal sample and completed a questionnaire before, during, and after three cycles of capecitabine. Several clinical characteristics, including tumour response, toxicity and antibiotic use were recorded. Intestinal microbiota were analysed by amplicon sequencing of the 16S rRNA V4 gene-region.

      Results

      Thirty-three patients were included. After three cycles of capecitabine, six patients (18%) achieved a partial response, 25 (76%) showed stable disease, and one (3%) experienced progressive disease. Of the 90 faecal samples were collected. Microbial diversity (α-diversity), community structure (β-diversity), and bacterial abundance on phylum and genus level were not significantly different between responders and non-responders and were not significantly affected by three cycles of capecitabine.

      Conclusion

      This is the first clinical study with longitudinal intestinal microbiota sampling in mCRC patients that explores the role of the intestinal microbiota during treatment with capecitabine. Intestinal microbiota composition and diversity before, during, and after three cycles of capecitabine were not associated with response in this study population. Capecitabine did not induce significant changes in the microbiota composition and diversity during the treatment period. Individual effects of antibiotics during capecitabine treatment were observed.

      Keywords

      Introduction

      Colorectal cancer (CRC) is the third most common cancer in the world.
      • Rawla P
      • Sunkara T
      • Barsouk A.
      Epidemiology of colorectal cancer: incidence, mortality, survival, and risk factors.
      Despite recent developments in systemic therapy, classical chemotherapeutic agents such as fluoropyrimidines, for example, capecitabine, an oral prodrug of 5-fluorouracil (5-FU), remain the backbone of most systemic therapies. Capecitabine, with or without the vascular endothelial growth factor inhibitor bevacizumab
      • Cunningham D
      • Lang I
      • Marcuello E
      • et al.
      Bevacizumab plus capecitabine versus capecitabine alone in elderly patients with previously untreated metastatic colorectal cancer (AVEX): an open-label, randomised phase 3 trial.
      , is often applied in mCRC patients who are not eligible for intensive chemotherapy combinations because of comorbidity or impaired performance score, resulting in an objective response rate of only 21-25%.
      • Hoff PM
      • Ansari R
      • Batist G
      • et al.
      Comparison of oral capecitabine versus intravenous fluorouracil plus leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study.
      ,
      • Van Cutsem E
      • Findlay M
      • Osterwalder B
      • et al.
      Capecitabine, an oral fluoropyrimidine carbamate with substantial activity in advanced colorectal cancer: results of a randomized phase II study.
      Besides controlling tumour growth, capecitabine potentially induces toxicity, severely impacting quality of life. The most common CTCAE grade 3 toxic events are diarrhea (24%), hand-foot syndrome (18%), and stomatitis (3%)[3].
      In order to optimise treatment outcome, factors that impact individual response and safety profile to capecitabine need to be identified. During the last decade, evidence of the interaction between systemic cancer therapies and the human intestinal microbiota has rapidly expanded. The human intestinal microbiota consists of bacteria, archaea, viruses, and fungi.
      Team NIHHMPA
      A review of 10 years of human microbiome research activities at the US National Institutes of Health, Fiscal Years 2007-2016.
      It has been shown that trillions of intestinal bacteria stimulate the immune system, might be involved in carcinogenesis and influence human metabolism of dietary components and medication, including chemotherapeutic agents.
      • Helmink BA
      • Khan MAW
      • Hermann A
      • Gopalakrishnan V
      • Wargo JA.
      The microbiome, cancer, and cancer therapy.
      Pre-clinical microbiota studies indicate significant interactions between the intestinal microbiota and 5-FU or capecitabine. Sougiannis et al. demonstrated that 5-FU treatment affects intestinal microbiota composition, the colonic morphology and immune profile, as well as functional outcomes of fatigue in a mouse model of colon cancer.
      • Sougiannis AT
      • VanderVeen BN
      • Enos RT
      • et al.
      Impact of 5 fluorouracil chemotherapy on gut inflammation, functional parameters, and gut microbiota.
      Furthermore, Lactobacillus plantarum supernatant sensitized CRC cell lines to 5-FU and stimulated apoptosis in chemo-resistant cells.
      • An J
      • Ha EM.
      Combination therapy of lactobacillus plantarum supernatant and 5-fluouracil increases chemosensitivity in colorectal cancer cells.
      Administration of an antibiotic cocktail (vancomycin, ampicillin, neomycin, and metronidazol) reduced antitumour efficacy of 5-FU in mice.
      • Yuan L
      • Zhang S
      • Li H
      • et al.
      The influence of gut microbiota dysbiosis to the efficacy of 5-Fluorouracil treatment on colorectal cancer.
      Very recently, Zimmermann et al. provided the first in vitro evidence that capecitabine can be metabolised by several bacterial species.
      • Zimmermann M
      • Zimmermann-Kogadeeva M
      • Wegmann R
      • Goodman AL.
      Mapping human microbiome drug metabolism by gut bacteria and their genes.
      Clinical evidence for a potential influence of intestinal microbiota on chemotherapy efficacy or toxicity is limited. This is mainly due to a lack of studies with longitudinal microbiota sampling during chemotherapy.
      • Aarnoutse R
      • Ziemons J
      • Penders J
      • Rensen SS
      • de Vos-Geelen J
      • Smidt ML.
      The clinical link between human intestinal microbiota and systemic cancer therapy.
      ,
      • Li J
      • Li J
      • Lyu N
      • et al.
      Composition of fecal microbiota in low-set rectal cancer patients treated with FOLFOX.
      With respect to CRC and capecitabine, no clinical studies are available. Only one study, in which 31 patients with rectal cancer were treated with a combination of 5-FU and oxaliplatin (FOLFOX), partly supports the pre-clinical data.
      • Li J
      • Li J
      • Lyu N
      • et al.
      Composition of fecal microbiota in low-set rectal cancer patients treated with FOLFOX.
      We hypothesised that pre-treatment intestinal microbiota composition and diversity and its changes during capecitabine therapy are associated with response and/or therapy-related toxicity in mCRC patients. We conducted a prospective study to evaluate changes in intestinal microbiota composition and diversity during chemotherapy, assessing chemotherapy toxicity and response to capecitabine in mCRC patients.

      Patients and Methods

       Patients

      Between March 2017 and September 2019, patients were prospectively enrolled in four Dutch Hospitals.
      • Aarnoutse R
      • de Vos-Geelen JMPGM
      • Penders J
      • et al.
      Study protocol on the role of intestinal microbiota in colorectal cancer treatment: a pathway to personalized medicine 2.0.
      Patients with histologically proven mCRC to be treated with capecitabine with or without bevacizumab, aged 18 years or older were eligible. Exclusion criteria included microsatellite instability (MSI), impaired renal function as defined by creatinine clearance (Cockroft-Gault) <30 ml/min, abdominal radiotherapy within two weeks prior to starting capecitabine, systemic cancer therapy within four weeks prior to starting capecitabine, and therapeutic antibiotics use within three months prior to starting capecitabine.
      The study was registered in the Dutch Trial Register (NTR6957) and approved by the Medical Ethics Committee azM/UM (METC 16-4-234.1) and was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. Each patient provided written informed consent.

       Treatment

      During the study period, patients received three cycles of capecitabine (1000-1250 mg/m2 orally, twice daily on days 1-14 in a 3 week cycle) with or without bevacizumab (7.5 mg/kg intravenously on day 1 every 3 weeks).

       Materials and Methods

      According to the previous published study protocol
      • Aarnoutse R
      • de Vos-Geelen J
      • Penders J
      • et al.
      Study protocol on the role of intestinal microbiota in colorectal cancer treatment: a pathway to personalized medicine 2.0.
      , patients collected pure faecal samples in preservation free faeces tubes (Sarstedt) and completed questionnaires at three time points: before the start of the first capecitabine cycle (T1, one or two days before the start of the cycle), between days 7 and 14 of the third cycle (T2), and at day 20 or 21 of the third cycle (T3) (Figure S1). After collection, samples were immediately stored in the freezer at home and transported to the hospital in a cooled container (Sarstedt), where samples were stored at -20°C first and at -80°C for long-term storage. Patient characteristics including history of gastrointestinal surgery, Karnofsky performance score, nutritional status assessed with the Malnutrition Universal Screening Tool, chemotherapy compliance, dose reductions, antibiotic/prebiotic/probiotic use, and the use of nutritional supportive drinks were registered.

       Response Measurement

      Tumour response was assessed using CT or MRI scans before and at the end of three cycles of capecitabine by means of RECIST (Response Evaluation Criteria in Solid Tumours) version 1.1.
      • Eisenhauer EA
      • Therasse P
      • Bogaerts J
      • et al.
      New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).
      Response was defined as complete response (CR): disappearance of all target lesions and partial response (PR): ≥30% decrease in the sum of of the target lesions. Non-response was defined as progressive disease (PD): ≥20% increase in the sum of target lesions and stable disease (SD): small changes that do not meet above criteria.
      • Eisenhauer EA
      • Therasse P
      • Bogaerts J
      • et al.
      New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).
      Toxicity was scored with CTCAE version 4.0.
      National Institutes of Health
      Common Terminology Criteria for Adverse Events (CTCEA).
      The following aspects were scored: diarrhea with or without colostomy, peripheral sensory neuropathy, hand-foot syndrome, fatigue, nausea, oral mucositis, vomiting, and constipation.

       Faecal Microbiota Analyses

      Metagenomic DNA was isolated using the Ambion MagMax Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific) and consisted of mechanical disruption with bead-beating, as well as chemical and thermal disruption. The manual pre-processing was followed by automated nucleic acid purification with the KingFisher FLEX (Thermo Fisher Scientific). Upon PCR-amplification of the 16S ribosomal RNA (rRNA) hypervariable V4 gene-region according to current international accepted standards
      • Liu PY
      • Wu WK
      • Chen CC
      • Panyod S
      • Sheen LY
      • Wu MS.
      Evaluation of Compatibility of 16S rRNA V3V4 and V4 Amplicon Libraries for Clinical Microbiome Profiling.
      , amplicons were sequenced on a MiSeq platform, as described previously.
      • Galazzo G
      • van Best N
      • Benedikter BJ
      • et al.
      How to count our microbes? the effect of different quantitative microbiome profiling approaches.
      Pre-processing of the sequencing data was performed using R. A standardised in-house pipeline using the software package DADA2 (R version 4.0.3) was applied.
      • Callahan BJ
      • McMurdie PJ
      • Rosen MJ
      • Han AW
      • Johnson AJ
      • Holmes SP.
      DADA2: High-resolution sample inference from Illumina amplicon data.
      After pre-processing, 908 taxa remained for downstrean analysis. For further details on DNA isolation, sequencing, and data pre-processing see the supplementary methods.

       Statistical Analysis of Clinical Data

      Baseline characteristics were analyzed in IBM SPSS version 26. For continuous data, normality was tested using the Shapiro-Wilk test. Depending on whether the variable was normally distributed or not, an unpaired t test or the non-parametric Mann-Whitney U test was applied. Levene's test was used to test for equal variances. For categorical variables, the non-parametric Chi-square test or a Fisher's exact test, in case of low frequencies for binary variables, was performed. For longitudinal analysis with two time points of quantitative variables, a paired sample t test or the non-parametric Wilcoxon signed-rank sum test was used. For longitudinal analysis with three time points, repeated-measures ANOVA (sphericity assumed) or Friedman's ANOVA were used for normally and non-normally distributed data, respectively. Significant results were subjected to a post hoc Wilcoxon signed-rank sum tests with Bonferroni correction. Two-tailed tests were use and P-values below .05 were considered statistically significant.

       Statistical Analysis of Intestinal Microbiota Data

      Bioinformatic analysis of the sequencing data was performed using R version 4.0.3.
      Team RC.
      R: A language and environment for statistical computing.
      For the calculation of α-diversity indices on Amplicon Sequencing Variant level (Shannon effective and observed richness) and prior data normalization, the standard script and settings of the Rhea pipeline were used.
      • Lagkouvardos I
      • Fischer S
      • Kumar N
      • Clavel T.
      Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.
      Testing the assumptions of normality, homogeneity of variance and subsequent statistical testing was performed as described for clinical data.
      In order to quantify microbial community structure (β-diversity), generalized UniFrac and Bray-Curtis distances were calculated on Amplicon Sequencing Variant level, using Rhea.
      • Lagkouvardos I
      • Fischer S
      • Kumar N
      • Clavel T.
      Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.
      and the R packages GUniFrac
      • GUniFrac Chen J.
      Generalized UniFrac distances.
      and phyloseq
      • McMurdie PJ
      • Holmes S.
      phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data.
      respectively. Temporal (in)stability of microbial community structure was expressed as generalized UniFrac/Bray-Curtis distances between T1/T2, T2/T3 and T1/T3 within the same patient. Mann Whitney U test was used to compare differences between responders and non-responders at all time points. The R packages, phyloseq
      • McMurdie PJ
      • Holmes S.
      phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data.
      , vegan
      • FGB Jari Oksanen
      • Friendly Michael
      • Kindt Roeland
      • et al.
      Eduard Szoecs, Wagner aH. vegan: community ecology package.
      , microbiome

      Lahti L, Ernst FGM, Huang R, et al. microbiome R package. 2020.

      , dplyr
      • Wickham H
      • François R
      • Henry L
      • Müller K.
      dplyr: A grammar of data manipulation.
      , ggplot2
      • Wickham H.
      ggplot2: elegant graphics for data analysis.
      , and microViz
      • Barnett DJM
      • Arts ICW
      • Penders J.
      microViz: an R package for microbiome data visualization and statistics.
      were used for ordination and visualisation of taxonomic composition. Taxa present in less than 5 samples were filtered out for ordination and all subsequent analyses. Permutational multivariate analysis of variance (PERMANOVA) was applied to examine associations between variation in overall microbial community structure and treatment response and study time point variables. Aitchison distance on phylum and genus level was used for ordination as well as for PERMANOVA. Differential abundance analysis of individual microbial taxa was conducted using the workflow of ANCOM v.2.1 which accounts for the underlying structure of microbiota data and the presence of zeros.
      • Mandal S
      • Van Treuren W
      • White RA
      • Eggesbø M
      • Knight R
      • Peddada SD.
      Analysis of composition of microbiomes: a novel method for studying microbial composition.
      We tested for differential abundance between responders and non-responders at T1 and T2, and for differential abundance over time within individuals. We set P < .05 at 70% of comparisons as a threshold for significance.

      Results

      In total, 33 patients with mCRC treated with capecitabine (+/- bevacizumab) were included. Baseline characteristics were stratified by response evaluation (Table 1 and S1). After three cycles of capecitabine, six patients (18%) achieved a partial response, 25 (76%) showed stable disease, and one (3%) had progressive disease. In one patient (3%), response could not be evaluated due to withdrawal of study participation. Consequently, 6 patients were classified as responders and 26 patients as non-responders. In total, 90 faecal samples were collected. Figure S2 provides an overview of all samples available for 16S rRNA gene sequencing.
      Table 1Clinical Characteristics
      Baseline characteristicsTotal (N = 33)Responders (n = 6)Non-responders (n = 26)P-value
      Age – Years

      Median (IQR)
      75 (14)74 (19)75 (13)0.981
      BMI – kg/m2

      Mean (SD)
      27 (±5)26 (±7)27 (± 4)0.439
      CTCAE Unintentional weight loss – No. (%)
      Percentages do not add up to 100% due to rounding.


      Gr. 0

      Gr. 1
      28 (88)

      4 (13)
      6 (100)

      0 (0)
      21 (84)

      4 (16)
      0.561
      Male – No. (%)25 (76)2 (33)22 (85)0.023
      Sidedness tumor – No. (%)

      Left sided

      Right sided
      21 (66)

      11 (34)
      5 (83)

      1 (17)
      16 (64)

      9 (36)
      0.634
      Time to metastasis – No. (%)

      Synchronous

      Metachronous
      20 (61)

      13 (39)
      3 (50)

      3 (50)
      16 (62)

      10 (38)
      0.666
      Colorectal surgery in the past – No. (%)29 (88)6 (100)22 (85)0.566
      Colostoma in situ at T1– No. (%)10 (30)2 (33)8 (31)1.000
      Previous systemic treatment – No. (%)16 (48)4 (66)12 (46)0.654
      MUST score – No (%)

      Low risk

      Medium risk

      High risk
      26 (79)

      4 (12)

      3 (9)
      5 (83)

      0 (0)

      1 (17)
      21 (81)

      3 (11)

      2 (8)
      0.823
      Karnofsky Performance Score – No (%)*

      50-60

      70-80

      90-100
      3 (10)

      9 (29)

      19 (62)
      1 (17)

      1 (17)

      4 (67)
      2 (8)

      8 (34)

      14 (58)
      0.372
      Response could not be evaluated in one patient.
      a Percentages do not add up to 100% due to rounding.

       Baseline Characteristics

      Median age was 75 years. Mean BMI was 27 kg/m2. Men (76%) were predominant in the total group. Most patients presented with synchronous metastatic disease, of which eight patients had metastasis at one site and 25 had multiple organs involved. Twenty-one (66%) patients had a left-sided tumor. In total 88% underwent resection of the primary tumor (Table 1). A low anterior resection was performed in twelve patients, a sigmoid resection in five patients, a left-sided hemicolectomy in two patients, an extended left-sided hemicolectomy in one patient, and a right-sided hemicolectomy in eight patients. Of the patients who underwent resection of the primary tumor, 30% still had a colostomy at the time of inclusion in the current study. Nearly half of the patients (48%) received previous systemic therapy in any setting with any type of chemotherapy. In the year prior to inclusion, 24% of the patients used therapeutic antibiotics (none within three months before T1). The mean time in days between the last intake of antibiotics and the baseline faecal sample collection was 197 days. In total, 30% used prophylactic antibiotics in the last year, with a mean of 96 days between the last intake of prophylactic antibiotics and faecal sample collection. None of the patients used prednisone (one month), prebiotics, or probiotics (1 year) prior to T1. Men were predominant in the non-responders group (85%, P = .023). All other baseline characteristics were not significantly different between responders and non-responders (Table 1 and S1).

       Clinical Characteristics Before, During, and After Three Cycles of Capecitabine

      During capecitabine treatment, there were no significant differences in capecitabine dose intensity, compliance, and antibiotic use between responders and non-responders (Table S2). In total, 83% of the responders and 81% of the non-responders received co-treatment with bevacizumab.
      After three cycles of capecitabine, non-responders indicated significantly higher grades of fatigue compared to responders (P = .026). All other toxicity measures were not significantly different between responders and non-responders before, during or after three cycles of capecitabine (Table S3-S5). Toxicity grades of peripheral sensory neuropathy, hand foot syndrome, oral mucositis, and bone marrow toxicity increased significantly over the study period (Figure 1, Table S6 and S7). All other toxicity measures, including diarrhea, did not change during three cycles of capacitabine (Table S6).
      Figure 1
      Figure 1Stacked bar charts presenting percentage toxicity grades before, during, and after three cycles of capecitabine. For peripheral sensory neuropathy and oral mucositis significant differences were observed between T1-T2 and T1-T3. For hand foot syndrome significant differences were observed between all-time points.
      Compared to baseline, Karnofsky performance score was significantly lower after three cycles of capecitabine (P = .002) (Table S8). The Malnutrition Universal Screening Tool score was not significantly different before, during or after three cycles of capecitabine (Table S8).

       Intestinal Microbiota Composition and Diversity

      Similar α-diversity in responders and non-responders
      Before (Figure S3 and Table S9) and during (Figure 2A and Table S10) three cycles of capecitabine, Shannon effective as well as observed richness were similar between responders and non-responders. In addition, both α-diversity indices did not significantly change over the course of three cycles of capecitabine (Figure 2B and Table S11).
      Figure 2
      Figure 2α-diversity measures A: Microbial diversity and richness of responders and non-responders at T2, measured in terms of Shannon effective (P = .301) and observed richness (P = .145) (Table S10). B: α-diversity before, during, and after three cycles of capecitabine, measured in terms of Shannon effective (P = .640) and observed richness (P = .240) (Table S11). Numbers presented in median (IQR).
      Microbial community structure (β-diversity) and abundance of specific bacteria

       No Differences Between Responders and Non-Responders

      Principal Component Analysis showed large heterogeneity in individual microbial community structures. PERMANOVA revealed that there was no statistically significant association between treatment response and the overall microbial community structure at T2 on phylum (P = .07) and genus (P = .41) level (Figure 3). However, on phylum level, responders tended to cluster in the direction of Proteobacteria and Actinobacteria (Figure 3A). In addition, in the entire population the abundance of Euryarcheota and Verrucomicrobia had a major contribution to the first and second Principal Component Analysis axis, respectively (Figure 3A). On genus level, Lachnospiraceae ND3007 group, Dialister, Veillonella, Anerostipes, and Flavonifractor contributed the most to the variation in the overall microbiota community structure (Figure 3B). At T1, there was also a large heterogeneity and no association between treatment response and overall microbial community structure on phylum (P = .38) and genus (P = .73) level (Figure S4). Furthermore, there were no differences found between responders and non-responders concerning within-subject temporal (in)stability of β-diversity between the various time points, using generalised UniFrac as well as Bray-Curtis distances. P08 showed considerably large instability between T2 to T3 and T1 to T3 (Figure S5 and Table S12).
      Figure 3
      Figure 3Ordination plots derived from unconstrained Principal Components Analysis (PCA), showing overall composition of the microbial community on phylum (A) and genus level (B) at T2. Aitchison distance was used. 10 phyla and 150 genera were included for this analysis. Data were transformed using centre-log-ratio transformation. Names are given for genera which contributed most to overall microbial variation.
      Differential abundance analysis on phylum and genus level identified no taxa which were differentially abundant between responders and non-responders at T1 and T2.

       Large Intra-Individual Microbiota Alterations During Capecitabine Treatment

      In the present research population, Firmicutes were the most abundant phylum, followed by Bacteroidetes and Actinobacteria. At phylum level, no major shifts were observed during the course of three cycles of capecitabine (Figure 4A). This was confirmed by PERMANOVA, which revealed no association between sampling time point and microbial community structure on phylum (P = .96) level.
      Figure 4
      Figure 4(A) Composition plot at phylum level, before, during, and after three cycles of capecitabine, indicating relative abundance of the most common phyla. (B) Changes in relative abundance of the most common genera before, during, and after three cycles of capecitabine indicate a large inter-individual heterogeneity and no prominent universal capecitabine-induced effect.
      Figure 4B shows the most abundant genera before, during, and after three cycles of capecitabine. We observed large inter-individual heterogeneity but no prominent universal capecitabine-induced pattern. On group level, PERMANOVA revealed no association between sampling time point and microbial community structure on genus (P = 1.0) level. In line with this, ANCOM analysis with treatment response as covariate identified no phyla or genera that significantly differed in abundance before, during, and after three cycles of capecitabine.
      During the study period, large intra-individual shifts of the intestinal microbiota composition were observed (Figure 4B), which could partly be explained by clinical data. P20 displayed a high relative abundance of bifidobacteria. This patient showed partial response (40% decrease in the sum of target lesions) after three cycles of capecitabine. P08 received oral amoxicillin/ciprofloxacin before collection of the last faecal sample and showed relatively high levels of Bacteroides and Streptococcus in this sample. P01 received oral ciprofloxacin 24 days before the second faecal sample collection, resulting in relatively high levels of Streptococcus.

      Discussion

      This is the first clinical study with longitudinal intestinal microbiota sampling in mCRC patients that explored the role of the intestinal microbiota during treatment with capecitabine (without surgery, radiation or chemotherapy or combinations thereof). Intestinal microbiota composition and diversity before, during or after three cycles of capecitabine were not associated with treatment response in the current small study population. Furthermore, capecitabine treatment did not alter the microbiota composition and diversity during the course of three cycles of capecitabine. In contrast to the minor effect of capecitabine on the intestinal microbiota, individual effects of antibiotic treatment during capecitabine treatment were observed in two patients.
      In this study, we showed that longitudinal faecal sample collection is feasible in mCRC patients. Our baseline characteristics indicate that we included a representative mCRC population; the disease control rate after three cycles of capecitabine is comparable to the study of Cutsem et al.
      • Van Cutsem E
      • Findlay M
      • Osterwalder B
      • et al.
      Capecitabine, an oral fluoropyrimidine carbamate with substantial activity in advanced colorectal cancer: results of a randomized phase II study.
      In the current study population, microbial α-diversity was not significantly different between responders and non-responders and did not diminish during the course of three cycles of capecitabine. This is partially in line with data described by Li et al
      • Li J
      • Li J
      • Lyu N
      • et al.
      Composition of fecal microbiota in low-set rectal cancer patients treated with FOLFOX.
      . Li et al studied rectal cancer patients without metastasis who received a combination of 5-FU and oxaliplatin (FOLFOX). They also did not observe a difference in α-diversity between responders and non-responders before FOLFOX treatment. However, a decrease in α-diversity after FOLFOX treatment in the responder group was found in that study. These different study outcomes could be due to an already altered intestinal microbiota at baseline in our study population. Nearly half of the patients (48%) received previous chemotherapy (more than one month before inclusion), which is associated with extensive hospitalization and lifestyle changes (desirable and undesirable).
      • Balhareth A
      • Aldossary MY
      • McNamara D.
      Impact of physical activity and diet on colorectal cancer survivors' quality of life: a systematic review.
      As a consequence, microbial dysbiosis might have been already present at baseline, leading to only minor capecitabine related effects. It is possible that the potential capecitabine-induced effects on the microbiota diversity would be higher if the patients were included and collected faecal samples at primary diagnosis.
      In line with the extensive medical history of these patients, we observed considerable heterogeneity in individual microbial community structure (β-diversity) before and during three cycles of capecitabine. This might have contributed to the lack of association between treatment response and microbial community structure.
      Abundance of taxa at phylum and genus level did not significantly differ before, during or after chemotherapy in the whole group. This is in contrast with results from Sze et al
      • Sze MA
      • Baxter NT
      • MTt Ruffin
      • Rogers MAM
      • Schloss PD.
      Normalization of the microbiota in patients after treatment for colonic lesions.
      who performed longitudinal microbiota analysis in patients with primary diagnosed CRC (n = 26). After treatment, they observed a change in community structure and a shift towards a microbiota comparable to the profile of healthy controls. These findings were based on a heterogeneously treated group including surgery, with or without eight different types of chemotherapy, with or without radiation.
      • Sze MA
      • Baxter NT
      • MTt Ruffin
      • Rogers MAM
      • Schloss PD.
      Normalization of the microbiota in patients after treatment for colonic lesions.
      Another study in patients with rectal cancer without metastasis treated with FOLFOX showed therapy-induced changes in genus abundances, which were more pronounced in the patients achieving a partial or complete response.
      • Li J
      • Li J
      • Lyu N
      • et al.
      Composition of fecal microbiota in low-set rectal cancer patients treated with FOLFOX.
      Additionally, they identified specific species (Coprobacter fastidiosus, Alistipes finegoldii, Gemella unclassified, Granulicatella adiacens, Parvimonas micra, and Clostridium ramosum) associated with the outcome of FOLFOX treatment, which might potentially be useful as a biomarker to predict therapy outcome.
      • Li J
      • Li J
      • Lyu N
      • et al.
      Composition of fecal microbiota in low-set rectal cancer patients treated with FOLFOX.
      After different types of chemotherapy (n = 23), Zwielehner et al. showed decreased levels of Clostridium cluster IV, Bacteroides, bifidobacteria, as well as Clostridium cluster XIVa in patients (n=17) with different types of cancer (n = 13).
      • Zwielehner J
      • Lassl C
      • Hippe B
      • et al.
      Changes in human fecal microbiota due to chemotherapy analyzed by TaqMan-PCR, 454 sequencing and PCR-DGGE fingerprinting.
      Recently, Zimmermann et al. provided in-vitro evidence that capecitabine can be metabolised by several bacterial species including Bifidobacterium ruminatum, Bacteroides xylanisolvens DSM18836, and Salmonella Typhimurium LT2.
      • Zimmermann M
      • Zimmermann-Kogadeeva M
      • Wegmann R
      • Goodman AL.
      Mapping human microbiome drug metabolism by gut bacteria and their genes.
      Our results are not in line with these previous studies, which may be related to the complex medical history of our patients in combination with the relatively mild form of systemic therapy with capecitabine in contrast to FOLFOX treatment.
      • Cunningham D
      • Lang I
      • Marcuello E
      • et al.
      Bevacizumab plus capecitabine versus capecitabine alone in elderly patients with previously untreated metastatic colorectal cancer (AVEX): an open-label, randomised phase 3 trial.
      • Hoff PM
      • Ansari R
      • Batist G
      • et al.
      Comparison of oral capecitabine versus intravenous fluorouracil plus leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study.
      • Van Cutsem E
      • Findlay M
      • Osterwalder B
      • et al.
      Capecitabine, an oral fluoropyrimidine carbamate with substantial activity in advanced colorectal cancer: results of a randomized phase II study.
      ,
      • Li J
      • Li J
      • Lyu N
      • et al.
      Composition of fecal microbiota in low-set rectal cancer patients treated with FOLFOX.
      Although there were no differences in microbiota composition between responders and non-responders, specific patients showed remarkable microbiota shifts during therapy, which could be explained based on clinical data. Two patients (P08/P01) received ciprofloxacin during chemotherapy. As a consequence of this broad-spectrum antibiotic, relatively high levels of possibly ciprofloxacin-resistant Streptococcus were observed.
      • Dubinsky V
      • Reshef L
      • Bar N
      • et al.
      Predominantly antibiotic-resistant intestinal microbiome persists in patients with pouchitis who respond to antibiotic therapy.
      These individual changes indicate that the impact of antibiotics was substantial compared to the impact of the relatively mild chemotherapeutic capecitabine.
      • Cunningham D
      • Lang I
      • Marcuello E
      • et al.
      Bevacizumab plus capecitabine versus capecitabine alone in elderly patients with previously untreated metastatic colorectal cancer (AVEX): an open-label, randomised phase 3 trial.
      • Hoff PM
      • Ansari R
      • Batist G
      • et al.
      Comparison of oral capecitabine versus intravenous fluorouracil plus leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study.
      • Van Cutsem E
      • Findlay M
      • Osterwalder B
      • et al.
      Capecitabine, an oral fluoropyrimidine carbamate with substantial activity in advanced colorectal cancer: results of a randomized phase II study.
      Since antibiotics are commonly applied in mCRC patients receiving palliative chemotherapy due to several comorbidities, this should be taken into account for future studies in this field. The faecal sample of P20 contained a relatively high relative abundance of bifidobacteria. Surprisingly, this patient also showed the highest tumour response (40% decrease in the sum of target lesions). Bifidobacteria are known to have immune-modulating effects and contribute to the production of the short-chain fatty acid for example, acetate.
      • Riviere A
      • Selak M
      • Lantin D
      • Leroy F
      • De Vuyst L.
      Bifidobacteria and butyrate-producing colon bacteria: importance and strategies for their stimulation in the human gut.
      These observations in individual patients are interesting but surely need further investigation in larger groups in order to have clinical relevance. Furthermore, the potential role of short-chain fatty acid -producing microbiota underlines the importance of performing functional microbiota analysis by performing metagenomic sequencing or measuring microbial metabolites in the future. Furthermore, it would be worthwhile to use full length 16S sequencing or metagenomic sequencing to acquire even higher taxonomic resolutions when studies evolve from explorative pilot studies to more causal designs.
      • Durazzi F
      • Sala C
      • Castellani G
      • Manfreda G
      • Remondini D
      • De Cesare A.
      Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota.
      In general, our study was limited by the small group size and an unequal distribution between responders (n = 6) and non-responders (n = 26). Large heterogeneity concerning inter and intra-individual microbiota composition and diversity further complicated the detection of differences on group level. This heterogeneity is most likely caused by a diverse medical history and other strong microbiota-modulating factors, such as the living environment, diet, and antibiotics.
      • Balhareth A
      • Aldossary MY
      • McNamara D.
      Impact of physical activity and diet on colorectal cancer survivors' quality of life: a systematic review.
      ,
      • Manor O
      • Dai CL
      • Kornilov SA
      • et al.
      Health and disease markers correlate with gut microbiome composition across thousands of people.
      Furthermore, the relatively mild cytotoxic effects of capecitabine might have contributed to the lack of association between capecitabine treatment and microbiota modulation.
      • Reigner B
      • Blesch K
      • Weidekamm E.
      Clinical pharmacokinetics of capecitabine.
      It is known that capecitabine is converted in tumor tissue to its cytotoxic moiety 5-FU and that approximately 3% of the dose is excreted via the faeces
      • Reigner B
      • Blesch K
      • Weidekamm E.
      Clinical pharmacokinetics of capecitabine.
      , thereby passing the colon. Compared to other chemotherapeutics, gastrointestinal toxicity was relatively low in the present study population. A possible explanation might be that the study period ended after the third cycle of capecitabine. It is probable that manifestation of gastrointestinal toxicity takes more time and that it would be beneficial to extend the study period in future studies.
      • van Hellemond IEG
      • Thijs AM
      • Creemers GJ.
      Capecitabine-associated terminal ileitis.
      ,

      FDA. Description Xeloda (capecitabine) tablets for oral use. 2015. p. 7.

      Due to the low prevalence of gastrointestinal toxicity in the current cohort, the association between gastrointestinal toxicity and the intestinal microbiota was not analyzed in the present study, but is considered to be highly relevant for future research.
      Before proceeding to clinical interventions studies with pre- and/or probiotics or even faecal microbiota transplantation in mCRC patients with a complex medical history, changes in intestinal microbiota composition and diversity should be evaluated in studies with larger and more equal group sizes between responders and non-responders supported with functional microbiota analysis. In view of the fact that the maintenance of quality of life is essential for mCRC patients, the design of targeted interventions improving treatment response and decreasing gastrointestinal toxicity for these patients is considered to be pivotal. Our study provides insights into potential challenges and points of attention for the design of upcoming microbiota studies in this complex patient population.
      In conclusion, intestinal microbiota composition and diversity before, during, and after three cycles of capecitabine were not associated with response in the current small study population. High inter- and intra-individual microbiota variations were observed during capecitabine treatment. This is most likely due to an extensive medical history in this complex patient group. This highly variable microbiota composition and diversity is a great challenge for the application of personalized medicine and microbiota-based therapies. It should be noted that the results of the current study are limited by the small group sizes and large heterogeneity. However, we provide a framework and insights into potential challenges and points of attention for future studies in mCRC patients. Additional longitudinal studies using larger and equal cohorts will be highly relevant to further explore microbiota-therapy interactions in mCRC patients. Upcoming research should also focus on functional microbiota analysis by performing metagenomic sequencing or measuring microbial metabolites. This knowledge could support future interventions with pre- or probiotics and/or faecal microbiota transplantations.

       Clinical Practice Points

      With the current study, we provide a framework and insights into potential challenges and points of attention for future studies in mCRC patients.
      Additional longitudinal studies using larger and equal cohorts will be highly relevant to further explore microbiota-therapy interactions in mCRC patients.
      Upcoming research should also focus on functional microbiota analysis by performing metagenomic sequencing or measuring microbial metabolites. This knowledge could support future interventions with pre- or probiotics and/or faecal microbiota transplantations.

      Author Contributions

      Romy Aarnoutse: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, Project administration, Funding acquisition. Janine Ziemons: Methodology, Validation, Formal analysis, Investigation, Writing - Original Draft, Writing - Review & Editing, Visualization, Project administration. Judith de Vos-Geelen: Conceptualization, Methodology, Resources, Data Curation, Writing - Review & Editing, Supervision. Liselot Valkenburg-van Iersel: Resources, Data Curation, Writing - Review & Editing. Aurelia Wildeboer: Data Curation, Writing - Review & Editing. Anne Vievermans: Data Curation, Writing - Review & Editing. Geert-Jan Creemers: Resources, Data Curation, Writing - Review & Editing. Arnold Baars: Resources, Data Curation, Writing - Review & Editing. Hanneke Vestjens: Resources, Data Curation, Writing - Review & Editing. Giang Le: Software, Formal analysis, Writing - Review & Editing. David Barnett: Software, Formal analysis, Writing - Review & Editing. Sander Rensen: Conceptualization, Methodology, Validation, Writing - Review & Editing. John Penders: Conceptualization, Methodology, Validation, Formal analysis, Resources, Writing - Review & Editing, Supervision. Marjolein Smidt: Conceptualization, Methodology, Validation, Writing - Review & Editing, Supervision, Funding acquisition.

      Disclosure

      JdVG has served as a consultant for Amgen, AstraZeneca, MSD, Pierre Fabre, and Servier. All outside the submitted work. LV has served as a consultant for MSD, Pierre Fabre, and Servier. All outside the submitted work. JdVG, MLS, and RA has received institutional research funding from Servier. All outside the submitted work. The other authors declare no potential conflicts of interest.

      Acknowledgment

      The authors thank all patients who participated in this study. Medical oncologists and research nurses from Catharina Hospital, VieCuri Medical Centre, Hospital Gelderse Vallei, and MUMC+ assisted with patient inclusion and sample collection. Especially, we are grateful to Marjan Laven, Ramon Bax, Sanne Achten, Eva de Jong, Alina van de Vendel, Janneke van den Brink, Ilona van Rooij-Tieleman, Wendy Heuts, Nicol Pepels, and Kim Puts-van der Burgt. The authors wish to acknowledge Christel Driessen and Wesley Nix for supporting the laboratory analyses. We also thank Renée Granzier for providing support for data analysis. This work was supported by the Stichting Jules Coenegracht Sr.

      Appendix. Supplementary materials

      References

        • Rawla P
        • Sunkara T
        • Barsouk A.
        Epidemiology of colorectal cancer: incidence, mortality, survival, and risk factors.
        Prz Gastroenterol. 2019; 14: 89-103
        • Cunningham D
        • Lang I
        • Marcuello E
        • et al.
        Bevacizumab plus capecitabine versus capecitabine alone in elderly patients with previously untreated metastatic colorectal cancer (AVEX): an open-label, randomised phase 3 trial.
        Lancet Oncol. 2013; 14: 1077-1085
        • Hoff PM
        • Ansari R
        • Batist G
        • et al.
        Comparison of oral capecitabine versus intravenous fluorouracil plus leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study.
        J Clin Oncol. 2001; 19: 2282-2292
        • Van Cutsem E
        • Findlay M
        • Osterwalder B
        • et al.
        Capecitabine, an oral fluoropyrimidine carbamate with substantial activity in advanced colorectal cancer: results of a randomized phase II study.
        J Clin Oncol. 2000; 18: 1337-1345
        • Team NIHHMPA
        A review of 10 years of human microbiome research activities at the US National Institutes of Health, Fiscal Years 2007-2016.
        Microbiome. 2019; 7: 31
        • Helmink BA
        • Khan MAW
        • Hermann A
        • Gopalakrishnan V
        • Wargo JA.
        The microbiome, cancer, and cancer therapy.
        Nat Med. 2019; 25: 377-388
        • Sougiannis AT
        • VanderVeen BN
        • Enos RT
        • et al.
        Impact of 5 fluorouracil chemotherapy on gut inflammation, functional parameters, and gut microbiota.
        Brain Behav Immun. 2019; 80: 44-55
        • An J
        • Ha EM.
        Combination therapy of lactobacillus plantarum supernatant and 5-fluouracil increases chemosensitivity in colorectal cancer cells.
        J Microbiol Biotechnol. 2016; 26: 1490-1503
        • Yuan L
        • Zhang S
        • Li H
        • et al.
        The influence of gut microbiota dysbiosis to the efficacy of 5-Fluorouracil treatment on colorectal cancer.
        Biomed Pharmacother. 2018; 108: 184-193
        • Zimmermann M
        • Zimmermann-Kogadeeva M
        • Wegmann R
        • Goodman AL.
        Mapping human microbiome drug metabolism by gut bacteria and their genes.
        Nature. 2019; 570: 462-467
        • Aarnoutse R
        • Ziemons J
        • Penders J
        • Rensen SS
        • de Vos-Geelen J
        • Smidt ML.
        The clinical link between human intestinal microbiota and systemic cancer therapy.
        Int J Mol Sci. 2019; 20: 4145
        • Li J
        • Li J
        • Lyu N
        • et al.
        Composition of fecal microbiota in low-set rectal cancer patients treated with FOLFOX.
        Ther Adv Chronic Dis. 2020; 112040622320904293
        • Aarnoutse R
        • de Vos-Geelen JMPGM
        • Penders J
        • et al.
        Study protocol on the role of intestinal microbiota in colorectal cancer treatment: a pathway to personalized medicine 2.0.
        Int J Colorectal Dis. 2017; 32: 1077-1084
        • Aarnoutse R
        • de Vos-Geelen J
        • Penders J
        • et al.
        Study protocol on the role of intestinal microbiota in colorectal cancer treatment: a pathway to personalized medicine 2.0.
        Int J Colorectal Dis. 2017; 32: 1077-1084
        • Eisenhauer EA
        • Therasse P
        • Bogaerts J
        • et al.
        New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).
        Eur J Cancer. 2009; 45: 228-247
        • National Institutes of Health
        Common Terminology Criteria for Adverse Events (CTCEA).
        Department of Health and Human Services, U.S.2010
        • Liu PY
        • Wu WK
        • Chen CC
        • Panyod S
        • Sheen LY
        • Wu MS.
        Evaluation of Compatibility of 16S rRNA V3V4 and V4 Amplicon Libraries for Clinical Microbiome Profiling.
        bioRxiv. 2020;
        • Galazzo G
        • van Best N
        • Benedikter BJ
        • et al.
        How to count our microbes? the effect of different quantitative microbiome profiling approaches.
        Front Cell Infect Microbiol. 2020; 10: 403
        • Callahan BJ
        • McMurdie PJ
        • Rosen MJ
        • Han AW
        • Johnson AJ
        • Holmes SP.
        DADA2: High-resolution sample inference from Illumina amplicon data.
        Nat Methods. 2016; 13: 581-583
        • Team RC.
        R: A language and environment for statistical computing.
        R Foundation for Statistical Computing, Vienna, Austria2020
        • Lagkouvardos I
        • Fischer S
        • Kumar N
        • Clavel T.
        Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.
        PeerJ. 2017; 5: e2836
        • GUniFrac Chen J.
        Generalized UniFrac distances.
        R package version 1. 2018; 1
        • McMurdie PJ
        • Holmes S.
        phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data.
        PLoS One. 2013; 8: e61217
        • FGB Jari Oksanen
        • Friendly Michael
        • Kindt Roeland
        • et al.
        Eduard Szoecs, Wagner aH. vegan: community ecology package.
        R package version 2.5-6. 2019;
      1. Lahti L, Ernst FGM, Huang R, et al. microbiome R package. 2020.

        • Wickham H
        • François R
        • Henry L
        • Müller K.
        dplyr: A grammar of data manipulation.
        R package version 1.0. 2 (2020). 2020;
        • Wickham H.
        ggplot2: elegant graphics for data analysis.
        springer, 2016
        • Barnett DJM
        • Arts ICW
        • Penders J.
        microViz: an R package for microbiome data visualization and statistics.
        J Open Source Software. 2021; 6: 3201
        • Mandal S
        • Van Treuren W
        • White RA
        • Eggesbø M
        • Knight R
        • Peddada SD.
        Analysis of composition of microbiomes: a novel method for studying microbial composition.
        Microb Ecol Health Dis. 2015; 26: 27663
        • Balhareth A
        • Aldossary MY
        • McNamara D.
        Impact of physical activity and diet on colorectal cancer survivors' quality of life: a systematic review.
        World J Surg Oncol. 2019; 17: 153
        • Sze MA
        • Baxter NT
        • MTt Ruffin
        • Rogers MAM
        • Schloss PD.
        Normalization of the microbiota in patients after treatment for colonic lesions.
        Microbiome. 2017; 5: 150
        • Zwielehner J
        • Lassl C
        • Hippe B
        • et al.
        Changes in human fecal microbiota due to chemotherapy analyzed by TaqMan-PCR, 454 sequencing and PCR-DGGE fingerprinting.
        PLoS One. 2011; 6: e28654
        • Dubinsky V
        • Reshef L
        • Bar N
        • et al.
        Predominantly antibiotic-resistant intestinal microbiome persists in patients with pouchitis who respond to antibiotic therapy.
        Gastroenterology. 2020; 158 (e13): 610-624
        • Riviere A
        • Selak M
        • Lantin D
        • Leroy F
        • De Vuyst L.
        Bifidobacteria and butyrate-producing colon bacteria: importance and strategies for their stimulation in the human gut.
        Front Microbiol. 2016; 7: 979
        • Durazzi F
        • Sala C
        • Castellani G
        • Manfreda G
        • Remondini D
        • De Cesare A.
        Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota.
        Sci Rep. 2021; 11: 3030
        • Manor O
        • Dai CL
        • Kornilov SA
        • et al.
        Health and disease markers correlate with gut microbiome composition across thousands of people.
        Nat Commun. 2020; 11: 5206
        • Reigner B
        • Blesch K
        • Weidekamm E.
        Clinical pharmacokinetics of capecitabine.
        Clin Pharmacokinet. 2001; 40: 85-104
        • van Hellemond IEG
        • Thijs AM
        • Creemers GJ.
        Capecitabine-associated terminal ileitis.
        Case Rep Oncol. 2018; 11: 654-659
      2. FDA. Description Xeloda (capecitabine) tablets for oral use. 2015. p. 7.