Analysis of Intestinal Mucosal Microbiome Changes before and after Chemoradiation in Locally Advanced Rectal Cancer Patients

Incheol Seo; Sung Uk Bae; Shin Kim; Woon Kyung Jeong; Seong Kyu Baek

doi:10.4167/jbv.2019.49.4.162

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Seo, Bae, Kim, Jeong, and Baek: Analysis of Intestinal Mucosal Microbiome Changes before and after Chemoradiation in Locally Advanced Rectal Cancer Patients

Original Article

Journal of Bacteriology and Virology 2019; 49(4): 162-175.

Published online: 31 December 2019

DOI: https://doi.org/10.4167/jbv.2019.49.4.162

Analysis of Intestinal Mucosal Microbiome Changes before and after Chemoradiation in Locally Advanced Rectal Cancer Patients

Incheol Seo¹, Sung Uk Bae², Shin Kim³, Woon Kyung Jeong², Seong Kyu Baek²

¹Dasa Geriatric Hospital, Daegu, Korea.

²Department of Surgery, School of Medicine, Keimyung University and Dongsan Medical Center, Daegu, Korea.

³Department of Immunology, School of Medicine, Keimyung University, Daegu, Korea.

Corresponding: Sung Uk Bae, M.D. Department of Surgery, Keimyung University School of Medicine 1095 Dalgubeol-daero, Dalseo-gu, Daegu 42601, Korea. Phone: +82-53-258-4701, Fax: +82-53-258-4701, sabiston0000@hanmail.net

Received 21 August 2019 Revised 10 October 2019 Accepted 15 October 2019

The Korean Society for Microbiology and The Korean Society of Virology

(open-access):

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/).

Abstract

Purpose

Dysbiosis of gut microbiota has been reported to participate in the pathogenesis of colorectal cancer, but changes in microbiota due to radiotherapy have not been studied. In this study, we tried to elucidate the changes in the microbiome in rectal cancer after chemoradiotherapy using RNA sequencing analysis.

Materials and methods

We included 11 pairs of human rectal cancer tissues before and after irradiation between August 2016 and December 2017 and performed RNA sequencing analysis. Mapped reads to human reference genomes were used for pair-wise transcriptome comparisons, and unmapped (non-human) reads were then mapped to bacterial marker genes using PathSeq.

Results

At microbiome level, interindividual variability of mucosal microbiota was greater than the change in microbial composition during radiotherapy. This indicates that rapid homeostatic recovery of the mucosal microbial composition takes place short after radiotherapy. At single microbe level, Prevotella and Fusobacterium, which were identified as important causative microbes of the initiation and progression of rectal cancer were decreased by radiotherapy. Moreover, changes in Prevotella were associated with changes in the human transcriptome of rectal cancer. We also found that there was a gene cluster that increased and decreased in association with changes in microbial composition by chemoradiation.

Conclusion

This study revealed changes in tumor-associated microbial community by irradiation in rectal cancer. These findings can be used to develop a new treatment strategy of neoadjuvant therapy for locally advanced rectal cancer by overcoming radio-resistance or facilitating radio-sensitivity.

Keywords: Microbiome, Rectal cancer, Radiotherapy

Figures and Tables

Figure 1

Microbial composition before and after chemoradiation in rectal cancer patients. The relative abundance of OTU is shown for each taxonomic rank. The paired bar plots on the right show the microbial composition before and after chemoradiation in the same patients. The numbers on the bar plot represent the patient identification numbers. The skyline plots on the left show the bar plot in ascending order based on the most abundant OTU. The y-axis of both charts indicates the relative abundance (%). “PRE” and “PRERT” represent samples before chemoradiation therapy. “POST” and “POSTRT” represent samples after chemoradiation therapy. OTUs that differ in ANCOM analysis are marked with an asterisk and the W-statistics are shown in parentheses. (A) Phylum (B) Class (C) Order and (D) Family.

Figure 2

Principal component analysis of the microbiota before and after chemoradiation in rectal cancer patients. The numbers represent the patient identification numbers. “PRERT” and “POSTRT” represent samples before and after chemoradiation therapy, respectively. (A) Phylum (B) Class (C) Order (D) Family.

Figure 3

Changes in the abundance of Prevotellaceae family after chemoradiation and concomitant changes in the human transcriptome. (A) Box plot shows the relative abundance of Prevotellaceae before and after chemoradiotherapy. “PRERT” and “POSTRT” represent samples before and after chemoradiation therapy, respectively. (B) Bubble chart shows the functional annotation of host gene expression highly correlated with changes in relative abundance of Prevotellaceae before and after chemoradiotherapy. Gene-Enrichment and Functional Annotation Analysis was performed using DAVID. The y-axis represents the functional term defined in GOTERM_MF_DIRECT. The numbers in parentheses are the identification numbers of the Gene Ontology. Fold enrichment, ratio of the number of genes involved the function term to the total number of inputs genes, and p-value are represented by x-axis, bubble size, and bubble color, respectively.

Table 1

Patient characteristics

ID^a: patient identification number, BMI^b: body mass index, Diff^c: differentiation (1 = well and moderately differentiated, 2 = poorly differentiated), Bud^d: presence of tumor budding, PNI^e: presence of perineural invasion, ETD^f: presence of extranodal tumor deposit, MSI^g: microsatellite instability (H: high, L: low), KRAS^h: presence of KRAS exon 2 mutation, BRAFⁱ: presence of BRAF mutation, TRG^j: Mandard tumor regression grade (NR = non-responder, R = responder), pT^k: primary tumor categories, pN^l: regional lymph node categories.

Table 2

Read count statistics

The numbers in parentheses, excluding the “PathSeq mapped” column, represent the percentage of read count in the previous step. The number in parentheses in the “PathSeq mapped” column is the percentage of non-human read counts which unmapped to hg38. ID^a: sample identification number (The number before hyphen indicates patient identification number. “PRE” and “POST” represent samples before and after chemoradiation therapy.), Raw^b: raw read counts, Trimmed^c: read counts after quality control. STAR hg38 mapped^d: read counts mapped to hg38 human reference genome using STAR aligner. PathSeq mapped^e: read counts mapped to bacterial marker genes using PathSeq.

Table 3

PERMANOVA results testing the effect of chemoradiation on microbial composition

Three distance metrics were used to test the differences in microbial composition before and after chemoradiation therapy in rectal adenocarcinoma patients. PERMANOVA^a: permutational multivariate analysis of variance.

Table 4

ANCOM analysis between before and after chemoradiotherapy

ANCOM: Analysis of composition of microbiomes, W^a: ANCOM W-statistics, N_feat ^b: Total number of OTUs detected within the same taxonomic rank, D_0.9^c~D_0.6^f: ANCOM significance with cut-offs from 0.9 to 0.6.

Table 5

Top 10 genes showing the strongest positive correlation with the abundance of Prevotellaceae

Table 6

Top 10 genes showing the strongest negative correlation with the abundance of Prevotellaceae

Notes

No potential conflict of interest relevant to this article was reported.

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