Gut microbiome is associated with the clinical response to anti-PD-1 based immunotherapy in hepatobiliary cancers

Jinzhu Mao, Dongxu Wang, Junyu Long, Xu Yang, Jianzhen Lin, Yiwei Song, Fucun Xie, Ziyu Xun, Yanyu Wang, Yunchao Wang, Yiran Li, Huishan Sun, Jingnan Xue, Yang Song, Bangyou Zuo, Junwei Zhang, Jin Bian, Ting Zhang, Xiaobo Yang, Lei Zhang, Xinting Sang, Haitao Zhao, Jinzhu Mao, Dongxu Wang, Junyu Long, Xu Yang, Jianzhen Lin, Yiwei Song, Fucun Xie, Ziyu Xun, Yanyu Wang, Yunchao Wang, Yiran Li, Huishan Sun, Jingnan Xue, Yang Song, Bangyou Zuo, Junwei Zhang, Jin Bian, Ting Zhang, Xiaobo Yang, Lei Zhang, Xinting Sang, Haitao Zhao

Abstract

Background: The gut microbiome is associated with the response to immunotherapy for different cancers. However, the impact of the gut microbiome on hepatobiliary cancers receiving immunotherapy remains unknown. This study aims to investigate the relationship between the gut microbiome and the clinical response to anti-programmed cell death protein 1 (PD-1) immunotherapy in patients with advanced hepatobiliary cancers.

Methods: Patients with unresectable hepatocellular carcinoma or advanced biliary tract cancers who have progressed from first-line chemotherapy (gemcitabine plus cisplatin) were enrolled. Fresh stool samples were collected before and during anti-PD-1 treatment and analyzed with metagenomic sequencing. Significantly differentially enriched taxa and prognosis associated taxa were identified. The Kyoto Encyclopedia of Genes and Genomes database and MetaCyc database were further applied to annotate the differentially enriched taxa to explore the potential mechanism of the gut microbiome influencing cancer immunotherapy.

Results: In total, 65 patients with advanced hepatobiliary cancers receiving anti-PD-1 treatment were included in this study. Seventy-four taxa were significantly enriched in the clinical benefit response (CBR) group and 40 taxa were significantly enriched in the non-clinical benefit (NCB) group. Among these taxa, patients with higher abundance of Lachnospiraceae bacterium-GAM79 and Alistipes sp Marseille-P5997, which were significantly enriched in the CBR group, achieved longer progression-free survival (PFS) and overall survival (OS) than patients with lower abundance. Higher abundance of Ruminococcus calidus and Erysipelotichaceae bacterium-GAM147 enriched in the CBR group was also observed in patients with better PFS. In contrast, worse PFS and OS were found in patients with higher abundance of Veillonellaceae, which was significantly enriched in the NCB group. Functional annotation indicated that the taxa enriched in the CBR group were associated with energy metabolism while the taxa enriched in the NCB group were associated with amino acid metabolism, which may modulate the clinical response to immunotherapy in hepatobiliary cancers. In addition, immunotherapy-related adverse events were affected by the gut microbiome diversity and relative abundance.

Conclusions: We demonstrate that the gut microbiome is associated with the clinical response to anti-PD-1 immunotherapy in patients with hepatobiliary cancers. Taxonomic signatures enriched in responders are effective biomarkers to predict the clinical response and survival benefit of immunotherapy, which might provide a new therapeutic target to modulate the response to cancer immunotherapy.

Keywords: immunotherapy; liver neoplasms; tumor biomarkers.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1
Figure 1
Study workflow. BTC, biliary tract cancer; CBR, clinical benefit response; HCC, hepatocellular carcinoma; NCB, non-clinical benefit; OS, overall survival; PD-1, programmed cell death protein 1; PFS, progression-free survival.
Figure 2
Figure 2
Gut microbiome composition of 65 patients with hepatobiliary cancers. (A) Gut microbiome composition at the order level in the 65 patients with hepatobiliary cancers (ordered by the most abundant taxa, Bacteroideles order). (B) Relative abundance comparison of Bacteroidales, Enterobacterales and Veillonellales in the CBR group and NCB group at the order level (Wilcoxon test). (C) Relative abundance comparison of Lachnospiraceae bacterium-GAM79, Ruminococcus callidus, Eubacterium siraeum, Gemmiger formicilis and Faecalibacterium genus in the CBR group and NCB group (Wilcoxon test). (D) Dynamic microbial composition in the CBR group and NCB group at the phylum level. CBR, clinical benefit response; NCB, non-clinical benefit.
Figure 3
Figure 3
Differential taxa were enriched in the CBR group and NCB group of the 65 patients with hepatobiliary cancers. (A) PCoA showed the beta diversity evaluated by Bray-Curtis distance between the CBR group and NCB group. (B) Common and unique taxa at the specie level between the CBR group and NCB group. (C) Taxonomic cladogram from LEfSe showed different taxa enriched in the CBR group and NCB group (LDA>3, p3, p

Figure 4

Differentially enriched taxa were associated…

Figure 4

Differentially enriched taxa were associated with the survival benefit of anti-programmed cell death…

Figure 4
Differentially enriched taxa were associated with the survival benefit of anti-programmed cell death protein 1 immunotherapy in patients with hepatobiliary cancers. The Kaplan-Meier method with log-rank test estimates the median progression-free survival (A) and median overall survival (B) for patients with higher or lower abundance of Lachnospiraceae bacterium-GAM79, Alistipes sp Marseille-P5997 and Veillonellaceae family.

Figure 5

Functional annotation of the gut…

Figure 5

Functional annotation of the gut microbiome metagenomic sequencing data. (A) Different KOs enriched…

Figure 5
Functional annotation of the gut microbiome metagenomic sequencing data. (A) Different KOs enriched in the CBR group and NCB group identified by LEfSe (LDA>2, p2, p

Figure 6

Immunotherapy-related adverse events were affected…

Figure 6

Immunotherapy-related adverse events were affected by the gut microbiome. (A) The Kaplan-Meier method…

Figure 6
Immunotherapy-related adverse events were affected by the gut microbiome. (A) The Kaplan-Meier method with log-rank test estimates the mPFS for patients with severe or mild diarrhea. (B) Gut microbiome alpha diversity comparison between the severe and mild diarrhea groups (Wilcoxon test). (C) Relative abundance comparison of significantly different taxa between patients with severe diarrhea and mild diarrhea (Wilcoxon test). mPFS, median progression-free survival.

Figure 7

The gut microbiome distribution was…

Figure 7

The gut microbiome distribution was affected by clinical factors. (A) RDA with permutation…

Figure 7
The gut microbiome distribution was affected by clinical factors. (A) RDA with permutation test showed the clinical factors associated with the distribution of patients with hepatobiliary cancers. (B) RDA with permutation test showed the clinical factors associated with the different taxa enriched in the CBR group and NCB group. (C) Relative abundance comparison of Eubacterium hallii in patients with large tumors (≥5 cm) and small tumors (≥5 cm) (Wilcoxon test). (D) Relative abundance comparison of the Clostridium genus in patients with elevated bile acid and normal bile acid (Wilcoxon test). CBR, clinical benefit response; ECOG-PS, Eastern Cooperative Oncology Group performance status; NCB, non-clinical benefit; RDA, redundancy analysis.
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Figure 4
Figure 4
Differentially enriched taxa were associated with the survival benefit of anti-programmed cell death protein 1 immunotherapy in patients with hepatobiliary cancers. The Kaplan-Meier method with log-rank test estimates the median progression-free survival (A) and median overall survival (B) for patients with higher or lower abundance of Lachnospiraceae bacterium-GAM79, Alistipes sp Marseille-P5997 and Veillonellaceae family.
Figure 5
Figure 5
Functional annotation of the gut microbiome metagenomic sequencing data. (A) Different KOs enriched in the CBR group and NCB group identified by LEfSe (LDA>2, p2, p

Figure 6

Immunotherapy-related adverse events were affected…

Figure 6

Immunotherapy-related adverse events were affected by the gut microbiome. (A) The Kaplan-Meier method…

Figure 6
Immunotherapy-related adverse events were affected by the gut microbiome. (A) The Kaplan-Meier method with log-rank test estimates the mPFS for patients with severe or mild diarrhea. (B) Gut microbiome alpha diversity comparison between the severe and mild diarrhea groups (Wilcoxon test). (C) Relative abundance comparison of significantly different taxa between patients with severe diarrhea and mild diarrhea (Wilcoxon test). mPFS, median progression-free survival.

Figure 7

The gut microbiome distribution was…

Figure 7

The gut microbiome distribution was affected by clinical factors. (A) RDA with permutation…

Figure 7
The gut microbiome distribution was affected by clinical factors. (A) RDA with permutation test showed the clinical factors associated with the distribution of patients with hepatobiliary cancers. (B) RDA with permutation test showed the clinical factors associated with the different taxa enriched in the CBR group and NCB group. (C) Relative abundance comparison of Eubacterium hallii in patients with large tumors (≥5 cm) and small tumors (≥5 cm) (Wilcoxon test). (D) Relative abundance comparison of the Clostridium genus in patients with elevated bile acid and normal bile acid (Wilcoxon test). CBR, clinical benefit response; ECOG-PS, Eastern Cooperative Oncology Group performance status; NCB, non-clinical benefit; RDA, redundancy analysis.
All figures (7)
Figure 6
Figure 6
Immunotherapy-related adverse events were affected by the gut microbiome. (A) The Kaplan-Meier method with log-rank test estimates the mPFS for patients with severe or mild diarrhea. (B) Gut microbiome alpha diversity comparison between the severe and mild diarrhea groups (Wilcoxon test). (C) Relative abundance comparison of significantly different taxa between patients with severe diarrhea and mild diarrhea (Wilcoxon test). mPFS, median progression-free survival.
Figure 7
Figure 7
The gut microbiome distribution was affected by clinical factors. (A) RDA with permutation test showed the clinical factors associated with the distribution of patients with hepatobiliary cancers. (B) RDA with permutation test showed the clinical factors associated with the different taxa enriched in the CBR group and NCB group. (C) Relative abundance comparison of Eubacterium hallii in patients with large tumors (≥5 cm) and small tumors (≥5 cm) (Wilcoxon test). (D) Relative abundance comparison of the Clostridium genus in patients with elevated bile acid and normal bile acid (Wilcoxon test). CBR, clinical benefit response; ECOG-PS, Eastern Cooperative Oncology Group performance status; NCB, non-clinical benefit; RDA, redundancy analysis.

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