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Intestinal microbiota signatures of clinical response and immune-related adverse events in melanoma patients treated with anti-PD-1

Abstract

Ample evidence indicates that the gut microbiome is a tumor-extrinsic factor associated with antitumor response to anti-programmed cell death protein-1 (PD-1) therapy, but inconsistencies exist between published microbial signatures associated with clinical outcomes. To resolve this, we evaluated a new melanoma cohort, along with four published datasets. Time-to-event analysis showed that baseline microbiota composition was optimally associated with clinical outcome at approximately 1 year after initiation of treatment. Meta-analysis and other bioinformatic analyses of the combined data show that bacteria associated with favorable response are confined within the Actinobacteria phylum and the Lachnospiraceae/Ruminococcaceae families of Firmicutes. Conversely, Gram-negative bacteria were associated with an inflammatory host intestinal gene signature, increased blood neutrophil-to-lymphocyte ratio, and unfavorable outcome. Two microbial signatures, enriched for Lachnospiraceae spp. and Streptococcaceae spp., were associated with favorable and unfavorable clinical response, respectively, and with distinct immune-related adverse effects. Despite between-cohort heterogeneity, optimized all-minus-one supervised learning algorithms trained on batch-corrected microbiome data consistently predicted outcomes to programmed cell death protein-1 therapy in all cohorts. Gut microbial communities (microbiotypes) with nonuniform geographical distribution were associated with favorable and unfavorable outcomes, contributing to discrepancies between cohorts. Our findings shed new light on the complex interaction between the gut microbiome and response to cancer immunotherapy, providing a roadmap for future studies.

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Fig. 1: Compositional differences in the fecal microbiome of anti-PD-1-treated patients with melanoma are associated with differential progression-free survival.
Fig. 2: Relationship between microbiota composition and associated host variables in relation to clinical response.
Fig. 3: Fecal microbial signatures are differentially associated with immune-related adverse events and progression-free survival in PD-1-treated patients with melanoma.
Fig. 4: Gut microbiome meta-analysis of five independent cohorts of patients with melanoma treated with anti-PD-1 identifies organisms and microbial genes differentially enriched in responders and nonresponders.
Fig. 5: Machine learning shows significant prediction of cohort response using models trained on other cohorts combined.
Fig. 6: Mapping of combined 16S rRNA gene amplicon sequencing data from PD-1-treated patients with melanoma to the American Gut Project dataset identifies favorable and unfavorable enteric microbiotypes.

Data availability

The corresponding authors will comply with all requests for raw and analyzed data and materials after verification whether the request is subject to any patients’ confidentiality obligation. Patient-related data not included in the paper were generated as part of clinical trials and might be subject to patient confidentiality. All sequencing (human and microbiome) data and de-identified metadata that support the findings have been deposited in NCBI databases and are all accessible via BioProject accession no. PRJNA762360. AGP data are available at the ENA database (https://www.ebi.ac.uk/) under accession no. PRJEB11419. Access to publicly available sequencing data of the other cohorts analyzed in this study was obtained through BioProject accession nos. PRJNA399742 (Chicago), PRJNA541981 (New York), PRJNA397906 (Dallas) and PRJEB22893 (Houston). Source data are provided with this paper. The Sequence Read Archive accession numbers for each sample from each of these cohorts is the stated name of the sample on the spreadsheet in the source data. All other data are provided within the paper and its Supplementary Information files.

Code availability

All codes used for shotgun sequencing analysis can be found within the in-house JAMS_BW package, version 1.5.7, publicly available on GitHub (https://github.com/johnmcculloch/JAMS_BW/). GSEA analysis was done in R using fgsea package 1.19.4. Codes for transkingdom network analysis are available at https://github.com/richrr/TransNetDemo/. Additional codes used are part of the packages mentioned in the text or can be found on GitHub at https://github.com/trinchierilab/microbiotapd1melanoma2021/.

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Acknowledgements

We acknowledge all patients and families affected by metastatic melanoma. We acknowledge J. Wargo for providing unpublished metadata of the Houston melanoma patient cohort. This work was supported in part by the Intramural Research Program of the NIH, NCI, Center for Cancer Research. D.D. is supported by the Melanoma Breakthrough Foundation Breakthrough Consortium. H.M.Z. is supported by the NIH/NCI (R01 CA228181 AND R01 CA222203) and the James W. and Frances G. McGlothlin Chair in Melanoma Immunotherapy Research. M.V. was supported by an Irvington postdoctoral fellowship from the Cancer Research Institute. Work of the University of Pittsburgh Medical Center HCC Microbiome Shared Facility and Cytometry Facility is supported by the NIH NCI Comprehensive Cancer Center Support Core grant (P30 CA047904). This research was supported in part by the University of Pittsburgh Center for Research Computing and Unified Flow Cytometry Core of the University of Pittsburgh’s Department of Immunology through the resources provided.

Author information

Authors and Affiliations

Authors

Contributions

J.A.M., D.D., R.R.R., H.M.Z., G.T. and A.K.D. conceived the study; D.D., A.M.C., M.V., S.P., M.R.F., R.G.F.C., W.Y., R.S., S.R., R.N.D., J.-M.C., Q.D., B.Z., A.L., S.C., W.G., O.P., S.J.E., A.R., N.K.N. and A.K.D. were involved in sample collection, processing, preparation and sequencing; J.A.M., R.R.R., J.H.B., J.R.F., E.B., R.M.M., N.K.N., A.M. and A.K.D. performed computational analysis; H.M.Z., G.T. and A.K.D. supervised the entirety of the project. All authors approved the manuscript.

Corresponding authors

Correspondence to Hassane M. Zarour, Giorgio Trinchieri or Amiran K. Dzutsev.

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Competing interests

D.D. reports the following disclosures: Arcus, Bristol-Myers Squibb, Checkmate Pharmaceuticals, CellSight Technologies, Merck, GlaxoSmithKline/Tesaro (research support); Array Biopharma, Checkmate Pharmaceuticals, Finch, Incyte, Immunocore, Merck; Shionogi (consulting); and Vedanta Biosciences (scientific advisory board). H.M.Z. reports the following disclosures: Bristol-Myers Squibb, Checkmate Pharmaceuticals, GlaxoSmithKline (research support); Bristol-Myers Squibb, Checkmate Pharmaceuticals, GlaxoSmithKline, Vedanta (consulting). D.D., H.M.Z., J.A.M., R.R.R., G.T. and A.K.D. are inventors on a patent application (US patent no. 63/208,719) submitted by the University of Pittsburgh that covers methods to enhance checkpoint blockade therapy by the microbiome. The other authors declare no competing interests.

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Nature Medicine thanks R. Dummer, A. Bhatt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Editor Recognition Statement: Javier Carmona and Saheli Sadanand are the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Kaplan-Meier plots of progression-free survival and overall survival in the Pittsburgh early sample cohort and progression-free survival after dichotomization for abundance of select bacterial species.

a and b. Kaplan-Meier plots of probability of progression-free survival (PFS) (a) and overall survival (OS) (b) of PD-1-treated Pittsburgh early cohort melanoma patients. Vertical ticks show censored data. Central line is median OS or PFS probability, shaded area shows 95% confidence interval. c. Optimal cutpoints of bacterial abundance determined using Evaluate Cutpoints. Different plots show the effect on PFS of abundance (high vs. low) of the top four most significantly increased (left) and decreased (right) individual bacterial species in non-progressors at 10 months, determined using Mann-Whitney U test (Fig. 1c). Number of people at risk in in either group (high vs. low abundance) is shown below each panel. Vertical ticks show censored data. Hazard Ratio (HR) and score (logrank) test two-tailed p-value from Cox proportional hazards regression analysis.

Extended Data Fig. 2 Microbiota composition of non-progressing patients in the Pittsburgh cohort whose stool samples were collected 4–41 months after initiation of therapy is not predictive of late therapy failure but is enriched for similar bacterial taxa as observed in the initial microbiome of patients who did not progress at 10 months.

a. Plot of time of stool sample acquisition from 31 patients whose samples were collected after >4 months from therapy initiation. b. Progressor (P) and non-progressor (NP) groups identified at serial timepoints after late stool collection (top panel) were used to calculate the significance (two-tailed p-value) of compositional differences of the late-collected fecal microbiome using PERMANOVA (bottom panel). Fecal microbiota composition was determined using metagenomic sequencing. Progression during continued therapy was evaluated using RECIST v1.1 every 3 months or by clinical observation during follow-up visits. Number of patients on follow-up at each timepoint in relation to response status is shown in top panel. c. t-distributed uniform manifold approximation and projection (t-UMAP) plot depicts fecal microbiota compositional differences between early-collected patients who progressed (red) or did not progress (blue) in the first 10 months after initiation of therapy and late-collected long-term responders (green). Distance between centroids calculated as described in Fig. 1a, and significance (two-tailed p-value) of the differences by PERMANOVA are shown in lower table. d. Heatmap shows differentially abundant taxa (p < 0.05 and FC > 2) between the late Pittsburgh cohort compared with Ps (top) and NPs (bottom) at 10 months from the early Pittsburgh cohort. Columns denote patients grouped by each cohort before clustering; rows denote bacterial taxa enriched (black) or depleted (red) in early-sampled P versus late-sampled long-term NP clustered based on microbiota composition. Two-tailed p-values were calculated using two-tailed Mann-Whitney U test. e. ROC curve for manual model trained on the organisms associated with increased and decreased PFS in the Pittsburgh cohort from Supplementary Tables 4 and 5. Note that the model predicts late Pittsburgh samples well even though they were not included in the data used in training.

Extended Data Fig. 3 Entire time-to-event progression data analysis by Cox regression method of baseline fecal microbiome composition identifies additional favorable and unfavorable taxa linked with response to anti-PD-1 immunotherapy.

a. Volcano plot depicting bacteria identified by effect on progression-free survival (PFS) in the Pittsburgh early sample cohort using Cox regression analysis in Evaluate Cutpoints software. Taxa with q < 0.05 are shown as red dots. b. Cladogram visualization (favorable taxa – blue; unfavorable taxa – red) of bacterial taxa at different phylogenetic levels identified using approach described in (a).

Extended Data Fig. 4 Differential abundance analysis reveals relationship of baseline gut microbial taxa with high vs. low neutrophil-lymphocyte ratio in Pittsburgh early sample cohort.

a. t-distributed uniform manifold approximation and projection (t-UMAP) plots depicting fecal microbiota compositional differences between patient groups with high (≥3.82; orange) and low (<3.82; green) pre-treatment neutrophil-lymphocyte ratio (NLR). Optimal cutoff for NLR (3.82) was determined by time serial PERMANOVA as shown in Fig. 1a. Two-tailed p-value was calculated using PERMANOVA. b. Heatmap of differentially abundant taxa (p < 0.05 and FC > 2) in high-pre-treatment NLR (orange) and low-NLR (green) patients, using optimal cutoff (3.82). Columns denote patients grouped by NLR status and clustered within each group; rows denote bacterial taxa enriched (red) in patients with high NLR clustered based on microbiota composition; no bacterial taxa significantly enriched in the low-NLR patients were identified. Statistical significance was calculated using two tailed Mann‐Whitney U test. Bar plot to left of heatmap indicates extent of association between corresponding taxa and PFS probability [scaled hazard ratio (HR)] with Storey’s q-values <0.1 displayed within cells. Proportion of Gram-negative bacteria among those associated with high NLR was 58%, significantly higher than the average proportion of Gram-negative in patients’ fecal microbiome (28%, Chi-squared p = 0.0004).

Extended Data Fig. 5 Gut microbial gene differences discriminate between non-progressors and progressors during anti-PD-1 therapy in the Pittsburgh early sample cohort.

a. t-distributed uniform manifold approximation and projection (t-UMAP) plot depicting genetic differences of gut microbiomes between non-progressors (NPs; blue) and progressors (Ps; red) at time of maximal difference from start of therapy (10 months). Filled circles represent centroids, with connecting lines corresponding to samples from each group. Two-tailed p-value was calculated using PERMANOVA. b. Metagenomic shotgun sequencing of fecal microbiota samples identifies differentially abundant genes in Ps vs. NPs at 10 months from start of therapy. Heatmap shows differentially abundant genes identified by metagenomic shotgun sequencing (FDR < 0.2 and FC > 1.5). Columns denote patients grouped by progression status and clustered within P/NP groups; rows denote bacterial genes significantly upregulated (red) or downregulated (blue) in Ps versus NPs. c and d. Select genes involved in representative microbial processes of lipopolysaccharide (LPS) processing (c) and iron metabolism (d).

Extended Data Fig. 6 Metagenomic sequencing identifies distinct taxa associated with various immune-related adverse events in PD-1-treated melanoma patients in Pittsburgh early cohort.

Heatmap depicts metagenomic compositional differences between patients with a given immune-related adverse event (irAE) as compared to patients with other irAEs using scaled fold differences (high – red; low – blue) in abundances of specific bacteria. Values in individual cells represent unadjusted p-values calculated using two-tailed Mann-Whitney U test, with p-values <0.1 displayed within cells. Bar plot to left of heatmap indicates extent of association between corresponding taxa and progression-free survival probability [scaled hazard ratio (HR)], with Storey q-values <0.1 displayed within cells (from Supplementary Tables 4 and 5).

Extended Data Fig. 7 Reanalysis of four previously published individual cohorts using the same bioinformatic pipeline.

a. Analysis of α-diversity from five PD-1-treated melanoma patient cohorts (n = 185), including the Pittsburgh early sample cohort (n = 63), using either shotgun metagenomic (5 cohorts, red) or 16S rRNA gene amplicon (4 cohorts, black) sequencing. Details of each individual cohort are summarized in Supplementary Table 3. Forest plots depict α-diversity-based association tests including inverse Simpson, Shannon, and observed operational taxonomic units. Within each fixed-effect plot, names of each cohort are shown on a separate line, while log odds ratio of α-diversity (squares, size proportional to sample size used in meta-analysis) and associated 95% confidence intervals (bars) are shown, along with the dotted vertical line of no effect. The p-values reported for each cohort are two-tailed p values computed from the z statistic. To control for unobserved heterogeneity, we separately evaluated α-diversity using a random effects model on both pooled shotgun and 16S sequencing data from the 5 cohorts and performed I2 test for heterogeneity as shown. The p-value reported for heterogeneity is a one-tailed Cochran’s Q-test. b. t-distributed uniform manifold approximation and projection (t-UMAP) plot before (left) and after (right) correction for study-related batch effect using ComBat R package for all cohorts together including Pittsburgh cohort. P-values were calculated using PERMANOVA. c. t-UMAP plot of batch-corrected pooled metagenomic sequencing data from five separate cohorts of melanoma patients treated with anti-PD-1 therapy depicting fecal microbiota compositional differences with two-tailed p-value calculated using PERMANOVA between responders (Rs) and non-responders (NRs). d. Heatmap of differentially (p-values were calculated using non-parametric two-tailed Mann-Whitney U test) abundant gut microbiome taxa (p < 0.05, FC > 2) evaluated with shotgun sequencing in five melanoma patient cohorts, including Pittsburgh early sample cohort. Study-related batch effect was removed using ComBat R package. Response to therapy in published cohorts was determined as described in each study (Supplementary Table 3). Response to therapy in the Pittsburgh early sample cohort was defined as non-progression at 10 months after initiation of treatment. Columns represent patients grouped by clinical response and clustered within R/NR groups; rows depict bacterial taxa enriched (black) or depleted (red) in Rs versus NRs clustered based on gut microbiota composition.

Extended Data Fig. 8 Meta-analysis of all cohorts using random effects model identifies organisms differentially enriched in melanoma patients treated with anti-PD-1 therapy in separate cohorts by response status.

a. Random effect model meta-analysis of differentially abundant bacteria between responders (Rs) and non-responders (NRs) from five cohorts (n = 185) including Pittsburgh early sample cohort (n = 63) using shotgun metagenomic sequencing. All significant bacterial taxa enriched in Rs and NRs are shown. b. Forest plots depict association of representative bacterial species with response to anti-PD-1 therapy. Within each plot, names of various cohorts are shown on separate lines, while Hedge’s g (squares, standardized mean differences, size proportional to sample size) and associated 95% confidence intervals (bars) are shown, along with the dotted vertical line of no effect. To control for unobserved heterogeneity, we separately evaluated Hedge’s g using random effect model on metagenomic data and performed I2 test for heterogeneity as shown. P-values were calculated using random effect model.

Extended Data Fig. 9 Expression levels of selected taxa in different American Gut Project enteric microbiotypes.

t-distributed stochastic neighbor embedding (t-SNE) plots depicting American Gut Project (AGP) dataset with visualization of abundances of select taxa (blue – low; red – high).

Extended Data Fig. 10 Geographic differences determine sampling variability between cohorts.

a. t-distributed stochastic neighbor embedding (t-SNE) plots depicting mapping of individual melanoma patient cohorts to American Gut Project (AGP) dataset revealed distinct compositional differences between them. Each cohort is represented by a different color that is maintained in the overlay. Colors in the overlay are semi-translucent and were stratified starting from the Pittsburgh to New York cohort. Gut microbiota compositions of the different cohorts were significantly different (PERMANOVA, two-tailed p < 0.001). b. and c. Heatmaps represent scaled abundances of each enteric microbiotypes across 28 states from which AGP data were available on the left, with the four states from which anti-PD-1-treated melanoma cohorts originated separately on the right using three individuals per county per cluster as a cutoff. Data were scaled by number of individuals per state and per cluster and are depicted in relation to the 28 states that met the cutoff and in relation to the four states from which the four studied cohorts originated. b. Heatmaps are (b) scaled only by number of samples from state (to reflect local abundance of microbiotypes) or (c) scaled by both number of samples per state and by number of samples per microbiotype (to reflect distribution of different microbiotypes across the US). d. Geographic representation in the US of four representative enteric microbiotypes, with most uneven distribution between four states (right panel in c).

Supplementary information

Supplementary Information

Supplementary Figs. 1–14 and Supplementary Tables 1–10

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Source data for microbiome analysis

Clinical metadata and microbiome analysis in all the five cohorts analyzed.

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McCulloch, J.A., Davar, D., Rodrigues, R.R. et al. Intestinal microbiota signatures of clinical response and immune-related adverse events in melanoma patients treated with anti-PD-1. Nat Med 28, 545–556 (2022). https://doi.org/10.1038/s41591-022-01698-2

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