Presented by Jacob Miller Gut microbiome modulates response to anti–PD-1 immunotherapy in melanoma patients Presented by Jacob Miller

Absract Pre-clinical mouse models suggest the gut microbiome tumor responds to checkpoint immunotherapy but hasn’t been well-characterized in humans. Melanoma patients’ oral and gut biome were examined while going through anti-PD-1 immunotherapy. Significant differences of composition and diversity in responders (R) vs non responders (NR) Metagenomic studies showed functional differences in R Shows implications for treatment of melanoma patients

Reasons Therapies targeting CTLA-4 and PD-1 proteins are heterogeneous and not durable Factors beyond the tumor genomics influence therapeutic responses, i.e. gut microbiome Gut microbiome may influence anti-tumor responses but have not been extensively studied in cancer patients.

Methods Oral and fecal samples where collected from patients undergoing anti-PD- 1 therapy Blood samples and tumor biopsies were collected and matched pre- treatment Taxonomic profiling via 16S rRNA gene sequencing and WGS Patients classified as R or NR via RECIST 1.1

Figure 1 Timeline of patient sampling Phylogenic composition of common bacteria Inverse Simpson diversity score Phylogenic composition at family level KM plot of PFS Principle coordinate analysis of fecal samples

Discussion 1 Found that patients with metastatic melanoma have high diversity microbiomes. Diversity in the gut microbiome was higher in R patients compared to NR patients High diversity fecal microbiome had prolong PFS compared to low diversity Gut microbiome was found to have notable clustering effect, oral did not.

Figure 2 Heatmap of OTU abundances Phylogenic composition of OTUs Taxonomic cladogram from LEfSe LDA score computed for differentially-abundant taxa in fecal microbiome Differentially-abundant gut bacteria Pairwise comparison of abundance of MGS

Discussion 2 No significant differences in enrichment were noted in the oral microbiome Type 1 and Type 2 communities were identified Type 1: Completely R patients, Clostridiales were enriched Type 2: Mixture of patients, Bacteroidales were enriched Showed differentially abundant bacteria in fecal microbiome of R vs NR Gut microbiome was shown to be relatively stable over time

Figure 3 Top: hierarchical clustering of complete linkage. Bottom: stacked bar pot of abundance at order level Association of community types with response to anit-PD-1 Comparison KM PFS curves Hierarchical clustering of pathway class enrichment

Discussion 3 Begin to focus on Faecalibacterium genis in R and bacteroidales order in NR Faecalibacterium and prior immunotherapy were used for the multivariate model Abunance of Faecalibacterium and Bacteroidales outperformed relevant clinical variables in ROC analysis Next step was to look at the mechanism the gut microbiome may influence response to anit-PD1 therapy

Figure 4 Quantification by IHC of the CD8+ cells Pairwise Spearmen rank correlation heatmap Univariate linear regression between CD8+ counts Pairwise Spearman rank correlation heatmap of different fecal taxa Representative multiplex IHC images Frequency of various immune cells Experimental design of studies in GF mice Difference in size of tumors Representative tumor growth curves Quantification of CD8+ density in tumor Quantification of CD8+ density in gut

Discussion 4 Fecal Microbiome Transplant was used in ger free mice Mice with FMT from R patients had significantly less tumor growth Responses to anti-PD1 immunotherapy were improved Faecalilbacterium had higher levels of effector CD4+ and CD8+ T cells higher density of immune cells

Final Discussion/Results “Favorable” gut microbiome have enhanced anti-tumor immune responses “Unfavorable” gut microbiomes have impaired anti-tumor immune responses Warrant evaluation of gut microbiome in cancer patients through clinical trials

Reference Gopalakrishnan, V., Spencer, C. N., Nezi, L., Reuben, A., Andrews, M. C., Karpinets, T. V., … Wargo, J. A. (2018). Gut microbiome modulates response to anti–PD-1 immunotherapy in melanoma patients. Science (New York, N.Y.), 359(6371), 97–103. http://doi.org/10.1126/science.aan4236