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9568 Background: Immune-checkpoint inhibitor (ICI) immunotherapy has increased survival in patients with melanoma. However, only half of the patients respond, and many experience immune-related adverse events (irAEs). Recent evidence suggests that modification of the gut microbiome may increase response to ICIs and decrease toxicity. Here we describe the first results of a clinical trial to determine if the microbiome can predict the response or toxicity during the first 16 weeks of ICI treatment. Methods: We enrolled patients aged 18 or older in a prospective observational cohort study at The Ohio State University Comprehensive Cancer Center Skin Cancer Clinic (OSUCCC-SCC) who were to receive treatment with pembrolizumab or nivolumab alone or in combination with other treatments (e.g. nivolumab and ipilimumab) for melanoma. Patients receiving systemic or oral corticosteroids at the start of ICI cycle 1 were excluded but were eligible if receiving adrenal physiologic replacement. Patients collected stool samples at baseline, within 2 days of an adverse event (if applicable), and at 12 weeks. The response to ICIs was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST v1.1) at a 12-week computed tomography scan. Metagenomic whole-genome shotgun sequencing was performed on an Illumina NovaSeq 6000 and then classified using HUMAnN3. The effect of microbe relative abundances on potential irAEs was modeled by logistic regression with the R package glmm. Results: In total, 88 patients consented to the trial. Pre-treatment microbiome samples were collected from 49 patients. Potential irAEs were observed in 16 out of the 49 patients for whom pre-treatment microbiome samples were collected. There was no significant difference in the ages (p = 0.150, genders (p = 0.2), stages (p = 0.2) or treatments (p = 0.07) of those who developed potential irAEs. Pretreatment abundance of the family Ruminococaceae was most strongly associated with the development of a potential irAE (p = 0.03), followed by a taxon in an unclassified order within the phylum Firmicutes (p = 0.05). The family Bacteroidaceae was most strongly associated with no potential irAE (p = 0.05). Conclusions: Longitudinal and event-driven biospecimen collection in the context of treatment with immunotherapies was feasible in the OSUCCC-SCC. The abundance of the two high-taxonomic rank microbe groups was significantly associated with potential irAEs. The association with Ruminococaceae is consistent with previous studies where it was associated with response to ICIs and, in separate studies, development of an irAE was associated with a better response. The unclassified taxon is potentially a new biomarker for the prediction of toxicity and a therapeutic target to reduce treatment side effects. Future analyses will associate microbes with treatment response and test for consistent microbiome changes at the time of irAE development. Clinical trial information: NCT05102773.

Rational manipulation of the gut microbiome by diet or other interventions is a promising approach to improving the efficacy and safety of cancer immunotherapy. However, linking lifestyle variables to specific microbial populations and the host immune response to cancer therapies is challenging due to their complexity. We established an experimental system to examine such complexity by linking a human dietary intervention and fecal biospecimens to mouse models to test the response to immunotherapy. Longitudinal human microbiome samples collected before and after a specific dietary or other intervention allow us to test the hypothesis that an intervention alters the response to immunotherapy via impact on the host microbiome. One set of samples was taken from the BEWELL study, which examined the impact of 2 × 80 mL black raspberry (BRB) drink boxes per day for 4 weeks in people at high risk for lung cancer. Participant samples were chosen based on enrichment of specific taxa. Pre- and post-BRB intervention samples were gavaged into mice. Mouse mc38 cells were injected subcutaneously and treated with anti-PD1 Ab or isotype control. Tumor size was monitored, and at the end of the study, tumor immune cell composition data were collected. Tumor growth over time was modeled using a linear mixed-effects model with tumor volume as the outcome variable and the predictor variables of time and treatment (PD1 vs. IgG) interacting with gavage and quadratic time to accommodate non-linear tumor growth and including random effects by mouse. For sample 68, enriched for Roseburia CAG 309, the interaction between time, gavage post-BRB, and Anti-PD1 treatment significantly affected tumor volume (p < 0.05) relative control. The same significant effect (p < 0.05) was also seen for sample 79, enriched for Lachnospira pectinoschiza, and samples 84 and 85, enriched for Blautia obeum. To determine the mechanism by which this might occur, we analyzed the tumor's immune cell composition. Post-BRB mice treated with Anti-PD1 showed a significant increase in tumor-infiltrating CD8+ immune cells relative control, as compared to mice gavaged with pre-BRB stool. Modeling results showed samples whose response was improved slightly after the BRB dietary intervention. These samples were associated with the enrichment of the taxon Roseburia CAG 309. A black-raspberry dietary intervention in humans modified the microbiomes of several participants in a way that is hypothesized to improve response to PD1 treatment. Future directions include supplementing individual microbes into pre-intervention gavages to confirm which taxa improve response. These results suggest that this modeling platform is an effective system for testing microbiome modification on tumor growth and assessing mechanisms by which this might occur and is extendable to other lifestyle-based interventions where pre- and post-intervention specimens are collected. Citation Format: Aaditya Pallerla, Bailey Conrad, Amna Bibi, Nyelia Williams, Caroline Wheeler, Rebecca Hoyd, Shankar Suman, Joseph Amann, Yangyang Liu, Marisa Bittoni, Shiqi Zhang, Madison Grogan, Alvin Anand, Najma Afrah, Carolyn Presley, Fred K. Tabung, Lang Li, Yael Vodovotz, Jiangjiang Zhu, David P. Carbone, Steven K. Clinton, Daniel Spakowicz. A causal modeling platform for testing lifestyle interventions on the microbiome and response to immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4435.

The gut microbiome changes with age and affects many aspects of human health, including response to cancer treatments. Cancer survival rates have improved with new treatment options, including immune checkpoint blockade (ICB); however, the objective response rate remains low. Manipulation of the microbiome is a promising approach to improving cancer outcomes, but the effect of age is understudied. Here, we sought to understand whether (1) specific microbes are associated with treatment response in older adults with non-small cell lung cancer (NSCLC) and (2) whether these microbes are the same as for younger adults. Next, we explored the causal effects of the microbiome on ICB response in mouse models and the relationship with blood-based markers of T-cell senescence. We conducted a prospective cohort study of adults ≥60 years with a new diagnosis of NSCLC who received any treatment modality. Stool was collected, and metagenomic whole-genome shotgun sequencing was performed. Blood T-cells were isolated, the RNA purified and then assessed for markers of senescence by nanostring. Response to treatment was determined by RECIST v1.1 criteria. Generalized linear regression was used to relate baseline microbiome abundances to treatment response and non-parametric correlations associated with CDKN2A (p16) expression to microbe abundances. To assess the causal role of the gut microbiome in ICB response, we gavaged gut microbiome samples from responders and non-responders into C57BL/6 mice to create human-microbiome avatar models. The mice were then injected with MC38 cancer cells and treated with anti-PD1 or isotype control antibodies, and tumor volume was measured over time. Biospecimens and best response data at three months were captured from 23 patients, of which five had a complete response, eight had a partial response, eight had stable disease, and two had progressive disease. Over 50 microbes were associated with a response after p-value adjustment. Responder stool was enriched for microbes associated with youth and ICB response (Bifidobacterium adolescentis, p = 2.64e-20). However, microbial taxa associated with response differed from those reported in younger populations (Firmicutes sp. CAG 145, p = 1.58e-20, Oscillibacter sp. 57-20, p = 7.96e-24). Stool from non-responders (NRs) was enriched in taxa previously linked to treatment-related toxicities and shorter progression-free survival (Streptococcus lutetiensis, p = 4.55E-24) but also contained microbes previously linked to response in younger adults (e.g., Roseburia sp. CAG 309, p = 5.16e-15). The T cell senescence marker, p16, correlated with the most enriched taxon in non-responders NRs (Streptococcus thermophilus, r = 0.45, p = 0.02), suggesting a connection between immune aging and the microbiome. Preliminary fecal transplant studies in mice showed improved ICB response in mice engrafted with stool from responders versus non-responders. Together, these data identify potential differences in the gut microbiomes of young and older adult NSCLC patients who respond to ICB. Citation Format: Daniel Spakowicz, Rebecca Hoyd, Caroline E. Wheeler, Nyelia Williams, Amna Bibi, Marium Husain, Srichandhana Rajamouli, Shankar Suman, Joseph Amann, Madison Grogan, Pooja Vibhakar, Dwight H. Owen, David P. Carbone, Ashley Rosko, Christin E. Burd, Carolyn J. Presley. Older adult-specific microbes correlate with treatment response and markers of T-cell senescence in NSCLC [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR004.