Abstract 3058: Characterization of the tumor microbiome in patients with locally advanced rectal cancer treated with neoadjuvant therapy
Authors: Vanderbilt Chad M. , Chatila Walid K. , Khalil Danny N. , Chen Chin-Tung , Kim Jin K. , Wu Fan , Schultz Nikolaus , Weiser Martin R. , Garcia-Aguilar Julio , Sanchez-Vega Francisco 
Published: June 2022

Abstract
The gut microbiota is a component of the tumor microenvironment that has the potential to influence sensitivity or resistance to neoadjuvant therapy through modulation of the host immune response in patients diagnosed with locally advanced rectal cancer (LARC, stage II-III).
We performed DNA and RNA sequencing of pre-treatment endoscopic biopsies from 89 LARC patients treated with neoadjuvant therapy (NAT). Our cohort included 84 microsatellite stable and 5 microsatellite unstable (MSI) patients. We identified the presence of microbial species by mapping non-human RNA read sequences to a large catalogue of viral and bacterial microorganisms. Our methodology has been validated and determined to have comparable accuracy to reference microbial tests for both viruses and bacteria. We analyzed correlations between presence of specific microbial species and (1) clinicopathological variables such as age, stage, anatomic location and MSI status, (2) genomic variables such as tumor mutational burden, chromosomal instability and somatic alterations and (3) response to NAT. We identified 152 species with at least 100 mapped reads in 10% or more of our cohort. The median number of species per patient was 37 [IQR 16-66], with only 6/89, 7% patients having no detected species at all. The most frequently detected species included well-known intestinal bacteria such as Escherichia coli, Fusobacterium nucleatum, Faecalibacterium prausnitzii and several members of the Bacteroides genus. The Parvimonas micra coccus was more often detected in tumors from the lower rectum (13/22, 59%, 0-4 cm from the anal verge) than the middle (8/36, 22%, 4-8 cm) and upper rectum (5/30, 17%, 8-12 cm) (p&lt;0.01). Several species from the Gordonia and Rhodococcus genera were associated with increased tumor mutational burden (p&lt;0.001) and significantly higher numbers of mapped reads in samples from the CMS1 molecular subtype (p&lt;0.01), which is enriched in MSI tumors. RNA-Seq based estimates of neutrophil abundance were positively correlated with detection of several Ruminococcus and Prevotella species (p&lt;0.001), highlighting the cross-talk between host immunity and microbiota. Opportunistic bacteria previously reported to benefit from a patient’s weakened immune system such as Williamsia muralis and several species of Agrobacterium were associated with lower rates of complete response (30% vs. 15%, n.s.) and significantly shorter disease free survival following NAT (3-year disease free survival was 25% in 8 Williamsia positive patients vs. 42% in 12 Agrobacterium fabrum patients vs. 87% in the rest of the cohort; pairwise log-rank p&lt;0.001). Our data shows that standard RNA sequencing platforms can be used to detect microbial species in endoscopic biopsies from LARC patients and that the presence of specific species is associated with clinical and genomic features of translational relevance.
Citation Format: Chad M. Vanderbilt, Walid K. Chatila, Danny N. Khalil, Chin-Tung Chen, Jin K. Kim, Fan Wu, Nikolaus Schultz, Martin R. Weiser, Julio Garcia-Aguilar, Francisco Sanchez-Vega. Characterization of the tumor microbiome in patients with locally advanced rectal cancer treated with neoadjuvant therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3058.