Association between intra-tumoral microbiome and clinical benefit (CB) from immunotherapy (IO) in patients with metastatic renal cell carcinoma (mRCC).
Authors: Meza Luis A , Choi Yongwook , Zhang Jiaming , Dizman Nazli , Zengin Zeynep Busra , Ebrahimi Hedyeh , Barragan-Carrillo Regina , Hsu Joann , Chawla Neal Shiv , Chehrazi-Raffle Alex , Tripathi Abhishek , Castro Daniela V. , Mercier Benjamin D , Govindarajan Ameish , Chan Agnes , Schork Nicholas , Byron Sara A. , Pal Sumanta Kumar 
Published: June 2023

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Background: Specific bacteria and microbial diversity in the gut may drive response to IO in patients (pts) with mRCC (Routy et al Science 2018; Salgia et al Eur Urol 2020). Despite the evidence of bacterial proliferation in RCC tumors (Heidler et al Urol Int 2020), the impact of the intra-tumoral microbiome in RCC has been poorly delineated. Here, we explored its association to CB in mRCC pts receiving IO-based regimens. Methods: We identified pts with mRCC and available RNA sequencing (RNA-seq) data from tumor tissue collected prior to start of IO.Intra-tumoral microbiome analysis was performed on formalin-fixed paraffin-embedded samples. Following quality and adapter trimming, RNA-seq reads were mapped to a human genome to filter host reads using the STAR alignment tool. Taxonomic classification was performed using Kraken2 and Bracken. Following removal of common contaminants, the relative abundances among all non-human species were calculated. Microbial diversity was assessed using the Shannon index and statistical analysis performed using a t-test. Relative microbial abundance was compared between tumors from pts with or without CB from 1st line IO treatment using the Kruskal-Wallis test. P-values less than 0.05 were considered significant. Results: A total of 96 pts (71:25, M:F) with mRCC were included in this analysis. Of these, 85 (89%) had clear cell histology and 68 (71%) were IMDC intermediate/poor risk. The median age at the time of tissue sampling was 62.7 yrs old and most of the samples analyzed (57%) were collected from the primary site. IO, alone or in combination, was received as 1st line treatment in 57 pts (59%), with the most common regimen being nivolumab/ipilimumab (47%). CB from 1st line IO was observed in 39 (68%) pts. Tumors from pts achieving CB from 1st line IO had elevated microbial diversity compared to tumors from pts without CB (p=0.01). Tumor samples from these pts also had a significantly higher relative abundance of several bacterial genera compared to tumors from pts without CB. These included Pasteurella, Escherichia, and Cloacibacterium (p<0.05). No significant differences in bacterial diversity were observed between CB and no-CB pts receiving IO as 2nd line or beyond. Conclusions: This is the largest study exploring the association between the intra-tumoral microbiome of mRCC and CB from IO. In line with gut microbiome studies, an increased intra-tumoral Shannon diversity index was also associated with CB to IO in our 1st line cohort, further suggesting that bacterial diversity could be a driver of response. Additionally, several genera associated with CB are particularly relevant. For instance, members of the Pasteurella genus have been implicated in the activation of c-myc, a proto-oncogene that has been shown to induce PD-L1 expression.