Spatiotemporal evolution of the ccRCC microenvironment links intra-tumoral heterogeneity to immune escape

Background Intratumoral heterogeneity (ITH) is a hallmark of clear cell renal cell carcinoma (ccRCC) that reflects the trajectory of evolution and influences clinical prognosis. Here we seek to elucidate how ITH and tumor evolution during immune checkpoint inhibitor (ICI) treatment can lead to therapy resistance. Methods Here, we spatiotemporally profiled the genomic and immunophenotypic characteristics of 29 ccRCC patients, including pre- and post-therapy samples from 17 ICI treated patients. Deep multi-regional whole exome and transcriptome sequencing were performed on 29 patients at different time points before and after ICI therapy. T cell repertoire was also monitored from tissue and peripheral blood collected from a subset of patients to study T cell clonal expansion during ICI therapy. Results Angiogenesis, lymphocytic infiltration, and myeloid infiltration varied significantly across regions of the same patient, potentially confounding their utility as biomarkers of ICI response. Elevated ITH associated with a constellation of both genomic features (HLA LOH, CDKN2A/B loss) and microenvironmental features, including elevated myeloid expression, reduced peripheral T cell receptor (TCR) diversity, and putative neoantigen depletion. Hypothesizing that ITH may itself play a role in shaping ICI response, we derived a transcriptomic signature associated with neoantigen depletion that strongly associated with response to ICI and targeted therapy treatment in several independent clinical trial cohorts. Conclusions These results argue that genetic and immune heterogeneity jointly co-evolve and influence response to ICI in ccRCC. Trial registration We completed a single-arm pilot study at Memorial Sloan Kettering Cancer Center (MSKCC; [ClinicalTrials.gov][1] identifier [NCT02595918][2]) to examine the safety and feasibility of neoadjuvant nivolumab in patients with localized RCC. ### Competing Interest Statement M.G., R.V., M.L.S., J.G., T.P., R.M., C.Z., S.Z., L.L., are current employees and shareholders of Illumina Inc. T.A.C. and L.G.T.M. are inventors on a patent held by Memorial Sloan Kettering related to the use of TMB in cancer immunotherapy. MSK has licensed the use of TMB for the identification of patients that benefit from immune checkpoint therapy to PGDx. L.G.T.M. reports laboratory research funding from AstraZeneca. T.A.C. is a co-founder of Gritstone Oncology and holds equity. T.A.C. holds equity in An2H. T.A.C. acknowledges grant funding from Bristol-Myers Squibb, AstraZeneca, Illumina, Pfizer, An2H, and Eisai. T.A.C. has served as an advisor for Bristol-Myers Squibb, Illumina, Eisai, and An2H. R.J.M. reports consulting fees from Aveo, Calithera, Eisai, Eli Lilly, EMD Serono, Genentech, Merck, Novartis AG, Pfizer, and Roche, and contracted research to employer MSKCC for Bristol Myers Squibb, Eisai, Exelixis, Genentech, Merck, Pfizer, and Roche. A.A.H. is on the advisory board for Merck. M.H. receives commercial research grants from Bristol-Myers Squibb, Pfizer and Genentech/Roche, honoraria from Novartis, Bristol-Myers Squibb, travel/accommodation from Astra Zeneca, Eisai, Novartis and Takeda, and is a consultant/advisory board member for Alexion Pharmaceuticals, Aveo, Calithera Biosciences, Corvus Pharmaceuticals, Exelixis, Eisai, GlaxoSmithKline, Merck, Natera; Onquality Pharmaceuticals; Novartis and Pfizer. Other authors declare no competing interests. ### Clinical Trial NCT02595918 ### Funding Statement We thank members of the Chan lab for their suggestions and critical reading of the manuscript. We acknowledge funding sources including NIH R01 CA205426 (T.A.C.), NIH R35 CA232097 (T.A.C.), DOD grant KC180165, NIH R01 DE027738 (to L.G.T.M.), the NIH/NCI Cancer Center Support Grant P30 CA008748 (to MSKCC), P30 core grants (to MSKCC), Ludwig institute (A.A.H.), Weiss family fund (A.A.H.), Department of Defense (A.A.H.) and Illumina Inc. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Informed consent and institutional review board approval were acquired at Memorial Sloan Kettering Cancer Center (MSK). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors * TCGA : The Cancer Genome Atlas WES : Whole-exome sequencing WTS : Whole-transcriptome sequencing SCNA : Somatic copy number alterations LOH : Loss of heterozygosity ITH : Intra-tumoral heterogeneity TCR : T cell receptor ccRCC : clear cell Renal Cell Carcinoma TME : Tumor microenvironment ICI : Immune checkpoint inhibitor TKI : Tyrosine kinase inhibitor HERV : Human endogenous retrovirus PBMC : peripheral blood mononuclear cells TIL : Tumor infiltrating lymphocytes WGCNA : Weighted gene co-expression network analysis ssGSEA : single sample gene set enrichment analysis [1]: http://ClinicalTrials.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02595918&atom=%2Fmedrxiv%2Fearly%2F2022%2F07%2F13%2F2022.07.11.22277322.atom