Signatures of defective DNA repair and replication in early-onset renal cancer patients referred for germline genetic testing

Early-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined. Here, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes. We performed whole-exome sequencing (WES) and found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of γH2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate PGVs in Caki RCC cells increased γH2AX foci. Immortalized patient-derived B cells bearing candidate PGVs in DNA polymerase genes ( POLD1, POLH, POLE, POLK ) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities. Together, these results suggest that constitutional defects in DNA repair such as DNA replication repair underlie a subset of eoRCC cases. These findings may provide opportunities for use of the DNA repair targeting agents for eoRCC treatment. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Significance Statement Screening for DNA repair variation may provide a more comprehensive risk assessment for eoRCC patients. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC. ### Competing Interest Statement M.J.H. performs collaborative research (with no funding) with the following: Myriad Genetics, Invitae Corporation, Ambry Genetics, Foundation Medicine Inc. He also performs collaborative research (with no funding) and is part of a Precision Oncology Alliance funded by Caris Life Sciences (cover travel and meals at meetings). S.A. performs collaborative research (with no funding) with Caris Life Sciences, Foundation Medicine, Inc., Ambry Genetics, and Invitae Corporation. S.A. spouse is employed by Akoya Biosciences and has stocks in Akoya Biosciences, HTG Molecular Diagnostics, Abcam Plc., and Senzo Health. S.A., M.J.H., E.A.G., I.G.S. have patents and/or pending patents related to cancer diagnostics/treatment. All other authors declare no competing interests. ### Funding Statement All Fox Chase Cancer Center affiliated authors are in part supported by the NCI Core Grant, P30 CA006927, to the Fox Chase Cancer Center. S.A. was supported by the DOD W81XWH-18-1-0148, and a CEP Award from the Yale Head and Neck Cancer SPORE. M.J.H. was supported by funding from the American Cancer Society. M.B.D was supported by the NIH U01 CA164920, R01 CA207365 grants. E.A.G. was supported by NIH R01 DK108195. E.V.D. and I.G.S. were partially supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030). R.J.D. was supported by NIH R35 GM122517. R.T.P. was supported by NIH grants 1R01GM130889 and 1R01GM137124. ### 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: Ethics committee/IRB of Fox Chase Cancer Center gave ethical approval for this work. 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 work are contained in the manuscript. Only the DNA sequence reads data is not available for sharing as consent was not obtained for sharing. * 8-oxoG, : 8-oxoGuanine; BWA, : Burrows-Wheeler aligner; ccRCC, : clear cell renal cell carcinoma; CTB, : Cell Titer Blue; CldU, : 5-Chloro-2’-deoxyuridine; DSBs, : double-strand breaks; eoRCC, : early-onset renal cell carcinoma; DDR, : DNA damage response; EBV, : Epstein-Barr virus; ExAC, : Exome Aggregation Consortium; ExoD, : exonuclease domain; FCCC, : Fox Chase Cancer Center; FDR, : false discovery rate; GATK, : genome analysis toolkit; GnomAD, : Genome Aggregation Database; GO, : gene ontology; IdU, : 5-Iodo-2′-deoxyuridine; LOH, : loss of heterozygosity; MMR, : mismatch repair; MNNG, : 1-Methyl-3-nitro-1-nitrosoguanidine; MSI, : microsatellite instability; MSS, : microsatellite stability; NCCN, : National Comprehensive Cancer Network; PBMCs, : peripheral blood monocytes; PGVs, : pathogenic germline variants; Pt, : patient; RAP, : risk assessment program; RCC, : renal cell carcinoma; SNPs, : single nucleotide polymorphisms; TLS, : translesion synthesis; TMB, : tumor mutation burden; VUS, : variant of uncertain significance; WebGestalt, : WEB-based GEne SeT AnaLysis Toolkit; WES, : whole-exome sequencing; wt, : wild type.