Prevalence of pathogenic variants in DNA damage response genes in patients undergoing cancer risk assessment and reporting a personal history of early-onset renal cancer

Purpose Pathogenic variants (PVs) in a number of genes are known to increase the risk of hereditary renal cancer (hRC). However, many early-onset RC (eoRC) patients undergoing genetic testing lack PVs in hRC genes; thus, their genetic risk remains undefined. To determine if PVs in DNA damage response (DDR) genes are enriched in a convenience sample of eoRC patients undergoing genetic testing. Materials and Methods Retrospective review of results for 844 unselected eoRC patients, undergoing genetic testing with a multi-gene cancer panel by Ambry Genetics [between July 2012 and December 2016]. The patients were tested with CancerNext and/or CancerNext Expanded panels for a variety of indications. Identified PVs were compared with patient characteristics. Results Mean age of RC diagnosis was 48 years [range 24-60]. In addition to eoRC, 57.9% patients tested reported at least one additional cancer; breast cancer being the most common (40.1% of females, 2.5% of males). PVs in cancer risk genes were identified in 12.8% of patients—3.7% in RC-specific genes, and 8.55% in DDR genes. DDR gene PVs were most commonly identified in CHEK2, BRCA1/2, and ATM. Among the 2.1% of patients with a BRCA1/2 PV, <50% reported a personal history of hereditary breast/ovarian-associated cancer. No association between age of RC diagnosis, and prevalence of PVs in RC-specific or DDR genes was observed. Conclusions Multi-gene panel testing including DDR genes may provide a more comprehensive risk assessment in unselected eoRC patients, and their families. Validation in larger datasets is needed to characterize the association with eoRC.


INTRODUCTION
Renal cancer (RC) often develops with no signs or symptoms and is referred to as the "silent disease" (1). While factors including smoking, environmental factors, obesity, and race have been linked to increased risk of RC, inherited factors are the most well-validated source of increased risk (1). Hereditary RC (hRC) syndromes, typically associated with early-onset disease and a clinically significant family history of cancer, result from germline pathogenic variants (PV) in high-penetrance 'RC-specific' genes including VHL, MET, FLCN, TSC1, TSC2, FH, SDH, PTEN and BAP1 (2-4). Previous report of an early-onset RC (eoRC) cohort screened with an RC-specific panel found 6.1% of individuals had a PV in an RC-specific gene (4).
However, for most eoRC patients a PV in an RC-specific gene is not identified, leaving many eoRC genetically undefined. Thus, there is a need to identify additional genes related to eoRC risk. Currently, there are no National Comprehensive Cancer Network (NCCN) guidelines for detection, prevention, or risk reduction in individuals who present with an eoRC but lack a PV in a defined RC-specific gene (5).
DNA damage response (DDR) genes play an important role in maintaining genome integrity, but also increase cancer risk (6). PVs in DDR genes confer susceptibility to a variety of cancer types, but are not typically considered risk factors for eoRC; however, in some cases, eoRCs arise in patients with personal or family histories of other forms of cancer. This population is particularly likely to be interested in evaluation of genetic risk. Further, germline PVs in some DDR genes have been observed in RC, including the DNA mismatch repair (Lynch syndrome) genes MSH2 and MLH1 in renal urothelial carcinoma, and CHEK2 in advanced renal cell carcinoma (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). To address the hypothesis that PVs in DDR genes may contribute to the missing heritability of eoRC, we analyzed germline sequencing data from a cohort of 844 eoRC, many of whom had additional non-RC primary tumors or a family history of non-RC cancers.

Statistical Analysis
To identify potential correlations between PVs and characteristics such as tumor pathology, additional primary tumor type, and age of diagnosis, genes were combined into pathways/groups of interest, histology's were grouped, and cancer types were grouped. Each individual was categorized as having a variant in one of the genes within the group or no variant in the group. Gene categories were used for comparison of RC diagnosis with a DDR gene or an RC-specific gene. For additional methods, see Supplemental file.

Patient characteristics
We first benchmarked the eoRC study cohort to the reported incidence of RC in SEER data for the general US population to provide context. In the study cohort, 40% of cases were between 50-59 years of age, and median age of diagnosis was 48 years. As expected, a higher percentage of RC cases were diagnosed between 20-44 years of age as compared to patients ≤ 60 diagnosed with RC in the general US population (SEER) (35%, versus 21.9%) (Fig. 1A).

High incidence of other cancers in study cohort
57.9% (n=489/844) of the cases in the study cohort reported at least one additional primary cancer (Fig. 1D, 1E, 1F, Table 1, Table S2). Each of the primary cancer types is also represented at a higher level in the study cohort than in the general US population as reported by the SEER database (Fig. 1D). For female-specific cancers, 40.1% of females (227/566) also had breast cancer, in comparison to the 4.3% breast cancer rate in women ≤ 60 in the general population (SEER) (Fig. 1D & 1E, Table S2). The rate of additional primary cancer in the study cohort (57.9%) is much higher than the rate of each cancer type observed in SEER cases with eoRC (21.6%) (Fig. 1F).
The overall gene variation rate in the full study cohort (n=844) is presented in Table S1.
The full study cohort was not tested for all 49 genes. The largest panel was tested in 491 cases, and consisted of 49 genes, which included 15 RC-specific genes, and 34 other-cancer associated genes including 19 DDR genes (Table S1). Here, 12.8% (63/491) of cases had PVs.

DDR PVs are similarly enriched in patients diagnosed with eoRC alone, or with eoRC and other cancers
To test the hypothesis that DDR PVs might be associated with the phenotype of multiple We performed similar analysis for patients who presented with eoRC plus one or more additional cancer (53.2%, n=261/491). Among the patients with RC and at least one additional cancer, PVs were identified in 12.7% cases (33/261, Fig. 2F). Among these 12.7% of cases, PVs in other-cancer associated genes, including DDR genes, were found in 8.8% of cases Overall, these data suggest that DDR PVs are enriched similarly in individuals diagnosed with eoRC alone or eoRC plus at least one additional primary cancer, but that the frequency of PVs in DDR genes, in either group, exceeded that in the control populations tested (gnomAD/ExAc) (Fig. 2, Table S5A). The specific PVs identified were similar in number and frequency to those identified in the full patient cohort (n=844), with CHEK2 the most highly represented DDR gene (Fig. 2). To gain additional insight into the prevalence of these PVs in cancer patients, we surveyed ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and found that multiple PVs from this study ( Table S4) have been reported in hereditary cancer predisposing syndromes (HCPS, summarized in Table S7). HCPS reflects a pattern of cancers in a family characterized by earlier onset, with individuals not necessarily having the same tumor and/or having more than one primary tumor, and having tumors that are more likely to be multicentric.

No correlation between age of RC diagnosis and type of PV in eoRC
To determine if identification of specific classes of germline PV correlated with age of diagnosis in this cohort, genes were divided into four broad (overlapping) categories: all genes tested, RCspecific genes, non-RC associated genes (including DDR genes) and DDR genes (see Supplementary Methods). The groups were compared to median age at first RC diagnosis of <48 or >48 years. Given the invariable early-onset of Wilms tumor, the 20 individuals with this diagnosis were excluded from the analysis. Within this eoRC cohort, there was no significant association between age at diagnosis of RC and the type of PV for any of the four broad categories above (Fig. 3A).

Correlation of renal histologies with PVs in specific genes
Of the 243 clear cell cases in this cohort, 13.6% (33/243) had a PV, of which 2.9% were RCassociated PVs. Similar findings were observed for the cases described as renal cell carcinoma, Odds ratios for identified PVs in specific renal tumor histologies were calculated.
Specifically, for PVs in DDR genes, ATM correlated with mixed papillary histology, BRCA1, MUTYH, and CHEK2 with Wilms tumor, BRCA2 with papillary renal cell, and CHEK2, and NBN with chromophobe histology ( Table S8). None of the associations were statistically significant.

DISCUSSION
This study for the first time demonstrates that PVs in multiple DDR genes occur in patients with eoRC. Importantly, this study found that DDR PVs were highly represented both in cases diagnosed with eoRC and additional cancers, and also cases diagnosed with eoRC alone. Comparison with a large control population indicated that germline PVs in DDR genes were more common in this study cohort than in the control population, although further studies 1 0 are required to confirm this finding and predict the penetrance of PVs in DDR genes for eoRC. We also found that germline testing using an RC-specific panel would have identified only 3.7% BRCA PVs, including non-breast tumors (24,25). Also, screening and prevention of HBOCsyndrome cancers will be increased significantly in the proband and family members found to have the same PV. In the study cohort, females were overrepresented, even though more males are typically diagnosed with RC (26). This difference may reflect likelihood of seeking genetic testing, given the frequency of HBOC in the cohort.
In RC, a number of germline PVs have been associated with treatment response. For example, bevacizumab with everolimus or erlotinib were added as treatment options for RC cases with germline PVs in FH (27,28). Currently, the predictive and prognostic significance of PVs in DDR genes is not clinically defined for RC. There is an urgent need to study the biological impact of PVs in DDR genes in renal tissue. Such work may also lead to improved understanding of RC pathogenesis. Studies are in progress to assess cancer risk in different tissue types, and response to treatment due to a germline defect in DDR genes (29). A recent study showed that VHL inactivation in RC led to reduced expression of DDR genes (such as BRCA1/2), and thereby increased sensitivity to PARP inhibitors (30). These results indicate that RC tumors with DDR vulnerabilities may be responsive to PARP or other DDR inhibitors under development. Ongoing clinical trials are assessing the effect of PARP inhibitor, olaparib, in patients with somatic DDR variants in the setting of metastatic RC (NCT03786796). Finally, it is also important to evaluate the penetrance of DDR PVs to clinically define RC risk. These studies will assist in genetic counseling of RC patients and their families.
The limitations of this study include the following: This is a relatively small cohort, and all cases were not tested for all 49 genes. The cohort is a highly selected sample, as these patients likely had clinical characteristics (e.g. high rate of additional primary cancers) or family history that led to expanded panel testing. Although twice as many men are typically diagnosed with RC than women, the study cohort had more females than males. This may reflect the observation that women are more likely to pursue genetic testing than men, or the fact that 34.4% of cases also had a diagnosis of breast/ovarian/uterine cancer. Alternatively, men diagnosed with RC might be considered high-risk due to smoking or other environmental factors that lead their physician to be less suspicious of a hereditary component. A large percentage (34.7%) of tumors from the study cohort was listed as "unknown subtypes", limiting comparison of PVs and RC histology types. Finally, matched (such as age, gender) comparisons were not possible using the control population, and we made no adjustment for population stratification.
Differences in the study cohort, and control population ascertainment strategies and data collection (i.e. bioinformatic pipeline for variant calling/filtering, sequence coverage, race/ethnicities) prevent us from making any conclusions about the relationship between the PVs and RC risk. The comparisons performed in this manuscript were not adjusted for multiple testing.

CONCLUSIONS
This study is the first to indicate a role for PVs in multiple DDR genes in eoRC. These results need to be validated in other large data sets. Additionally, to fully elucidate the biological relevance of DDR to RC, family and functional studies are needed as a next step to quantify the associated risks.

Ethics approval and consent to participate
The Fox Chase Cancer Center Institutional Review Board (IRB) approved all work (IRB-14-831).

Consent for publication
The patients in the study did not consent to sharing their raw DNA sequence data.

Availability of data materials
All data generated or analysed during this study are included in this published article [and its supplementary files].