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PURPOSEDespite advances in DNA sequencing technology and expanded medical guidelines, the vast majority of individuals carrying pathogenic variants of common cancer susceptibility genes have yet to be identified. An alternative to population-wide genetic screening of healthy individuals would exploit the trend for genetic testing at the time of cancer diagnosis to guide therapy and prevention, combined with augmented familial diffusion or “cascade” of genomic risk information.METHODSUsing a multiple linear regression model, we derived the time interval to detect an estimated 3.9 million individuals in the United States with a pathogenic variant in 1 of 18 cancer susceptibility genes. We analyzed the impact of the proportion of incident patients sequenced, varying observed frequencies of pathogenic germline variants in patients with cancer, differential rates of diffusion of genetic information in families, and family size.RESULTSThe time to detect inherited cancer predisposing variants in the population is affected by the extent of cascade to first-, second-, and third-degree relatives (FDR, SDR, TDR, respectively), family size, prevalence of mutations in patients with cancer, and the proportion of patients with cancer sequenced. In a representative scenario, assuming a 7% prevalence of pathogenic variants across cancer types, an average family size of 3 per generation, and 15% of incident patients with cancer in the United States undergoing germline testing, the time to detect all 3.9 million individuals with pathogenic variants in 18 cancer susceptibility genes would be 46.2, 22.3, 13.6, and 9.9 years if 10%, 25%, 50%, and 70%, respectively, of all FDR, SDR, and TDR were tested for familial mutations.CONCLUSIONPeridiagnostic and cascade cancer genetic testing offers an alternative strategy to achieve population-wide identification of cancer susceptibility mutations.

Background Constitutional or somatic mosaic epimutations are increasingly recognized as a mechanism of gene dysregulation resulting in cancer susceptibility. Beckwith‐Wiedemann syndrome is the cancer predisposition syndrome most commonly associated with epimutation and is extremely variable in its phenotypic presentation, which can include isolated tumors. Because to the authors' knowledge large‐scale germline DNA sequencing studies have not included methylation analysis, the percentage of pediatric cancer predisposition that is due to epimutations is unknown. Methods Germline methylation testing at the 11p15.5 locus was performed in blood for 24 consecutive patients presenting with hepatoblastoma (3 patients) or Wilms tumor (21 patients). Results Six individuals with Wilms tumor and 1 patient with hepatoblastoma were found to have low‐level gain of methylation at imprinting control 1, and a child with hepatoblastoma was found to have loss of methylation at imprinting control 2. The loss of methylation at imprinting control 2 was found to be maternally inherited, despite not being associated with any detectable genomic alteration. Conclusions Overall, 33% of patients (8 of 24 patients) with Wilms tumor or hepatoblastoma were found to have an epigenetic susceptibility that was detectable in the blood. It is interesting to note that low‐level gain of methylation at imprinting control 1 predominantly was detected in females with bilateral Wilms tumors. Further studies in larger cohorts are needed to determine the efficacy of testing all patients with Wilms tumor or hepatoblastoma for 11p15.5 epimutations in the blood as part of DNA analysis because this hallmark of predisposition will not be detected by sequencing‐based approaches and detecting a cancer predisposition may modify treatment., In the current study, all patients presenting with Wilms tumor or hepatoblastoma undergo 11p15.5 methylation analysis. Approximately one‐third are found to have an epimutation at this locus that is detectable in peripheral blood.

Introduction: Germline pathogenic variants in genes in the homologous recombination (HR) pathway such as BRCA1, BRCA2, ATM, PALB2, RAD51C and RAD51D confer an increased risk of breast and ovarian cancers. Screening for germline variants in these cancer susceptibility genes is critical as prophylactic measures and monitoring can be performed in these individuals to minimize their risk of developing breast and/or ovarian cancers. More recently, it has been recognized that cancers arising in carriers of germline pathogenic (P)/ likely pathogenic (LP) variants in HR genes often show a homologous recombination (HR) deficient (HRD) phenotype and can be targeted with platinum agents or PARP inhibitors. RAD51B is a RAD51 paralog that binds to RAD51C and functions as a heterodimer in the HR pathway. Whilst RAD51B germline variants have been reported in isolated cases of breast and ovarian cancers, it is unclear as to whether RAD51B P/LP germline variants would confer predisposition to these cancer types. Materials and Methods: We screened 9,287 consecutive unselected cancer patients who consented for both tumor and germline testing using the MSK-IMPACT platform for the presence of RAD51B germline truncating variants. Selected RAD51B germline mutant breast cancers identified by MSK-IMPACT were subjected to whole-exome sequencing (WES) analysis to determine the dominant mutational signatures using DeconstructSigs. Functional assays were performed to ascertain the impact of RAD51B loss on HR DNA repair and PARP inhibitor sensitivity using genome editing methods in non-malignant breast epithelial cell lines. Results: Out of the 9,287 cancer patients, we detected likely pathogenic loss of function RAD51B germline variants in 11 patients (0.12%), which was similar to the frequency detected in the gnomAD database (0.09%). All female carriers of RAD51B loss of function variants (n=8) had breast or ovarian cancers (8/1,619 breast or ovarian cancers, 0.5%). The observed carrier frequencies of germline truncating variants in RAD51B was statistically enriched in breast and ovarian cancer patients compared to individuals in the gnomAD database (0.5% vs 0.09% P=0.0003; odds ratio = 5.06 (95% CI: 2.1-10.3)). Although segregation studies were not available, 9/11 of the RAD51B germline loss of function variant carriers had a personal or family history of breast or ovarian cancers. All five breast and ovarian cancers from RAD51B mutation carriers investigated by WES were found to harbor RAD51B bi-allelic inactivation through loss of heterozygosity of the RAD51B wild-type allele. In addition, these five cases were found to display genomic features of HRD including high large-scale state transition scores and a dominant mutational signature 3. CRISPR/Cas9 genome editing of RAD51B in a non-malignant breast epithelial cell model revealed that RAD51B deficient cells display PARP inhibitor sensitivity similar to that reported in BRCA1 and BRCA2 deficient cell lines. Conclusion: RAD51B loss-of-function germline variants confer susceptibility to breast and ovarian cancer development. Breast and ovarian cancers occurring in the context of RAD51B germline mutations harbor bi-allelic inactivation of RAD51B through loss-of-heterozygosity of the wild-type allele, genomic features of HRD and are likely sensitive to PARP inhibition. Albeit rarely germline mutated, RAD51B should be considered as an addition to clinical germline testing panels for hereditary breast and ovarian cancer syndrome patients. Citation Format: Diana SReis-FilhoReis-Filho Mandelker, Semanti Mukherjee, Jeremy Setton, Pier Selenica, Yelena Kemel, Ozge Ceyhan-Birsoy, Margaret Sheehan, Kaitlyn Tkachuk, David N Brown, Simon Powell, Britta Weigelt, Mark E Robson, Nadeem Riaz, Kenneth Offit, Jorge S Reis-Filho. RAD51B loss-of-function variants confer susceptibility to hereditary breast and ovarian cancers and result in tumors with genomic features of homologous recombination repair defects [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-09-01.

Acquired somatic mutations in hematopoietic stem and progenitor cells (clonal hematopoiesis or CH) are associated with advanced age, increased risk of cardiovascular and malignant diseases, and decreased overall survival. These adverse sequelae may be mediated by altered inflammatory profiles observed in patients with CH. A pro-inflammatory immunologic profile is also associated with worse outcomes of certain infections, including SARS-CoV-2 and its associated disease Covid-19. Whether CH predisposes to severe Covid-19 or other infections is unknown. Among 525 individuals with Covid-19 from Memorial Sloan Kettering (MSK) and the Korean Clonal Hematopoiesis (KoCH) consortia, we show that CH is associated with severe Covid-19 outcomes (OR = 1.85, 95%=1.15–2.99, p = 0.01), in particular CH characterized by non-cancer driver mutations (OR = 2.01, 95% CI = 1.15–3.50, p = 0.01). We further explore the relationship between CH and risk of other infections in 14,211 solid tumor patients at MSK. CH is significantly associated with risk of Clostridium Difficile (HR = 2.01, 95% CI: 1.22–3.30, p = 6×10−3) and Streptococcus/Enterococcus infections (HR = 1.56, 95% CI = 1.15–2.13, p = 5×10−3). These findings suggest a relationship between CH and risk of severe infections that warrants further investigation., Clonal haematopoiesis (CH) has been associated with altered inflammatory profiles and increased risk of cardiovascular and malignant diseases. Here, the authors analyze patient data from two different cohorts and show that CH is associated with severe infections and severe Covid19.

Pathogenic germline mutations in the RAD51 paralog genes RAD51C and RAD51D, are known to confer susceptibility to ovarian and triple-negative breast cancer. Here, we investigated whether germline loss-of-function variants affecting another RAD51 paralog gene, RAD51B, are also associated with breast and ovarian cancer. Among 3422 consecutively accrued breast and ovarian cancer patients consented to tumor/germline sequencing, the observed carrier frequency of loss-of-function germline RAD51B variants was significantly higher than control cases from the gnomAD population database (0.26% vs 0.09%), with an odds ratio of 2.69 (95% CI: 1.4–5.3). Furthermore, we demonstrate that tumors harboring biallelic RAD51B alteration are deficient in homologous recombination DNA repair deficiency (HRD), as evidenced by analysis of sequencing data and in vitro functional assays. Our findings suggest that RAD51B should be considered as an addition to clinical germline testing panels for breast and ovarian cancer susceptibility.

Background: Cancer survivors are developing more subsequent tumors. We sought to characterize patients with multiple (≥2) primary cancers (MPC) to assess associations and genetic mechanisms. Methods: Patients were prospectively consented (01/2013–02/2019) to tumor-normal sequencing via a custom targeted panel (MSK-IMPACT). A subset consented to return of results of ≥76 cancer predisposition genes. International Agency for Research on Cancer (IARC) 2004 rules for defining MPC were applied. Tumor pairs were created to assess relationships between cancers. Age-adjusted, sex-specific, standardized incidence ratios (SIR) for first to second cancer event combinations were calculated using SEER rates, adjusting for confounders and time of ascertainment. Associations were made with germline and somatic variants. Results: Of 24,241 patients, 4,340 had MPC (18%); 20% were synchronous. Most (80%) had two primaries; however, 4% had ≥4 cancers. SIR analysis found lymphoma–lung, lymphoma–uterine, breast–brain, and melanoma–lung pairs in women and prostate–mesothelioma, prostate–sarcoma, melanoma–stomach, and prostate–brain pairs in men in excess of expected after accounting for synchronous tumors, known inherited cancer syndromes, and environmental exposures. Of 1,580 (36%) patients who received germline results, 324 (21%) had 361 pathogenic/likely pathogenic variants (PV), 159 (44%) in high penetrance genes. Of tumor samples analyzed, 55% exhibited loss of heterozygosity at the germline variant. In those with negative germline findings, melanoma, prostate, and breast cancers were common. Conclusions: We identified tumor pairs without known predisposing mutations that merit confirmation and will require novel strategies to elucidate genetic mechanisms of shared susceptibilities. Impact: If verified, patients with MPC with novel phenotypes may benefit from targeted cancer surveillance.

PURPOSE Tumor mutational profiling is increasingly performed in patients with advanced cancer. We determined the extent to which germline mutation profiling guides therapy selection in patients with advanced cancer. METHODS Patients with cancer undergoing tumor genomic profiling were prospectively consented for germline cancer predisposition gene analysis (2015-2019). In patients harboring germline likely pathogenic or pathogenic (LP/P) alterations, therapeutic actionability was classified using a precision oncology knowledge base. Patients with metastatic or recurrent cancer receiving germline genotype–directed therapy were determined. RESULTS Among 11,947 patients across > 50 malignancies, 17% (n = 2,037) harbored a germline LP/P variant. By oncology knowledge base classification, 9% (n = 1042) had an LP/P variant in a gene with therapeutic implications (4% level 1; 4% level 3B; < 1% level 4). BRCA1/2 variants accounted for 42% of therapeutically actionable findings, followed by CHEK2 (13%), ATM (12%), mismatch repair genes (11%), and PALB2 (5%). When limited to the 9,079 patients with metastatic or recurrent cancer, 8% (n = 710) harbored level 1 or 3B genetic findings and 3.2% (n = 289) received germline genotype–directed therapy. Germline genotype–directed therapy was received by 61% and 18% of metastatic cancer patients with level 1 and level 3B findings, respectively, and by 54% of BRCA1/2, 75% of mismatch repair, 43% of PALB2, 35% of RAD51C/D, 24% of BRIP1, and 19% of ATM carriers. Of BRCA1/2 patients receiving a poly(ADP-ribose) polymerase inhibitor, 45% (84 of 188) had tumors other than breast or ovarian cancer, wherein the drug, at time of delivery, was delivered in an investigational setting. CONCLUSION In a pan-cancer analysis, 8% of patients with advanced cancer harbored a germline variant with therapeutic actionability with 40% of these patients receiving germline genotype–directed treatment. Germline sequence analysis is additive to tumor sequence analysis for therapy selection and should be considered for all patients with advanced cancer.

Purpose: Nucleotide excision repair (NER) gene alterations constitute potential cancer therapeutic targets. We explored the prevalence of NER gene alterations across cancers and putative therapeutic strategies targeting these vulnerabilities. Experimental Design: We interrogated our institutional dataset with mutational data from more than 40,000 patients with cancer to assess the frequency of putative deleterious alterations in four key NER genes. Gene-edited isogenic pairs of wild-type and mutant ERCC2 or ERCC3 cell lines were created and used to assess response to several candidate drugs. Results: We found that putative damaging germline and somatic alterations in NER genes were present with frequencies up to 10% across multiple cancer types. Both in vitro and in vivo studies showed significantly enhanced sensitivity to the sesquiterpene irofulven in cells harboring specific clinically observed heterozygous mutations in ERCC2 or ERCC3. Sensitivity of NER mutants to irofulven was greater than to a current standard-of-care agent, cisplatin. Hypomorphic ERCC2/3-mutant cells had impaired ability to repair irofulven-induced DNA damage. Transcriptomic profiling of tumor tissues suggested codependencies between DNA repair pathways, indicating a potential benefit of combination therapies, which were confirmed by in vitro studies. Conclusions: These findings provide novel insights into a synthetic lethal relationship between clinically observed NER gene deficiencies and sensitivity to irofulven and its potential synergistic combination with other drugs. See related commentary by Jiang and Greenberg, p. 1833

Purpose: Evaluate response of mismatch repair–deficient (dMMR) rectal cancer to neoadjuvant chemotherapy. Experimental Design: dMMR rectal tumors at Memorial Sloan Kettering Cancer Center (New York, NY) were retrospectively reviewed for characteristics, treatment, and outcomes. Fifty patients with dMMR rectal cancer were identified by IHC and/or microsatellite instability analysis, with initial treatment response compared with a matched MMR-proficient (pMMR) rectal cancer cohort. Germline and somatic mutation analyses were evaluated. Patient-derived dMMR rectal tumoroids were assessed for chemotherapy sensitivity. Results: Of 21 patients receiving neoadjuvant chemotherapy (fluorouracil/oxaliplatin), six (29%) had progression of disease. In comparison, no progression was noted in 63 pMMR rectal tumors (P = 0.0001). Rectal cancer dMMR tumoroids reflected this resistance to chemotherapy. No genomic predictors of chemotherapy response were identified. Of 16 patients receiving chemoradiation, 13 (93%) experienced tumor downstaging; one patient had stable disease, comparable with 48 pMMR rectal cancers. Of 13 patients undergoing surgery, 12 (92%) had early-stage disease. Forty-two (84%) of the 50 patients tested positive for Lynch syndrome with enrichment of germline MSH2 and MSH6 mutations when compared with 193 patients with Lynch syndrome–associated colon cancer (MSH2, 57% vs 36%; MSH6, 17% vs 9%; P < 0.003). Conclusions: Over one-fourth of dMMR rectal tumors treated with neoadjuvant chemotherapy exhibited disease progression. Conversely, dMMR rectal tumors were sensitive to chemoradiation. MMR status should be performed upfront in all locally advanced rectal tumors with careful monitoring for response on neoadjuvant chemotherapy and genetic testing for Lynch syndrome in patients with dMMR rectal cancer.

1502 Background: 17% of patients (pts) with a cancer diagnosis in the U.S. have a prior malignancy. We sought to characterize pts with multiple (≥2) primary cancers (MPC) & identify potential drivers of cancer risk to guide management. Methods: Pts prospectively consented (1/2013-2/2019) to tumor-normal sequencing via custom targeted NGS panel. A subset consented to testing of >76 germline cancer predisposition genes. IARC 2004 rules for defining MPC were applied.Age adjusted gender specific standardized incidence ratios (SIR) for cancer event combinations occurring in at least 5pt were calculated using R statistical package. Results: Of 24417 pts sequenced, 4341 had MPC (18%). (Table) 3465 (80%) had 2, 4% had >4 cancers. Cancer pairs where SIR of 2nd cancer was higher than expected included: colon-colon, prostate-pancreas, bladder-prostate in men & lung-lung, breast-pancreas, thyroid-pancreas in women. 1580 (36%) pts had germline testing; 324 (21%) had 361 pathogenic/likely pathogenic (P/LP) variants (vts). Of these, 157 (48%), 66(20%), pts had high, moderate penetrance vts. The remainder had low penetrance, recessive or vts of uncertain utility. Of pts with high penetrance vt, 132 (84%) had at least one tumor type concordant with germline findings. Conclusions: 18% of pts in this cohort had MPC. There was a significant excess over expected incidence in some cancer combinations. Of pts with germline testing, 21% had a P/LP vt, with most (69%) being high or moderate penetrance. Assessment for loss of heterozygosity in tumor & germline sequencing of the full MPC cohort is ongoing. [Table: see text]

A proportion of cancer patients carry inherited pathogenic variants or somatic alterations in the DNA helicase genes ERCC2 and ERCC3, important components of the nucleotide excision repair (NER) pathway. Cancer risk associated with mutations in nucleotide excision repair is not very well understood and there are no targeted therapies available for patients with NER gene mutations. We have recently shown that functionally significant cancer-associated mutations in ERCC3 exhibit remarkable sensitivity against the fungal sesquiterpene IlludinS. Irofulven, a derivative of IlludinS, has previously been evaluated as an antitumor agent in multiple clinical trials and has shown antitumor activity in a subset of patients. Based on the observation that ERCC3 mutant cell lines are highly sensitive to IlludinS we explored the role of Irofulven as a potential therapeutic agent to treat patients with hypomorphic mutations in nucleotide excision repair genes. We used CRISPR/Cas9 to create isogenic pairs of wildtype and mutant ERCC2 or ERCC3 cell lines and assessed in vitro and in vivo response to Irofulven. In vitro studies showed significantly increased sensitivity of the ERCC3 mutant cells towards Irofulven, when compared to commonly used drugs such as PARP inhibitors or alkylating agents. ERCC2 mutant cells showed elevated in vitro sensitivity to Irofulven even surpassing response to the current standard of care agent Cisplatin. Additionally, in vitro experiments show impaired ability of ERCC2/3 mutant cells to repair Irofulven induced DNA damage. The enhanced Irofulven sensitivity of ERCC3 mutants was replicated in vivo by xenograft experiments. Transcriptome analyses of the tumor tissues gave insights into pathways deregulated in the mutant background and revealed novel biology mediating Irofulven sensitivity suggesting potential for the development of combinatorial therapies. Similar to tumors arising in the background of homologous repair defects, mutations in nucleotide excision repair genes such as ERCC2 and ERCC3 could constitute potential biomarkers of therapeutic response. The present study provides pre-clinical evidence that a subset of tumors with mutations in nucleotide excision repair pathway genes exhibits exceptional sensitivity to Irofulven. Citation Format: Sabine Topka, Zoe Steinsnyder, Kaitlyn Tkachuk, Vignesh Ravichandran, Mogens Winkel Madsen, Helena Furberg-Barnes, Gopakumar Iyer, Elisa De Stanchina, Vijai Joseph, Kenneth Offit. Targeting nucleotide excision repair defects in cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A07. doi:10.1158/1535-7163.TARG-19-LB-A07