e22604 Background: The success of PARP inhibitors in BRCA1/2 mutant tumors represents the convergence of germline and somatic molecular profiling as part of routine management of clinical cancer care. Other predictive molecular alterations with matched precision oncology drugs in the germline setting have also emerged. The expansion of germline genetic testing to guide therapy selection has increasingly shifted the burden of germline cancer risk assessment to the point-of-care oncologists who now have the additional challenge of ensuring appropriate interpretation of genetic test results. We therefore sought to expand OncoKB, MSK’s FDA-recognized precision oncology knowledge base, to incorporate annotations for germline variants by partnering with MSK’s Clinical Genetics and Diagnostic Molecular Genetics (DMG) services to provide clinicians and researchers worldwide with an automated, evidence-based tool for interpreting the pathogenicity and clinical actionability of germline variants. Methods: We reviewed data compiled by MSK’s DMG for over 2,600 unique germline variants in known cancer susceptibility genes. For each variant, we standardized information on pathogenicity, penetrance, mechanism of inheritance, and associated inherited syndromes to enable integration into OncoKB. Using the OncoKB therapeutic levels of evidence framework, we assigned clinical actionability to individual germline variants based on supporting evidence as to whether the variant is predictive of response to standard care or investigational targeted therapy. Additionally, we expanded the OncoKB API to include endpoints for germline variants, enabling the programmatic annotation of germline sequencing reports and research cohorts. Results: To date, OncoKB has incorporated information on 2631 unique germline variants across 91 genes implicated in germline cancer predisposition and detected by MSK-IMPACT. 24 genes across >10 cancer types are considered Level 1 standard care biomarkers in the germline setting. To date, more than 3400 patients in the MSK-IMPACT cohort have germline alterations that can be annotated with germline OncoKB. Conclusions: OncoKB’s new annotation of germline variants provides a novel and easily accessible tool for clinicians who utilize germline genetic testing to programmatically determine the effect of the variant based on MSK clinical expertise. Public release of this data is expected in 2025.

PURPOSE:To determine the genetic predisposition underlying pancreatic acinar cell carcinoma (PACC) and characterize its genomic features. METHODS:Both somatic and germline analyses were performed using an Food and Drug Administration-authorized matched tumor/normal sequencing assay on a clinical cohort of 28,780 patients with cancer, 49 of whom were diagnosed with PACC. For a subset of PACCs, whole-genome sequencing (WGS; n = 12) and RNA sequencing (n = 6) were performed. RESULTS:Eighteen of 49 (36.7%) PACCs harbored germline pathogenic variants in homologous recombination (HR) and DNA damage response (DDR) genes, including BRCA1 (n = 1), BRCA2 (n = 12), PALB2 (n = 2), ATM (n = 2), and CHEK2 (n = 1). Thirty-one PACCs displayed pure, and 18 PACCs harbored mixed acinar cell histology. Fifteen of 31 (48%) pure PACCs harbored a germline pathogenic variant affecting HR-/DDR-related genes. BRCA2 germline pathogenic variants (11 of 31, 35%) were significantly more frequent in pure PACCs than in pancreatic adenocarcinoma (86 of 2,739, 3.1%; P < .001), high-grade serous ovarian carcinoma (67 of 1,318, 5.1%; P < .001), prostate cancer (116 of 3,401, 3.4%; P < .001), and breast cancer (79 of 3,196, 2.5%; P < .001). Genomic features of HR deficiency (HRD) were detected in 7 of 12 PACCs undergoing WGS, including 100% (n = 6) of PACCs with germline HR-related pathogenic mutations and 1 of 6 PACCs lacking known pathogenic alterations in HR-related genes. Exploratory analyses revealed that in PACCs, the repertoire of somatic driver genetic alterations and the load of neoantigens with high binding affinity varied according to the presence of germline pathogenic alterations affecting HR-/DDR-related genes and/or HRD. CONCLUSION:In a large pan-cancer cohort, PACC was identified as the cancer type with the highest prevalence of both BRCA2 germline pathogenic variants and genomic features of HRD, suggesting that PACC should be considered as part of the spectrum of BRCA-related malignancies.

Abstract Background Germline variants in the DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) cause Lynch syndrome, an autosomal dominant hereditary cancer susceptibility syndrome. The risk for endometrial cancer is significantly higher in women with MSH6 pathogenic/likely pathogenic (P/LP) variants compared with that for MLH1 or MSH2 variants. Methods The proband was tested via a clinical testing, Memorial Sloan Kettering‐Integrated Mutation Profiling of Actionable Cancer Targets (MSK‐IMPACT). RT‐PCR was performed using patient's blood DNA and cDNA was analyzed by DNA sequencing and a cloning approach. Results We report a 56‐year‐old female with endometrial cancer who carries a germline variant, MSH6 c.4001G > C, located at the last nucleotide of exon 9. While the pathogenicity of this variant was previously unknown, functional studies demonstrated that this variant completely abolished normal splicing and caused exon 9 skipping, which is expected to lead to a prematurely truncated or abnormal protein. Conclusion Our results indicate that this variant likely contributes to cancer predisposition through disruption of normal splicing, and is classified as likely pathogenic.

Traditional genetic testing for patients with gastrointestinal stromal tumors (GISTs) focus on those with syndromic features. To assess whether expanded genetic testing of GIST patients could identify hereditary cancer predisposition, we analyzed matched tumor-germline sequencing results from 103 patients with GISTs over a 6-year period. Germline pathogenic/likely pathogenic (P/LP) variants in GIST-associated genes (SDHA, SDHB, SDHC, NF1, KIT) were identified in 69% of patients with KIT/PDGFRA-wildtype GISTs, 63% of whom did not have any personal or family history of syndromic features. To evaluate the frequency of somatic versus germline variants identified in tumor-only sequencing of GISTs, we analyzed 499 de-identified tumor-normal pairs. P/LP variants in certain genes (e.g., BRCA1/2, SDHB) identified in tumor-only sequencing of GISTs were almost exclusively germline in origin. Our results provide guidance for genetic testing of GIST patients and indicate that germline testing should be offered to all patients with KIT/PDGFRA-wildtype GISTs regardless of their history of syndromic features.

TP53 is one of the most ubiquitously altered genes in human cancer. The biological impact of rare variants, particularly those located within noncoding regions, remains poorly understood. From interrogation of clinical massively parallel sequencing data from over 55,000 tumors, which included 23,330 tumors with known TP53 mutations, TP53 intron 4 nucleotide substitutions at position c.375+5G were identified in 45 tumors (0.2% of TP53-mutated cancers), comprising cancers of different organ sites. Loss-of-heterozygosity or a second-hit somatic TP53 mutation was observed in 34 of 40 (85%) informative cases. RT-PCR analysis showed the c.375+5G>T variant to be associated with aberrantly spliced TP53 mRNA transcripts with concomitant loss of the normal transcript. Immunohistochemical staining for p53 was performed on a representative subset of tumors with TP53 c.375+5G variants (n = 14), all of which showed loss of protein expression (100%; n = 13 complete loss, n = 1 subclonal loss). Our data are consistent with classification of TP53 c.375+5G variants as deleterious intronic mutations that interfere with proper mRNA splicing, ultimately resulting in loss of expression of functional p53 protein. The clinical scenario of a tumor with loss of p53 immunohistochemical staining, yet lacking a detectable TP53 exonic mutation, should therefore prompt consideration of splice-altering intronic variants.

Background Genetic testing (GT) for hereditary cancer predisposition is traditionally performed on selected genes based on established guidelines for each cancer type. Recently, expanded GT (eGT) using large hereditary cancer gene panels uncovered hereditary predisposition in a greater proportion of patients than previously anticipated. We sought to define the diagnostic yield of eGT and its clinical relevance in a broad cancer patient population over a 5-year period. Methods A total of 17,523 cancer patients with a broad range of solid tumors, who received eGT at Memorial Sloan Kettering Cancer Center between July 2015 to April 2020, were included in the study. The patients were unselected for current GT criteria such as cancer type, age of onset, and/or family history of disease. The diagnostic yield of eGT was determined for each cancer type. For 9187 patients with five common cancer types frequently interrogated for hereditary predisposition (breast, colorectal, ovarian, pancreatic, and prostate cancer), the rate of pathogenic/likely pathogenic (P/LP) variants in genes that have been associated with each cancer type was analyzed. The clinical implications of additional findings in genes not known to be associated with a patients’ cancer type were investigated. Results 16.7% of patients in a broad cancer cohort had P/LP variants in hereditary cancer predisposition genes identified by eGT. The diagnostic yield of eGT in patients with breast, colorectal, ovarian, pancreatic, and prostate cancer was 17.5%, 15.3%, 24.2%, 19.4%, and 15.9%, respectively. Additionally, 8% of the patients with five common cancers had P/LP variants in genes not known to be associated with the patient’s current cancer type, with 0.8% of them having such a variant that confers a high risk for another cancer type. Analysis of clinical and family histories revealed that 74% of patients with variants in genes not associated with their current cancer type but which conferred a high risk for another cancer did not meet the current GT criteria for the genes harboring these variants. One or more variants of uncertain significance were identified in 57% of the patients. Conclusions Compared to targeted testing approaches, eGT can increase the yield of detection of hereditary cancer predisposition in patients with a range of tumors, allowing opportunities for enhanced surveillance and intervention. The benefits of performing eGT should be weighed against the added number of VUSs identified with this approach.

BACKGROUND AND OBJECTIVE:Pancreatic ductal adenocarcinoma (PDAC) is characterized by the occurrence of pathogenic variants in BRCA1/2 in 5-6% of patients. Biallelic loss of BRCA1/2 enriches for response to platinum agents and poly (ADP-ribose) polymerase 1 inhibitors. There is a dearth of evidence on the mechanism of inactivation of the wild-type BRCA1 allele in PDAC tumors with a germline BRCA1 (gBRCA1) pathogenic or likely pathogenic variant (P/LPV). Herein, we examine promotor hypermethylation as a "second hit" mechanism in patients with gBRCA1-PDAC.METHODS:We evaluated patients with PDAC who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) somatic and germline testing from an institutional database. DNA isolated from tumor tissue and matched normal peripheral blood were sequenced by MSK-IMPACT. In patients with gBRCA1-PDAC, we examined the somatic BRCA1 mutation status and promotor methylation status of the tumor BRCA1 allele via a methylation array analysis. In patients with sufficient remaining DNA, a second methylation analysis by pyrosequencing was performed.RESULTS:Of 1012 patients with PDAC, 19 (1.9%) were identified to harbor a gBRCA1 P/LPV. Fifteen patients underwent a methylation array and the mean percentage of BRCA1 promotor methylation was 3.62%. In seven patients in whom sufficient DNA was available, subsequent pyrosequencing confirmed an unmethylated BRCA1 promotor. Loss of heterozygosity was detected in 12 of 19 (63%, 95% confidence interval 38-84) patients, demonstrating loss of heterozygosity is the major molecular mechanism of BRCA1 inactivation in PDAC. Two (10.5%) cases had a somatic BRCA1 mutation.CONCLUSIONS:In patients with gBRCA1-P/LPV-PDAC, loss of heterozygosity is the main inactivating mechanism of the wild-type BRCA1 allele in the tumor, and methylation of the BRCA1 promoter is a distinctly uncommon occurrence.

Public access to genetic information is increasing, and community dermatologists may progressively encounter patients interested in genetic testing for melanoma risk. Clarifying potential utility will help plan for this inevitability. We determined interest and uptake of genetic risk feedback based on melanocortin receptor gene (MC1R) variants, immediate (two weeks) responses to risk feedback, and test utility at three months in patients (age ≥ 18, with a history of nonmelanoma skin cancer). Participants (N = 50) completed a baseline survey and were invited to consider MC1R testing via the study website. Testing interest and uptake were assessed through registration of test decision, request of a saliva test kit, and kit return (all yes/no). Immediate responses to risk feedback included feedback-relevant thoughts, emotions, communication, and information seeking after result receipt; test utility outcomes included family and physician communication and information seeking. Results indicated good retention at both time points (76%; 74%). Half (48%) logged onto the study website, and of these, most (92%) chose testing and (95%) returned a saliva sample. After two weeks, most (94%) had read all the risk feedback information and distress was low (M = 8.81, 7–28, SD = 2.23). Many (69%) had talked with their family about the results. By three months, most had spoken with family (92%) and physicians (80%) about skin cancer risk. Physician communication was higher (70%) in those tested versus those not tested (40%, p=0.02). The substantial interest and promising outcomes associated with MC1R genetic testing in dermatology patients inform intervention strategies to enhance benefits and minimize risks of skin cancer genetic testing.

BACKGROUND Li-Fraumeni syndrome (LFS) genetic testing is performed using blood specimens from patients selected based on phenotype-dependent guidelines. This approach is problematic for understanding LFS clinical spectrum, because patients with non-classical presentations are missed, clonal hematopoiesis (CH)-related somatic blood mutations cannot be distinguished from germline variants, and unrelated tumors cannot be differentiated from those driven by germline TP53 defects. METHODS To provide insights into LFS-related cancer spectrum, we analyzed paired tumor-blood DNA sequencing results in 17,922 cancer patients, and distinguished CH-related, mosaic, and germline TP53 variants. Loss-of-heterozygosity (LOH) and TP53 mutational status were assessed in tumors, followed by immunohistochemistry for p53 expression on a subset to identify those lacking biallelic TP53 inactivation. RESULTS Pathogenic/likely pathogenic TP53 variants were identified in 50 patients, 12 (24.0%) of which were CH-related and four (8.0%) were mosaic. Twelve (35.3%) of 34 patients with germline TP53 variants did not meet LFS testing criteria. LOH of germline TP53 variant was observed in 96.0% (95% CI = 79.7-99.9%) of core LFS-spectrum type tumors versus 45.5% (95% CI = 16.8-76.6%) of other tumors, and 91.3% (95% CI = 72.0-98.9%) of tumors from patients who met LFS testing criteria versus 61.5% (95% CI = 31.6-86.1%) of tumors from patients who did not. Tumors retaining wild-type TP53 allele exhibited wild-type p53 expression. CONCLUSIONS Our results indicate that some TP53 variants identified in blood-only sequencing are not germline and a substantial proportion of LFS patients are missed by current testing guidelines. Additionally, a subset of tumors from LFS patients do not have biallelic TP53 inactivation and may represent cancers unrelated to their germline TP53 defect.

The spectrum of germline predisposition in pediatric cancer continues to be realized. Here we report 751 patients with solid tumors who underwent prospective matched tumor–normal DNA sequencing with downstream clinical use (ClinicalTrials.gov NCT01775072 ). Germline pathogenic and likely pathogenic (P/LP) variants were reported. One or more P/LP variants were found in 18% (138/751) of individuals when including variants in low-, moderate- and high-penetrance dominant or recessive genes or in 13% (99/751) of individuals in moderate- and high-penetrance dominant genes. Thirty-four percent of high- or moderate-penetrance variants were unexpected based on the patient’s diagnosis and previous history. Seventy-six percent of patients with positive results completed a clinical genetics visit, and 21% had at least one relative undergo cascade testing as a result of this testing. Clinical actionability additionally included screening, risk reduction in relatives, reproductive use and targeted therapies. Germline testing should be considered for all children with cancer. Walsh and colleagues use prospective sequencing in a large cohort of pediatric patients with solid tumors to detect mutations in cancer predisposition genes and guide downstream clinical care.

Germline mutations in the DNA mismatch repair (MMR) genes cause Lynch syndrome (LS). In this study, we identified and characterized a novel SINE-VNTR-Alu (SVA) insertion in exon 12 of MSH2 in an individual with early-onset colorectal cancer and a very strong LS family history. RT-PCR analysis indicated a larger aberrant MSH2 transcript in one of the family members. MSK-IMPACT next-generation sequencing and long-range PCR analyses revealed an insertion in MSH2 exon 12 at the c.1972 position in an antisense orientation. The insertion was further characterized as an SVA element approximately 3 kb in length, belonging to the SVA_F1 family of retrotransposons. This variant also segregated with LS related cancers in four affected family members in this family. Based on this evidence, this MSH2 SVA insertion is considered pathogenic.

Purpose Mutations in RAD51D are associated with a predisposition to primary ovarian, fallopian tube, and peritoneal carcinoma. Our study aims to characterize a RAD51D missense variant in a hereditary ovarian cancer family. Methods The effects of the RAD51D c.82G>A (p.Val28Met) variant on mRNA splicing were evaluated and characterized using RT-PCR, cloning and DNA sequencing. Results This variant completely disrupts normal splicing and results in the loss of 3 ' end of 5 ' UTR and the entire exon 1 (c.-86_c.82), which presumably leads to loss of the RAD51D protein. The RAD51D c.82G>A (p.Val28Met) variant is clinically significant and classified as likely pathogenic. Conclusions Our results indicate that the RAD51D c.82G>A (p.Val28Met) variant contributes to cancer predisposition through disruption of normal mRNA splicing. The identification of this variant in an individual affected with high-grade serous fallopian tube cancer suggests that the RAD51D variant may contribute to predisposition to the ovarian cancer in this family.

BACKGROUND:Genetic testing for Li-Fraumeni syndrome (LFS) is performed by using blood specimens from patients selected based on phenotype-dependent guidelines. This approach is problematic for understanding the LFS clinical spectrum because patients with nonclassical presentations are missed, clonal hematopoiesis-related somatic blood alterations cannot be distinguished from germline variants, and unrelated tumors cannot be differentiated from those driven by germline TP53 defects. METHODS:To provide insights into the LFS-related cancer spectrum, we analyzed paired tumor-blood DNA sequencing results in 17 922 patients with cancer and distinguished clonal hematopoiesis-related, mosaic, and germline TP53 variants. Loss of heterozygosity and TP53 mutational status were assessed in tumors, followed by immunohistochemistry for p53 expression on a subset to identify those lacking biallelic TP53 inactivation. RESULTS:Pathogenic/likely pathogenic TP53 variants were identified in 50 patients, 12 (24.0%) of which were clonal hematopoiesis related and 4 (8.0%) of which were mosaic. Twelve (35.3%) of 34 patients with germline TP53 variants did not meet LFS testing criteria. Loss of heterozygosity of germline TP53 variant was observed in 96.0% (95% confidence interval [CI] = 79.7% to 99.9%) of core LFS spectrum-type tumors vs 45.5% (95% CI = 16.8% to 76.6%) of other tumors and 91.3% (95% CI = 72.0% to 98.9%) of tumors from patients who met LFS testing criteria vs 61.5% (95% CI = 31.6% to 86.1%) of tumors from patients who did not. Tumors retaining the wild-type TP53 allele exhibited wild-type p53 expression. CONCLUSIONS:Our results indicate that some TP53 variants identified in blood-only sequencing are not germline and a substantial proportion of patients with LFS are missed based on current testing guidelines. Additionally, a subset of tumors from patients with LFS do not have biallelic TP53 inactivation and may represent cancers unrelated to their germline TP53 defect.

Germline mutations in the DNA mismatch repair (MMR) genes cause Lynch syndrome. Classification and interpretation of intronic variants, especially those outside the consensus ± 1 ~ 2 splice sites are challenging as it is uncertain whether such variants would affect splicing accuracy and efficiency. The assessment of the pathogenicity of splice site variants in MLH1 is further complicated by the various isoforms due to alternative splicing. In this report, we describe a 42-year-old female with Lynch syndrome who carries a germline variant, MLH1 c.678-3T>A, in the splice acceptor site of intron 8. Functional studies and semiquantitative analysis demonstrated that this variant causes a significant increase in the transcripts with exon 9 or exon 9 and 10 deletions, which presumably leads to premature protein truncation or abnormal protein. In addition, we also observed MSI-H and loss of MLH1 by IHC in patient's tumor tissue. This variant also segregated with Lynch Syndrome related cancers in three affected family members. Based on these evidence, the MLH1 c.678-3T>A variant is considered pathogenic.

Fumarate hydratase (FH) mutations underpin the autosomal recessive syndrome. FH deficiency and the autosomal dominant syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). The FH c.1431_1433dupAAA (p.Lys477dup) genomic alteration has been conclusively shown to contribute to FH deficiency when occurring with another FH germline alteration. However, a sufficiently large dataset has been lacking to conclusively determine its clinical significance to cancer predisposition in the heterozygous state. We reviewed a series of 7,571 patients with cancer who received germline results through MSK-IMPACT testing at the Memorial Sloan Kettering Cancer Center. The FH c.1431_1433dupAAA (p.Lys477dup) variant was detected in 24 individuals, none of whom was affected with renal cancer. Eleven of the 372 patients with renal cancer were identified to carried pathogenic FH variants associated with HLRCC. None of these 372 patients with renal cancer carried the FH c.1431_1433dupAAA variant. Our data indicate the FH c.1431_1433dupAAA is not associated with cancer including renal cell carcinoma.

The Blm10/PA200 family of proteasome activators modulates the peptidase activity of the core particle (20S CP). They participate in opening the 20S CP gate, thus facilitating the degradation of unstructured proteins such as tau and Dnm1 in a ubiquitin- and ATP-independent manner. Furthermore, PA200 also participates in the degradation of acetylated histones. In our study, we use a combination of yeast and human cell systems to investigate the role of Blm10/PA200 in the degradation of N-terminal Huntingtin fragments (N-Htt). We demonstrate that the human PA200 binds to N-Htt. The loss of Blm10 in yeast or PA200 in human cells results in increased mutant N-Htt aggregate formation and elevated cellular toxicity. Furthermore, Blm10 in vitro accelerates the proteasomal degradation of soluble N-Htt. Collectively, our data suggest N-Htt as a new substrate for Blm10/PA200-proteasomes and point to new approaches in Huntington's disease (HD) research.

PURPOSE:Mutations in PALB2 have been associated with a predisposition to breast and pancreatic cancers. This study aims to characterize a novel PALB2 synonymous variant c.18G>T (p.Gly6=) identified in a family with pancreatic and breast cancers.METHODS:The PALB2 c.18G>T (p.Gly6=) variant in this family was identified using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT™). RT-PCR and subsequent cloning were performed to investigate whether this variant affects normal splicing.RESULTS:This variant completely disrupts normal splicing and leads to several abnormal transcripts, which presumably leads to premature protein truncation. The major abnormal transcript resulted in a deletion of 32 base pairs in exon 1 and frameshift.CONCLUSIONS:Our results indicate that the PALB2 c.18G>T (p.Gly6=) variant is likely pathogenic. This study provided important laboratory evidence for classification of this variant and guided improved patient management.

Mutations in succinate dehydrogenase complex genes predispose to familial paraganglioma-pheochromocytoma syndrome (FPG) and gastrointestinal stromal tumors (GIST). Here we describe cancer patients undergoing agnostic germline testing at Memorial Sloan Kettering Cancer Center and found to harbor germline SDHA mutations. Using targeted sequencing covering the cancer census genes, we identified 10 patients with SDHA germline mutations. Cancer diagnoses for these patients carrying SDHA germline mutations included neuroblastoma (n = 1), breast (n = 1), colon (n = 1), renal (n = 1), melanoma and uterine (n = 1), prostate (n = 1), endometrial (n = 1), bladder (n = 1), and gastrointestinal stromal tumor (GIST) (n = 2). Immunohistochemical staining and assessment of patient tumors for second hits and loss of heterozygosity in SDHA confirmed GIST as an SDHA-associated tumor and suggests SDHA germline mutations may be a driver in neuroblastoma tumorigenesis.