Germline and Somatic Fumarate Hydratase Testing in Atypical Uterine Leiomyomata.

PREVENTION RELEVANCE: Women diagnosed with fumarate hydratase (FH)-deficient uterine leiomyomata are at increased risk of renal cancer. This work suggests a more standardized pathology-genetic counseling referral pathway for these patients, and that research on underlying causes of FH-deficient uterine leiomyomata in the absence of germline FH pathogenic/likely pathogenic variants is needed.

Advancing Precision Oncology in Hereditary Paraganglioma-Pheochromocytoma Syndromes: Integrated Interpretation and Data Sharing of the Germline and Tumor Genomes.

Standard methods of variant assessment in hereditary cancer susceptibility genes are limited by the lack of availability of key supporting evidence. In cancer, information derived from tumors can serve as a useful source in delineating the tumor behavior and the role of germline variants in tumor progression. We have previously demonstrated the value of integrating tumor and germline findings to comprehensively assess germline variants in hereditary cancer syndromes. Building on this work, herein, we present the development and application of the INT2GRATE|HPPGL platform. INT2GRATE (INTegrated INTerpretation of GeRmline And Tumor gEnomes) is a multi-institution oncology consortium that aims to advance the integrated application of constitutional and tumor data and share the integrated variant information in publicly accessible repositories. The INT2GRATE|HPPGL platform enables automated parsing and integrated assessment of germline, tumor, and genetic findings in hereditary paraganglioma-pheochromocytoma syndromes (HPPGLs). Using INT2GRATE|HPPGL, we analyzed 8600 variants in succinate dehydrogenase (SDHx) genes and their associated clinical evidence. The integrated evidence includes germline variants in SDHx genes; clinical genetics evidence: personal and family history of HPPGL-related tumors; tumor-derived evidence: somatic inactivation of SDHx alleles, KIT and PDGFRA status in gastrointestinal stromal tumors (GISTs), multifocal or extra-adrenal tumors, and metastasis status; and immunohistochemistry staining status for SDHA and SDHB genes. After processing, 8600 variants were submitted programmatically from the INT2GRATE|HPPGL platform to ClinVar via a custom-made INT2GRATE|HPPGL variant submission schema and an application programming interface (API). This novel integrated variant assessment and data sharing in hereditary cancers aims to improve the clinical assessment of genomic variants and advance precision oncology.

Genomic disparity impacts variant classification of cancer susceptibility genes in Turkish breast cancer patients.

OBJECTIVE: Turkish genome is underrepresented in large genomic databases. This study aims to evaluate the effect of allele frequency in the Turkish population in determining the clinical utility of germline findings in breast cancer, including invasive lobular carcinoma (ILC), mixed invasive ductal and lobular carcinoma (IDC-L), and ductal carcinoma (DC). METHODS: Two clinic-based cohorts from the Umraniye Research and Training Hospital (URTH) were used in this study: a cohort consisting of 132 women with breast cancer and a non-cancer cohort consisting of 492 participants. The evaluation of the germline landscape was performed by analysis of 27 cancer genes. The frequency and type of variants in the breast cancer cohort were compared to those in the non-cancer cohort to investigate the effect of population genetics. The variant allele frequencies in Turkish Variome and gnomAD were statistically evaluated. RESULTS: The genetic analysis identified 121 variants in the breast cancer cohort (actionable = 32, VUS = 89) and 223 variants in the non-cancer cohort (actionable = 25, VUS = 188). The occurrence of 21 variants in both suggested a possible genetic population effect. Evaluation of allele frequency of 121 variants from the breast cancer cohort showed 22% had a significantly higher value in Turkish Variome compared to gnomAD (p < 0.0001, 95% CI) with a mean difference of 60 times (ranging from 1.37-354.4). After adjusting for variant allele frequency using the ancestry-appropriate database, 6.7% (5/75) of VUS was reclassified to likely benign. CONCLUSION: To our knowledge, this is the first study of population genetic effects in breast cancer subtypes in Turkish women. Our findings underscore the need for a large genomic database representing Turkish population-specific variants. It further highlights the significance of the ancestry-appropriate population database for accurate variant assessment in clinical settings.

Multiple TP53 p.R337H haplotypes and implications for tumor susceptibility.

The germline TP53 p.R337H mutation is reported as the most common germline TP53 variant. It exists at a remarkably high frequency in the population of southeast Brazil as founder mutation in two distinct haplotypes with the most frequent co-segregating with the p.E134∗ variant of the XAF1 tumor suppressor and an increased cancer risk. Founder mutations demonstrate linkage disequilibrium with neighboring genetic polymorphic markers that can be used to identify the founder variant in different geographic regions and diverse populations. We report here a shared haplotype among Brazilian, Portuguese, and Spanish families and the existence of three additional distinct TP53 p.R337H alleles. Mitochondrial DNA sequencing and Y-STR profiling of Brazilian carriers of the founder TP53 p.R337H allele reveal an excess of Native American haplogroups in maternal lineages and exclusively European haplogroups in paternal lineages, consistent with communities established through male European settlers with extensive intermarriage with Indigenous women. The identification of founder and independent TP53 p.R337H alleles underlines the importance for considering the haplotype as a functional unit and the additive effects of constitutive polymorphisms and associated variants in modifier genes that can influence the cancer phenotype.

Racial differences in knowledge, attitudes, and sources of information about germline cancer genetic testing in the U.S.A.: An analysis of the health information National Trends Survey System.

PURPOSE: To understand racial disparities in germline cancer genetic testing and the role of prior knowledge, attitudes, and sources of information. METHODS: A cross-sectional analysis of the Health Information National Trends Survey 5 (HINTS 5) was conducted between February 24th and June 15th, 2020. The study aimed to investigate knowledge and receipt of genetic testing, attitudes toward the importance of genetic testing in preventing, detecting, and treating cancer, and information sources of genetic testing in the United States of America. RESULTS: Non-Hispanic Black (NHB) and Hispanic race/ethnicity were associated with lower odds of being informed about genetic testing, whereas those of NHB race were more likely to endorse the importance of genetic testing in cancer prevention and treatment. Regarding sources of information about genetic testing: Non-Hispanic Asians were less likely to be informed about genetic testing from television (Mean Predicted Probability (MPP) 0.38 95%CI; 0.21-0.55, (Adjusted Risk Difference) ARD vs. Non-Hispanic White (NHW); -0.228, p = 0.01), NHB were less likely to report being informed about genetic testing from social media (MPP 0.27 95%CI; 0.20-0.34, ARD vs. NHW; -0.139, p < 0.01). CONCLUSIONS: NHB and Hispanic groups face unequal access to information about genetic testing. There are significant race-based differences in information sources. These differences could be used to promote equitable access to cancer genetic testing.

Pretest Video Education Versus Genetic Counseling for Patients With Prostate Cancer: ProGen, A Multisite Randomized Controlled Trial.

PURPOSE: Germline genetic testing (GT) is recommended for men with prostate cancer (PC), but testing through traditional models is limited. The ProGen study examined a novel model aimed at providing access to GT while promoting education and informed consent. METHODS: Men with potentially lethal PC (metastatic, localized with a Gleason score of ≥8, persistent prostate-specific antigen after local therapy), diagnosis age ≤55 years, previous malignancy, and family history suggestive of a pathogenic variant (PV) and/or at oncologist's discretion were randomly assigned 3:1 to video education (VE) or in-person genetic counseling (GC). Participants had 67 genes analyzed (Ambry), with results disclosed via telephone by a genetic counselor. Outcomes included GT consent, GT completion, PV prevalence, and survey measures of satisfaction, psychological impact, genetics knowledge, and family communication. Two-sided Fisher's exact tests were used for between-arm comparisons. RESULTS: Over a 2-year period, 662 participants at three sites were randomly assigned and pretest VE (n = 498) or GC (n = 164) was completed by 604 participants (VE, 93.1%; GC, 88.8%), of whom 596 participants (VE, 98.9%; GC, 97.9%) consented to GT and 591 participants completed GT (VE, 99.3%; GC, 98.6%). These differences were not statistically significant although subtle differences in satisfaction and psychological impact were. Notably, 84 PVs were identified in 78 participants (13.2%), with BRCA1/2 PV comprising 32% of participants with a positive result (BRCA2 n = 21, BRCA1 n = 4). CONCLUSION: Both VE and traditional GC yielded high GT uptake without significant differences in outcome measures of completion, GT uptake, genetics knowledge, and family communication. The increased demand for GT with limited genetics resources supports consideration of pretest VE for patients with PC.

Pathogenic variants among females with breast cancer and a non-breast cancer reveal opportunities for cancer interception.

PURPOSE: Herein, we report the frequency and distribution of germline pathogenic variants (PVs) among females with breast cancer (BC) and at least one other non-BC who underwent multi-gene panel testing (MGPT). Among females with PVs diagnosed first with BC or ovarian cancer (OC), we sought to enumerate the frequency of subsequent PV-associated cancers. METHODS: Females with BC and cancer of ≥ 1 other site (multiple primary cancers, MPC) who underwent MGPT through Ambry Genetics from March 2012 to December 2016 were included if they had testing of at least 21 genes of interest (ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, STK11, and TP53). Phenotypic data were abstracted from test requisition forms and clinical notes. RESULTS: Of 6,617 evaluable patients, most were White (70.8%) and median age at first cancer, second cancer, and MGPT was 49 (interquartile range [IQR]: 18), 59 (IQR: 16), and 63 (IQR: 16) years, respectively. PVs were found among 14.1% (932/6617) of the overall cohort and in 16.4% (440/2687) of females who were diagnosed first with BC. Among those, 55.2% (243/440) had an actionable PV associated with a subsequent cancer diagnosis including 150 OCs. Of the 2443 females with breast and ovarian cancer, few (n = 97, 9.5%) were diagnosed first with OC, limiting our analysis. CONCLUSIONS: Females with MPC, including BC, have a high frequency of germline PVs (14.1%). These data delineate the opportunities for intercepting subsequent cancers associated with genetic risk among females diagnosed first with BC.

Racial Differences in Germline Genetic Testing for Prostate Cancer: A Systematic Review.

PURPOSE: Testing for pathogenic variants can aid in oncologic risk stratification and identification of targeted therapies. Despite known disparities in access to prostate cancer (PCa) care, little has been written about access to germline genetic testing (GGT) for Black men and other historically marginalized populations. This systematic review sought to delineate racial/ethnic disparities in GGT for PCa. METHODS: This systematic review identified articles published from January 1996 through May 2021 in PubMed, Web of Science, and Embase. We included studies that reported rates of GGT in men with PCa in the United States by race/ethnicity as reflective of routine clinical care or research. A narrative synthesis was performed. RESULTS: Of 4,309 unique records, 91 studies examining 50 unique study populations met inclusion criteria. Of these, four populations included men who received GGT through routine clinical care, accounting for 4,415 men (72.6% White and 7.2% Black). The other 46 populations included men who received GGT as part of a research study, accounting for 30,824 men (64.3% White and 21.6% Black). Of these 46 research populations, 19 used targeted methods to increase recruitment from a specific demographic. CONCLUSION: Most studies that report GGT rates by race/ethnicity are in research settings. Many of these studies used targeted recruitment methods and subsequently have a greater proportion of Black men than clinical and US population-based studies. Other historically marginalized populations are not well represented. There remains a knowledge gap regarding the extent of racial disparities in the use of GGT, particularly in the clinical setting.

Development and evaluation of INT2GRATE: a platform for comprehensive assessment of the role of germline variants informed by tumor signature profile in Lynch syndrome.

The presence of variants of uncertain significance (VUS) in DNA mismatch repair (MMR) genes leads to uncertainty in the clinical management of patients being evaluated for Lynch syndrome (LS). Currently, there is no platform to systematically use tumor-derived evidence alongside germline data for the assessment of VUS in relation to LS. We developed INT2GRATE (INTegrated INTerpretation of GeRmline And Tumor gEnomes) to leverage information from the tumor genome to inform the potential role of constitutional VUS in MMR genes. INT2GRATE platform has two components: a comprehensive evidence-based decision tree that integrates well-established clinico-genomic data from both the tumor and constitutional genomes to help inform the potential relevance of germline VUS in LS; and a web-based user interface (UI). With the INT2GRATE decision tree operating in the backend, INT2GRATE UI enables the front-end collection of comprehensive clinical genetics and tumor-derived evidence for each VUS to facilitate INT2GRATE assessment and data sharing in the publicly accessible ClinVar database. The performance of the INT2GRATE decision tree was assessed by qualitative retrospective analysis of genomic data from 5057 cancer patients with MMR alterations which included 52 positive control cases. Of 52 positive control cases with LS and pathogenic MMR alterations, 23 had all the testing parameters for the evaluation by INT2GRATE. All these variants were correctly categorized as INT2GRATE POSITIVE. The stringent INT2GRATE decision tree flagged 29 of positive cases by identifying the absence or unusual presentation of specific evidence, highlighting the conservative INT2GRATE logic in favor of a higher degree of confidence in the results. The remaining 99% of cases were correctly categorized as INCONCLUSIVE due to the absence of LS criteria and ≥1 tumor parameters. INT2GRATE is an effective platform for clinical and genetics professionals to collect and assess clinical genetics and complimentary tumor-derived information for each germline VUS in suspected LS patients. Furthermore, INT2GRATE enables the collation of integrated tumor-derived evidence relevant to germline VUS in LS, and sharing them with a large community, a practice that is needed in precision oncology.

Concurrent Pathogenic Variants of BRCA1, MUTYH and CHEK2 in a Hereditary Cancer Family.

Concurrent pathogenic variants (PVs) in cancer predisposition genes have been reported in 0.1-2% of hereditary cancer (HC) patients. Determining concurrent PVs is crucial for the diagnosis, treatment, and risk assessment of unaffected family members. Next generation sequencing based diagnostic tests, which are widely used in HCs, enable the evaluation of multiple genes in parallel. We have screened the family members of a patient with bilateral breast cancer who was found to have concurrent PVs in BRCA1 (NM_007294.3;c.5102_5103del, p.Leu1701Glnfs*14) and MUTYH (NM_001128425.1;c.884C>T, p.Pro295Leu). Further analysis revealed concurrent PVs in CHEK2 (NM_007194.4;c.1427C>T, p.Thr476Met) and MUTYH (NM_001128425.1;c.884C>T, p.Pro295Leu) in the maternal uncle of the index case. Eight additional family members were found to have PVs in BRCA1 and MUTYH among 26 tested relatives. The sister and the brother of the index case who were diagnosed with breast and colon cancers, respectively, presented with the same genotype as the index case. Each family member was evaluated individually for clinical care and surveillance. This is the first report describing a family with BRCA1, MUTYH and CHEK2 concurrent PVs. Our findings provide valuable information for the assessment and management considerations for families with concurrent PVs.

Differences in Cancer Phenotypes Among Frequent CHEK2 Variants and Implications for Clinical Care-Checking CHEK2.

Importance: Germline CHEK2 pathogenic variants (PVs) are frequently detected by multigene cancer panel testing (MGPT), but our understanding of PVs beyond c.1100del has been limited. Objective: To compare cancer phenotypes of frequent CHEK2 PVs individually and collectively by variant type. Design, Setting, and Participants: This retrospective cohort study was carried out in a single diagnostic testing laboratory from 2012 to 2019. Overall, 3783 participants with CHEK2 PVs identified via MGPT were included. Medical histories of cancer in participants with frequent PVs, negative MGPT (wild type), loss-of-function (LOF), and missense were compared. Main Outcomes and Measures: Participants were stratified by CHEK2 PV type. Descriptive statistics were summarized including median (IQR) for continuous variables and proportions for categorical characteristics. Differences in age and proportions were assessed with Wilcoxon rank sum and Fisher exact tests, respectively. Frequencies, odds ratios (ORs), 95% confidence intervals were calculated, and P values were corrected for multiple comparisons where appropriate. Results: Of the 3783 participants with CHEK2 PVs, 3473 (92%) were female and most reported White race. Breast cancer was less frequent in participants with p.I157T (OR, 0.66; 95% CI, 0.56-0.78; P<.001), p.S428F (OR, 0.59; 95% CI. 0.46-0.76; P<.001), and p.T476M (OR, 0.74; 95% CI, 0.56-0.98; P = .04) PVs compared with other PVs and an association with nonbreast cancers was not found. Following the exclusion of p.I157T, p.S428F, and p.T476M, participants with monoallelic CHEK2 PV had a younger age at first cancer diagnosis (P < .001) and were more likely to have breast (OR, 1.83; 95% CI, 1.66-2.02; P < .001), thyroid (OR, 1.63; 95% CI, 1.26-2.08; P < .001), and kidney cancer (OR, 2.57; 95% CI, 1.75-3.68; P < .001) than the wild-type cohort. Participants with a CHEK2 PV were less likely to have a diagnosis of colorectal cancer (OR, 0.62; 95% CI, 0.51-0.76; P < .001) compared with those in the wild-type cohort. There were no significant differences between frequent CHEK2 PVs and c.1100del and no differences between CHEK2 missense and LOF PVs. Conclusions and Relevance: CHEK2 PVs, with few exceptions (p.I157T, p.S428F, and p.T476M), were associated with similar cancer phenotypes irrespective of variant type. CHEK2 PVs were not associated with colorectal cancer, but were associated with breast, kidney, and thyroid cancers. Compared with other CHEK2 PVs, the frequent p.I157T, p.S428F, and p.T476M alleles have an attenuated association with breast cancer and were not associated with nonbreast cancers. These data may inform the genetic counseling and care of individuals with CHEK2 PVs.

An integrated somatic and germline approach to aid interpretation of germline variants of uncertain significance in cancer susceptibility genes.

Genomic profiles of tumors are often unique and represent characteristic mutational signatures defined by DNA damage or DNA repair response processes. The tumor-derived somatic information has been widely used in therapeutic applications, but it is grossly underutilized in the assessment of germline genetic variants. Here, we present a comprehensive approach for evaluating the pathogenicity of germline variants in cancer using an integrated interpretation of somatic and germline genomic data. We have previously demonstrated the utility of this integrated approach in the reassessment of pathogenic germline variants in selected cancer patients with unexpected or non-syndromic phenotypes. The application of this approach is presented in the assessment of rare variants of uncertain significance (VUS) in Lynch-related colon cancer, hereditary paraganglioma-pheochromocytoma syndrome, and Li-Fraumeni syndrome. Using this integrated method, germline VUS in PMS2, MSH6, SDHC, SHDA, and TP53 were assessed in 16 cancer patients after genetic evaluation. Comprehensive clinical criteria, somatic signature profiles, and tumor immunohistochemistry were used to re-classify VUS by upgrading or downgrading the variants to likely or unlikely actionable categories, respectively. Going forward, collation of such germline variants and creation of cross-institutional knowledgebase datasets that include integrated somatic and germline data will be crucial for the assessment of these variants in a larger cancer cohort.

Addition of Germline Testing to Tumor-Only Sequencing Improves Detection of Pathogenic Germline Variants in Men With Advanced Prostate Cancer.

PURPOSE: Guidelines recommend somatic and germline testing for men with advanced prostate cancer (PCa). Barriers to widespread implementation result in underutilization of germline testing. Somatic testing alone risks missing pathogenic germline variants (PGVs). We sought to determine whether the addition of germline testing to tumor-only sequencing improves detection of PGVs in men with advanced PCa. Secondarily, we sought to define the added value of combining somatic and germline testing to optimize detection of clinically actionable alterations. PATIENTS AND METHODS: We analyzed results of independent germline testing and tumor-only sequencing from 100 men with advanced PCa from a prospective clinical trial (ClinicalTrials.gov identifier: NCT03328091). The primary outcome was the proportion of PGVs not reported with tumor-only sequencing. The secondary outcome was the association of locus-specific loss of heterozygosity for PGVs in homologous recombination genes with clinical-genomic features. RESULTS: In the 100 men who underwent germline testing and tumor-only sequencing, 24 PGVs were identified, 17 of which were clinically actionable, in 23 patients. Tumor-only sequencing failed to report four (17%) of the PGVs. One additional PGV (4.2%) had variant allele frequency on tumor-sequencing below the threshold for follow-up germline testing. When integrating tumor-only sequencing with germling testing results, 33% of patients harbored clinically actionable alterations. Rates of locus-specific loss of heterozygosity were higher for BRCA2 PGVs in castration-resistant PCa than PGVs in other homologous recombination genes in hormone-sensitive PCa (P = .029). CONCLUSION: Tumor-only sequencing failed to report more than 20% of PGVs in men with advanced PCa. These findings strongly support guideline recommendations for universal germline and somatic testing in this population. Combining tumor and germline sequencing doubled the chance of detecting a clinically actionable alteration.

Incidence of Germline Variants in Familial Bladder Cancer and Among Patients With Cancer Predisposition Syndromes.

BACKGROUND: The familial aggregation of bladder cancers has been observed, but the incidence and association of familial bladder cancer with germline pathogenic and likely pathogenic (P/LP) variants is unknown. PATIENTS AND METHODS: A retrospective analysis was conducted of patients with bladder cancer treated at the Dana-Farber Cancer Institute to identify those with a first-degree relative with bladder cancer. A second cohort of patients referred to DFCI for suspicion of a cancer predisposition syndrome was analyzed for candidate P/LP germline variants. Descriptive statistics were generated. RESULTS: Among 885 patients with bladder cancer, 38 patients (4.3%) had a family history of bladder cancer in a first-degree relative. No significant association of age of diagnosis was observed between patients with and without a first-degree family history of bladder cancer (P = .3). In the second cohort, 27 of 80 (34%) patients with bladder cancer evaluated for cancer predisposition syndromes harbored a P/LP germline variant. P/LP variants were identified most commonly in the following genes: BRCA1 (n = 5), MSH2 (n = 5), MLH1 (n = 4), ATM (n = 3), and CHEK2 (n = 2). Of the 27 patients with identified germline P/LP variants, 20 (74%) had a family history of a tumor component syndrome in a first- or second-degree relative and 3 were subsequently diagnosed with another genetically-linked associated cancer. CONCLUSION: Familial bladder cancer defined as bladder cancer in the proband and a first-degree relative, was present in 4.3% of patients with bladder cancer and was not associated with age of diagnosis. Additionally, among patients suspected to have a familial cancer syndrome, one-third harbored a germline P/LP variant. Further study of germline variants in patients with familial bladder cancer including somatic testing for loss of heterozygosity may provide insights regarding disease pathogenesis and inform therapy.

Impact of Genetic Counseling on Patient-Reported Electronic Cancer Family History Collection.

BACKGROUND: Cancer family history is a vital part of cancer genetic counseling (GC) and genetic testing (GT), but increasing indications for germline cancer GT necessitate less labor-intensive models of collection. We evaluated the impact of GC on patient pedigrees generated by an electronic cancer family history questionnaire (eCFHQ). METHODS: An Institutional Review Board-approved review of pedigrees collected through an eCFHQ was conducted. Paired pre-GC and post-GC pedigrees (n=1,113 each group) were analyzed independently by cancer genetic counselors for changes in patient-reported clinical history and to determine whether the pedigrees met NCCN GT criteria. Discrepancy in meeting NCCN GT criteria between pre-GC and post-GC pedigrees was the outcome variable of logistic regressions, with patient and family history characteristics as covariates. RESULTS: Overall, 780 (70%) patients had cancer (affected), 869 (78%) were female, and the median age was 57 years (interquartile range, 45-66 years; range, 21-91 years). Of the 1,113 pairs of pre-GC and post-GC pedigrees analyzed, 85 (8%) were blank, 933 (84%) were not discrepant, and 95 (9%) were discrepant in meeting any NCCN GT criteria. Of the discrepant pedigrees, n=79 (83%) became eligible for testing by at least one of the NCCN GT criteria after GC. Patients with discrepant pedigrees were more likely to report no or unknown history of GT (odds ratio [OR], 4.54; 95% CI, 1.66-18.70; P=.01, and OR, 18.47; 95% CI, 5.04-88.73; P<.0001, respectively) and belonged to racially and/or ethnically underrepresented groups (OR, 1.91; 95% CI, 1.08-3.25; P=.02). CONCLUSIONS: For most patients (84%), a standalone eCFHQ was sufficient to determine whether NCCN GT criteria were met. More research is needed on the performance of the eCFHQ in diverse patient populations.

An optimized protocol for evaluating pathogenicity of VHL germline variants in patients suspected with von Hippel-Lindau syndrome: Using somatic genome to inform the role of germline variants.

The interpretation of hereditary genetic sequencing variants is often limited due to the absence of functional data and other key evidence to assess the role of variants in disease. Cancer genetics is unique, as two sets of genomic information are often available from a cancer patient: somatic and germline. Despite the progress made in the integrated analysis of somatic and germline findings, the assessment of pathogenicity of germline variants in high penetrance genes remains grossly underutilized. Indeed, standard ACMG/AMP guidelines for interpreting germline sequence variants do not address the evidence derived from tumor data in cancer. Previously, we have demonstrated the utility of somatic tumor data as supporting evidence to elucidate the role of germline variants in patients suspected with VHL syndrome and other cancers. We have leveraged the key elements of cancer genetics in these cases: genes with expected high disease penetrance and those with a known biallelic mechanism of tumorigenicity. Here we provide our optimized protocol for evaluating the pathogenicity of germline VHL variants using informative somatic profiling data. This protocol provides details of case selection, assessment of personal and family evidence, somatic tumor profiles, and loss of heterozygosity (LOH) as supporting evidence for the re-evaluation of germline variants.