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Importance: Personalized surveillance, prophylaxis, and cancer treatment options for individuals with hereditary cancer predisposition are informed by results of germline genetic testing. Improvements to genomic technology, such as the availability of RNA sequencing, may increase identification of individuals eligible for personalized interventions by improving the accuracy and yield of germline testing., Objective: To assess the cumulative association of paired DNA and RNA testing with detection of disease-causing germline genetic variants and resolution of variants of uncertain significance (VUS)., Design, Setting, and Participants: Paired DNA and RNA sequencing was performed on individuals undergoing germline testing for hereditary cancer indication at a single diagnostic laboratory from March 2019 through April 2020. Demographic characteristics, clinical data, and test results were curated as samples were received, and changes to variant classification were assessed over time. Data analysis was performed from May 2020 to June 2023., Main Outcomes and Measures: Main outcomes were increase in diagnostic yield, decrease in VUS rate, the overall results by variant type, the association of RNA evidence with variant classification, and the corresponding predicted effect on cancer risk management., Results: A total of 43 524 individuals were included (median [range] age at testing, 54 [2-101] years; 37 373 female individuals [85.7%], 6224 male individuals [14.3%], and 2 individuals of unknown sex [<0.1%]), with 43 599 tests. A total of 2197 (5.0%) were Ashkenazi Jewish, 1539 (3.5%) were Asian, 3077 (7.1%) were Black, 2437 (5.6%) were Hispanic, 27 793 (63.7%) were White, and 2049 (4.7%) were other race, and for 4507 individuals (10.3%), race and ethnicity were unknown. Variant classification was impacted in 549 individuals (1.3%). Medically significant upgrades were made in 97 individuals, including 70 individuals who had a variant reclassified from VUS to pathogenic/likely pathogenic (P/LP) and 27 individuals who had a novel deep intronic P/LP variant that would not have been detected using DNA sequencing alone. A total of 93 of 545 P/LP splicing variants (17.1%) were dependent on RNA evidence for classification, and 312 of 439 existing splicing VUS (71.1%) were resolved by RNA evidence. Notably, the increase in positive rate (3.1%) and decrease in VUS rate (-3.9%) was higher in Asian, Black, and Hispanic individuals combined compared to White individuals (1.6%; P = .02; and -2.5%; P < .001)., Conclusions and Relevance: Findings of this diagnostic study demonstrate that the ability to perform RNA sequencing concurrently with DNA sequencing represents an important advancement in germline genetic testing by improving detection of novel variants and classification of existing variants. This expands the identification of individuals with hereditary cancer predisposition and increases opportunities for personalization of therapeutics and surveillance.

Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C-/- cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case-control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18-7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71-30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers., Significance: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants., (©2023 The Authors; Published by the American Association for Cancer Research.)

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., (© 2023. The Author(s).)

Purpose: With the availability of multigene panel testing (MGPT) for hereditary cancer risk assessment, clinicians need to assess the likelihood of pathogenic germline variants (PGVs) across numerous genes in parallel. This study's aim was to develop and validate a clinical prediction model (PREMMplus) for MGPT risk assessment., Materials and Methods: PREMMplus was developed in a single-institution cohort of 7,280 individuals who had undergone MGPT. Logistic regression models with Least Absolute Shrinkage and Selection Operator regularization were used to examine candidate predictors (age, sex, ethnicity, and personal/family history of 18 cancers/neoplasms) to estimate one's likelihood of carrying PGVs in 19 genes (broadly categorized by phenotypic overlap and/or relative penetrance: 11 category A [ APC , BRCA1 / 2 , CDH1 , EPCAM , MLH1 , MSH2 , MSH6 , biallelic MUTYH , PMS2 , and TP53 ] and eight category B genes [ ATM , BRIP1 , CDKN2A , CHEK2 , PALB2 , PTEN , RAD51C , and RAD51D ]). Model performance was validated in nonoverlapping data sets of 8,691 and 14,849 individuals with prior MGPT ascertained from clinic- and laboratory-based settings, respectively., Results: PREMMplus (score ≥ 2.5%) had 93.9%, 91.7%, and 89.3% sensitivity and 98.3%, 97.5%, and 97.8% negative-predictive value (NPV) for identifying category A gene PGV carriers in the development and validation cohorts, respectively. PREMMplus assessment (score ≥ 2.5%) had 89.9%, 85.6%, and 84.2% sensitivity and 95.0%, 93.5%, and 93.5% NPV, respectively, for identifying category A/B gene PGV carriers. Decision curve analyses support MGPT for individuals predicted to have ≥ 2.5% probability of a PGV., Conclusion: PREMMplus accurately identifies individuals with PGVs in a diverse spectrum of cancer susceptibility genes with high sensitivity/NPV. Individuals with PREMMplus scores ≥ 2.5% should be considered for MGPT.

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.

DNA germline genetic testing can identify individuals with cancer susceptibility. However, DNA sequencing alone is limited in its detection and classification of mRNA splicing variants, particularly those located far from coding sequences. Here we address the limitations of splicing variant identification and interpretation by pairing DNA and RNA sequencing and describe the mutational and splicing landscape in a clinical cohort of 43,524 individuals undergoing genetic testing for hereditary cancer predisposition., (© 2022. The Author(s).)

Loss-of-function variants in the BRCA1 and BRCA2 susceptibility genes predispose carriers to breast and/or ovarian cancer. The use of germline testing panels containing these genes has grown dramatically, but the interpretation of the results has been complicated by the identification of many sequence variants of undefined cancer relevance, termed "Variants of Uncertain Significance (VUS)." We have developed functional assays and a statistical model called VarCall for classifying BRCA1 and BRCA2 VUS. Here we describe a multifactorial extension of VarCall, called VarCall XT, that allows for co-analysis of multiple forms of genetic evidence. We evaluated the accuracy of models defined by the combinations of functional, in silico protein predictors, and family data for VUS classification. VarCall XT classified variants of known pathogenicity status with high sensitivity and specificity, with the functional assays contributing the greatest predictive power. This approach could be used to identify more patients that would benefit from personalized cancer risk assessment and management., (© 2022. The Author(s).)

Background: Practice guidelines to identify individuals with hereditary pheochromocytomas and paragangliomas (PPGLs) advocate for sequential gene testing strategy guided by specific clinical features and predate the routine use of multigene panel testing (MGPT)., Objective: To describe results of MGPT for hereditary PPGL in a clinically and ancestrally diverse cohort., Setting: Commercial laboratory based in the United States., Methods: Clinical data and test results were retrospectively reviewed in 1727 individuals who had targeted MGPT from August 2013 through December 2019 because of a suspicion of hereditary PPGL., Results: Overall, 27.5% of individuals had a pathogenic or likely pathogenic variant (PV), 9.0% had a variant of uncertain significance, and 63.1% had a negative result. Most PVs were identified in SDHB (40.4%), followed by SDHD (21.1%), SDHA (10.1%), VHL (7.8%), SDHC (6.7%), RET (3.7%), and MAX (3.6%). PVs in FH, MEN1, NF1, SDHAF2, and TMEM127 collectively accounted for 6.5% of PVs. Clinical predictors of a PV included extra-adrenal location, early age of onset, multiple tumors, and positive family history of PPGL. Individuals with extra-adrenal PGL and a positive family history were the most likely to have a PV (85.9%). Restricting genetic testing to SDHB/C/D misses one-third (32.8%) of individuals with PVs., Conclusion: Our data demonstrate a high diagnostic yield in individuals with and without established risk factors, a low inconclusive result rate, and a substantial contribution to diagnostic yield from rare genes. These findings support universal testing of all individuals with PPGL and the use of concurrent MGPT as the ideal platform., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Purpose: Multigene panels allow simultaneous testing of genes involved in cancer predisposition. Thyroid cancer (TCa) is a component tumor of several cancer predisposition syndromes, but the complete landscape of germline variants predisposing to TCa remains to be determined., Methods: Clinical information and genetic test results were reviewed from over 170,000 individuals who had multigene panel testing for hereditary cancer at a single diagnostic laboratory. Germline pathogenic and likely pathogenic variants ("pathogenic variants") were examined among individuals with TCa. A cohort with breast cancer (BCa) was examined to serve as a comparison group and to determine the added contribution of TCa to the ascertainment of genetic risk., Results: Of 3134 individuals with TCa, 291 (9.3%) were found to have one or more pathogenic variant(s). Among 904 individuals with TCa alone, 7.5% had one or more pathogenic variant(s), similar to those with BCa alone (8.4%). In all groups, CHEK2 was the gene with the highest number of pathogenic variants identified, with a significantly increased frequency among individuals with a history of both thyroid and BCa compared to BCa alone., Conclusions: A high prevalence of germline pathogenic variants was observed among individuals with TCa referred for hereditary cancer genetic testing, even in the absence of other cancer diagnoses. These data suggest that TCa may be an under-recognized component of cancer predisposition syndromes., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Multiple primary cancers (MPCs) are a hallmark of cancer predisposition syndromes. Here the frequency of germline pathogenic variants (PVs) among patients with MPCs is reported., Methods: Patients with MPCs who underwent multigene panel testing from March 2012 to December 2016 were studied. Eligible patients had an analysis of 21 genes: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, STK11, and TP53. The frequencies of PVs by sex, number of cancers, and age at diagnosis were compared with 2-sided χ 2 tests or Fisher exact tests when the number was <10., Results: Among the 9714 patients analyzed, most were female (91.1%) and White (71.0%); the median age at testing was 63 years, and the median ages at first and second cancer diagnoses were 49 and 58 years, respectively. Overall, 1320 (13.6%) had PVs. The prevalence of PVs increased with the number of primary cancers (PCs): 13.1% with 2 PCs, 15.9% with 3 PCs, and 18.0% with ≥4 PCs (P = .00056). Differences in the prevalence of PVs by age at diagnosis were significant: 14.7% with 2 PCs at an age < 50 years, 15.8% with 1 PC at an age < 50 years, and 12.0% with all PCs at an age ≥ 50 years (P = 2.07E-05). PVs by the age at second cancer diagnosis were also significant: 14.7% at an age < 50 years, 13.9% at an age of 50 to 69 years, and 11.4% at an age ≥ 70 years (P for trend = .005)., Conclusions: Among patients with MPCs, there is a high frequency of germline PVs, with a higher frequency found among patients with a higher number of PCs. These findings suggest that genetic testing should be considered even among patients who are older at the diagnosis of an additional primary malignancy., (© 2021 American Cancer Society.)

Background: With increased adoption of multi-gene panel testing (MGPT) for hereditary cancer, management guidelines now include a wider range of predisposition genes. Yet little is known about whether MGPT results prompt changes to clinicians' risk management recommendations and whether those recommendations adhere to guidelines., Methods: We assessed cancer risk management recommendations made by clinicians ordering MGPT for hereditary cancer at a diagnostic laboratory using an internet-based survey. We received paired pre- and posttest responses for 2172 patients (response rate = 14.3%). Unpaired posttest responses were received in 168 additional patients with positive results. All tests were 2-sided., Results: Clinicians reported a change in risk management recommendations for 76.6% of patients who tested positive for a pathogenic or likely pathogenic variant, with changes to surveillance being most common (71.1%), followed by surgical (33.6%), chemoprevention (15.1%), and clinical trial (9.4%) recommendations. Clinicians recommended risk-reducing interventions more often for patients with pathogenic variants in high-risk than moderate-risk genes (P < .001), whereas surveillance recommendations were similar for high-risk and moderate-risk genes. Guideline adherence was high for surveillance (86.3%) and surgical (79.6%) recommendations. Changes to risk management recommendations occurred in 8.8% and 7.6% of patients with uncertain and negative results, respectively., Conclusions: Clinicians report frequent changes to cancer risk management recommendations based on positive results in both high-risk and moderate-risk genes. Reported introduction of interventions in patients with inconclusive and negative results is rare and adherence to practice guidelines is high in patients with positive results, suggesting a low probability of harm resulting from MGPT., (© The Author(s) 2022. Published by Oxford University Press.)

MUTYH -associated polyposis (MAP) is an autosomal recessive disorder characterized by the development of multiple adenomatous colonic polyps and an increased lifetime risk of colorectal cancer. Germline biallelic pathogenic variants in MUTYH are responsible for MAP. The MUTYH c.934-2A > G (NM&#95;001128425.1) variant, which is also known as c.850-2A > G for NM&#95;001048174.2, has been identified in our laboratory in more than 800 patients, including homozygous and compound heterozygote carriers. The variant was initially classified as a variant of uncertain significance (VUS) because of lack of a MAP phenotype in biallelic carriers. In two unrelated female patients who were heterozygous carriers of this variant, further testing by RNA sequencing identified an aberrant transcript with a deletion of 9 nt at the start of exon 11 ( MUTYH r.934&#95;942del9). This event is predicted to lead to an in-frame loss of three amino acids in a noncritical domain of the protein. This was the only splice defect identified in these patients that was not present in the controls, and the aberrant transcript is derived exclusively from the variant allele, strongly supporting the cause of this splice defect as being the intronic variant, MUTYH c.934-2A > G. The splicing analysis demonstrating a small in-frame skipping of three amino acids in a noncritical domain, along with the absence of a MAP phenotype in our internal cohort of biallelic carriers, provides evidence that the variant is likely benign and not of clinical significance., (© 2022 Hernandez et al.; Published by Cold Spring Harbor Laboratory Press.)

Purpose: The weight of the evidence to attach to observation of a novel rare missense variant in SDHB or SDHD in individuals with the rare neuroendocrine tumors, pheochromocytomas and paragangliomas (PCC/PGL), is uncertain., Methods: We compared the frequency of SDHB and SDHD very rare missense variants (VRMVs) in 6328 and 5847 cases of PCC/PGL, respectively, with that of population controls to generate a pan-gene VRMV likelihood ratio (LR). Via windowing analysis, we measured regional enrichments of VRMVs to calculate the domain-specific VRMV-LR (DS-VRMV-LR). We also calculated subphenotypic LRs for variant pathogenicity for various clinical, histologic, and molecular features., Results: We estimated the pan-gene VRMV-LR to be 76.2 (54.8-105.9) for SDHB and 14.8 (8.7-25.0) for SDHD. Clustering analysis revealed an SDHB enriched region (ɑɑ 177-260, P = .001) for which the DS-VRMV-LR was 127.2 (64.9-249.4) and an SDHD enriched region (ɑɑ 70-114, P = .000003) for which the DS-VRMV-LR was 33.9 (14.8-77.8). Subphenotypic LRs exceeded 6 for invasive disease (SDHB), head-and-neck disease (SDHD), multiple tumors (SDHD), family history of PCC/PGL, loss of SDHB staining on immunohistochemistry, and succinate-to-fumarate ratio >97 (SDHB, SDHD)., Conclusion: Using methodology generalizable to other gene-phenotype dyads, the LRs relating to rarity and phenotypic specificity for a single observation in PCC/PGL of a SDHB/SDHD VRMV can afford substantial evidence toward pathogenicity., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To determine the contribution of germline pathogenic variants (PVs) in hereditary cancer testing panel genes to invasive lobular carcinoma (ILC) of the breast., Materials and Methods: The study included 2,999 women with ILC from a population-based cohort and 3,796 women with ILC undergoing clinical multigene panel testing (clinical cohort). Frequencies of germline PVs in breast cancer predisposition genes ( ATM , BARD1 , BRCA1 , BRCA2 , BRIP1 , CDH1 , CHEK2 , PALB2 , PTEN , RAD51C , RAD51D , and TP53 ) were compared between women with ILC and unaffected female controls and between women with ILC and infiltrating ductal carcinoma (IDC)., Results: The frequency of PVs in breast cancer predisposition genes among women with ILC was 6.5% in the clinical cohort and 5.2% in the population-based cohort. In case-control analysis, CDH1 and BRCA2 PVs were associated with high risks of ILC (odds ratio [OR] > 4) and CHEK2 , ATM , and PALB2 PVs were associated with moderate (OR = 2-4) risks. BRCA1 PVs and CHEK2 p.Ile157Thr were not associated with clinically relevant risks (OR < 2) of ILC. Compared with IDC, CDH1 PVs were > 10-fold enriched, whereas PVs in BRCA1 were substantially reduced in ILC., Conclusion: The study establishes that PVs in ATM , BRCA2 , CDH1 , CHEK2 , and PALB2 are associated with an increased risk of ILC, whereas BRCA1 PVs are not. The similar overall PV frequencies for ILC and IDC suggest that cancer histology should not influence the decision to proceed with genetic testing. Similar to IDC, multigene panel testing may be appropriate for women with ILC, but CDH1 should be specifically discussed because of low prevalence and gastric cancer risk., Competing Interests: Jeffrey N. WeitzelSpeakers' Bureau: AstraZeneca Celine M. VachonStock and Other Ownership Interests: Exact Sciences (I)Research Funding: GRAILPatents, Royalties, Other Intellectual Property: Breast Density softwareTravel, Accommodations, Expenses: GRAIL Allison W. KurianResearch Funding: Myriad GeneticsOther Relationship: Ambry Genetics, Color Genomics, GeneDx/BioReference, InVitae, Genentech Rachid KaramEmployment: Ambry GeneticsResearch Funding: Ambry GeneticsTravel, Accommodations, Expenses: Ambry Genetics Elizabeth ChaoEmployment: Ambry Genetics Amal YussufEmployment: Ambry Genetics Tina PesaranEmployment: Ambry Genetics/Konica Minolta Jill S. DolinskyEmployment: Ambry Genetics Holly LaDucaEmployment: Ambry GeneticsStock and Other Ownership Interests: Ambry Genetics Eric C. PolleyResearch Funding: GRAIL Susan M. DomchekHonoraria: AstraZeneca, Clovis Oncology, Bristol Myers SquibbResearch Funding: AstraZeneca, Clovis Oncology Fergus J. CouchConsulting or Advisory Role: AstraZenecaSpeakers' Bureau: Ambry Genetics, QiagenResearch Funding: GRAILTravel, Accommodations, Expenses: GRAIL, QiagenOther Relationship: Ambry GeneticsNo other potential conflicts of interest were reported.