A substantial proportion of apparently heterozygous TP53 pathogenic variants detected with a next-generation sequencing hereditary pan-cancer panel are acquired somatically.

Previous analysis of next-generation sequencing (NGS) hereditary pan-cancer panel testing demonstrated that approximately 40% of TP53 pathogenic and likely pathogenic variants (PVs) detected have NGS allele frequencies between 10% and 30%, indicating that they likely are acquired somatically. These are seen more frequently in older adults, suggesting that most result from normal aging-related clonal hematopoiesis. For this analysis, apparent heterozygous germline TP53 PV carriers (NGS allele frequency 30-70%) were offered follow-up testing to confirm variant origin. Ninety-eight probands had samples submitted for follow-up family member testing, fibroblast testing, or both. The apparent heterozygous germline TP53 PV was not detected in 32.6% (15/46) of submitted fibroblast samples, indicating that it was acquired somatically, either through clonal hematopoiesis or via constitutional mosaicism. Notably, no individuals with confirmed germline or likely germline TP53 PVs met classic Li-Fraumeni syndrome (LFS) criteria, only 41% met Chompret LFS criteria, and 59% met neither criteria, based upon provider-reported personal and family cancer history. Comprehensive reporting of TP53 PVs detected using NGS, combined with follow-up analysis to confirm variant origin, is advised for clinical testing laboratories. These findings underscore the investment required to provide individuals and family members with clinically accurate genetic test results pertaining to their LFS risk.

Hereditary cancer testing challenges: assembling the analytical pieces to solve the patient clinical puzzle.

Expanded genetic test utilization to guide cancer management has driven the development of larger gene panels and greater diversity in the patient population pursuing testing, resulting in increased identification of atypical or technically challenging genetic findings. To ensure appropriate patient care, it is critical that genetic tests adequately identify and characterize these findings. We describe genetic testing challenges frequently encountered by our laboratory and the methodologies we employ to improve test accuracy for the identification and characterization of atypical genetic findings. While these findings may be individually rare, 15,745 (9%) individuals tested by our laboratory for hereditary cancer risk had an atypical genetic finding, highlighting the importance of employing highly accurate and comprehensive methods in clinical genetic testing.

An independent validation of a gene expression signature to differentiate malignant melanoma from benign melanocytic nevi.

BACKGROUND: Recently, a 23-gene signature was developed to produce a melanoma diagnostic score capable of differentiating malignant and benign melanocytic lesions. The primary objective of this study was to independently assess the ability of the gene signature to differentiate melanoma from benign nevi in clinically relevant lesions. METHODS: A set of 1400 melanocytic lesions was selected from samples prospectively submitted for gene expression testing at a clinical laboratory. Each sample was tested and subjected to an independent histopathologic evaluation by 3 experienced dermatopathologists. A primary diagnosis (benign or malignant) was assigned to each sample, and diagnostic concordance among the 3 dermatopathologists was required for inclusion in analyses. The sensitivity and specificity of the score in differentiating benign and malignant melanocytic lesions were calculated to assess the association between the score and the pathologic diagnosis. RESULTS: The gene expression signature differentiated benign nevi from malignant melanoma with a sensitivity of 91.5% and a specificity of 92.5%. CONCLUSIONS: These results reflect the performance of the gene signature in a diverse array of samples encountered in routine clinical practice. Cancer 2017;123:617-628. © 2016 American Cancer Society.

Identification of a Variety of Mutations in Cancer Predisposition Genes in Patients With Suspected Lynch Syndrome.

BACKGROUND & AIMS: Multigene panels are commercially available tools for hereditary cancer risk assessment that allow for next-generation sequencing of numerous genes in parallel. However, it is not clear if these panels offer advantages over traditional genetic testing. We investigated the number of cancer predisposition gene mutations identified by parallel sequencing in individuals with suspected Lynch syndrome. METHODS: We performed germline analysis with a 25-gene, next-generation sequencing panel using DNA from 1260 individuals who underwent clinical genetic testing for Lynch syndrome from 2012 through 2013. All patients had a history of Lynch syndrome-associated cancer and/or polyps. We classified all identified germline alterations for pathogenicity and calculated the frequencies of pathogenic mutations and variants of uncertain clinical significance (VUS). We also analyzed data on patients' personal and family history of cancer, including fulfillment of clinical guidelines for genetic testing. RESULTS: Of the 1260 patients, 1112 met National Comprehensive Cancer Network (NCCN) criteria for Lynch syndrome testing (88%; 95% confidence interval [CI], 86%-90%). Multigene panel testing identified 114 probands with Lynch syndrome mutations (9.0%; 95% CI, 7.6%-10.8%) and 71 with mutations in other cancer predisposition genes (5.6%; 95% CI, 4.4%-7.1%). Fifteen individuals had mutations in BRCA1 or BRCA2; 93% of these met the NCCN criteria for Lynch syndrome testing and 33% met NCCN criteria for BRCA1 and BRCA2 analysis (P = .0017). An additional 9 individuals carried mutations in other genes linked to high lifetime risks of cancer (5 had mutations in APC, 3 had bi-allelic mutations in MUTYH, and 1 had a mutation in STK11); all of these patients met NCCN criteria for Lynch syndrome testing. A total of 479 individuals had 1 or more VUS (38%; 95% CI, 35%-41%). CONCLUSIONS: In individuals with suspected Lynch syndrome, multigene panel testing identified high-penetrance mutations in cancer predisposition genes, many of which were unexpected based on patients' histories. Parallel sequencing also detected a high number of potentially uninformative germline findings, including VUS.

Frequency of mutations in individuals with breast cancer referred for BRCA1 and BRCA2 testing using next-generation sequencing with a 25-gene panel.

BACKGROUND: Next-generation sequencing (NGS) allows for simultaneous sequencing of multiple cancer susceptibility genes and, for an individual, may be more efficient and less expensive than sequential testing. The authors assessed the frequency of deleterious germline mutations among individuals with breast cancer who were referred for BRCA1 and BRCA2 (BRCA1/2) gene testing using a panel of 25 genes associated with inherited cancer predisposition. METHODS: This was a cross-sectional study using NGS in 2158 individuals, including 1781 who were referred for commercial BRCA1/2 gene testing (cohort 1) and 377 who had detailed personal and family history and had previously tested negative for BRCA1/2 mutations (cohort 2). RESULTS: Mutations were identified in 16 genes, most frequently in BRCA1, BRCA2, CHEK2, ATM, and PALB2. Among the participants in cohort 1, 9.3% carried a BRCA1/2 mutation, 3.9% carried a mutation in another breast/ovarian cancer susceptibility gene, and 0.3% carried an incidental mutation in another cancer susceptibility gene unrelated to breast or ovarian cancer. In cohort 2, the frequency of mutations in breast/ovarian-associated genes other than BRCA1/2 was 2.9%, and an additional 0.8% had an incidental mutation. In cohort 1, Lynch syndrome-related mutations were identified in 7 individuals. In contrast to BRCA1/2 mutations, neither age at breast cancer diagnosis nor family history of ovarian or young breast cancer predicted for other mutations. The frequency of mutations in genes other than BRCA1/2 was lower in Ashkenazi Jews compared with non-Ashkenazi individuals (P=.026). CONCLUSIONS: Using an NGS 25-gene panel, the frequency of mutations in genes other than BRCA1/2 was 4.3%, and most mutations (3.9%) were identified in genes associated with breast/ovarian cancer.

Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk.

BACKGROUND: Germline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer. Traditional methods using Sanger sequencing focus on small groups of genes and therefore are unable to screen for numerous genes from several patients simultaneously. The goal of the present study was to validate a 25-gene panel to assess genetic risk for cancer in 8 different tissues using next generation sequencing (NGS) techniques. METHODS: Twenty-five genes associated with hereditary cancer syndromes were selected for development of a panel to screen for risk of these cancers using NGS. In an initial technical assessment, NGS results for BRCA1 and BRCA2 were compared with Sanger sequencing in 1864 anonymized DNA samples from patients who had undergone previous clinical testing. Next, the entire gene panel was validated using parallel NGS and Sanger sequencing in 100 anonymized DNA samples. Large rearrangement analysis was validated using NGS, microarray comparative genomic hybridization (CGH), and multiplex ligation-dependent probe amplification analyses (MLPA). RESULTS: NGS identified 15,877 sequence variants, while Sanger sequencing identified 15,878 in the BRCA1 and BRCA2 comparison study of the same regions. Based on these results, the NGS process was refined prior to the validation of the full gene panel. In the validation study, NGS and Sanger sequencing were 100% concordant for the 3,923 collective variants across all genes for an analytical sensitivity of the NGS assay of >99.92% (lower limit of 95% confidence interval). NGS, microarray CGH and MLPA correctly identified all expected positive and negative large rearrangement results for the 25-gene panel. CONCLUSION: This study provides a thorough validation of the 25-gene NGS panel and indicates that this analysis tool can be used to collect clinically significant information related to risk of developing hereditary cancers.

Clinical validation of a gene expression signature that differentiates benign nevi from malignant melanoma.

BACKGROUND: Histopathologic examination is sometimes inadequate for accurate and reproducible diagnosis of certain melanocytic neoplasms. As a result, more sophisticated and objective methods have been sought. The goal of this study was to identify a gene expression signature that reliably differentiated benign and malignant melanocytic lesions and evaluate its potential clinical applicability. Herein, we describe the development of a gene expression signature and its clinical validation using multiple independent cohorts of melanocytic lesions representing a broad spectrum of histopathologic subtypes. METHODS: Using quantitative reverse-transcription polymerase chain reaction (PCR) on a selected set of 23 differentially expressed genes, and by applying a threshold value and weighting algorithm, we developed a gene expression signature that produced a score that differentiated benign nevi from malignant melanomas. RESULTS: The gene expression signature classified melanocytic lesions as benign or malignant with a sensitivity of 89% and a specificity of 93% in a training cohort of 464 samples. The signature was validated in an independent clinical cohort of 437 samples, with a sensitivity of 90% and specificity of 91%. CONCLUSIONS: The performance, objectivity, reliability and minimal tissue requirements of this test suggest that it could have clinical application as an adjunct to histopathology in the diagnosis of melanocytic neoplasms.

Mutation Update for UBE3A variants in Angelman syndrome.

Angelman syndrome is a neurodevelopmental disorder caused by a deficiency of the imprinted and maternally expressed UBE3A gene. Although de novo genetic and epigenetic imprinting defects of UBE3A genomic locus account for majority of Angelman diagnoses, approximately 10% of individuals affected with Angelman syndrome are a result of UBE3A loss-of-function mutations occurring on the expressed maternal chromosome. The variants described in this manuscript represent the analysis of 2,515 patients referred for UBE3A gene sequencing at our institution, along with a comprehensive review of the UBE3A mutation literature. Of these, 267 (10.62%) patients had a report issued for detection of a UBE3A gene nucleotide variant, which in many cases involved family studies resulting in reclassification of variants of unknown clinical significance (VUS). Overall, 111 (4.41%) probands had a nucleotide change classified as pathogenic or strongly favored to be pathogenic, 29 (1.15%) had a VUS, and 126 (5.0%) had a nucleotide change classified as benign or strongly favored to be benign. All variants and their clinical interpretations are submitted to NCBI ClinVar, a freely accessible human variation and phenotype database.

Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay.

Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.

Comprehensive sequencing of PALB2 in patients with breast cancer suggests PALB2 mutations explain a subset of hereditary breast cancer.

BACKGROUND: This study sought to determine the prevalence of PALB2 mutations in a cohort referred for diagnostic testing for hereditary breast cancer. METHODS: Sanger sequencing was used to analyze the entire coding region and flanking introns of PALB2 in anonymized DNA samples from 1479 patients. Samples were stratified into a "high-risk" group, 955 samples from individuals predicted to have a high probability of carrying a mutation in BRCA1 or BRCA2 based on their personal and family history, and a "lower-risk" group consisting of 524 samples from patients with breast cancer, but fewer risk factors for being a BRCA1 or BRCA2 mutation carrier. All patients were known to be negative for deleterious sequence mutations and large rearrangements in BRCA1 and BRCA2. RESULTS: We identified 12 disease-associated PALB2 mutations among the 1479 patients (0.8%). The PALB2 mutations included 8 nonsense, 3 frameshift mutations and a splice-site mutation. The mutation prevalence for the high-risk population was 1.05% (95% CI = 0.5-1.92), whereas that for the lower-risk population was 0.38% (95% CI = 0.05-1.37). We identified 59 PALB2 variants of uncertain significance (VUS) among 57 of the 1479 patients (3.9%). CONCLUSIONS: These results suggest that PALB2 mutations occur at a frequency of ~1% in patients with hereditary breast cancer.

Development and validation of a new algorithm for the reclassification of genetic variants identified in the BRCA1 and BRCA2 genes.

BRCA1 and BRCA2 sequencing analysis detects variants of uncertain clinical significance in approximately 2 % of patients undergoing clinical diagnostic testing in our laboratory. The reclassification of these variants into either a pathogenic or benign clinical interpretation is critical for improved patient management. We developed a statistical variant reclassification tool based on the premise that probands with disease-causing mutations are expected to have more severe personal and family histories than those having benign variants. The algorithm was validated using simulated variants based on approximately 145,000 probands, as well as 286 BRCA1 and 303 BRCA2 true variants. Positive and negative predictive values of ≥99 % were obtained for each gene. Although the history weighting algorithm was not designed to detect alleles of lower penetrance, analysis of the hypomorphic mutations c.5096G>A (p.Arg1699Gln; BRCA1) and c.7878G>C (p.Trp2626Cys; BRCA2) indicated that the history weighting algorithm is able to identify some lower penetrance alleles. The history weighting algorithm is a powerful tool that accurately assigns actionable clinical classifications to variants of uncertain clinical significance. While being developed for reclassification of BRCA1 and BRCA2 variants, the history weighting algorithm is expected to be applicable to other cancer- and non-cancer-related genes.

Mutation of TDP1, encoding a topoisomerase I-dependent DNA damage repair enzyme, in spinocerebellar ataxia with axonal neuropathy.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs covalently bound topoisomerase I-DNA complexes and is essential for preventing the formation of double-strand breaks that result when stalled topoisomerase I complexes interfere with DNA replication in yeast. Here we show that a deficiency of this DNA repair pathway in humans does not predispose to neoplasia or dysfunctions in rapidly replicating tissues, but instead causes spinocerebellar ataxia with axonal neuropathy (SCAN1) by affecting large, terminally differentiated, non-dividing neuronal cells. Using genome-wide linkage mapping and a positional candidate approach in a Saudi Arabian family affected with autosomal recessive SCAN1, we identified a homozygous mutation in TDP1 (A1478G) that results in the substitution of histidine 493 with an arginine residue. The His493 residue is conserved in TDP1 across species and is located in the active site of the enzyme. Protein modeling predicts that mutation of this amino acid to arginine will disrupt the symmetric structure of the active site. We propose that loss-of-function mutations in TDP1 may cause SCAN1 either by interfering with DNA transcription or by inducing apoptosis in postmitotic neurons.

Discordance between germline genetic findings and abnormal tumor immunohistochemistry staining of mismatch repair proteins in individuals with suspected Lynch syndrome.

Background and Aims: Tumor immunohistochemical staining (IHC) of DNA mismatch repair (MMR) proteins is often used to guide germline genetic testing and variant classification for patients with suspected Lynch syndrome. This analysis examined the spectrum of germline findings in a cohort of individuals showing abnormal tumor IHC. Methods: We assessed individuals with reported abnormal IHC findings and referred for testing with a six-gene syndrome-specific panel (n=703). Pathogenic variants (PVs) and variants of uncertain significance (VUS) in MMR genes were designated expected/unexpected relative to IHC results. Results: The PV positive rate was 23.2% (163/703; 95% confidence interval [CI], 20.1%-26.5%); 8.0% (13/163; 95% CI, 4.3%-13.3%) of PV carriers had a PV in an unexpected MMR gene. Overall, 121 individuals carried VUS in MMR genes expected to be mutated based on IHC results. Based on independent evidence, in 47.1% (57/121; 95% CI, 38.0%-56.4%) of these individuals the VUSs were later reclassified as benign and in 14.0% (17/121; 95% CI, 8.4%-21.5%) of these individuals the VUSs were reclassified as pathogenic. Conclusions: Among patients with abnormal IHC findings, IHC-guided single-gene genetic testing may miss 8% of individuals with Lynch syndrome. In addition, in patients with VUS identified in MMR genes predicted to be mutated by IHC, extreme caution must be taken when the IHC results are considered in variant classification.

Clinical significance of large rearrangements in BRCA1 and BRCA2.

BACKGROUND: Current estimates of the contribution of large rearrangement (LR) mutations in the BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 2, early onset) genes responsible for hereditary breast and ovarian cancer are based on limited studies of relatively homogeneous patient populations. The prevalence of BRCA1/2 LRs was investigated in 48,456 patients with diverse clinical histories and ancestries, referred for clinical molecular testing for suspicion of hereditary breast and ovarian cancer. METHODS: Sanger sequencing analysis was performed for BRCA1/2 and LR testing for deletions and duplications using a quantitative multiplex polymerase chain reaction assay. Prevalence data were analyzed for patients from different risk and ethnic groups between July 2007 and April 2011. Patients were designated as "high-risk" if their clinical history predicted a high prior probability, wherein LR testing was performed automatically in conjunction with sequencing. "Elective" patients did not meet the high-risk criteria, but underwent LR testing as ordered by the referring health care provider. RESULTS: Overall BRCA1/2 mutation prevalence among high-risk patients was 23.8% versus 8.2% for the elective group. The mutation profile for high-risk patients was 90.1% sequencing mutations versus 9.9% LRs, and for elective patients, 94.1% sequencing versus 5.9% LRs. This difference may reflect the bias in high-risk patients to carry mutations in BRCA1, which has a higher penetrance and frequency of LRs compared with BRCA2. There were significant differences in the prevalence and types of LRs in patients of different ancestries. LR mutations were significantly more common in Latin American/Caribbean patients. CONCLUSIONS: Comprehensive LR testing in conjunction with full gene sequencing is an appropriate strategy for clinical BRCA1/2 analysis.

Interpretation of BRCA2 Splicing Variants: A Case Series of Challenging Variant Interpretations and the Importance of Functional RNA Analysis.

A substantial proportion of pathogenic variants associated with an increased risk of hereditary cancer are sequence variants affecting RNA splicing. The classification of these variants can be complex when both non-functional and functional transcripts are produced from the variant allele. We present four BRCA2 splice site variants with complex variant interpretations (BRCA2 c.68-3T>G, c.68-2A>G, c.425G>T, c.8331+2T>C). Evidence supporting a pathogenic classification is available for each variant, including in silico models, absence in population databases, and published functional data. However, comprehensive RNA analysis showed that some functional transcript may be produced by each variant. BRCA2 c.68-3T>G results in a partial splice defect. For BRCA2 c.68-2A>G and c.425G>T, aberrant splicing was shown to produce a potentially functional, in-frame transcript. BRCA2 c.8331+2T>C may utilize a functional GC donor in place of the wild-type GT donor. The severity of cancer history for carriers of these variants was also assessed using a history weighting algorithm and was not consistent with pathogenic controls (carriers of known pathogenic variants in BRCA2). Due to the conflicting evidence, our laboratory classifies these BRCA2 variants as variants of uncertain significance. This highlights the importance of evaluating new and existing evidence to ensure accurate variant classification and appropriate patient care.

Development and Validation of a Breast Cancer Polygenic Risk Score on the Basis of Genetic Ancestry Composition.

PURPOSE: Polygenic risk scores (PRSs) for breast cancer (BC) risk stratification have been developed primarily in women of European ancestry. Their application to women of non-European ancestry has lagged because of the lack of a formal approach to incorporate genetic ancestry and ancestry-dependent variant frequencies and effect sizes. Here, we propose a multiple-ancestry PRS (MA-PRS) that addresses these issues and may be useful in the development of equitable PRSs across other cancers and common diseases. MATERIALS AND METHODS: Women referred for hereditary cancer testing were divided into consecutive cohorts for development (n = 189,230) and for independent validation (n = 89,126). Individual genetic composition as fractions of three reference ancestries (African, East Asian, and European) was determined from ancestry-informative single-nucleotide polymorphisms. The MA-PRS is a combination of three ancestry-specific PRSs on the basis of genetic ancestral composition. Stratification of risk was evaluated by multivariable logistic regression models controlling for family cancer history. Goodness-of-fit analysis compared expected with observed relative risks by quantiles of the MA-PRS distribution. RESULTS: In independent validation, the MA-PRS was significantly associated with BC risk in the full cohort (odds ratio, 1.43; 95% CI, 1.40 to 1.46; P = 8.6 × 10-308) and within each major ancestry. The top decile of the MA-PRS consistently identified patients with two-fold increased risk of developing BC. Goodness-of-fit tests showed that the MA-PRS was well calibrated and predicted BC risk accurately in the tails of the distribution for both European and non-European women. CONCLUSION: The MA-PRS uses genetic ancestral composition to expand the utility of polygenic risk prediction to non-European women. Inclusion of genetic ancestry in polygenic risk prediction presents an opportunity for more personalized treatment decisions for women of varying and mixed ancestries.

Comprehensive Breast Cancer Risk Assessment for CHEK2 and ATM Pathogenic Variant Carriers Incorporating a Polygenic Risk Score and the Tyrer-Cuzick Model.

PURPOSE: Breast cancer risks for CHEK2 and ATM pathogenic variant (PV) carriers are modified by an 86-single nucleotide polymorphism polygenic risk score (PRS) and individual clinical factors. Here, we describe comprehensive risk prediction models for women of European ancestry combining PV status, PRS, and individual clinical variables. MATERIALS AND METHODS: This study included deidentified clinical records from 358,095 women of European ancestry who received testing with a multigene panel (September 2013 to November 2019). Model development included CHEK2 PV carriers (n = 4,286), ATM PV carriers (n = 2,666), and women negative for other breast cancer risk gene PVs (n = 351,143). Odds ratios (ORs) were calculated using multivariable logistic regression with adjustment for familial cancer history. Risk estimates incorporating PV status, PRS, and Tyrer-Cuzick v7.02 were calculated using a Fixed-Stratified method that accounts for correlations between risk factors. Stratification of PV carriers into risk categories on the basis of remaining lifetime risk (RLR) was assessed in independent cohorts of PV carriers. RESULTS: ORs for association of PV status with breast cancer were 2.01 (95% CI, 1.88 to 2.16) and 1.83 (95% CI, 1.68 to 2.00) for CHEK2 and ATM PV carriers, respectively. ORs for PRS per one standard deviation were 1.51 (95% CI, 1.37 to 1.66) and 1.45 (95% CI, 1.30 to 1.64) in CHEK2 and ATM PV carriers, respectively. Using the combined model (PRS plus Tyrer-Cuzick plus PV status), RLR was low (≤ 20%) for 24.2% of CHEK2 PV carriers, medium (20%-50%) for 63.8%, and high (> 50%) for 12.0%. Among ATM PV carriers, RLR was low for 31.5% of patients, medium for 58.5%, and high for 9.7%. CONCLUSION: In CHEK2 and ATM PV carriers, risk assessment including PRS, Tyrer-Cuzick, and PV status has the potential for more precise direction of screening and prevention strategies.

Integrating Clinical and Polygenic Factors to Predict Breast Cancer Risk in Women Undergoing Genetic Testing.

PURPOSE: Screening and prevention decisions for women at increased risk of developing breast cancer depend on genetic and clinical factors to estimate risk and select appropriate interventions. Integration of polygenic risk into clinical breast cancer risk estimators can improve discrimination. However, correlated genetic effects must be incorporated carefully to avoid overestimation of risk. MATERIALS AND METHODS: A novel Fixed-Stratified method was developed that accounts for confounding when adding a new factor to an established risk model. A combined risk score (CRS) of an 86-single-nucleotide polymorphism polygenic risk score and the Tyrer-Cuzick v7.02 clinical risk estimator was generated with attenuation for confounding by family history. Calibration and discriminatory accuracy of the CRS were evaluated in two independent validation cohorts of women of European ancestry (N = 1,615 and N = 518). Discrimination for remaining lifetime risk was examined by age-adjusted logistic regression. Risk stratification with a 20% risk threshold was compared between CRS and Tyrer-Cuzick in an independent clinical cohort (N = 32,576). RESULTS: Simulation studies confirmed that the Fixed-Stratified method produced accurate risk estimation across patients with different family history. In both validation studies, CRS and Tyrer-Cuzick were significantly associated with breast cancer. In an analysis with both CRS and Tyrer-Cuzick as predictors of breast cancer, CRS added significant discrimination independent of that captured by Tyrer-Cuzick (P < 10-11 in validation 1; P < 10-7 in validation 2). In an independent cohort, 18% of women shifted breast cancer risk categories from their Tyrer-Cuzick-based risk compared with risk estimates by CRS. CONCLUSION: Integrating clinical and polygenic factors into a risk model offers more effective risk stratification and supports a personalized genomic approach to breast cancer screening and prevention.

Development and Validation of a Clinical Polygenic Risk Score to Predict Breast Cancer Risk.

PURPOSE: Women with a family history of breast cancer are frequently referred for hereditary cancer genetic testing, yet < 10% are found to have pathogenic variants in known breast cancer susceptibility genes. Large-scale genotyping studies have identified common variants (primarily single-nucleotide polymorphisms [SNPs]) with individually modest breast cancer risk that, in aggregate, account for considerable breast cancer susceptibility. Here, we describe the development and empirical validation of an SNP-based polygenic breast cancer risk score. METHODS: A panel of 94 SNPs was examined for association with breast cancer in women of European ancestry undergoing hereditary cancer genetic testing and negative for pathogenic variants in breast cancer susceptibility genes. Candidate polygenic risk scores (PRSs) as predictors of personal breast cancer history were developed through multivariable logistic regression models adjusted for age, cancer history, and ancestry. An optimized PRS was validated in 2 independent cohorts (n = 13,174; n = 141,160). RESULTS: Within the training cohort (n = 24,259), 4,291 women (18%) had a personal history of breast cancer and 8,725 women (36%) reported breast cancer in a first-degree relative. The optimized PRS included 86 variants and was highly predictive of breast cancer status in both validation cohorts (P = 6.4 × 10-66; P < 10-325). The odds ratio (OR) per unit standard deviation was consistent between validations (OR, 1.45 [95% CI, 1.39 to 1.52]; OR 1.47 [95% CI, 1.45 to 1.49]). In a direct comparison, the 86-SNP PRS outperformed a previously described PRS of 77 SNPs. CONCLUSION: The validation and implementation of a PRS for women without pathogenic variants in known breast cancer susceptibility genes offers potential for risk stratification to guide surveillance recommendations.

Association of a Polygenic Risk Score With Breast Cancer Among Women Carriers of High- and Moderate-Risk Breast Cancer Genes.

Importance: To date, few studies have examined the extent to which polygenic single-nucleotide variation (SNV) (formerly single-nucleotide polymorphism) scores modify risk for carriers of pathogenic variants (PVs) in breast cancer susceptibility genes. In previous reports, polygenic risk modification was reduced for BRCA1 and BRCA2 PV carriers compared with noncarriers, but limited information is available for carriers of CHEK2, ATM, or PALB2 PVs. Objective: To examine an 86-SNV polygenic risk score (PRS) for BRCA1, BRCA2, CHEK2, ATM, and PALB2 PV carriers. Design, Setting, and Participants: A retrospective case-control study using data on 150 962 women tested with a multigene hereditary cancer panel between July 19, 2016, and January 11, 2019, was conducted in a commercial testing laboratory. Participants included women of European ancestry between the ages of 18 and 84 years. Main Outcomes and Measures: Multivariable logistic regression was used to examine the association of the 86-SNV score with invasive breast cancer after adjusting for age, ancestry, and personal and/or family cancer history. Effect sizes, expressed as standardized odds ratios (ORs) with 95% CIs, were assessed for carriers of PVs in each gene as well as for noncarriers. Results: The median age at hereditary cancer testing of the population was 48 years (range, 18-84 years); there were 141 160 noncarriers in addition to carriers of BRCA1 (n = 2249), BRCA2 (n = 2638), CHEK2 (n = 2564), ATM (n = 1445), and PALB2 (n = 906) PVs included in the analysis. The 86-SNV score was associated with breast cancer risk in each of the carrier populations (P < 1 × 10-4). Stratification was more pronounced for noncarriers (OR, 1.47; 95% CI, 1.45-1.49) and CHEK2 PV carriers (OR, 1.49; 95% CI, 1.36-1.64) than for carriers of BRCA1 (OR, 1.20; 95% CI, 1.10-1.32) or BRCA2 (OR, 1.23; 95% CI, 1.12-1.34) PVs. Odds ratios for ATM (OR, 1.37; 95% CI, 1.21-1.55) and PALB2 (OR, 1.34; 95% CI, 1.16-1.55) PV carrier populations were intermediate between those for BRCA1/2 and CHEK2 noncarriers. Conclusions and Relevance: In this study, the 86-SNV score was associated with modified risk for carriers of BRCA1, BRCA2, CHEK2, ATM, and PALB2 PVs. This finding supports previous reports of reduced PRS stratification for BRCA1 and BRCA2 PV carriers compared with noncarriers. Modification of risk in CHEK2 carriers associated with the 86-SNV score appeared to be similar to that observed in women without a PV. Larger studies are needed to provide more refined estimates of polygenic modification of risk for women with PVs in other moderate-penetrance genes.