High risk of breast cancer in women with biallelic pathogenic variants in CHEK2.

PURPOSE: Compared to breast cancer risk genes such as BRCA2, ATM, PALB2, and NBN, no defined phenotype is currently associated with biallelic pathogenic variants (PVs) in CHEK2. This study compared the prevalence of breast and other cancers in women with monoallelic and biallelic CHEK2 PVs. METHODS: CHEK2 PV carriers were identified through commercial hereditary cancer panel testing (09/2013-07/2019). We compared cancer histories of 6473 monoallelic carriers to 31 biallelic carriers. Breast cancer risks were estimated using multivariate logistic regression and are reported as odds ratios (OR) with 95% confidence intervals (CI). RESULTS: Breast cancer frequency was higher among biallelic CHEK2 PV carriers (80.6%, 25/31) than monoallelic carriers (41.2%, 2668/6473; p < 0.0001). Biallelic carriers were more likely to be diagnosed at or before age 50 (61.3%, 19/31) and to have a second breast cancer diagnosis (22.6%, 7/31) compared to monoallelic carriers (23.9%, 1548/6473; p < 0.0001 and 8.1%, 523/6473; p = 0.0107, respectively). Proportionally more biallelic carriers also had any cancer diagnosis and > 1 primary diagnosis. Compared to women with no PVs, biallelic PV carriers had a higher risk of developing ductal invasive breast cancer (OR 8.69, 95% CI 3.69-20.47) and ductal carcinoma in situ (OR 4.98, 95% CI 2.00-12.35) than monoallelic carriers (OR 2.02, 95% CI 1.90-2.15 and OR 1.82, 95% CI 1.66-2.00, respectively). CONCLUSIONS: These data suggest that biallelic CHEK2 PV carriers have a higher risk for breast cancer, are more likely to be diagnosed younger, and to have multiple primary breast cancers compared to monoallelic carriers. Biallelic carriers also appear to have a higher risk of cancer overall. Therefore, more aggressive management may be appropriate for women with biallelic PVs in CHEK2 compared with current recommendations for monoallelic carriers.

Prevalence of Variant Reclassification Following Hereditary Cancer Genetic Testing.

Importance: Variant reclassification is an important component of hereditary cancer genetic testing; however, there are few published data quantifying the prevalence of reclassification. Objective: Retrospective cohort study of individuals who had genetic testing from 2006 through 2016 at a single commercial laboratory. Design, Setting, and Participants: A retrospective cohort of individuals who had genetic testing between 2006 and 2016 at a single commercial laboratory was assessed. Variants were classified as benign, likely benign, variant of uncertain significance, likely pathogenic, or pathogenic. Retrospective chart reviews were conducted for patients from the University of Texas Southwestern (UTSW) Medical Center. Exposures: Hereditary cancer genetic testing. Main Outcomes and Measures: Frequency of and time to amended reports; frequency and types of variant reclassification. Results: From 2006 through 2018, 1.45 million individuals (median [interquartile range] age at testing, 49 years [40.69-58.31 years], 95.6% women) had genetic testing, and 56.6% (n = 821 724) had a personal history of cancer. A total of 1.67 million initial tests were reported and 59 955 amended reports were issued due to variant reclassification. Overall, 6.4% (2868 of 44 777) of unique variants were reclassified. Reclassification to a different clinical category was rare among unique variants initially classified as pathogenic or likely pathogenic (0.7%, 61 of 9112) or benign or likely benign (0.2%, 15 of 8995). However, 7.7% (2048 of 26 670) of unique variants of uncertain significance were reclassified: 91.2% (1867 of 2048) were downgraded to benign or likely benign (median time to amended report, 1.17 years), 8.7% (178 of 2048) were upgraded to pathogenic or likely pathogenic variants (median time to amended report, 1.86 years). Because most variants were observed in more than 1 individual, 24.9% (46 890 of 184 327) of all reported variants of uncertain significance were reclassified. Conclusions and Relevance: Following hereditary cancer genetic testing at a single commercial laboratory, 24.9% of variants of uncertain significance were reclassified, which included both downgrades and upgrades. Further research is needed to assess generalizability of the findings for other laboratories, as well as the clinical consequences of the reclassification as a component of a genetic testing program.

Clinical Variant Classification: A Comparison of Public Databases and a Commercial Testing Laboratory.

BACKGROUND: There is a growing move to consult public databases following receipt of a genetic test result from a clinical laboratory; however, the well-documented limitations of these databases call into question how often clinicians will encounter discordant variant classifications that may introduce uncertainty into patient management. Here, we evaluate discordance in BRCA1 and BRCA2 variant classifications between a single commercial testing laboratory and a public database commonly consulted in clinical practice. MATERIALS AND METHODS: BRCA1 and BRCA2 variant classifications were obtained from ClinVar and compared with the classifications from a reference laboratory. Full concordance and discordance were determined for variants whose ClinVar entries were of the same pathogenicity (pathogenic, benign, or uncertain). Variants with conflicting ClinVar classifications were considered partially concordant if ≥1 of the listed classifications agreed with the reference laboratory classification. RESULTS: Four thousand two hundred and fifty unique BRCA1 and BRCA2 variants were available for analysis. Overall, 73.2% of classifications were fully concordant and 12.3% were partially concordant. The remaining 14.5% of variants had discordant classifications, most of which had a definitive classification (pathogenic or benign) from the reference laboratory compared with an uncertain classification in ClinVar (14.0%). CONCLUSION: Here, we show that discrepant classifications between a public database and single reference laboratory potentially account for 26.7% of variants in BRCA1 and BRCA2. The time and expertise required of clinicians to research these discordant classifications call into question the practicality of checking all test results against a database and suggest that discordant classifications should be interpreted with these limitations in mind. IMPLICATIONS FOR PRACTICE: With the increasing use of clinical genetic testing for hereditary cancer risk, accurate variant classification is vital to ensuring appropriate medical management. There is a growing move to consult public databases following receipt of a genetic test result from a clinical laboratory; however, we show that up to 26.7% of variants in BRCA1 and BRCA2 have discordant classifications between ClinVar and a reference laboratory. The findings presented in this paper serve as a note of caution regarding the utility of database consultation.

Update on pediatric cancer predisposition syndromes.

Hereditary cancer syndromes in children and adolescents are becoming more recognized in the field of pediatric hematology/oncology. A recent workshop held at the American Society of Pediatric Hematology/Oncology (ASPHO) 2012 Annual Meeting included several interactive sessions related to specific familial cancer syndromes, genetic testing and screening, and ethical issues in caring for families with inherited cancer risk. This review highlights the workshop presentations, including a brief background about pediatric cancer predisposition syndromes and the importance of learning about them for the practicing pediatric hematologists/oncologists. This is followed by a brief summary of the newly described cancer predisposition syndromes including Rhabdoid Tumor Predisposition Syndrome, Hereditary Paragangliomas and Pheochromocytoma Syndrome, and Familial Pleuropulmonaryblastoma Tumor Predisposition (DICER1) Syndrome. The next section covers genetic testing and screening for pediatric cancer predisposition syndromes. Ethical issues are also discussed including preimplantation genetic diagnosis or testing (PGD/PGT), suspicious lesions found on tumor screening, and incidental mutations discovered by whole genome sequencing. Finally, the perspective of a family with Li-Fraumeni Syndrome is shared.

Breast and colorectal cancer risks among over 6,000 CHEK2 pathogenic variant carriers: A comparison of missense versus truncating variants.

BACKGROUND AND AIMS: Heterozygous truncating pathogenic variants (PVs) in CHEK2 confer a 1.5 to 3-fold increased risk for breast cancer and may elevate colorectal cancer risks. Less is known regarding missense variants. Here we compared the cancer associations with truncating and missense PVs in CHEK2 across breast and colorectal cancer. METHODS: This was a retrospective analysis of 705,797 patients who received single laboratory multigene panel testing between 2013 and 2020. Multivariable logistic regression models determined cancer risk associated with CHEK2 variants as odds ratios (ORs) and 95% confidence intervals (CIs) after adjusting for age at diagnosis, cancer history, and ancestry. Breast and colorectal cancer analyses were performed using 6255 CHEK2 PVs, including truncating PVs (N = 4505) and missense PVs (N = 1750). RESULTS: CHEK2 PVs were associated with an increased risk of ductal invasive breast cancer (p < 0.001) and ductal carcinoma in situ (DCIS) (p < 0.001), with no statistically significant differences when truncating PVs (p < 0.001) and missense PVs (p < 0.001) were evaluated separately. All CHEK2 variants assessed conferred little to no risk of colorectal cancer. CONCLUSIONS: In our large cohort, CHEK2 truncating and missense PVs conferred similar risks for breast cancer and did not seem to elevate risk for colorectal cancer.

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.

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.

Assessment of in silico protein sequence analysis in the clinical classification of variants in cancer risk genes.

Missense variants represent a significant proportion of variants identified in clinical genetic testing. In the absence of strong clinical or functional evidence, the American College of Medical Genetics recommends that these findings be classified as variants of uncertain significance (VUS). VUSs may be reclassified to better inform patient care when new evidence is available. It is critical that the methods used for reclassification are robust in order to prevent inappropriate medical management strategies and unnecessary, life-altering surgeries. In an effort to provide evidence for classification, several in silico algorithms have been developed that attempt to predict the functional impact of missense variants through amino acid sequence conservation analysis. We report an analysis comparing internally derived, evidence-based classifications with the results obtained from six commonly used algorithms. We compiled a dataset of 1118 variants in BRCA1, BRCA2, MLH1, and MSH2 previously classified by our laboratory's evidence-based variant classification program. We compared internally derived classifications with those obtained from the following in silico tools: Align-GVGD, CONDEL, Grantham Analysis, MAPP-MMR, PolyPhen-2, and SIFT. Despite being based on similar underlying principles, all algorithms displayed marked divergence in accuracy, specificity, and sensitivity. Overall, accuracy ranged from 58.7 to 90.8% while the Matthews Correlation Coefficient ranged from 0.26-0.65. CONDEL, a weighted average of multiple algorithms, did not perform significantly better than its individual components evaluated here. These results suggest that the in silico algorithms evaluated here do not provide reliable evidence regarding the clinical significance of missense variants in genes associated with hereditary cancer.

High prevalence of mismatch repair deficiency in prostate cancers diagnosed in mismatch repair gene mutation carriers from the colon cancer family registry.

The question of whether prostate cancer is part of the Lynch syndrome spectrum of tumors is unresolved. We investigated the mismatch repair (MMR) status and pathologic features of prostate cancers diagnosed in MMR gene mutation carriers. Prostate cancers (mean age at diagnosis = 62 ± SD = 8 years) from 32 MMR mutation carriers (23 MSH2, 5 MLH1 and 4 MSH6) enrolled in the Australasian, Mayo Clinic and Ontario sites of the Colon Cancer Family Registry were examined for clinico-pathologic features and MMR-deficiency (immunohistochemical loss of MMR protein expression and high levels of microsatellite instability; MSI-H). Tumor MMR-deficiency was observed for 22 cases [69 %; 95 % confidence interval (CI) 50-83 %], with the highest prevalence of MMR-deficiency in tumors from MSH2 mutation carriers (19/23, 83 %) compared with MLH1 and MSH6 carriers combined (3/9, 33 %; p = 0.01). MMR-deficient tumors had increased levels of tumor infiltrating lymphocytes compared with tumors without MMR-deficiency (p = 0.04). Under the assumption that tumour MMR-deficiency occurred only because the cancer was caused by the germline mutation, mutation carriers are at 3.2-fold (95 % CI 2.0-6.3) increased risk of prostate cancer, and when assessed by gene, the relative risk was greatest for MSH2 carriers (5.8, 95 % CI 2.6-20.9). Prostate cancer was the first or only diagnosed tumor in 37 % of carriers. MMR gene mutation carriers have at least a twofold or greater increased risk of developing MMR-deficient prostate cancer where the risk is highest for MSH2 mutation carriers. MMR IHC screening of prostate cancers will aid in identifying MMR gene mutation carriers.

Genetics of Hirschsprung disease and anorectal malformations.

Hirschsprung disease (HD) and anorectal malformations (ARMs) result from alterations in hindgut development. It has long been recognized that both recur in families and thus result, at least in part, from genetic factors. Progress in the understanding of the genetic basis of HD has been made by the application of findings from genetic animal models of altered enteric nervous system development to human beings. Several genes have been shown to be important for human enteric nervous system development, and current work is progressing to identify genetic interactions that may explain the variable phenotype of HD. By contrast, understanding of the genetic factors underlying ARMs is much less developed. We and others have shown that genetic factors play an important role in the pathogenesis of ARMs, and many mouse genetic models suggest molecular pathways that may be altered in ARMs.