The landscape of BRCA1 and BRCA2 large rearrangements in an international cohort of over 20 000 ovarian tumors identified using next-generation sequencing.

BACKGROUND: Approximately half of ovarian tumors have defects within the homologous recombination repair pathway. Tumors carrying pathogenic variants (PVs) in BRCA1/BRCA2 are more likely to respond to poly-ADP ribose polymerase (PARP) inhibitor treatment. Large rearrangements (LRs) are a challenging class of variants to identify and characterize in tumor specimens and may therefore be underreported. This study describes the prevalence of pathogenic BRCA1/BRCA2 LRs in ovarian tumors and discusses the importance of their identification using a comprehensive testing strategy. METHODS: Sequencing and LR analyses of BRCA1/BRCA2 were conducted in 20 692 ovarian tumors received between March 18, 2016 and February 14, 2023 for MyChoice CDx testing. MyChoice CDx uses NGS dosage analysis to detect LRs in BRCA1/BRCA2 genes using dense tiling throughout the coding regions and limited flanking regions. RESULTS: Of the 2217 PVs detected, 6.3% (N = 140) were LRs. Overall, 0.67% of tumors analyzed carried a pathogenic LR. The majority of detected LRs were deletions (89.3%), followed by complex LRs (5.7%), duplications (4.3%), and retroelement insertions (0.7%). Notably, 25% of detected LRs encompassed a single or partial single exon. This study identified 84 unique LRs, 2 samples each carried 2 unique LRs in the same gene. We identified 17 LRs that occurred in multiple samples, some of which were specific to certain ancestries. Several cases presented here illustrate the intricacies involved in characterizing LRs, particularly when multiple events occur within the same gene. CONCLUSIONS: Over 6% of PVs detected in the ovarian tumors analyzed were LRs. It is imperative for laboratories to utilize testing methodologies that will accurately detect LRs at a single exon resolution to optimize the identification of patients who may benefit from PARP inhibitor treatment.

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.

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.

Detection of large rearrangements in a hereditary pan-cancer panel using next-generation sequencing.

BACKGROUND: Healthcare providers increasingly use information about pathogenic variants in cancer predisposition genes, including sequence variants and large rearrangements (LRs), in medical management decisions. While sequence variant detection is typically robust, LRs can be difficult to detect and characterize and may be underreported as a cause for hereditary cancer risk. This report describes the outcomes of hereditary cancer genetic testing using a comprehensive strategy that employs next-generation sequencing (NGS) for LR detection, coupled with LR confirmation using repeat hybrid capture NGS, microarray comparative genomic hybridization (microarray-CGH), and/or multiplex ligation-dependent probe amplification (MLPA). METHODS: Sequencing and LR analysis were conducted in a consecutive series of 376,159 individuals who received clinical testing with a hereditary pan-cancer gene panel from September 2013 through May 2017. NGS dosage analysis was used to evaluate potential deletions or duplications, with controls in place to exclude pseudogene reads. Samples positive for a putative LR based on NGS were confirmed using a comprehensive approach that included targeted microarray-CGH and/or MLPA analysis, with further examination as needed to ascertain the nature of the LR. RESULTS: A total of 3461 LRs were identified and classified as a deleterious mutation (DM), suspected deleterious mutation (SDM) or variant of uncertain significance. Pathogenic LRs (DM/SDM) accounted for the majority of LRs (67.7%), the largest proportion of which were deletions (86.1%), followed by duplications (11.3%), insertions (1.8%), triplications (0.5%), and inversions (0.3%). Several cases presented illustrate that the laboratory approach employed here can ensure consistent identification and accurate characterization of LRs. In the absence of this comprehensive testing strategy, 9% of LRs identified in this testing population might have been missed, potentially leading to inappropriate medical management in as many as 210 individuals referred for hereditary cancer testing. CONCLUSIONS: These data show that copy number analysis using NGS coupled with confirmatory testing reliably detects and characterizes LRs. Further, LRs comprise a substantial proportion (7.2%) of pathogenic variants identified by the test. A robust and accurate LR identification strategy is an essential component of a high-quality genetic testing program, enabling clinicians to optimize patient medical management decisions.

Prevalence and characteristics of likely-somatic variants in cancer susceptibility genes among individuals who had hereditary pan-cancer panel testing.

Next-generation sequencing (NGS) hereditary pan-cancer panel testing can identify somatic variants, which exhibit lower allele frequencies than do germline variants and may confound hereditary cancer predisposition testing. This analysis examined the prevalence and characteristics of likely-somatic variants among 348,543 individuals tested using a clinical NGS hereditary pan-cancer panel. Variants showing allele frequencies between 10% and 30% were interpreted as likely somatic and identified in 753 (0.22%) individuals. They were most frequent in TP53, CHEK2 and ATM, commonly as C-to-T transitions. Among individuals who carried a likely-somatic variant and reported no personal cancer history, 54.2% (78/144) carried a variant in TP53, CHEK2 or ATM. With a reported cancer history, this percentage increased to 81.1% (494/609), predominantly in CHEK2 and TP53. Their presence was associated with age (OR=3.1, 95% CI 2.5, 3.7; p<0.001) and personal history of cancer (OR=3.3, 95% CI 2.7, 4.0; p<0.001), particularly ovarian cancer. Germline ATM pathogenic variant carriers showed significant enrichment of likely-somatic variants (OR=2.8, 95% CI 1.6, 4.9; p = 0.005), regardless of cancer status. The appearance of likely-somatic variants is consistent with clonal hematopoiesis, possibly influenced by cancer treatment. These findings highlight the precision required of diagnostic laboratories to deliver accurate germline testing results.

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.

Identification of pathogenic retrotransposon insertions in cancer predisposition genes.

Cancer risks have been previously reported for some retrotransposon element (RE) insertions; however, detection of these insertions is technically challenging and very few oncogenic RE insertions have been reported. Here we evaluate RE insertions identified during hereditary cancer genetic testing using a comprehensive testing strategy. Individuals who had single-syndrome or pan-cancer hereditary cancer genetic testing from February 2004 to March 2017 were included. RE insertions were identified using Sanger sequencing, Next Generation Sequencing, or multiplex quantitative PCR, and further characterized using targeted PCR and sequencing analysis. Personal cancer history, ancestry, and haplotype were evaluated. A total of 37 unique RE insertions were identified in 10 genes, affecting 211 individuals. BRCA2 accounted for 45.9% (17/37) of all unique RE insertions. Several RE insertions were detected with high frequency in populations of conserved ancestry wherein up to 100% of carriers shared a high degree of haplotype conservation, suggesting founder effects. Our comprehensive testing strategy resulted in a substantial increase in the number of reported oncogenic RE insertions, several of which may have possible founder effects. Collectively, these data show that the detection of RE insertions is an important component of hereditary cancer genetic testing and may be more prevalent than previously reported.

Detection of somatic variants in peripheral blood lymphocytes using a next generation sequencing multigene pan cancer panel.

Next Generation Sequencing (NGS) multigene panels, which are routinely used to assess hereditary cancer risk, can detect both inherited germline variants and somatic variants in cancer-risk genes. We evaluated the frequency and distribution of likely somatic Pathogenic and Likely Pathogenic variants (PVs) detected in >220,000 individuals who underwent clinical testing with a 25-gene panel between September 2013 and March 2016. Likely somatic PVs are defined as variants with NGS read frequencies from 10% to 30%. Overall, 137 (0.06%) individuals were identified as carrying likely somatic PVs, most commonly in TP53 (73), CHEK2 (27), and ATM (20). Among this group, a second PV with a NGS read frequency consistent with a germline variant within the same gene or a different gene on the panel was detected in 21 individuals (15.3%), which is similar to the detection rate in our general testing population. Likely somatic PVs accounted for 38.8% of all PVs in TP53. In comparison, likely somatic PVs accounted for <1% of PVs in most other genes. Likely somatic PVs were more frequently identified in older individuals (p < 0.001). Additional studies are ongoing to further investigate the incidence and clinical implications of somatic variants, enabling the appropriate medical management for these patients.

Design and validation of an oligonucleotide microarray for the detection of genomic rearrangements associated with common hereditary cancer syndromes.

BACKGROUND: Conventional Sanger sequencing reliably detects the majority of genetic mutations associated with hereditary cancers, such as single-base changes and small insertions or deletions. However, detection of genomic rearrangements, such as large deletions and duplications, requires special technologies. Microarray analysis has been successfully used to detect large rearrangements (LRs) in genetic disorders. METHODS: We designed and validated a high-density oligonucleotide microarray for the detection of gene-level genomic rearrangements associated with hereditary breast and ovarian cancer (HBOC), Lynch, and polyposis syndromes. The microarray consisted of probes corresponding to the exons and flanking introns of BRCA1 and BRCA2 (≈1,700) and Lynch syndrome/polyposis genes MLH1, MSH2, MSH6, APC, MUTYH, and EPCAM (≈2,200). We validated the microarray with 990 samples previously tested for LR status in BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, MUTYH, or EPCAM. Microarray results were 100% concordant with previous results in the validation studies. Subsequently, clinical microarray analysis was performed on samples from patients with a high likelihood of HBOC mutations (13,124), Lynch syndrome mutations (18,498), and polyposis syndrome mutations (2,739) to determine the proportion of LRs. RESULTS: Our results demonstrate that LRs constitute a substantial proportion of genetic mutations found in patients referred for hereditary cancer genetic testing. CONCLUSION: The use of microarray comparative genomic hybridization (CGH) for the detection of LRs is well-suited as an adjunct technology for both single syndrome (by Sanger sequencing analysis) and extended gene panel testing by next generation sequencing analysis. Genetic testing strategies using microarray analysis will help identify additional patients carrying LRs, who are predisposed to various hereditary cancers.

DNA binding sites for putative methylation boundaries in the unmethylated region of the BRCA1 promoter.

Changes in DNA methylation patterns are frequently observed in human cancers and are associated with a decrease in tumor suppressor gene expression. Hypermethylation of the BRCA1 promoter has been reported in a portion of sporadic breast tumours that correspond to a reduction in BRCA1 transcription and expression. Questions remain concerning the maintenance of methylation free zones in promoter regions of tumor suppressor genes in normal tissues. Sodium bisulfite based analysis of the BRCA1 promoter defines a methylation free zone in normal breast tissue that starts 650 bp upstream of the transcription start site and extends for 1.4 kb through most of the BRCA1 CpG island. We provide data implicating 2 proteins, Sp1 and CTCF, in the maintenance of this methylation-free zone. Both of these proteins have been shown to function as methylation boundaries in other genes. Four Sp1 sites have been identified in the BRCA1 promoter by gel shift assays. In vivo binding of Sp1 has been confirmed at 2 of these sites in the BRCA1 promoter and at 2 CTCF sites that flank the unmethylated region. Our data suggest that these DNA binding sites for Sp1 and CTCF may act as boundary elements that are important in maintaining genomic integrity by delineating the normal methylation free BRCA1 promoter. Inactivation or disruption of these boundaries may facilitate an epigenetic "hit", in this case DNA methylation, leading to BRCA1 downregulation and contributing to tumorigenesis, particularly the genesis of sporadic breast tumours.