Germline Mutant Alleles Research Articles

Analysis of more than 400,000 women provides case-control evidence for BRCA1 and BRCA2 variant classification.

Clinical genetic testing identifies variants causal for hereditary cancer, information that is used for risk assessment and clinical management. Unfortunately, some variants identified are of uncertain clinical significance (VUS), complicating patient management. Case-control data is one evidence type used to classify VUS, and previous findings indicate that case-control likelihood ratios (LRs) outperform odds ratios for variant classification. As an initiative of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) Analytical Working Group we analyzed germline sequencing data of BRCA1 and BRCA2 from 96,691 female breast cancer cases and 303,925 unaffected controls from three studies: the BRIDGES study of the Breast Cancer Association Consortium, the Cancer Risk Estimates Related to Susceptibility consortium, and the UK Biobank. We observed 11,227 BRCA1 and BRCA2 variants, with 6,921 being coding, covering 23.4% of BRCA1 and BRCA2 VUS in ClinVar and 19.2% of ClinVar curated (likely) benign or pathogenic variants. Case-control LR evidence was highly consistent with ClinVar assertions for (likely) benign or pathogenic variants; exhibiting 99.1% sensitivity and 95.4% specificity for BRCA1 and 92.2% sensitivity and 86.6% specificity for BRCA2. This approach provides case-control evidence for 785 unclassified variants, that can serve as a valuable element for clinical classification.

Characterization of the zebrafish gabra1sa43718/sa43718 germline loss of function allele confirms a function for Gabra1 in motility and nervous system development

Mutation of the GABRA1 gene is associated with neurodevelopmental defects and epilepsy. GABRA1 encodes for the α1 subunit of the γ-aminobutyric acid type A receptor (GABAAR), which regulates the fast inhibitory impulses of the nervous system. Multiple model systems have been developed to understand the function of GABRA1, but these models have produced complex and, at times, incongruent data. Thus, additional model systems are required to validate and substantiate previous results. We sought to provide initial phenotypic analysis of a novel germline mutant allele. Our analysis provides a solid foundation for the future use of this allele to characterize gabra1 functionally and pharmacologically using zebrafish. We investigated the behavioral swim patterns associated with a nonsense mutation of the zebrafish gabra1 (sa43718 allele) gene. The sa43718 allele causes a decrease in gabra1 mRNA expression, which is associated with light induced hypermotility, one phenotype previously associated with seizure like behavior in zebrafish. Mutation of gabra1 was accompanied by decreased mRNA expression of gabra2, gabra3, and gabra5, indicating a reduction in the expression of additional α sub-units of the GABAAR. Although multiple sub-units were decreased, larvae continued to respond to pentylenetetrazole (PTZ), indicating that a residual GABAAR exists in the sa43718 allele. Proteomics analysis demonstrated that mutation of gabra1 is associated with abnormal expression of proteins that regulate synaptic vesicle fusion, vesicle transport, synapse development, and mitochondrial protein complexes. These data support previous studies performed in a zebrafish nonsense allele created by CRISPR/Cas9 and validate that loss of function mutations in the gabra1 gene result in seizure-like phenotypes with abnormal development of the GABA synapse. Our results add to the existing body of knowledge as to the function of GABRA1 during development and validate that zebrafish can be used to provide complete functional characterization of the gene.

Clinical implications of VUS reclassification in a single-centre series from application of ACMG/AMP classification rules specified for BRCA1/2

BackgroundBRCA1/2 testing is crucial to guide clinical decisions in patients with hereditary breast/ovarian cancer, but detection of variants of uncertain significance (VUSs) prevents proper management of carriers. The ENIGMA (Evidence-based...

BRCA1-specific machine learning model predicts variant pathogenicity with high accuracy.

Identification of novel BRCA1 variants outpaces their clinical annotation which highlights the importance of developing accurate computational methods for risk assessment. Therefore our aim was to develop a BRCA1-specific machine learning model to predict the pathogenicity of all types of BRCA1 variants and to apply this model and our previous BRCA2-specific model to assess BRCA variants of uncertain significance (VUS) among Qatari patients with breast cancer. We developed an XGBoost model that utilizes variant information such as position frequency and consequence as well as prediction scores from numerous in silico tools. We trained and tested the model with BRCA1 variants that were reviewed and classified by the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. In addition we tested the model's performance on an independent set of missense variants of uncertain significance with experimentally determined functional scores. The model performed excellently in predicting the pathogenicity of ENIGMA-classified variants (accuracy: 99.9%) and in predicting the functional consequence of the independent set of missense variants (accuracy: 93.4%). Moreover it predicted 2 115 potentially pathogenic variants among the 31 058 unreviewed BRCA1 variants in the BRCA exchange database. Using two BRCA-specific models we did not identify any pathogenic BRCA1 variants among those found in patients in Qatar but predicted four potentially pathogenic BRCA2 variants, which could be prioritized for functional validation.

Abstract P6-02-15: Don’t get lost in translation: Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) recommendations for reporting germline cancer susceptibility gene variants in 19 languages – breast cancer as a model

Abstract Genetic testing for cancer susceptibility is a cornerstone of precision cancer prevention and care. Major communication hurdles remain for the differently specialized professionals involved in the identification, counselling, and clinical management of at-risk individuals. This may be ascribed to gaps in the genetic/genomic literacy of health care providers and to an ambiguous lexicon used for variant description. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) international consortium endorses controlled terminology and a framework for interpretation and reporting of germline variants in cancer susceptibility genes (PMID: 30962250). However, for most ENIGMA affiliates a language other than English is used for written and verbal communication of genetic test results, potentially confounding local application of the published framework. The ENIGMA Clinical Working Group thus launched a Vocabulary Translation Project (VTP) to translate the ENIGMA recommendations into the various languages spoken by the membership. The VTP involved 65 ENIGMA members from 22 countries organized into 19 language-specific teams, covering Catalan, Chinese, Czech, Danish, Dutch, Finnish, French, Galician, German, Greek, Italian, Japanese, Malay, Norwegian, Polish, Portuguese, Spanish (Castilian), Swedish, and Tagalog. Excerpts from the original publication were selected for translation based on a majority consensus and included a glossary of terms and recommendations for interpreting and reporting germline sequence variants in (breast) cancer susceptibility genes. Using a two-step process, each team conducted the relevant translation followed by independent back-translation to English. The VTP proved useful to reappraise the reference text. It disclosed transnational issues, which prompted revision of the original source to emphasize that risk estimates and actionability were based on breast cancer as an exemplar. It also highlighted country-specific differences with regards to breast cancer risk assessment (e.g. different absolute/relative breast cancer risk cut points) and management. As a secondary outcome, via electronic survey of the participating teams we documented the perceived high value of the translation effort and its expected positive impact on more consistent clinical management of carrier individuals. The identified target audience encompasses medical geneticists, physicians of other specialties participating in multidisciplinary teams, genetic counselors, primary care physicians, as well as non-health care professionals, e.g. journalists and science communicators. The outreach program includes dissemination of the translations via local, regional, and especially national networks and their use for education and training purposes. Because French, Portuguese, and Spanish are widely used as official, co-official, or secondary languages, the reach of the VTP potentially extends to a greater number of countries and territories, mostly in Central and South America, Caribbean, and Africa. By moving a step forward towards terminological coherence across disciplines and borders, we will facilitate more precise delivery and clinical application of genetic test results for breast cancer predisposition. Our translated recommendations will improve interdisciplinary cross-talk and carriers’ awareness of the risks and implications associated with their status, contributing to more informed decision-making. We used breast cancer as a blueprint. Application of the model to other cancer types will require calibration on the cancer-specific absolute and relative risks. Citation Format: Arcangela De Nicolo, Diana M. Eccles, Sarah Louise Ariansen, Michela Biancolella, Miguel de la Hoya, Orland Diez, Hans Ehrencrona, Florentia Fostira, Tiara Hassan, Issei Imoto, Artur Kowalik, Fabienne Lesueur, Arjen R. Mensenkamp, Heli Nevanlinna, Joanne Ngeow, Edenir I. Palmero, Inge Søkilde Pedersen, Frances Que, Jana Soukupová, Yen Tan, Ana Vega, Amanda B. Spurdle, Paolo Radice. Don’t get lost in translation: Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) recommendations for reporting germline cancer susceptibility gene variants in 19 languages – breast cancer as a model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-02-15.

Gene-specific machine learning model to predict the pathogenicity of BRCA2 variants.

Background: Existing BRCA2-specific variant pathogenicity prediction algorithms focus on the prediction of the functional impact of a subtype of variants alone. General variant effect predictors are applicable to all subtypes, but are trained on putative benign and pathogenic variants and do not account for gene-specific information, such as hotspots of pathogenic variants. Local, gene-specific information have been shown to aid variant pathogenicity prediction; therefore, our aim was to develop a BRCA2-specific machine learning model to predict pathogenicity of all types of BRCA2 variants. Methods: We developed an XGBoost-based machine learning model to predict pathogenicity of BRCA2 variants. The model utilizes general variant information such as position, frequency, and consequence for the canonical BRCA2 transcript, as well as deleteriousness prediction scores from several tools. We trained the model on 80% of the expert reviewed variants by the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium and tested its performance on the remaining 20%, as well as on an independent set of variants of uncertain significance with experimentally determined functional scores. Results: The novel gene-specific model predicted the pathogenicity of ENIGMA BRCA2 variants with an accuracy of 99.9%. The model also performed excellently on predicting the functional consequence of the independent set of variants (accuracy was up to 91.3%). Conclusion: This new, gene-specific model is an accurate method for interpreting the pathogenicity of variants in the BRCA2 gene. It is a valuable addition for variant classification and can prioritize unreviewed variants for functional analysis or expert review.

BRCA1/2 NGS Somatic Testing in Clinical Practice: A Short Report.

High-grade serous ovarian carcinoma (HGSOC) is the most common subtype of all ovarian carcinomas. HGSOC harboring BRCA1/2 germline or somatic mutations are sensitive to the poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi). Therefore, detecting these mutations is crucial to identifying patients for PARPi-targeted treatment. In the clinical setting, next generation sequencing (NGS) has proven to be a reliable diagnostic approach BRCA1/2 molecular evaluation. Here, we review the results of our BRCA1/2 NGS analysis obtained in a year and a half of diagnostic routine practice. BRCA1/2 molecular NGS records of HGSOC patients were retrieved from our institutional archive covering the period from January 2020 to September 2021. NGS analysis was performed on the Ion S5™ System (Thermo Fisher Scientific, Waltham, MA, USA) with the Oncomine™ BRCA Research Assay panel (Thermo Fisher Scientific). Variants were classified as pathogenic or likely pathogenic according to the guidelines of the American College of Medical Genetics and Genomics by using the inspection of Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) and ClinVar (NCBI) databases. Sixty-five HGSOC patient samples were successfully analyzed. Overall, 11 (16.9%) out of 65 cases harbored a pathogenic alteration in BRCA1/2, in particular, six BRCA1 and five BRCA2 pathogenic variations. This study confirms the efficiency and high sensitivity of NGS analysis in detecting BRCA1/2 germline or somatic variations in patients with HGSOC.

BRIDGES, Breast Cancer Risk after Diagnostic Gene Sequencing, HORIZON2020

"Breast cancer affects more than 360,000 women per year in the EU and causes more than 90,000 deaths. Identification of women at high risk of the disease can lead to early detection or disease prevention through intensive screening, therapeutic and/or lifestyle preventive measures, or prophylactic surgery. Breast cancer risk is determined by a combination of genetic and lifestyle risk factors. The advent of next generation sequencing has opened the opportunity for testing in many disease genes, and diagnostic gene panel testing is being introduced in many EU countries. However, the cancer risks associated with most variants in most genes are unknown. This leads to a major problem in appropriate counselling and management of women undergoing panel testing. The BRIDGES and B-CAST projects are jointly building a knowledge base that will allow identification of women at high-risk of specific subtypes of breast cancer, through comprehensive evaluation of DNA variants in known and suspected breast cancer genes. The effort exploits the huge resources established through the Breast Cancer Association Consortium (BCAC) and ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles). Existing datasets will be expanded by sequencing all known breast cancer susceptibility genes in >100,000 breast cancer cases and controls from population-based studies. Risk factor and tumour genome data have been collected for 10,000 cases. Jointly, the data will allow us to generate a comprehensive risk model with unprecedented discriminative power, that can provide personalised risk estimates. We will develop online tools to aid the interpretation of gene variants and provide risk estimates in a user-friendly format, to help genetic counsellors and patients worldwide to make informed clinical decisions for risk management. We will evaluate the acceptability and utility of comprehensive gene panel testing in the clinical genetics context."

Abstract 1992: Improved somatic classification and detection of tumor heterogeneity using ctDNA based liquid biopsy compared to tumor-only tissue in colorectal cancer

Abstract Purpose Tumor cells and white blood cells shed DNA into the blood at different rates, resulting in easily distinguishable somatic and germline mutant allele frequency (MAF)s in plasma. Liquid biopsy (LBx) can robustly detect somatic and germline variants enabling paired germline-somatic evaluation. Previous comparisons of plasma and tissue samples mainly focused on somatic variant calls; we investigate the clinical utility of LBx by comparing somatic and germline variant agreement between matched plasma and tissue samples in colorectal cancer (CRC). Procedure Circulating free DNA was extracted from plasma of 25 treatment-naïve patients with stage III/IV CRC and tested using the GuardantOMNI™ (500 gene, 2.145Mb) panel for reporting SNVS, Indels, CNVs, fusions, MSI-High status, and tumor mutation burden. Somatic classification & status for SNVs and Indels is performed by a beta-binomial model that incorporates genomic context and MAF (Nance AACR 2018). Matched FFPE tissue was analyzed by whole Exome sequencing (WES). To determine variant-level agreement between plasma and tissue, results from both assays were restricted to the common panel space. For SNVs and Indels, agreement was defined as the presence of identical alterations at a given position. Digital droplet PCR (ddPCR) was performed to assess selected clinically relevant somatic variants which were detected only in plasma by GuardantOMNI . Data Summary ctDNA was detected in 23/25 (92%) samples after excluding variants commonly associated with clonal hematopoiesis. In the common panel space, 108 somatic mutations were detected only in plasma across the 23 plasma samples at an average MAF of 4.35%. For variants detected only in plasma, ddPCR was conducted on four clinically relevant mutations thus far; PIK3CA C420R, FBXW7 R505C, KRAS G12D, and TP53 R273C. GuardantOMNI reported these mutations at MAFs of 29.15%, 60.39%, 28.91%, and 49.95%, respectively; ddPCR detected these variants at 27.8%, 62.8%, 33.7%, and 51.6%, respectively. ddPCR assessment of additional lower MAF variants is ongoing. 225 variants called germline by GuardantOMNI were also called in tissue, however, 176 (78%) were called somatic in tissue. In plasma, these variants were easily distinguishable from the tumor fraction (TF), with an average MAF difference of 40% higher than the TF as estimated by Max MAF. Conclusion: GuardantOMNI panel determines somatic status by leveraging local genomic context of common germline MAFs that provides a holistic view to reflect tumor heterogeneity. The ddPCR confirmatory results suggest that tissue of origin&lt metastasis may attribute to the observed disagreement between plasma ctDNA and matched tissue. Data also suggest that LBx and multiple site tumor tissue biopsy can serve as an alternative cross reference for corroborating metastasis and therapeutically relevant genetic evolution. Citation Format: Zheng Feng, Dennis Merkle, Danyi Wang, Dennis O'Rourke, Allysia Mak, Juergen Scheuenpflug. Improved somatic classification and detection of tumor heterogeneity using ctDNA based liquid biopsy compared to tumor-only tissue in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1992.

Incorporation of semi-quantitative analysis of splicing alterations for the clinical interpretation of variants in BRCA1 and BRCA2 genes.

BRCA1 and BRCA2 (BRCA1/2) genetic variants that disrupt messenger RNAsplicing are commonly associated with increased risks of developing breast/ovarian cancer. The majority of splicing studies published to date rely on qualitative methodologies (i.e., Sanger sequencing), but it is necessary to incorporate semi-quantitative or quantitative approaches to accurately interpret the clinical significance of spliceogenic variants. Here, we characterize the splicing impact of 31 BRCA1/2 variants using semi-quantitative capillary electrophoresis of fluorescent amplicons (CE), Sanger sequencing and allele-specific assays. A total of 14 variants were found to disrupt splicing. Allelic-specific assays could be performed for BRCA1 c.302-1G>A and BRCA2 c.516+2T>A, c.1909+1G>A, c.8332-13T>G, c.8332-2A>G, c.8954-2A>T variants, showing a monoallelic contribution to full-length transcript expression that was concordant with semi-quantitative data. The splicing fraction of alternative and aberrant transcripts was also measured by CE, facilitating variant interpretation. Following Evidence-based Network for the Interpretation of Germline Mutant Alleles criteria, we successfully classified eight variants as pathogenic (Class 5), five variants as likely pathogenic (Class 4), and 14 variants as benign (Class 1). We also provide splicing data for four variants classified as uncertain (Class 3), which produced a "leaky" splicing effect or introduced a missense change in the protein sequence, that will require further assessment to determine their clinical significance.

BRCA Challenge: BRCA Exchange as a global resource for variants in BRCA1 and BRCA2.

The BRCA Challenge is a long-term data-sharing project initiated within the Global Alliance for Genomics and Health (GA4GH) to aggregate BRCA1 and BRCA2 data to support highly collaborative research activities. Its goal is to generate an informed and current understanding of the impact of genetic variation on cancer risk across the iconic cancer predisposition genes, BRCA1 and BRCA2. Initially, reported variants in BRCA1 and BRCA2 available from public databases were integrated into a single, newly created site, www.brcaexchange.org. The purpose of the BRCA Exchange is to provide the community with a reliable and easily accessible record of variants interpreted for a high-penetrance phenotype. More than 20,000 variants have been aggregated, three times the number found in the next-largest public database at the project’s outset, of which approximately 7,250 have expert classifications. The data set is based on shared information from existing clinical databases—Breast Cancer Information Core (BIC), ClinVar, and the Leiden Open Variation Database (LOVD)—as well as population databases, all linked to a single point of access. The BRCA Challenge has brought together the existing international Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium expert panel, along with expert clinicians, diagnosticians, researchers, and database providers, all with a common goal of advancing our understanding of BRCA1 and BRCA2 variation. Ongoing work includes direct contact with national centers with access to BRCA1 and BRCA2 diagnostic data to encourage data sharing, development of methods suitable for extraction of genetic variation at the level of individual laboratory reports, and engagement with participant communities to enable a more comprehensive understanding of the clinical significance of genetic variation in BRCA1 and BRCA2.

Genetic Testing and Clinical Management Practices for Variants in Non-BRCA1/2 Breast (and Breast/Ovarian) Cancer Susceptibility Genes: An International Survey by the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) Clinical Working Group.

To describe a snapshot of international genetic testing practices, specifically regarding the use of multigene panels, for hereditary breast/ovarian cancers. We conducted a survey through the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium, covering questions about 16 non-BRCA1/2 genes. Data were collected via in-person and paper/electronic surveys. ENIGMA members from around the world were invited to participate. Additional information was collected via country networks in the United Kingdom and in Italy. Responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers, but only six (PALB2, TP53, PTEN, CHEK2, ATM, and BRIP1) were tested regularly. US centers tested the genes most often, whereas United Kingdom and Italian centers with no direct ENIGMA affiliation at the time of the survey were the least likely to regularly test them. Most centers tested the 16 genes through multigene panels; some centers tested TP53, PTEN, and other cancer syndrome-associated genes individually. Most centers reported (likely) pathogenic variants to patients and would test family members for such variants. Gene-specific guidelines for breast and ovarian cancer risk management were limited and differed among countries, especially with regard to starting age and type of imaging and risk-reducing surgery recommendations. Currently, a small number of genes beyond BRCA1/2 are routinely analyzed worldwide, and management guidelines are limited and largely based on expert opinion. To attain clinical implementation of multigene panel testing through evidence-based management practices, it is paramount that clinicians (and patients) participate in international initiatives that share panel testing data, interpret sequence variants, and collect prospective data to underpin risk estimates and evaluate the outcome of risk intervention strategies.

Quantitative Analysis of BRCA1 and BRCA2 Germline Splicing Variants Using a Novel RNA-Massively Parallel Sequencing Assay

Clinical genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming widespread. However, the interpretation of variants of unknown significance (VUS) in HBOC genes, such as the clinically actionable genes BRCA1 and BRCA2, remain a challenge. Among the variants that are frequently classified as VUS are those with unclear effects on splicing. In order to address this issue we developed a high-throughput RNA-massively parallel sequencing assay—CloneSeq—capable to perform quantitative and qualitative analysis of transcripts in cell lines and HBOC patients. This assay is based on cloning of RT-PCR products followed by massive parallel sequencing of the cloned transcripts. To validate this assay we compared it to the RNA splicing assays recommended by members of the ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium. This comparison was performed using well-characterized lymphoblastoid cell lines (LCLs) generated from carriers of the BRCA1 or BRCA2 germline variants that have been previously described to be associated with splicing defects. CloneSeq was able to replicate the ENIGMA results, in addition to providing quantitative characterization of BRCA1 and BRCA2 germline splicing alterations in a high-throughput fashion. Furthermore, CloneSeq was used to analyze blood samples obtained from carriers of BRCA1 or BRCA2 germline sequence variants, including the novel uncharacterized alteration BRCA1 c.5152+5G>T, which was identified in a HBOC family. CloneSeq provided a high-resolution picture of all the transcripts induced by BRCA1 c.5152+5G>T, indicating it results in significant levels of exon skipping. This analysis proved to be important for the classification of BRCA1 c.5152+5G>T as a clinically actionable likely pathogenic variant. Reclassifications such as these are fundamental in order to offer preventive measures, targeted treatment, and pre-symptomatic screening to the correct individuals.

Abstract 4272: A novel approach to differentiation of somatic vs. germline variants in liquid biopsies using a betabinomial model

Abstract Background: A recent large study found that over 2% of advanced cancer patients have unidentified germline alterations found incidentally during next-generation sequencing (NGS) for targetable somatic alterations. However, tissue-based NGS cannot definitively distinguish germline from somatic mutations without comparison to normal tissue. Because somatic variants typically occur in plasma at allele fractions 1-2 orders of magnitude lower than germline, liquid biopsy often enables differentiation of incidental germline mutations. Not uncommonly, high tumor burden or allelic imbalance from copy number variation or loss of heterozygosity complicates simplistic differentiation based on assumption of germline 50% allele frequency. We developed a novel statistical method for discriminating between somatic and germline variants in deep sequencing data, and applied to data generated from the Guardant 360 targeted cfDNA assay. Our model dynamically incorporates prior knowledge of germline SNP variants and observed allele frequencies within a sample, and gains power in larger panels like the GuardantOmni 500-gene assay. Methods: Our method uses common heterozygous SNPs to model local germline allele count behavior, and calls variants somatic if they deviate significantly from the observed germline mutant allele fraction (MAF). A betabinomial model is well suited to this problem, because it models both the mean and variance of mutant allele counts at common SNPs. This is important since simpler methods like fixed MAF cutoffs or Poisson models may not represent the variance in molecule counts appropriately. Results: We evaluated our method against variants from 361 clinical samples that were manually annotated by reviewers with expertise in cancer genomics. Of the 11,679 heterozygous variants considered in these samples, 7,221 (62%) had two or more common SNPs in the same gene, so that a local germline heterozygous MAF could be estimated and the betabinomial model could be applied. Using the manual annotation as ground truth, we created ROC curves for a classifier based on the betabinomial model with varying p-value cutoffs, as well as a classifier based on MAF cutoffs. The AUC for the betabinomial model was 0.996, as compared with an AUC of 0.985 for MAF cutoffs. The ROC curve allowed us to choose a betabinomial p-value cutoff that maintained a similar level of specificity as a 15% MAF cutoff (99.2% for MAF cutoff, 99% for betabinomial), but with significantly increased sensitivity (89% for MAF cutoff, 97% for betabinomial). Conclusions: We have developed a novel method for differentiation of somatic versus germline variants in a single plasma sample. Identifying incidental germline alterations in advanced cancer patients may identify new opportunities to apply targeted therapy such as PARP inhibitors for BRCA1/2, and may also serve to alert physicians and their patients to familial risk. Citation Format: Tracy Nance, Elena Helman, Carlo Artieri, Jennifer Yen, Thomas P. Slavin, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. A novel approach to differentiation of somatic vs. germline variants in liquid biopsies using a betabinomial model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4272.

Identification of Eight Spliceogenic Variants in BRCA2 Exon 16 by Minigene Assays.

Genetic testing of BRCA1 and BRCA2 identifies a large number of variants of uncertain clinical significance whose functional and clinical interpretations pose a challenge for genetic counseling. Interestingly, a relevant fraction of DNA variants can disrupt the splicing process in cancer susceptibility genes. We have tested more than 200 variants throughout 19 BRCA2 exons mostly by minigene assays, 54% of which displayed aberrant splicing, thus confirming the utility of this assay to check genetic variants in the absence of patient RNA. Our goal was to investigate BRCA2 exon 16 with a view to characterizing spliceogenic variants recorded at the mutational databases. Seventy-two different BIC and UMD variants were analyzed with NNSplice and Human Splicing Finder, 12 of which were selected because they were predicted to disrupt essential splice motifs: canonical splice sites (ss; eight variants) and exonic/intronic splicing enhancers (four variants). These 12 candidate variants were introduced into the BRCA2 minigene with seven exons (14–20) by site-directed mutagenesis and then transfected into MCF-7 cells. Seven variants (six intronic and one missense) induced complete abnormal splicing patterns: c.7618-2A>T, c.7618-2A>G, c.7618-1G>C, c.7618-1G>A, c.7805G>C, c.7805+1G>A, and c.7805+3A>C, as well as a partial anomalous outcome by c.7802A>G. They generated at least 10 different transcripts: Δ16p44 (alternative 3’ss 44-nt downstream; acceptor variants), Δ16 (exon 16-skipping; donor variants), Δ16p55 (alternative 3’ss 55-nt downstream), Δ16q4 (alternative 5’ss 4-nt upstream), Δ16q100 (alternative 5’ss 4-nt upstream), ▾16q20 (alternative 5’ss 20-nt downstream), as well as minor (Δ16p93 and Δ16,17p69) and uncharacterized transcripts of 893 and 954 nucleotides. Isoforms Δ16p44, Δ16, Δ16p55, Δ16q4, Δ16q100, and ▾16q20 introduced premature termination codons which presumably inactivate BRCA2. According to the guidelines the American College of Medical Genetics and Genomics these eight variants could be classified as pathogenic or likely pathogenic whereas the Evidence-based Network for the Interpretation of Germline Mutant Alleles rules suggested seven class 4 and one class 3 variants. In conclusion, our study highlights the relevance of splicing functional assays by hybrid minigenes for the clinical classification of genetic variations. Hence, we provide new data about spliceogenic variants of BRCA2 exon 16 that are directly correlated with breast cancer susceptibility.

Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.

Abstract 1755: The fate of germline BRCA related mutations in breast tumor tissues

Abstract Background - aim: Germline mutation carriers in BRCA1 and related genes frequently develop triple-negative breast carcinomas (TNBC) that presumably preserve the inherited mutation. Herein, we examined the clinicopathologic features and prognostic impact of paired germline and tumor genotypes in this disease. Methods: We compared baseline germline and paraffin tumor genotype data (next generation [NGS] and Sanger sequencing) from 194 patients with operable TNBC who were treated with anthracyclines-taxanes based chemotherapy within adjuvant trials by our Group. Because tissue NGS did not target BRCA-related genes, we additionally interrogated the presence of germline mutations in tumor and non-cancerous breast tissues, in carriers with available tissue material (n=33). We validated all NGS germline mutations with Sanger sequencing and ensured matched germline/tumor identity with microsatellite testing. Results: We identified 50 (26%) germline mutation carriers, 39 in BRCA1, 4 in BRCA2 and the rest in RAD51C, BARD1, RAD50, NBN, MRE11A and BRIP1. BRCA1 carriers were younger as compared to non-BRCA1 carriers (median 42y.o. vs. 56y.o, respectively; p=0.020), premenopausal (p=0.014), and had lower nodal status (p<0.001). Somatic mutations were found in 136 tumors, TP53 in 113 (81%). The germline mutant allele was lost in 14/33 (42%) tumor and matched normal tissues (mut-LOH). Loss affected 12 BRCA1 mutations, among them the common pathogenic p.Q1756fsX74 (c.5266dupC) and p.G1738R (c.5212G>A), and 2 BRCA2 mutations. In one such tumor, the germline mutation was replaced by a somatic BRCA1 (p.Q1811X [c.5431C>T]). Tumors with mut-LOH were more often TP53 mutant as compared to those with preserved germline mutations, which had mutations in other genes as well (p=0.034). Overall though, TP53 mutations were not associated with germline status. Germline mutations and tumor TP53 mutations were not individually associated with patient disease-free survival (DFS) but interacted with each other: in non-carriers, TP53 status did not affect outcome; in carriers, those with mutant TP53 had unfavorable DFS compared to those with wild-type TP53 tumors (interaction p, multivariate analysis: 0.053). Conclusions: Germline BRCA-related TNBC may be distinguished into two groups with potentially different biological characteristics, those with preserved and lost inherited mutation. The interaction between germline status and tumor TP53 mutations may have treatment implications and seems worthy pursuing in larger studies. Combined germline/tumor genotype testing may be needed for TNBC patient classification, especially in the context of clinical trials. Citation Format: Vassiliki Kotoula, Florentia Fostira Fostira, Kyriaki Papadopoulou, Paraskevi Apostolou, Eleftheria Tsolaki, Georgios Lazaridis, Kyriaki Manoussou, Flora Zagouri Zagouri, Dimitrios Pectasides, Ioannis Vlachos, Ioannis Tikas, Sotiris Lakis, Irene Konstantopoulou, George Pentheroudakis, Helen Gogas, Pavlos Papakostas, Christos Christodoulou, Drakoulis Yannoukakos, George Fountzilas. The fate of germline BRCA related mutations in breast tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1755. doi:10.1158/1538-7445.AM2017-1755

Abstract 1450: Alternative splicing analysis identifies mutation hotspots in hereditary breast and ovarian cancer genes

Abstract Genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming increasingly widespread in the era of precision medicine. The implementation of next-generation sequencing (NGS) has resulted in an explosion of genetic data. While the majority of patients receive definitive results, germline unclassified variants with unknown function are regularly detected in thousands of patients. In particular, variants of unknown significance (VUS) in the HBOC susceptibility genes BRCA1 and BRCA2 pose a quandary to medical providers and patients because these genes are clinically actionable. A large percentage of VUS in BRCA1/2 are predicted to affect splicing. Previous efforts have focused on interrogating splicing VUS using low-throughput and/or imprecise techniques. Therefore, we developed and validated a method for using RT-PCR NGS to accurately and efficiently characterize germline splicing defects. The ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium recommends capillary electrophoresis and Sanger sequencing of subcloned transcripts as the gold standard to identify the BRCA1/2 transcripts that are present in patient samples. We followed the recommendations of the ENIGMA consortium and were able to largely replicate their results when we analyzed the cDNA of lymphoblastoid cell lines (LCLs) generated by the kConFab consortium from carriers of BRCA1 or BRCA2 variants known to be associated with splicing defects. In addition to LCLs, we analyzed RNA extracted from normal blood controls and normal breast tissue for the entire BRCA1 gene. We compared our capillary electrophoresis and Sanger sequencing results to our newly developed RT-PCR NGS assay. For this assay, we used RT-PCR NGS coupled to our custom in-house bioinformatics analysis to identify splicing events including exon skipping, novel intron insertion, and alternative 5’ donor and 3’ acceptor splicing sites. Capillary electrophoresis allowed us to roughly visualize the various transcripts present in our samples, though sequencing was needed to confidently identify the exact splicing event. Similar to Sanger sequencing, our RT-PCR NGS assay detected all major splicing events. Interestingly, we found that RT-PCR NGS allowed us to visualize more minor splicing events present in the samples due to our high coverage. We sequenced tens of thousands of reads with RT-PCR NGS, as opposed to Sanger sequencing, which was limited to low-throughput sequencing of single colonies containing subcloned RT-PCR products. In conclusion, RT-PCR NGS is a reliable high-throughput alternative to the gold-standard splicing assays. Our assay will greatly improve the interpretation of splicing VUS detected by clinical genetic tests for BRCA1/2. Citation Format: Suzette Farber-Katz, Vickie Hsuan, Jayne Hoo, Sitao Wu, Huy Vuong, Dong Xu, Hsiao-Mei Lu, Phillip Gray, Aaron Elliott, Rachid Karam. Alternative splicing analysis identifies mutation hotspots in hereditary breast and ovarian cancer genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1450. doi:10.1158/1538-7445.AM2017-1450

Assessment of the InSiGHT Interpretation Criteria for the Clinical Classification of 24 MLH1 and MSH2 Gene Variants.

Pathogenicity assessment of DNA variants in disease genes to explain their clinical consequences is an integral component of diagnostic molecular testing. The International Society for Gastrointestinal Hereditary Tumors (InSiGHT) has developed specific criteria for the interpretation of mismatch repair (MMR) gene variants. Here, we performed a systematic investigation of 24 MLH1 and MSH2 variants. The assessments were done by analyzing population frequency, segregation, tumor molecular characteristics, RNA effects, protein expression levels, and in vitro MMR activity. Classifications were confirmed for 15 variants and changed for three, and for the first time determined for six novel variants. Overall, based on our results, we propose the introduction of some refinements to the InSiGHT classification rules. The proposed changes have the advantage of homogenizing the InSIGHT interpretation criteria with those set out by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium for the BRCA1/BRCA2 genes. We also observed that the addition of only few clinical data was sufficient to obtain a more stable classification for variants considered as "likely pathogenic" or "likely nonpathogenic." This shows the importance of obtaining as many as possible points of evidence for variant interpretation, especially from the clinical setting.

Abstract 1859: Bioinformatic and experimental evaluation of regulatory variants in breast cancer susceptibility genes

Abstract Many germline DNA sequence variants in gene regulatory elements are associated with an increased risk of cancer. These include large genomic deletions of the promoter of the BRCA1 gene, through to single nucleotide variants in the promoter, UTRs and long-range enhancers of multiple other cancer related genes. Next generation sequence analysis of early onset and familial breast cancer cases is currently identifying an escalating number of variants in non-coding regions of the genome, however for most of these the significance in unknown. This project aims to determine the functional and clinical significance of breast cancer associated variants in regulatory regions of BRCA1 and BRCA2, as part of an international collaborative project arising from the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. To date over two hundred variants mapping to promoter, UTR and deep intronic regions have been identified in early onset or familial breast cancer patients that have no identifiable coding or splicing changes in BRCA1 or BRCA2. Through a pipeline of bioinformatics analyses using ENCODE datasets, these variants have been prioritized for experimental evaluation. This has lead to the identification of several variants that significantly alter the regulation of BRCA1 or BRCA2 expression, through mechanisms including altered binding of transcription factors and microRNAs. These studies will lay the groundwork for comprehensive statistical analyses, in which bioinformatics and experimental data is combined with clinical and genetic data to establish multifactorial risk prediction models that can ultimately be used in a clinical setting. Citation Format: Jan Sevcik, Leslie Burke, Gaetana Gambino, Brooke L. Brewster, Emma Tudini, Philip J. Whiley, Siranoush Manoukian, ENIGMA Consortium, Thomas van Overeem Hansen, Marta Santamariña Pena, Ana Vega, Maria A. Caligo, Paolo Radice, Paolo Peterlongo, Etienne Rouleau, Amanda B. Spurdle, Melissa A. Brown. Bioinformatic and experimental evaluation of regulatory variants in breast cancer susceptibility genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1859.