Next-Generation Sequencing in Order to Better Characterize a BRCA Variant of Uncertain Significance

BRCA germline mutations are the most common predisposing factor in familial breast-ovarian cancer syndrome families. However, many screened patients are identified as harboring BRCA variants of uncertain significance (VUS), rather than carrying deleterious germline mutations [Calo et al.: Cancers 2010; 2:1644–1660]. While such VUSs are typically reclassified as benign polymorphisms, this may occur years after the VUS is first identified [Murray et al.: Genet Med 2011; 13; 998–1005]. Loss of heterozygosity (LOH) of BRCA is nearly always the gatekeeper event in inherited BRCA-related breast cancer and LOH of BRCA is rare in sporadic cancers [Osorio et al.: Int J Cancer 2002; 99:305–309]. Here, we describe a patient identified as carrying a germline BRCA VUS. Tumor next-generation sequencing (NGS) demonstrated a very high mutation allelic frequency for that BRCA VUS, consistent with LOH. This case illustrates that since BRCA LOH is the typical mechanism of transformation in inherited BRCA-related breast cancers, NGS might be used to suggest that the BRCA VUS is actually cancer predisposing in a particular family. As a result, this may help patients make more informed decisions regarding screening and prophylactic therapy, long before official reclassification of the VUS occurs.

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Genetic counseling referrals after next generation sequencing testing.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.1515 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. 1515-1515

Author(s):

Rafael Gonzalez ◽

Emma Ryan ◽

Catherine Watson ◽

Gloria Broadwater ◽

Noah D. Kauff ◽

...

Keyword(s):

Genetic Counseling ◽

Next Generation Sequencing ◽

Hereditary Cancer ◽

Germline Mutations ◽

Tumor Board ◽

Hereditary Cancer Syndrome ◽

Next Generation ◽

Cancer Syndrome ◽

Generation Sequencing ◽

Germline Testing

1515 Background: Next generation sequencing (NGS) testing of tumor tissue or blood is performed to identify ‘actionable’ mutations that might guide patient care. NGS testing might incidentally identify germline mutations associated with cancer syndromes. No distinction is made between germline and somatic alterations on NGS reports, thus confirmatory germline testing is required. In this quality improvement (QI) initiative, we evaluated the frequency of referrals to genetic counseling (GC) for patients with potentially heritable germline mutations identified through NGS testing. Methods: We generated a list of high-risk mutations (HRMs) which merit GC referral based on NCCN guidelines. NGS test results for 3,400 consecutive patients with solid tumor malignancies were reviewed by the molecular tumor board from 1/2014-9/2019 and were screened for pathogenic HRMs. Basic demographic, oncologic, and GC data were retrospectively abstracted for each patient. The outcomes of interest were the frequency of HRMs identified through NGS testing, the proportion of patients subsequently referred to GC, and the proportion of patients ultimately diagnosed with a hereditary cancer syndrome. Results: 472 individual patients (14%) had NGS testing with one or more HRM identified; 465 patients were evaluable which corresponded to 519 HRMs that were included in the analysis (Table). Malignancies included were gastrointestinal 199 (42.8%), lung 83 (17.8%), genitourinary/renal 56 (12.0%), breast 49 (10.5%), gynecologic 35 (7.5%), and other 43 (9.2%). 75 (16.1%) patients had germline testing prior to NGS testing. Of those patients without prior germline genetic testing, 62 (15.9%) were referred to GC, and 19 (4.9%) patients were diagnosed with a hereditary cancer syndrome. Conclusions: Tumor NGS testing identifies HRMs that may represent an undiagnosed heritable germline mutation. Providers ordering NGS tests should review results for HRMs, refer to GC when appropriate, and offer confirmatory germline testing for patients and their families. [Table: see text]

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Abstract 2178: Spectrum of pathogenic germline mutations in Chinese esophageal squamous cell carcinoma patients through next-generation sequencing

10.1158/1538-7445.am2021-2178 ◽

2021 ◽

Author(s):

Ruijun Cai ◽

Yating Zheng ◽

Wenzhuan Xie ◽

Mengli Huang

Keyword(s):

Squamous Cell Carcinoma ◽

Next Generation Sequencing ◽

Esophageal Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Germline Mutations ◽

Next Generation ◽

Generation Sequencing

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Design and validation of a next generation sequencing assay for hereditaryBRCA1andBRCA2mutation testing

PeerJ ◽

10.7717/peerj.2162 ◽

2016 ◽

Vol 4 ◽

pp. e2162 ◽

Cited By ~ 13

Author(s):

Hyunseok P. Kang ◽

Jared R. Maguire ◽

Clement S. Chu ◽

Imran S. Haque ◽

Henry Lai ◽

...

Keyword(s):

Next Generation Sequencing ◽

Copy Number Variants ◽

Next Generation ◽

Single Nucleotide Variants ◽

Cancer Syndrome ◽

Cancer Screen ◽

Inherited Cancer ◽

Increased Risk ◽

Large Rearrangements ◽

Generation Sequencing

Hereditary breast and ovarian cancer syndrome, caused by a germline pathogenic variant in theBRCA1orBRCA2(BRCA1/2) genes, is characterized by an increased risk for breast, ovarian, pancreatic and other cancers. Identification of those who have aBRCA1/2mutation is important so that they can take advantage of genetic counseling, screening, and potentially life-saving prevention strategies. We describe the design and analytic validation of the Counsyl Inherited Cancer Screen, a next-generation-sequencing-based test to detect pathogenic variation in theBRCA1andBRCA2genes. We demonstrate that the test is capable of detecting single-nucleotide variants (SNVs), short insertions and deletions (indels), and copy-number variants (CNVs, also known as large rearrangements) with zero errors over a 114-sample validation set consisting of samples from cell lines and deidentified patient samples, including 36 samples withBRCA1/2pathogenic germline mutations.

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Complexities of Next-Generation Sequencing in Solid Tumors: Case Studies

Journal of the National Comprehensive Cancer Network ◽

10.6004/jnccn.2020.7569 ◽

2020 ◽

Vol 18 (9) ◽

pp. 1150-1155

Author(s):

Alexandra O. Sokolova ◽

Brian H. Shirts ◽

Eric Q. Konnick ◽

Ginger J. Tsai ◽

Bernardo H.L. Goulart ◽

...

Keyword(s):

Next Generation Sequencing ◽

Case Studies ◽

Checkpoint Inhibitors ◽

Hereditary Cancer Syndrome ◽

Next Generation ◽

Cancer Syndrome ◽

Expert Review ◽

Tumor Boards ◽

Tumor Sequencing ◽

Generation Sequencing

With the promise and potential of clinical next-generation sequencing for tumor and germline testing to impact treatment and outcomes of patients with cancer, there are also risks of oversimplification, misinterpretation, and missed opportunities. These issues risk limiting clinical benefit and, at worst, perpetuating false conclusions that could lead to inappropriate treatment selection, avoidable toxicity, and harm to patients. This report presents 5 case studies illustrating challenges and opportunities in clinical next-generation sequencing interpretation and clinical application in solid tumor oncologic care. First is a case that dissects the origin of an ATM mutation as originating from a hematopoietic clone rather than the tumor. Second is a case illustrating the potential for tumor sequencing to suggest germline variants associated with a hereditary cancer syndrome. Third are 2 cases showing the potential for variant reclassification of a germline variant of uncertain significance when considered alongside family history and tumor sequencing results. Finally, we describe a case illustrating challenges with using microsatellite instability for predicting tumor response to immune checkpoint inhibitors. The common theme of the case studies is the importance of examining clinical context alongside expert review and interpretation, which together highlight an expanding role for contextual examination and multidisciplinary expert review through molecular tumor boards.

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Spectrum of Pathogenic Germline Mutations in Chinese Lung Cancer Patients through Next-Generation Sequencing

Pathology & Oncology Research ◽

10.1007/s12253-019-00771-5 ◽

2019 ◽

Vol 26 (1) ◽

pp. 109-114 ◽

Cited By ~ 1

Author(s):

Panwen Tian ◽

Xiangyang Cheng ◽

Zhengyi Zhao ◽

Yuzi Zhang ◽

Celimuge Bao ◽

...

Keyword(s):

Lung Cancer ◽

Next Generation Sequencing ◽

Cancer Patients ◽

Germline Mutations ◽

Next Generation ◽

Lung Cancer Patients ◽

Generation Sequencing

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Identification of a spectrum of germline mutations for hereditary diffuse gastric cancer in the Russian population by next-generation sequencing

Annals of Oncology ◽

10.1093/annonc/mdz247.152 ◽

2019 ◽

Vol 30 ◽

pp. v316

Author(s):

I.U. Efimova ◽

E. Ignatova ◽

I. Bykov ◽

A.I. Kalinkin ◽

A.S. Tanas ◽

...

Keyword(s):

Gastric Cancer ◽

Next Generation Sequencing ◽

Germline Mutations ◽

Russian Population ◽

Hereditary Diffuse Gastric Cancer ◽

Diffuse Gastric Cancer ◽

Next Generation ◽

Generation Sequencing

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A novel CHEK2 variant identified by next generation sequencing in an Indian family with hereditary breast cancer syndrome

Cancer Genetics ◽

10.1016/j.cancergen.2019.05.003 ◽

2019 ◽

Vol 235-236 ◽

pp. 13-17

Author(s):

Pratibha Bhai ◽

Renu Saxena ◽

Samarth Kulshrestha ◽

Ishwar Chander Verma

Keyword(s):

Breast Cancer ◽

Next Generation Sequencing ◽

Hereditary Breast Cancer ◽

Next Generation ◽

Cancer Syndrome ◽

Indian Family ◽

Generation Sequencing

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Next-generation sequencing of BRCA1 and BRCA2 genes for rapid detection of germline mutations in hereditary breast/ovarian cancer

PeerJ ◽

10.7717/peerj.6661 ◽

2019 ◽

Vol 7 ◽

pp. e6661 ◽

Cited By ~ 7

Author(s):

Arianna Nicolussi ◽

Francesca Belardinilli ◽

Yasaman Mahdavian ◽

Valeria Colicchia ◽

Sonia D’Inzeo ◽

...

Keyword(s):

Next Generation Sequencing ◽

Sanger Sequencing ◽

Germline Mutations ◽

Ion Torrent ◽

Next Generation ◽

Training Set ◽

Brca1 And Brca2 ◽

Ion Torrent Pgm ◽

Validation Set ◽

Generation Sequencing

Background Conventional methods used to identify BRCA1 and BRCA2 germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment. Methods We first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set). Results By minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel BRCA2 pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis. Discussion Our experience strongly supports that the Ion Torrent PGM technology in BRCA1 and BRCA2 germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches.

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