BRCA1/2 somatic mutation detection in formalin-fixed paraffin embedded tissue by next-generation sequencing in Korean
Ahwon Lee1, Jun Kang2, Hyoungnam Lee2
1Department of Hospital Pathology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Cancer Research Institute, The Catholic University of Korea, Seoul, Republic of Korea.
Introduction:
The detection of BRCA1/2 mutations is important because PARP1 inhibitors are approved for germline and/or somatic BRCA-mutated advanced ovarian cancer. Next-generation sequencing (NGS) is increasingly used in clinical practice for BRCA1/2 mutations. The purpose of this study was to consider several conditions of NGS BRCA1/2 assay applicable to clinical laboratory tests, in particular for using formalin fixed paraffin embedded (FFPE) ovarian tissues.
Materials And Methods:
We selected 64 ovarian cancer patients and performed Oncomine™ BRCA assay using FFPE tissue. Effect of FFPE sample quality was analyzed by NGS quality parameters including deamination metric. Somatic variants were selected by removing germline variants of peripheral blood and interpreted as pathogenic, variants of unknown significance, and false positive.
Results:
We found a positive relationship between the number of variants over the deamination metric and FFPE age (P < 0.001) with a cutoff values of approximately 0.7 and 60 months, respectively. When comparing NGS results with Sanger sequencing, NGS misreported 3 of 15 variants using default parameters which were corrected after changing parameters. We detected somatic variants in eight patients and classified them into pathogenic (n = 3), VUS (n = 3) and false positive (n = 2).
Conclusions:
This study is important for improving BRCA1/2 mutation detection capabilities of NGS analytical pipelines and strategy to overcome their limitations using FFPE tissue in ovarian cancer patients.
Insights
Next-generation sequencing (NGS) can reliably detect BRCA1/2 mutations in formalin-fixed paraffin-embedded (FFPE) ovarian tissues. Optimizing NGS parameters improves accuracy for clinical use in ovarian cancer diagnostics.
Area of Science:
- Oncology
- Genetics
- Molecular Diagnostics
Background:
- BRCA1/2 mutations are crucial targets for PARP1 inhibitor therapy in advanced ovarian cancer.
- Next-generation sequencing (NGS) is a standard method for detecting BRCA1/2 mutations in clinical settings.
- Formalin-fixed paraffin-embedded (FFPE) tissues are commonly used but present challenges for molecular analysis.
Purpose of the Study:
- To evaluate the performance of an NGS-based BRCA1/2 assay using FFPE ovarian tissues.
- To identify optimal conditions for clinical laboratory testing of BRCA1/2 mutations in FFPE samples.
- To understand the impact of FFPE sample quality on NGS assay results.
Main Methods:
- Sixty-four ovarian cancer patients' FFPE tissues were analyzed using the Oncomine™ BRCA assay.
- NGS quality parameters, including the deamination metric, were assessed to evaluate FFPE sample quality.
- Somatic variants were identified by excluding germline variants from peripheral blood and classified into pathogenic, unknown significance (VUS), and false positive categories.
Main Results:
- A significant correlation was observed between the deamination metric, FFPE age, and the number of variants detected (P < 0.001).
- NGS analysis with default parameters misreported 3 out of 15 variants compared to Sanger sequencing, which were corrected by adjusting parameters.
- Somatic variants were identified in eight patients, classified as pathogenic (n=3), VUS (n=3), and false positive (n=2).
Conclusions:
- This study provides strategies to enhance BRCA1/2 mutation detection using NGS analytical pipelines with FFPE tissues.
- Optimizing NGS parameters is essential for accurate variant classification and overcoming FFPE-related limitations.
- The findings support the clinical utility of NGS for BRCA1/2 mutation testing in ovarian cancer patients using FFPE samples.
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