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Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
Simultaneous detection of BRCA mutations and large genomic rearrangements in germline DNA and FFPE tumor samples
Márton Zsolt Enyedi1, Gábor Jaksa2, Lajos Pintér2
1Institute of Genetics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged 6726, Hungary.
Abstract:
The development of breast and ovarian cancer is strongly connected to the inactivation of the BRCA1 and BRCA2 genes by different germline and somatic alterations, and their diagnosis has great significance in targeted tumor therapy, since recently approved PARP inhibitors show high efficiency in the treatment of BRCA-deficient tumors. This raises the need for new diagnostic methods that are capable of performing an integrative mutation analysis of the BRCA genes not only from germline DNA but also from formalin-fixed and paraffin-embedded (FFPE) tumor samples. Here we describe the development of such a methodology based on next-generation sequencing and a new bioinformatics software for data analysis. The diagnostic method was initially developed on an Illumina MiSeq NGS platform using germline-mutated stem cell lines and then adapted for the Ion Torrent PGM NGS platform as well. We also investigated the usability of NGS coverage data for the detection of copy number variations and exon deletions as a replacement of the conventional MLPA technique. Finally, we tested the developed workflow on FFPE samples from breast and ovarian cancer patients. Our method meets the sensitivity and specificity requirements for the genetic diagnosis of breast and ovarian cancers both from germline and FFPE samples.
Insights
A new next-generation sequencing method enables comprehensive BRCA1/BRCA2 mutation analysis in both germline DNA and FFPE tumor samples, crucial for targeted cancer therapies. This diagnostic approach aids in identifying BRCA-deficient tumors for effective treatment strategies.
Area of Science:
- Genetics
- Oncology
- Molecular Diagnostics
Background:
- BRCA1 and BRCA2 gene mutations are key drivers in breast and ovarian cancer development.
- Accurate diagnosis of BRCA mutations is vital for targeted therapy, particularly with PARP inhibitors effective in BRCA-deficient tumors.
- Current diagnostic methods require improvements for analyzing both germline and formalin-fixed, paraffin-embedded (FFPE) tumor samples.
Purpose of the Study:
- To develop and validate a novel methodology for integrative mutation analysis of BRCA1 and BRCA2 genes.
- To enable genetic diagnosis from both germline DNA and FFPE samples.
- To assess the utility of next-generation sequencing (NGS) for copy number variation and exon deletion detection.
Main Methods:
- Development of a diagnostic workflow utilizing next-generation sequencing (NGS) and custom bioinformatics software.
- Initial platform development on Illumina MiSeq, followed by adaptation to Ion Torrent PGM.
- Evaluation of NGS coverage data for detecting copy number variations and exon deletions, as an alternative to MLPA.
- Validation of the workflow on FFPE samples from breast and ovarian cancer patients.
Main Results:
- A sensitive and specific NGS-based method for BRCA1/BRCA2 mutation analysis was successfully developed.
- The methodology demonstrated effectiveness for both germline DNA and FFPE samples.
- NGS coverage data proved reliable for detecting copy number variations and exon deletions.
Conclusions:
- The developed NGS workflow provides a robust tool for the genetic diagnosis of breast and ovarian cancers.
- This method facilitates comprehensive BRCA gene analysis from FFPE samples, supporting personalized medicine approaches.
- The diagnostic approach meets clinical requirements for sensitivity and specificity in cancer genetic testing.
