Targeted capture-based NGS is superior to multiplex PCR-based NGS for hereditary BRCA1 and BRCA2 gene analysis in FFPE tumor samples

Abstract

Background: With the introduction of Olaparib treatment for BRCA-deficient recurrent ovarian cancer, testing for somatic and/or germline mutations in BRCA1/2 genes in tumor tissues became essential for treatment decisions. In most cases only formalin-fixed paraffin-embedded (FFPE) samples, containing fragmented and chemically modified DNA of minor quality, are available. Thus, multiplex PCR-based sequencing is most commonly applied in routine molecular testing, which is predominantly focused on the identification of known hot spot mutations in oncogenes. Methods: We compared the overall performance of an adjusted targeted capture-based enrichment protocol and a multiplex PCR-based approach for calling of pathogenic SNVs and InDels using DNA extracted from 13 FFPE tissue samples. We further applied both strategies to seven blood samples and five matched FFPE tumor tissues of patients with known germline exon-spanning deletions and gene-wide duplications in BRCA1/2 to evaluate CNV detection based solely on panel NGS data. Finally, we analyzed DNA from FFPE tissues of 11 index patients from families suspected of having hereditary breast and ovarian cancer, of whom no blood samples were available for testing, in order to identify underlying pathogenic germline BRCA1/2 mutations. Results: The multiplex PCR-based protocol produced inhomogeneous coverage among targets of each sample and between samples as well as sporadic amplicon drop out, leading to insufficiently or non-covered nucleotides, which subsequently hindered variant detection. This protocol further led to detection of PCR-artifacts that could easily have been misinterpreted as pathogenic mutations. No such limitations were observed by application of an adjusted targeted capture-based protocol, which allowed for CNV calling with 86% sensitivity and 100% specificity. All pathogenic CNVs were confirmed in the five matched FFPE tumor samples from patients carrying known pathogenic germline mutations and we additionally identified somatic loss of the second allele in BRCA1/2. Furthermore we detected pathogenic BRCA1/2 variants in four the eleven FFPE samples from patients of whom no blood was available for analysis. Conclusions: We demonstrate that an adjusted targeted capture-based enrichment protocol is superior to commonly applied multiplex PCR-based protocols for reliable BRCA1/2 variant detection, including CNV-detection, using FFPE tumor samples.

Germline and Somatic BRCA1/2 Mutations in 172 Chinese Women With Epithelial Ovarian Cancer

“…The fact that the distribution of somatic mutations was consistent with other Chinese studies (36,48) and with a Polish study using FFPE tissues from HGSC patients (52) could guarantee the quality of our sample preparation. It was suggested that an adjusted targeted capture-based enrichment protocol was superior to commonly applied multiplex PCRbased protocols for reliable BRCA1/2 variant detection, including CNV detection, using FFPE tumor samples (53). As previously reported, the prevalence of BRCA1/2 mutations in patients with peritoneal carcinoma or fallopian tube carcinoma is comparable to that in EOC patients (54).…”

Optimizing Genetic Workup in Pheochromocytoma and Paraganglioma by Integrating Diagnostic and Research Approaches

“…Furthermore, the enrichment-based design can be adapted to be used, not only for high-quality DNA, but also for low-quality and fragmented DNA from paraffin-embedded (FFPE) tumor tissues. [26]…”

“…Copy number variants (CNVs), for instance, are typically not detected by NGS-based variant calling [26]. We therefore applied a CNV detection approach based on the sequencing data generated by our custom panel in order to complement single nucleotide variant (SNV) detection from both cryo-conserved tumor samples and paraffin embedded tissues.…”

Evaluation of DNA and RNA quality from archival formalin‐fixed paraffin‐embedded tissue for next‐generation sequencing – Retrospective study in Japanese single institution

“…However, comprehensive analysis by NGS enables the sequencing results of many amplicons with a short base pair length at the same time, demonstrating that genetic analysis can also be performed on DNA extracted from FFPE tissues, and making it easier to introduce genetic analysis into pathological diagnosis. [21][22][23][24][25] Despite FFPE use, there is a debate of whether FFPE applicable to genetic analysis can maintain satisfactory quality by avoiding damage to nucleic acids, due to formalin fixation and degradation of nucleic acids based on long-term storage in a paraffin block. Visual fragmentation confirmation by electrophoresis and Ct value by quantitative PCR are used as quality parameters for nucleic acids.…”