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.

Keywords: BRCA1; BRCA2; CNV detection; FFPE tissue; Genetic testing; HBOC; NGS; Pathogenic germline mutations; Targeted capture-based NGS.

Fig. 1

Comparison of normalized coverage of targeted capture-based NGS and multiplex PCR-based NGS in control blood and diagnostic FFPE tumor samples. a Normalized coverage (y-axis) of targeted capture-based and multiplex PCR-based NGS at single-base resolution along all concatenated BRCA1/2 targets (x-axis) of five blood control samples (Additional file 3: Table S2, K1 to K5). The five blood samples are color-coded. The vertical green line indicates the end of BRCA1 targets and the start of the BRCA2 targets (target number is increasing from left to right which corresponds to five to three prime orientation of the gene). The horizontal green line displays normalized coverage of 1.0. All target exons are separated via gray dotted vertical lines. Selected target exons are marked. Exemplary, two randomly chosen amplicon dropouts are marked by a red arrow. b Normalized coverage (y-axis) of targeted capture-based and multiplex PCR-based NGS of FFPE samples from patient 1 to 15 (Additional file 3: Table S2, P01 to P15) at single-base resolution along all concatenated BRCA1/2 targets (x-axis) Samples P1 to P13 are color encoded in blue. Exemplary, randomly chosen capture target dropouts and amplicon dropouts are marked by a red arrow. The vertical green line indicates the end of the BRCA2 targets (target number is increasing from left to right which corresponds to five to three prime orientation of the gene) and the start of the BRCA1 targets (decreasing from left to right). The horizontal green line displays normalized coverage of 1.0. All target exons are separated by gray dotted vertical lines. Selected target exons are marked. Exemplary, two randomly chosen amplicon dropouts are marked with a red arrow. c To illustrate the advantages of the targeted capture-based protocol over the multiplex PCR-based approach for analysis of low-quality DNA the normalized coverages (y-axis) of targets of capture-based and multiplex PCR-based NGS of two FFPE samples (P 5 and P10) are displayed at single-base resolution along all concatenated BRCA1/2 targets (x-axis). Exemplary, two randomly chosen amplicon dropouts are marked with a red arrow

Fig. 2

Advantages of the modified targeted capture-based protocol over traditionally applied multiplex PCR-based NGS of two low quality DNA-samples extracted from FFPE tissues of patients P5 and P10. a In contrast to the modified targeted capture (TP)-based approach, pathogenic BRCA1 variants were missed by the multiplex PCR (MP)-based approach. b DNA concentration of FFPE samples of patients P1 to P13. The FFPE samples of P5 and P10 are marked by a red dot. c Delta ct values of DNA from FFPE samples of patients P1 to P13. The DNA of FFPE samples of P5 and P10 are marked by a red dot.d Zero-covered nucleotides occur only with the multiplex PCR-based protocol. e Low-covered nucleotides (< 500 with the multiplex PCR-based approach and < 30 with the targeted capture-based approach) occur only with the multiplex PCR-based protocol. f Artifacts only occur with the multiplex PCR-based approach. g Polymorphisms were detected via both approaches. In patient 10 two additional polymorphisms were found with the targeted capture-based protocol. h False-positive pathogenic BRCA variants were detected in both patients using the multiplex PCR-based technique

Fig. 3

Example of NGS-based CNV detection in BRCA1 and BRCA2. Illustration of CNV detection by panelcn.MOPS as performed for patient 21 using a blood (a) and a FFPE tumor sample (b). Regions (x-axis) are labelled with the individual BRCA-specific targets. The log2 values of the normalized read counts (RCs) of each sample are symbolized by black dots. InDels are highlighted. a Heterozygous deletion of exons 12 to 18 in BRCA1 in the blood sample (pathogenic germline variant). b LOH of a complete BRCA1 allele in the tumor of the same patient. The allele with detected loss of exons 12–18 remains present in the tumor. This is represented by the lower log2 ratios of corresponding BRCA1 targets. c The duplication of BCRA2 exons E4-E13 of one BCRA2 allele in the blood of patient 24. No tumor tissue was available

Fig. 4

Pedigree of two families fulfilling the criteria for hereditary breast and ovarian cancer. a In this family, all patients that had developed cancer were deceased. We identified a pathogenic BRCA2-mutation NM_000059.3:c.7879A > T,p.(Ile2627Phe) in paraffin-embedded normal tissue from individual 1 (P27). The counselee (2) did not inherit the mutation and could therefore be relieved from her concern to have inherited the genetic predisposition from her mother. b In this family, patient 2, suffering from breast cancer, refused genetic testing. The pathogenic BRCA2-mutation NM_000059.3:c.8167G>C, p.(Asp2723His) was identified in tumor tissue available from individual 1 (P28). Targeted analysis revealed that the daughter (3) also carried the mutation, confirming that the variant identified in tumor tissue from the mother was indeed a germline variant. The daughter was therefore included in a high risk breast- and ovarian cancer screening program