Multigene panel testing beyond BRCA1/2 in breast/ovarian cancer Spanish families and clinical actionability of findings

Abstract

Purpose: Few and small studies have been reported about multigene testing usage by massively parallel sequencing in European cancer families. There is an open debate about what genes should be tested, and the actionability of some included genes is under research.

Methods: We investigated a panel of 34 known high/moderate-risk cancer genes, including 16 related to breast or ovarian cancer (BC/OC) genes, and 63 candidate genes to BC/OC in 192 clinically suspicious of hereditary breast/ovarian cancer (HBOC) Spanish families without pathogenic variants in BRCA1 or BRCA2 (BRCA1/2).

Results: We identified 16 patients who carried a high- or moderate-risk pathogenic variant in eight genes: 4 PALB2, 3 ATM, 2 RAD51D, 2 TP53, 2 APC, 1 BRIP1, 1 PTEN and 1 PMS2. These findings led to increased surveillance or prevention options in 12 patients and predictive testing in their family members. We detected 383 unique variants of uncertain significance in known cancer genes, of which 35 were prioritized in silico. Eighteen loss-of-function variants were detected in candidate BC/OC genes in 17 patients (1 BARD1, 1 ERCC3, 1 ERCC5, 2 FANCE, 1 FANCI, 2 FANCL, 1 FANCM, 1 MCPH1, 1 PPM1D, 2 RBBP8, 3 RECQL4 and 1 with SLX4 and XRCC2), three of which also carry pathogenic variants in known cancer genes.

Conclusions: Eight percent of the BRCA1/2 negative patients carry pathogenic variants in other actionable genes. The multigene panel usage improves the diagnostic yield in HBOC testing and it is an effective tool to identify potentially new candidate genes.

Keywords: BRCA1 or BRCA2 negative; Breast/ovarian hereditary cancer; Clinical actionability; Panel testing by massively parallel sequencing.

Fig. 1

Sequencing platform, bioinformatics and variant prioritization pipeline

Fig. 2

Summary of prioritized and classified variants found in the 192 analysed patients. MAF minor allele frequency, NFE Non-Finnish European, ExAc Exome Aggregation Consortium, 1000G 1000 Genomes Project Data

Fig. 3

Results of in vitro mRNA analysis of five variants leading to aberrant splicing. Sequencing results are shown for the patient carrying the variant and a negative control. A red X illustrates the position of the variant. Nucleotide and exon numbering for cDNA is based on NCBI entries NM_000546.4 (TP53), NM_000535.5 (PMS2), NM_020937.3 (FANCM), NM_000123.3 (ERCC5) and NM_024596.3 (MCPH1)

Fig. 4

Qualitative and semi-quantitative cDNA analyses of the RNA effect caused by the five variants leading to aberrant splicing. a Capillary electrophoresis in a QIAxcel instrument of RT-PCR products from one carrier and controls shows different isoforms for all of the genes, except for FANCM, where the difference between the aberrant transcript and the full-length is only 1 bp and QIAxcel has not enough resolution to differentiate. For the sake of simplicity, only one out of the eight controls analysed is shown. PMS2 c.989-2 A>G variant analysis showed a common band in carrier and controls. After Sanger sequencing of whole amplified sample, we could not find out the identity of this presumed transcript (data not shown). Consequently, we cannot confirm if it is an alternative RNA form of PMS2 or an unspecific transcript. b Full-length (FL) relative amounts measured in the carrier (red dots) and in eight healthy controls (blue dots). Healthy controls mean is indicated in dotted line. Error bars indicate standard error of the mean (SEM). c Bar graphs showing the mean relative abundance (splicing fraction) of each transcript detected, indicated by different colours

Fig. 5

Distribution of unique variants according to sequenced and analysed nucleotides of genes associated with cancer risk other than BRCA1/2. Genes are ordered for analysed region size showing pathogenic variants, VUS “in silico” prioritized and VUS “in silico” no prioritized

Fig. 6

Patient distribution according to the variant classification. *Not included patients with pathogenic variants. **Not included patients with potentially pathogenic VUS in risk cancer-associated genes