Germline mutations in BRCA1 and BRCA2 are highly penetrant genetic susceptibility factors that predispose carriers to develop breast and ovarian cancer. Across different populations, an estimated 5 – 10% of breast cancer arises in individuals who inherit mutations in BRCA1 and BRCA2 genes. The protein products of these two genes play important functions in DNA homologous recombination repair (HRR). In normal cells, the HRR pathway is activated in response to DNA double-stranded breaks. However in BRCA-deficient cells, HRR is abrogated and therefore the more error-prone DNA repair mechanisms, such as single-strand annealing and nonhomologous end joining (NHEJ) are activated. These latter pathways are particularly sensitive to the DNA damaging effects of chemotherapy. HRR defect due to BRCA-deficiency can be exploited as a therapeutic strategy by the use of poly (ADP-ribose) polymerase (PARP) inhibitors which inhibit the PARP proteins commonly PARP1 and 2 that take part in base excision repair (BER). While HRR or BER pathway disruption on its own is not lethal, the combined defect in both pathways lead to cell death through synthetic lethality. In normal cells of carriers heterozygous for BRCA gene mutation, the remaining wild-type allele is active and the residual protein product produced can repair double-stranded breaks through HRR. As a result, the treatment of BRCA gene mutation carriers with PARP inhibitors is highly specific for cancer cells and should spare normal cells.
Clinical significance of genetic testing
Genetic testing for BRCA1 and BRCA2 mutations is indicated for patients who fulfill inclusion criteria for hereditary and high-risk breast and ovarian cancer, to be followed by family study for carrier detection in positive index patients. Any genetic testing must be performed with consent before testing. Of equal importance is the provision of genetic counseling before and after genetic testing. To mitigate the risk of breast and ovarian cancer, enhanced surveillance, prophylactic surgery and chemoprevention may be offered to mutation carriers. Also for the patient the documentation of BRCA1 and BRCA2 mutation status may be of prognostic significance. A recent large scale analysis shows that, among patients with invasive epithelial ovarian cancer, having a germline mutation in BRCA1 or BRCA2 is associated with improved 5-year overall survival, and that BRCA2 carriers shows the best prognosis. Furthermore, in ovarian cancer a mutated BRCA1 or BRCA2 status predicts for platinum sensitivity and response to PARP-1 inhibitor therapy.
Genetic testing by conventional methods
Based on the lower incidence of breast cancer in Asia cohorts, high-risk female patients satisfying the clinical criteria for genetic testing are generally defined as those who: 1) had at least one first- or second-degree relative with breast and / or ovarian cancer, regardless of age; 2) were less than 45 years of age at diagnosis; 3) had bilateral breast cancer; 4) had triple negative (TN) or medullary type pathology; 5) had at least one relative with cancers other than breast and ovarian cancer such as stomach and prostate that are known to be related to BRCA mutations; 6) male breast cancer; or 7) they were ovarian cancer patients with a family history of breast cancer. Since 2015, ovarian cancer patients without family history are also accepted.
The conventional mutation detection methodology is a combination of BRCA1 and BRCA2 full gene DNA sequencing and multiplex ligation dependent probe amplification (MLPA). Mutation detection is performed on genomic DNA extracted from peripheral blood samples and mutation analysis is performed by direct DNA sequencing of all coding exons of BRCA1 and BRCA2 and partial flanking intronic sequences. Sequencing results are compared with the reference DNA sequences using variant reporter software such as Mutation Surveyor and then reviewed manually. Computational analysis for potential cryptic splice site mutation is performed using splice site prediction programs when sequence changes were identified. All mutation and sequence variants are named according to the recommendations for the description of sequence variants of Human Genome Variation Society (HGVS). DNA sequencing is supplemented by MLPA to detect large deletions or rearrangements of the BRCA1 and BRCA2 genes.
An important observation is the detection of recurrent and founder BRCA mutations in the Chinese population, many of which are hitherto unreported. The most common is the founder mutation c.3109C>T, p.Q1037X of the BRCA2 gene. Significantly, genetic screening methods such as high-resolution melting study or mini-sequencing that were simple, rapid and economical may be employed upfront to focus the detection of these recurrent and founder mutations before proceeding to detailed genetic analysis.
The era of next-generation sequencing (NGS)
The advent of NGS or massively parallel sequencing allows an increase in both the capacity (more patient samples) and capability (more genes to the exome or even genome level) of DNA sequencing at a lower cost than conventional Sanger sequencing. Since November 2011, our laboratory has started to utilize microfluidic access array for PCR amplification of a gene panel (BRCA1, BRCA2, TP53 and PTEN) for 454 pyrosequencing on the Roche GS Junior. A total of 91 PCR primer pairs are used to target all exons of the 4 genes and 10-bp intron-exon boundaries (except downstream of BRCA1 exon 2 where 8 bp was covered) for amplification of genomic DNA samples. Access Array System (Fluidigm) was used to generate separate pools of 91 amplicons from 48 DNA samples per run. A 10-bp barcode nucleotide sequence is incorporated in both ends of each amplicon for sample identification. Resulting amplicon pools of on average 12 patients are further pooled for sequencing library preparation. Each library is subjected to multiple sequencing runs of 454 GS Junior system so that each nucleotide position in target regions is sequenced at a minimum depth of 30-fold. It is also equivalent to the average depth of at least 150-fold. Since 2014, the NGS workflow has migrated from Roche GS Junior to Illumina MiSeq benchtop genome sequencer, for which the minimum depth is 50-fold and the average depth is 300-fold.
A robust and properly validated bioinformatics pipeline is the key to success in NGS diagnostics. In Roche GS Junior, sequencing reads are assigned to corresponding samples according to sequence barcodes using Amplicon Variant Analyzer (AVA). Reads are then aligned to reference sequence by AVA. Each patient-specific BAM sequence alignment was processed by three variant callers: 1) AVA using default settings, and 2) SAMtools and 3) an in-house algorithm for homopolymer variant detection. Multiplicity is essential and therefore the adoption of more than one variant caller (including in-house scripts) is useful. Variant effect on corresponding protein coding genes is annotated using Ensembl Variant Effect Predictor. Annotated variants are automatically prioritized for manual review according to standardized laboratory criteria Variants are designated according to the recommendations from HGVS. Putative mutations are validated by bi-directional Sanger sequencing. All patient samples subjected to massively parallel sequencing are simultaneously examined for large genomic rearrangement of BRCA1 and BRCA2 by MLPA. For MiSeq, BWA-MEM and variant callers SAMtools and GATK HaplotypeCaller are adopted.
Another important aspect of NGS is appropriate variant interpretation. Searching through the literature and various database can be time consuming. To characterize missense variants of unknown significance (VUS) identified in our patient cohort, the allele is checked against 107 healthy local individuals and 1000 Genomes project samples, and analyzed by in-silico prediction methods including PolyPhen and SIFT. Segregation of VUS with cancer phenotype is documented by family study as far as possible.
Among 948 high-risk breast and/or ovarian patients who are subject to genetic testing by NGS in Hong Kong, the prevalence of BRCA1 and BRCA2 germline mutations was 9.4% in our Chinese cohort, of which 48.8% of the mutations arose from hotspot mutations. The frequencies of PTEN and TP53 were 0.21% and 0.53% respectively. High-throughput NGS approach allows the incorporation of control cohort that provides an ethnicity-specific data for polymorphic variants. Our data suggest that hotspot mutations screening such as SNaPshot or mini-sequencing can be an effective preliminary screening alternative adopted in a standard clinical laboratory without NGS setup.
Recent studies from large consortium have resulted in the identification of additional breast cancer susceptibility loci through candidate gene or whole genome approaches. While familial breast cancer comprises approximately 20 – 30% of all breast cancers, the two major high penetrance genes BRCA1 and BRCA2 associated with hereditary breast and ovarian syndromes explain less than 10% of all breast cancer cases. Mutations in CHEK2 contribute to a substantial fraction of familial breast cancer. Carriers of TP53 mutations develop Li-Fraumeni syndrome and are at high risk of developing early-onset breast cancer, but these mutations are very rare. Susceptibility alleles in other genes such as PTEN, ATM, STK11/LKB1, MSH2/MLH1, BRIP1 and PALB2 are also rare causes of inherited breast cancer. Nevertheless, around half of the familial clustering of breast cancer remains unexplained. The susceptibility to breast cancer in this group is presumed to be due to additional but hitherto unidentified high-penetrance genes or variants at many moderate to low-penetrance loci each conferring a moderate risk of disease. The NGS approach can be harnessed to provide comprehensive gene panel testing that extends beyond BRCA mutations, especially for those high risk but BRCA negative breast cancer patients. It should be borne in mind that may only provide the information on genetic risk but not all the answers to the patient since the standard of care for many of these rare genetic susceptibility genes awaits more evidence for clinical practice to be generated.
PARP inhibitors induce synthetic lethality in tumours with HRR defect due to loss of function BRCA gene mutations. Irrespective of whether the origin of the BRCA mutation is germline or somatic, tumours in patients with a BRCAmutation should be sensitive to PRAP inhibition because of the loss of function of the gene within the tumour cells. The NGS approach can be applied to germline or somatic testing or both. Olaparib is a potent oral PARP inhibitor that has demonstrable anti-tumour activity in clinical trials of patients with BRCA-mutated or sporadic high-grade serous ovarian cancer. Also PARP inhibitors may have a wider application in the treatment of cancers defective in DNA damage repair pathway such as breast, prostate, endometrial and pancreatic cancer. Due to the therapeutic implication, testing for BRCAmutation status in breast and ovarian cancer is in high demand and the NGS is going to be the method of choice in this situation.