Ovarian cancer is the leading cause of death from gynaecological cancers in the Western world1. Next generation Sequencing (NGS) is quickly becoming a commonly used tool for analysis of mutations — both single nucleotide variants (SNVs) and indels — in genes associated with ovarian cancer.
The SureSeq™ Ovarian Cancer Panel has been developed with leading cancer experts and covers all coding exons of seven genes (Table 1). The panel allows detection of known and novel variants in tumour suppressor genes as well as genes involved in homologous repair to advance research into ovarian cancer treatment and for use in clinical trials to help the development of new targeted therapies.
* Exon examples not yet available
Mutations in certain genes can predispose an individual to develop cancer at some point during their lifetime. Screening for germline mutations in such genes allows research into familial risk of developing breast and ovarian cancer. On the other hand, assessment of somatic mutations in tumour samples can help research into drug response and the development of new therapies.
Various new generation drugs are being trialled to replace classical chemotherapy with the promise of improved efficacy and reduced side effects. One approach to select the right patients for clinical trials may be to test for somatic mutations within the tumour.
The SureSeq Ovarian Cancer Panel has been validated on DNA derived from FFPE tissue and whole blood to allow investigation of both germline and somatic mutations in ovarian cancer research.
Heterogeneous cancer samples pose significant challenges as alleles are likely to be present at a lower fraction than would be expected for standard germline variants. Samples typically contain a mixture of cancer and normal cells, moreover cancer can consist of several molecularly distinct clones. In order to detect alleles that contribute only a small percentage to the reads at any locus, a highly uniform and sensitive enrichment is required. Utilising hybridisation-based enrichment, the SureSeq Ovarian Cancer Panel delivers excellent run-to-run consistency and extremely uniform coverage across the whole region of interest to allow sensitive detection of variants present even at low variant allele fraction (VAF) (Table 1).
Table 1: Example mutations detected in FFPE clinical research samples using the SureSeq Ovarian Cancer Panel. The ability to detect VAFs as low as 1.13% gives added confidence in the variants being called and facilitates the exploration of tumour heterogeneity. Rows 1–4: low-frequency SNVs; rows 5–7: low-frequency indels. Samples kindly provided by Biopathology Department of Gustave Roussy, Villejuif, France.
Hybridisation-based enrichment is now well recognised as providing superior results over amplicon-based enrichment technology. To date, the protocol has required more DNA and the library preparation protocol has been longer and more complex. In combination with the OGT SureSeq Library Preparation Kit, these issues have been addressed. There are fewer hands-on steps, turnaround times have been significantly improved and the panel has been optimised to work with as little as 500 ng of DNA derived from FFPE* or whole blood.
Interpret — OGT’s powerful, standalone NGS data analysis package — is complimentary with the SureSeq Ovarian Cancer Panel and allows the conversion of FASTQ NGS files into an intuitive interactive report. The user friendly report allows for easy filtering of the variants without the need for additional in-house bioinformatics resource.
*For samples passing QC citeria.
Enrichment assay optimisation is a crucial step in ensuring accuracy and sensitivity of targeted sequencing. Where regions are poorly enriched, they will generate fewer sequencing reads. If a variant falls into a region not covered at all, or covered by only a few reads, that variant is likely to be missed. OGT’s expert bait design ensures efficient and more uniform capture of all targeted regions, so that all variants present can be called with maximum confidence (Figure 1). Uniform enrichment also allows proportionately lower sequencing depth to be used to identify low-frequency variants, potentially lowering sequencing costs and increasing sample throughput.
We were delighted with the performance of the SureSeq panel. It showed complete concordance with our other techniques, detecting all known mutations with excellent sensitivity down to 1% [MAF (minor allele frequency)], including, in one case, a JAK2 V617F mutation which was not detected by ddPCR due to a second mutation under the primer. The panel also demonstrated mutations in other genes in samples with low level JAK2 V617F and good correlation between allele frequencies and quantitative analysis by ddPCR. We are planning to adopt the panel in the near future.
Dr Anna Skowronska
R&D Scientist, Haemato-Oncology Team, West Midlands Regional Genetics Laboratory, Birmingham Women’s NHS Foundation Trust, UK