Molecular profiling of low grade serous ovarian tumours identifies novel candidate driver genes.

Low grade serous ovarian tumours are a rare and under-characterised histological subtype of epithelial ovarian tumours, with little known of the molecular drivers and facilitators of tumorigenesis beyond classic oncogenic RAS/RAF mutations. With a move towards targeted therapies due to the chemoresistant nature of this subtype, it is pertinent to more fully characterise the genetic events driving this tumour type, some of which may influence response to therapy and/or development of drug resistance. We performed genome-wide high-resolution genomic copy number analysis (Affymetrix SNP6.0) and mutation hotspot screening (KRAS, BRAF, NRAS, HRAS, ERBB2 and TP53) to compare a large cohort of ovarian serous borderline tumours (SBTs, n = 57) with low grade serous carcinomas (LGSCs, n = 19). Whole exome sequencing was performed for 13 SBTs, nine LGSCs and one mixed low/high grade carcinoma. Copy number aberrations were detected in 61% (35/57) of SBTs, compared to 100% (19/19) of LGSCs. Oncogenic RAS/RAF/ERBB2 mutations were detected in 82.5% (47/57) of SBTs compared to 63% (12/19) of LGSCs, with NRAS mutations detected only in LGSC. Some copy number aberrations appeared to be enriched in LGSC, most significantly loss of 9p and homozygous deletions of the CDKN2A/2B locus. Exome sequencing identified BRAF, KRAS, NRAS, USP9X and EIF1AX as the most frequently mutated genes. We have identified markers of progression from borderline to LGSC and novel drivers of LGSC. USP9X and EIF1AX have both been linked to regulation of mTOR, suggesting that mTOR inhibitors may be a key companion treatment for targeted therapy trials of MEK and RAF inhibitors.


Microdissection and DNA extraction
Matched normal DNA extracted from peripheral lymphocytes was used for comparison with all SBT cases except IC510, 8591 and 1408, where adjacent normal tissue was dissected from the tumour block. For the serous carcinomas, only 7/21 cases had matched normal DNA from lymphocyte available and sufficient quantities of normal tissue were not available.

Copy number data
Copy number analysis has previously been published for 24 of the SBTs 18 , indicated in supplementary  Table S1. Additional SNP6 data was obtained for 14 cases of LGSC and seven cases of high grade (grade 2-3) serous carcinoma (HGSC). Previously published raw SNP6 copy number data was obtained for four LGSCs and 33 HGSCs from Gorringe et al. (2010) 36 and for 316 HGSCs from The Cancer Genome Atlas (TCGA, 2011) 37 .

Copy number analysis
All samples were analysed using unpaired normalisation against a pool of reference samples consisting of all blood normals from the cohort. Samples with paired blood normals or stromal normals with no CNAs were also analysed using paired normalisation. Fraction of the genome altered (FGA) and intrachromosomal breakpoint counts were performed using the circular binary segmentation output (minimum of 10 probes) on total copy number in Partek. For these metrics regions < 1 Mb were removed to reduce the influence of germline copy number polymorphisms and noise.

p16 immunohistochemistry
The CDKN2A locus appears to be the primary target of the highly enriched copy number imbalance and loss events targeting the short arm of chromosome 9, accompanied by focal homozygous deletion events and truncating mutations. Immunohistochemical (IHC) staining for p16 INK4A was performed for 30 SBT,16 LGSC and 191 HGSC on TMAs, and 9 LGSC with whole sections; 26 of these cases have overlapping copy number data (Supplementary Table S1). p16 INK4A staining was typically found to be highly heterogeneous within each individual tumour of the SBT and LGSC cohorts, with ~40-50% of cases having strong patchy or weak diffuse staining (Supplementary Figures S1 and S2;  Supplementary Tables S4 and S5). There appeared to be a trend towards negative staining and homogeneous low level staining in the LGSCs compared to SBTs, and carcinomas with 9p loss had negative staining (Supplementary Table S5). In contrast 80% of HGSCs were scored with 100% positive or 100% negative staining (Supplementary Table S4). LGSC

Supplementary
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