Abstract B12: Targeted sequencing of ovarian clear cell carcinomas reveals intertumor heterogeneity and distinct clustering related to mutational load.
Authors: Jönsson Jenny-Maria , Arildsen Nicolai Skovbjerg , Ebbesson Anna , Westbom-Fremer Sofia , Måsbäck Anna , Malander Susanne , Nilbert Mef , Hedenfalk Ingrid 
Published: May 2016

Abstract
Objective: Ovarian clear cell carcinomas (OCCCs) constitute a rare ovarian cancer subtype. Despite relatively clear clinical features, OCCCs can be difficult to distinguish morphologically from other epithelial ovarian cancers. OCCC gene expression signatures capturing not only the morphology but also the biology of this tumor subtype have been described, and a previous study using comparative genomic hybridization showed that OCCCs are indeed heterogeneous. Still, only a few genes have been analyzed using gene sequencing techniques. In the present study we aimed to further characterize OCCCs and their histotype specific genetic changes using targeted sequencing of 60 well-established cancer genes. Increased knowledge about genetic aberrations in OCCCs may help to guide future development of targeted treatments and improve the outcome for these patients.
Methods: Formalin fixed, paraffin-embedded (FFPE) tumor tissue was collected from 11 primary OCCCs, diagnosed at the Skane University Hospital, Lund, Sweden (1998-2013). One sample was excluded due to poor DNA quality. For the remaining 10 samples, 600 ng genomic DNA/sample was used for single strand targeted DNA sequencing using the SureSeq™ solid tumor panel consisting of 60 key genes involved in cancer evolution, including ovarian cancer. The panel covers all coding exons in the included genes, and is validated for research use on FFPE samples. The publicly available ENSEMBL database was used to classify the mutations according to severity across the genes. Serious mutations, defined as major non-conservative amino acid changes, splice or frameshift variants or changes in stop codons, affecting single nucleotide variations (SNVs) or insertions or deletions of DNA bases (indels) were used for further analyses. The mutation call cut-off was set to 20% and the reading depth ≥300x, and unsupervised hierarchical clustering of the tumor samples was performed.
Results: &gt;99% of the target bases had a 30x coverage across all samples, and the mean target coverage was 534x. Using the 20% cut-off, approximately 300 mutations were detected in the 60 genes across the 10 tumor samples. Unsupervised hierarchical clustering revealed two distinct clusters related to mutational load. Mutations in genes previously reported to be affected in OCCCs, such as ARID1A and ERBB2, were identified. Whereas ARID1A was mainly affected in one cluster, mutations in genes signifying a highly malignant phenotype, such as BRCA1 and BRCA2 dominated in the other. Genes in signaling pathways often affected in malignant tumors, as e.g. the RAS pathway, however, were unaffected. Other mutations detected in this dataset were ZFHX3 and KMT2C. KMT2C was the only highly mutated gene across both clusters. The clustering pattern was not related to age of FFPE blocks or tumor cell content.
Conclusions: In the present study we show that OCCCs are genetically heterogeneous. The variation in mutation rate is in line with a previous publication, and points to differences in tumor biology that may be important for tumor behavior and thus patient outcome. The highly mutated genes may help in guiding trials of targeted treatments; the methyl transferase gene KMT2C, also known as MLL3, may potentially be one such target. Rearrangements of this gene are well-described in acute leukemias. Interestingly, KMT2C/MLL3 is also reported to be a mutational cancer driver gene in renal clear cell carcinomas according to the TCGA. Likewise, ZFHX3 has recently been reported in endometrial clear cell carcinomas. Despite the small size of this study it points to the well-established fact that ovarian cancer is not one single disease, and also confirms that even the rare OCCCs seem to harbor different biological features which may be of potential clinical importance.
Citation Format: Jenny-Maria Jönsson, Nicolai Skovbjerg Arildsen, Anna Ebbesson, Sofia Westbom-Fremer, Anna Måsbäck, Susanne Malander, Mef Nilbert, Ingrid Hedenfalk. Targeted sequencing of ovarian clear cell carcinomas reveals intertumor heterogeneity and distinct clustering related to mutational load. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B12.