Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms

Robertson Mackenzie, Stefan Kommoss, Boris J. Winterhoff, Benjamin R. Kipp, Joaquin J. Garcia, Jesse Voss, Kevin Halling, Anthony Karnezis, Janine Senz, Winnie Yang, Elena-Sophie Prigge, Miriam Reuschenbach, Magnus Von Knebel Doeberitz, Blake C. Gilks, David G. Huntsman, Jamie Bakkum-Gamez, Jessica N. McAlpine, Michael S. Anglesio

Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4% of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be "RAS-pathway alteration negative", using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations. Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously. We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9%) and mucinous borderline tumors (MBOT) (92.3%). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8% and 11.5%, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68% of MC and as many as 20% of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell populations, while consistency of KRAS allelic frequency in both ERBB2 amplified and non-amplified regions suggests this mutation occurred in advance of the amplification event. Overall, the prevalence of RAS-alteration and striking co-occurrence of pathway "double-hits" supports a critical role for tumor progression in this ovarian malignancy. Given the spectrum of RAS-activating mutations, it is clear that targeting this pathway may be a viable therapeutic option for patients with recurrent or advanced stage mucinous ovarian carcinoma, however caution should be exercised in selecting one or more personalized therapeutics given the frequency of non-redundant RAS-activating alterations.