To improve our understanding of the molecular basis of chemo-refractory high grade serous ovarian cancers (HGSOCs), we characterized the proteogenomic landscape of 242 (refractory and sensitive) HGSOCs, representing one discovery and two independent validation cohorts (spanning four academic centers) and utilizing two biospecimen types (formalin-fixed paraffin-embedded and frozen tumor biopsies). As the largest and most comprehensive study of refractory ovarian cancers to date, this effort identified a 64-protein signature that predicts a subset of HGSOCs refractory to initial platinum-based therapy with high specificity and is validated in two independent patient cohorts. We also detected significant association between lack of Ch17 loss-of-heterozygosity and refractory disease. Moreover, based on protein pathway expression, five novel clusters of HGSOC were identified and validated across two independent patient cohorts, as well as in patient-derived xenograft (PDX) models. These clusters may represent different mechanisms of refractoriness and implicate putative therapeutic targets.

As a technical validation, we designed, characterized and implemented an MRM assay targeting 84 peptides of intermediate abundance to 26 proteins, of which 70 peptides (to 22 proteins) targeted the predictor panel and 37 peptides (to 12 proteins) targeted the novel cluster panel (23 peptides to 8 proteins were in common across the two panels). The LLOQ, linearity, day-to-day reproducibility and stability of the assay was characterized.

The assay was applied to 102 individual HGSOC FFPE samples which were a subset of the original 158 HGSOC FFPE samples that were profiled in the discovery experiment. Results from the MRM assay were reported as the peak area ratio of the endogenous peptide to the heavy labeled peptide analog standard.