Abstract
Intense efforts are currently being directed toward profiling gene expression in the hope of developing better cancer markers and identifying potential drug targets. Here, we present a sensitive new approach for the identification of cancer signatures based on direct high-throughput reverse transcription-PCR validation of alternative splicing events. This layered and integrated system for splicing annotation (LISA) fills a gap between high-throughput microarray studies and high-sensitivity individual gene investigations, and was created to monitor the splicing of 600 cancer-associated genes in 25 normal and 21 serous ovarian cancer tissues. Out of >4,700 alternative splicing events screened, the LISA identified 48 events that were significantly associated with serous ovarian tumor tissues. In a further screen directed at 39 ovarian tissues containing cancer pathologies of various origins, our ovarian cancer splicing signature successfully distinguished all normal tissues from cancer. High-volume identification of cancer-associated splice forms by the LISA paves the way for the use of alternative splicing profiling to diagnose subtypes of cancer.
Highlights
Epithelial ovarian cancer is the second most common gynecologic cancer and the deadliest among gynecologic pelvic malignancies
These genes were entered into the layered and integrated system for splicing annotation (LISA) for the identification of alternative splicing events and experimental design
A transcript map for each gene was generated (Supplementary Fig. S1B) from publicly available mRNAs and ESTs, and sets of PCR primers and experiments were designed such that all putative exon-exon junctions and alternative splicing events were covered by at least two distinct PCR reactions
Summary
Epithelial ovarian cancer is the second most common gynecologic cancer and the deadliest among gynecologic pelvic malignancies. Inability to detect ovarian cancer at an early stage and its propensity for peritoneal metastasis are largely responsible for these low survival rates. The CA125 serum marker combined with transvaginal ultrasonography are the usual clinical tests offered to screen for early stages of ovarian cancer in high-risk populations. These tests are used as predictors of clinical recurrence of ovarian cancers, and to monitor response to anticancer therapy. Neither of these strategies, Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/)
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