Abstract - Rebecca Whelan

New Insights on an Ovarian Cancer Biomarker Enabled by Bioanalytical Chemistry... and the Questions that Remain

Despite its essential role in the clinical management of ovarian cancer, the CA125 biomarker—located on the mucin protein MUC16—is still not completely understood.  Questions remain about MUC16’s function and structure, and the identity and location of the CA125 epitopes remain undefined.  The first part of this talk will report on our most recent efforts to map the epitopes of CA125.  Using E. coli expression, we isolated nine repeats from the putative antigenic domain of CA125.  Amino acid composition of recombinant repeats was confirmed by high-resolution mass spectrometry.  We characterized the binding of four antibodies—OC125, M11, “OC125-like,” and “M11-like”—to nine recombinant repeats using Western blotting, indirect ELISA, and localized surface plasmon resonance (SPR) spectroscopy.  Each recombinant repeat was recognized by a different combination of CA125 antibodies. OC125 and “OC125-like” antibodies did not bind the same set of recombinant repeats, nor did M11 and “M11-like” antibodies.  Characterization of the interactions between MUC16 recombinant repeats and CA125 antibodies will contribute to ongoing efforts to identify the CA125 epitopes and improve our understanding of this important biomarker.  In the second part of the talk, we report a revised model of the tandem repeat region of CA125 (MUC16) based on third-generation DNA sequencing.  Samples of mRNA were isolated from three cancer cell lines (Kuramochi, OVCAR-3, and OVCAR-5), reverse transcribed and amplified with primers flanking the putative tandem repeat domain of CA125 (MUC16).  The ~10 kilobase pair product was sequenced on an Oxford Nanopore platform and the derived sequence was aligned to the full-length sequence.  An immunologically active region comprising only 19 tandem repeats was sequenced from three cell lines and three primary ovarian tumors from patients with high-grade serous ovarian cancer.  Shorter cDNA transcripts, coding for versions of CA125 (MUC16) with fewer than 19 tandem repeats, were also detected in the patient samples and may result from alternative splicing.  Observations from bottom-up proteomics experiments are consistent with the revised model.  A more accurate model of MUC16 is a significant advance in the molecular-level understanding of this informative ovarian cancer biomarker.