Purpose The TP53 tumor suppressor gene is mutated in >95% of high grade serous ovarian cancers. Detecting an autologous antibody response to TP53 might improve early detection. Experimental design An immunoassay was developed to measure TP53 autoantibody in sera from 378 cases of invasive epithelial ovarian cancer and in 944 age-matched healthy controls from the United States, Australia and the United Kingdom. Serial preclinical samples from cases and controls were also assayed from the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). Results Using a cut-off of 78 U/mL to achieve a specificity of 97.4%, TP53 autoantibody were elevated in 30% of 50 cases from MD Anderson, 21.3% of 108 cases from the Australian Ovarian Cancer Study and 21% of 220 cases from the UKCTOCS. Among 164 cases with rising CA125 detected with the UKCTOCS risk of ovarian cancer algorithm (ROCA), 20.7% had elevated TP53 autoantibody. In cases missed by the ROCA, 16% of cases had elevated TP53 autoantibody. Of the 34 ovarian cancer cases detected with the ROCA, TP53 autoantibody titers were elevated 11.0 months prior to CA125. In the 9 cases missed by the ROCA, TP53 autoantibody was elevated 22.9 months before cancer diagnosis. Similar sensitivity was obtained using assays with specific mutant and wild-type TP53. Conclusion TP53 autoantibody levels provide a biomarker with clinically significant lead time over elevation of CA125 or an elevated ROCA value. Quantitative assessment of autoantibodies in combination with CA125 hold promise for earlier detection of invasive epithelial ovarian cancer.

Point-of-care (POC) diagnostic platforms have the potential to enable low-cost, large-scale screening. As no single biomarker is shed by all ovarian cancers, multiplexed biomarker panels promise improved sensitivity and specificity to address the unmet need for early detection of ovarian cancer. We have configured the programmable bio-nano-chip (p-BNC) - a multiplexable, microfluidic, modular platform - to quantify a novel multimarker panel comprising CA125, HE4, MMP-7 and CA72-4. The p-BNC is a bead-based immunoanalyzer system with a credit-card-sized footprint that integrates automated sample metering, bubble and debris removal, reagent storage and waste disposal, permitting POC analysis. Multiplexed p-BNC immunoassays demonstrated high specificity, low cross-reactivity, low limits of detection suitable for early detection, and a short analysis time of 43 minutes. Day-to-day variability, a critical factor for longitudinally monitoring biomarkers, ranged between 5.4–10.5%, well below the biological variation for all four markers. Biomarker concentrations for 31 late-stage sera correlated well (R2 = 0.71 to 0.93 for various biomarkers) with values obtained on the Luminex® platform. In a 31 patient cohort encompassing early- and late-stage ovarian cancers along with benign and healthy controls, the multiplexed p-BNC panel was able to distinguish cases from controls with 68.7% sensitivity at 80% specificity. Utility for longitudinal biomarker monitoring was demonstrated with pre-diagnostic sera from 2 cases and 4 controls. Taken together, the p-BNC shows strong promise as a diagnostic tool for large-scale screening that takes advantage of faster results and lower costs while leveraging possible improvement in sensitivity and specificity from biomarker panels.

Objectives Longitudinal multi-marker combinations have the potential to improve sensitivity while maintaining the high specificity required for early detection of ovarian cancer. The use of multiple markers to improve sensitivity over CA125 in longitudinal algorithms for early ovarian cancer detection requires the selection of markers with optimal discriminatory power and low longitudinal variance relative to disease-initiated changes. Our objective was to identify a multi-marker panel suitable for ovarian cancer, where each individual marker has its own baseline, permitting longitudinal algorithm development. Materials and methods In this retrospective study, we measured CA125, HE4, MMP-7, CA72-4, CA19-9, CA15-3, CEA and s-VCAM concentrations using immunoassays in pre-treatment sera from 142 stage I ovarian cancer cases and 5 annual samples each from 217 healthy controls. Following random division into training and validation sets, all possible biomarker combinations were exhaustively explored using linear classifiers, to identify the panel with greatest sensitivity for stage I disease at a high specificity of 98%. To evaluate longitudinal performance of the individual markers, the within-person over time and the between-person coefficient of variation (CV) were estimated. Hierarchical modeling across women of log-concentrations enabled borrowing of information across subjects to moderate variance estimates, given the small number of observations per subject. Results The four marker panel comprising CA125, HE4, MMP-7 and CA72-4 performed with the highest sensitivity (83.2%) at 98% specificity. The within-person CVs were lower for CA125, HE4, MMP-7 and CA72-4 (15%, 25%, 25% and 21% respectively) compared to their corresponding between-person CV (49%, 20%, 35% and 84% respectively) indicating baselines in healthy volunteers. Following simple log-transformations, within-volunteer variation across volunteers was modeled with a normal distribution, permitting parsimonious hierarchical modeling. Conclusions The multiplex panel chosen is suitable for early detection of ovarian cancer and the individual markers have their own baseline permitting longitudinal algorithm development.

Early detection of ovarian cancer has the potential to impact mortality. A multi-modal screening strategy where rising CA125 values over time, analyzed with the risk of ovarian cancer algorithm (ROCA), triggers transvaginal sonography and possible surgery has high sensitivity and specificity, but still fails to detect the 20% of early stage cases that do not express CA125. Use of multiple biomarkers could detect cases missed by CA125. We have studied the sensitivity and lead time of a multi-marker panel (CA125, HE4, MMP-7 and CA 72–4) compared to CA125 alone. We used PRoBE design principles to select pre-clinical longitudinal specimens from 75 women (50 screen-positive, 25 screen-negative) who developed invasive epithelial ovarian cancer (3–5 serial specimens each) and 547 corresponding healthy controls (1–10 serial specimens each) from the ovarian cancer screening trial, UKCTOCS, in a blinded fashion. We measured the multi-marker concentrations in ultra-low serum volumes (16 μL) utilizing multiplexed bead-based immunoassays with low detection limits, high inter- and intra-assay precision, negligible cross-reactivity and good correlation with standard immunoassays. While, at least one of the complementary biomarkers rose with CA125 in 44% (22/50) of screen-positive cases, there was no advantage in lead time over CA125. Therefore, we developed single-marker longitudinal algorithms (ROCA-like) to determine the presence of a change point to distinguish between the cases and controls. Using these algorithms, at 98% specificity, HE4 and CA72–4 identified 16% (4/25) of screen-negative cases, while MMP-7 identified none. Taken together, HE4 and CA72–4 show promise as complementary biomarkers to CA125 for longitudinal screening.

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