Serum protein markers for early detection of ovarian cancer

Abstract

Early diagnosis of epithelial ovarian cancer (EOC) would significantly decrease the morbidity and mortality from this disease but is difficult in the absence of physical symptoms. Here, we report a blood test, based on the simultaneous quantization of four analytes (leptin, prolactin, osteopontin, and insulin-like growth factor-II), that can discriminate between disease-free and EOC patients, including patients diagnosed with stage I and II disease, with high efficiency (95%). Microarray analysis was used initially to determine the levels of 169 proteins in serum from 28 healthy women, 18 women newly diagnosed with EOC, and 40 women with recurrent disease. Evaluation of proteins that showed significant differences in expression between controls and cancer patients by ELISA assays yielded the four analytes. These four proteins then were evaluated in a blind cross-validation study by using an additional 106 healthy females and 100 patients with EOC (24 stage I/II and 76 stage III/IV). Upon sample decoding, the results were analyzed by using three different classification algorithms and a binary code methodology. The four-analyte test was further validated in a blind binary code study by using 40 additional serum samples from normal and EOC cancer patients. No single protein could completely distinguish the cancer group from the healthy controls. However, the combination of the four analytes exhibited the following: sensitivity 95%, positive predictive value (PPV) 95%, specificity 95%, and negative predictive value (NPV) 94%, a considerable improvement on current methodology.

Fig. 1.

Four tumor markers identified for which the RCA microarray data and ELISA data were completely concordant in classifying serum from normal and EOC individuals in the training cohort. Microarray data are expressed as median fluorescence intensity (mfl); ELISA data are expressed as average protein concentration (ng/ml).

Fig. 2.

Analyte concentration distributions in ELISA assays of leptin (graph 1), prolactin (graph 2), OPN (graph 3), and IGF-II (graph 4) from 106 normal (N) and 100 EOC (C) individuals. The average concentrations were as follows: leptin, 12 ng/ml (N) and 3 ng/ml (C); prolactin, 1 ng/ml (N) and 40 ng/ml (C); OPN, 11 ng/ml (N) and 49 ng/ml (C); and IGF-II, 716 ng/ml (N) and 350 ng/ml (C). No single assay can completely distinguish the cancer group from the normal group.

Fig. 3.

Four-analyte split-point analysis of serum from healthy women (Healthy) and women with EOC (Cancer). Correctly classified normal serum should have a score of 0 or 1, whereas samples from cancer patients have a score of 2, 3, or 4. False-positive and false-negative samples are readily detected.

Fig. 4.

Receiver operating characteristic (ROC) curves for each individual analyte and a combination of all four analytes. Marker 1, leptin; Marker 2, prolactin; Marker 3, OPN; Marker 4, IGF-II; Fisher, all four analytes using Fisher's linear discriminant analysis; Score, all four analytes using split-point analysis.