5542 Background: Given the low prevalence of ovarian cancer, a successful strategy for early detection will require both high sensitivity (>90%) and specificity (99.6%) to achieve a positive predictive value of 10%. The requisite specificity might be attained through a two stage strategy that utilizes an abnormal value for serum marker(s) to prompt the performance of transvaginal sonography (TVS) in a limited number (2–5%) of women, reducing the specificity required for serum markers to 95–98%. Optimal sensitivity might be achieved by combining CA125 with other biomarkers. Methods: A training set from MDACC that included serum samples from patients with epithelial ovarian cancer (22 stage I and 19 stage II), 40 with benign disease and 99 healthy individuals, was analyzed using the CA125II immunoassay and SELDI-TOF-MS protocols to measure 7 proteins [transthyretin, Apo-A1, transferrin, hepcidin, ß2M, CTAP3, and ITIH4]. Using normalized peak intensity data, statistical models were fit by logistic regression, followed by stepwise optimization of factors retained in the models determined by optimizing the Akaike Information Criterion. A validation set from the GOG included 136 stage I ovarian cancers, 140 benign pelvic masses and 50 healthy controls from the GOG supplemented with 124 healthy controls from MDACC. Results: In the training set analysis, CA125 (>35 U/mL) alone distinguished stage I patients from healthy women with 68 % sensitivity, the 7 biomarkers exhibited 50% sensitivity and a combination of CA125 and the panel of 7 biomarkers achieved a sensitivity of 80% at 98% specificity. The combination separated patients with stage I-II ovarian cancer from benign and normal samples with 85% sensitivity. In the validation set, CA125 (>35U/ml) exhibited a sensitivity of 79% for stage I, whereas the marker panel + CA125 produced a sensitivity of 87% at 95% specificity (P= 0.015, McNemar's test). The combined panel also detected 38% of benign lesions, but these should be further distinguished during a second stage of screening with TVS. Conclusions: Combining a panel of 7 proteomic markers with CA125 could provide a first step in a sequential two-stage strategy with TVS for early detection of ovarian cancer. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Vermillion, Inc. Fujirebio Diagnostics Inc Vermillion, Inc. Fujirebio Diagnostics Inc, Vermillion, Inc. Fujirebio Diagnostics Inc

5057 Background: Previously we discovered seven serum biomarkers: ITIH4, transthyretin, ApoA1, CTAPIII, hepcidin, transferrin, and SAA, for the detection of ovarian cancer. They are fragments, cleavages, or PTMs of circulating proteins that are difficult to measure by conventional immunoassays. Using optimzed mass spectrometry assays, we evaluated these biomarkers in a large-scale multi-center study. Methods: A total of 607 serum samples from five sites were analyzed using SELDI TOF-MS protocols optimized for the seven biomarkers. They included 234 women with benign gynecologic diseases, and 373 patients with invasive epithelial ovarian cancer (101 early stage, 231 late stage, and 40 stage unknown). Among them, 165 benigns and 228 cancers had a CA125 available at time of analysis. The median and quartiles of CA125 for benign, early stage, and late or unknown stage were 26/11/57 IU, 80/22/434 IU, and 234/40/1114 IU, respectively. The biomarkers were assessed individually using the Mann-Whitney U Test. A linear composite index was derived in an unsupervised fashion using data from one site and then calculated for the remaining data using the fixed formula. ROC curve analyses were performed on data from individual sites and all sites combined. Results: All seven biomarkers individually demonstrated statistically significant differentiating power, and the majority had p-value<0.00001. AUCs of the composite index in ROC analyses for the six sites were 0.602, 0.566, 0.821, 0.813, and 0.592 in detecting cancer at all stages from benign. On the combined data, the differences in AUC between the index and CA125 were not statistically significant for the detection of cancer at all stages (AUC=0.706 vs. 0.725) or early stages only (AUC=0.534 vs. 0.653). However, the index did better at the high-sensitivity range. At a fixed sensitivity of 86%, the specificity of the index was 34% (77/226) compared to CA125 at 26% (42/163). For early stage cases, at a fixed sensitivity of 84%, the specificity of the index was 24% (55/226) compared to CA125 at 14%% (22/163). Conclusions: We validated seven previously discovered biomarkers, individually and in combination. Prospective studies and assay development are under way to further characterize their clinical utility. [Table: see text]

Protein expression profiling has been increasingly used to discover and characterize biomarkers that can be used for diagnostic, prognostic or therapeutic purposes. Most proteomic studies published to date have identified relatively abundant host response proteins as candidate biomarkers, which are often dismissed because of an apparent lack of specificity. We demonstrate that 2 host response proteins previously identified as candidate markers for early stage ovarian cancer, transthyretin and inter-alpha trypsin inhibitor heavy chain 4 (ITIH4), are posttranslationally modified. These modifications include proteolytic truncation, cysteinylation and glutathionylation. Assays using Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS) may provide a means to confer specificity to these proteins because of their ability to detect and quantitate multiple posttranslationally modified forms of these proteins in a single assay. Quantitative measurements of these modifications using chromatographic and antibody-based ProteinChip&REG; array assays reveal that these posttranslational modifications occur to different extents in different cancers and that multivariate analysis permits the derivation of algorithms to improve the classification of these cancers. We have termed this process host response protein amplification cascade (HRPAC), since the process of synthesis, posttranslational modification and metabolism of host response proteins amplifies the signal of potentially low-abundant biologically active disease markers such as enzymes. &COPY; 2005 Wiley-Liss. Inc.