Sahasrabudhe, Deepak M.

11 Background: Currently there are no serum markers of response to AA. We performed a proteome based analysis of serum from prostate cancer patients, followed by mapping of candidate markers to networks based on known molecular interactions in Ingenuity Knowledge Base (IKB). Methods: Isobaric mass tags for relative and absolute quanititation (iTRAQ) analyzed by reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) was performed in serum of 3 non-localized prostate cancer cohorts. The first cohort included 15 paired untreated hormone-sensitive “pre AA” and 3-month “post AA” specimens; the second included 10 “early AA failure” (median failure time: 11 months) and the third included 10 “late AA failure” specimens (median failure time: 95 months). Differentially expressed candidate proteins were identified by comparisons of (i) paired pre/post AA proteomes and (ii) post AA proteome with the combined AA failure cohorts at a False Discovery Rate of 0.2. To facilitate biological interpretation of multiple candidates identified in the comparisons, IPA was used. Candidate markers implicated in IPA networks with statistical significance were pursued in a second, independent patient cohort for association with time to AA failure using univariate Cox regression analysis. Results: Median PSA for pre/post AA first cohort were 3.15 and 0.29 ng/ml. Median PSA for the second and third cohorts were 27.3 and 4.3 ng/ml. Between post AA and AA failure cohorts, 149 proteins were differentially expressed. Between early and late AA failure 98 proteins were differentially expressed; 47 proteins were common in both comparisons. Network enrichment analysis of the 47 proteins by IPA identified four interaction networks (p<0.01), one of which highlighted a role for 17-β-estradiol (E1). Gas chromatography used for measuring 3-month post AA initiation serum E1, estrone (E2) and testosterone levels (N=38) detected high E1, E2 levels associated with longer time to AA failure (P=0.07 for E1; P=0.08 for E2) in the independent cohort. Conclusions: A global proteomic analysis identified post AA initiation serum E1, E2 levels as potential response markers, which needs validation.

104 Background: Currently there are no serum predictive markers of response to AA. We used a proteomic based analytic approach to identify candidates. Methods: Serum from three non-localized prostate cancer cohorts was analyzed. The first included15 paired untreated hormone-sensitive “pre-AA” and 3-month “post-AA” specimens; the second included 10 “early AA failure” (median time to AA failure:11 months) and the third included 10 “late AA failure” specimens (median time to AA failure: 95 months). Proteomic analysis was performed with isobaric mass tags for relative and absolute quanititation (iTRAQ) analyzed by reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). Differentially expressed candidate proteins were identified by comparisons of (i) paired pre/post-AA proteomes and (ii) post-AA proteome with the combined AA failure cohorts at a False Discovery Rate of 0.2. ELISA assays were used to verify candidate markers in a second stored aliquot of first cohort specimens. This cohort was followed for AA failure. Association of post-AA ELISA levels of candidate markers with time to AA failure was performed using Cox proportional hazards regression, summarized as relative risk (RR) for AA failure. Results: Median PSA in pre/post-AA first cohort were 3.15 ng/ml and 0.29 ng/ml. Median PSA in the second and third cohorts were 27.3 and 4.3 ng/ml. Between post-AA and AA failure cohorts, 149 proteins were differentially expressed. Between early and late AA failure 98 proteins were differentially expressed; 47 proteins were common in both comparisons. ELISA assays verified expression levels of 2/47 proteins in the first cohort; zinc alpha-2 macroglobulin (ZAG), and Neuropilin-2 (NPL2). Median change in ZAG decreased by 2073.5 ng/ml (post versus pre-AA) while median change in NPL2 levels increased by 2.9 ng/ml. After a median follow-up of 43 months from the post-AA time-point, 4/15 first cohort subjects had failed AA. The RR of AA failure for ZAG levels below the median change was 3.8 (95% CI: 0.4-37) and 3.0 (95% CI: 0.3-29) for NPL2. Conclusions: A global proteomic analysis identified ZAG and NPL2 as candidate serum predictive markers of AA response which needs further validation.