8506 Background: Uncontrolled evidence demonstrates that adjuvant high-dose interferon alfa-2b (HDI) benefit in high-risk melanoma patients is predicted by autoimmunity induction, and the prognosis of melanoma has been correlated with levels of S-100 protein. We have evaluated these two serological markers in the context of a completed phase II study E2696. Methods: Patients with resectable AJCC stage IIB, III, and IV melanoma were randomly assigned to receive GM2-KLH/QS-1 (GMK vaccine) plus concurrent HDI (Arm A) or GMK plus sequential HDI (Arm B), or GMK without HDI (Arm C). Sera from 103 patients were banked at baseline and 3 additional time points, and have been tested for serum protein S100, and anti-nuclear, anti-thyroid peroxidase, anti-thyroglobulin, anti-mitochondrial, and total anti-cardiolipin autoantibodies (AA) using ELISA. Results: At a median follow-up of 96.5 mo (range: 17–109), the median relapse-free survival (RFS) was 28.3 mo, 95% CI (18.4, 43.6) (64 /103 patients relapsed). Median survival has not been reached (54/103 alive). AA were induced in 17 subjects (25%; n=69) receiving HDI and GMK versus 2 (6%; n=34) receiving GMK alone (2p-value=0.031). In HDI arms induced AA were detected = 12 weeks after therapy initiation. RFS was improved among HDI recipients with AA, but with the limited numbers in this phase II trial this trend does not achieve significance. In the multivariate (Cox regression model) analysis, adjusting for treatment (HDI vs. no HDI) and baseline S-100 value for later time points, S-100 level ≥0.08 microg/l was an independent prognostic factor for RFS at baseline (HR=1.96; p=0.0273), week 4–6 (HR=1.72; p=0.073), wk 12–14 (HR=1.81; p=0.048) and wk 52 (HR=4.3; p<0.001). The hazard ratio (HR) for OS was 1.86 at wk 12–14 (p=0.061), and 5.7 at wk 52 (p<.001). In the model using wk 52 S-100 value (at completion of HDI), the HR adjusted for treatment was 7.1 p=0.009. Conclusions: Autoimmunity is a predictive biomarker of RFS with HDI in the E2696 trial compared to GMK. Serum protein S-100 level ≥0.08 microg/l is an independent prognostic marker for RFS and OS most significant at baseline and 1 year of followup. Current studies will evaluate these markers in the larger phase III trial E1694 with 880 subjects. [Table: see text]