Background: Pemetrexed has demonstrated efficacy in the treatment of NSCLC, primarily in non-squamous cell carcinomas. One explanation may be that squamous cell carcinomas have a higher level of thymidylate synthase (TS); TS-inhibition is a key mechanism of action for pemetrexed. We conducted a phase III trial comparing pemetrexed/carboplatin (PC) with gemcitabine/carboplatin (GC) as 1st-line treatment in advanced NSCLC (Grønberg et al., JCO 2009). The aim of the present study was to investigate whether biomarkers characterize those patients who benefit the most from either regimen. Methods: Formalin-fixed, paraffin-embedded biopsies were collected. Levels of TTF1, TS, folate receptor (FR), FPGS, estrogen receptor (ER), RRM1, EGFR and PTEN were assessed using immunohistochemistry assays from Ventana Medical Systems. The percentages of positive/highly positive tumor cells and H-scores (range: 0-200) were assessed. A central pathology review was performed. Results: Biopsies from 370/436 pts enrolled onto the trial were collected - of which 239 were analyzable (PC: n=115, GC: n=124). Patient characteristics were well balanced between the treatment arms in this cohort. To date, we have assessed TTF1, TS, FR and FPGS. So far, no associations between these markers or histology (original and revised histological diagnoses) and outcomes of any of the two regimens have been revealed. TTF1 (Pos: 10.4 mo, Neg: 6.0 mo; p<.001), TS H-score (<100: 11.9 mo, >100: 6.3 mo; p<.001), but none of the FR- or FPGS-scores were prognostic factors. In multivariate analyses adjusting for other prognostic characteristics (gender, stage, performance status), TS but not TTF1 was a significant prognostic factor (p<.001). Conclusions: So far, no associations between TTF1, TS, FR or FPGS and outcomes of the PC- or GC-regimen have been detected. TTF1 and TS were significant prognostic factors for survival. Complete analyses including ER, EGFR, PTEN and RRM1 status will be presented at the meeting.