Background: In the Phase III LUX-H&N1 trial, afatinib improved PFS (median 2.6 vs 1.7 months; HR 0.80; p = 0.03) vs MTX in second-line R/M HNSCC pts. As there are no accepted predictive biomarkers in this setting, the association of pre-specified biomarkers with clinical outcomes was explored. Methods: Pts were randomized 2:1 to 40 mg/d afatinib or 40 mg/m²/wk MTX. The primary endpoint was PFS. Tumor biomarker analyses included human papillomavirus status assessed by p16 immunohistochemistry (IHC), EGFR amplification (FISH), HER3 (IHC) and PTEN (IHC) on archival tissue, and the VeriStrat proteomic signature (classifying pts likely to have good or poor outcomes with EGFR inhibitors) on plasma samples. Results: Of 483 ptsenrolled, 301 (62%) provided plasma and 234 (48%) provided tissue for biomarker analyses. A more pronounced PFS benefit with afatinib vs MTX was observed in p16-negative vs p16-positive, PTEN-high vs PTEN-low, and HER3-low vs HER3-high disease (Table). A trend towards prolonged PFS was observed with afatinib vs MTX in EGFR-amplified tumors. PFS was similar with afatinib and MTX in VeriStrat good and poor groups. Further tumor samples are being analyzed and updated results of PFS and OS will be presented. Conclusions: Subgroups of R/M HNSCC pts who may achieve increased benefit from afatinib vs MTX were preliminarily identified based on biomarkers. PFS benefit with afatinib was more pronounced in pts with p16-negative, PTEN-high, HER3-low, and EGFR-amplified disease. Clinical trial information: NCT01345682