Background: Afatinib (A) is an irreversible pan-HER inhibitor that improved PFS in second line RM SCCHN after platinum (LUX-H&N1 trial). In a retrospective biomarker analysis, subgroups of pts with p16 negative, PTEN high or EGFR amplification SCCHN seem to benefit the most. Other studies suggest that A has activity in tumors with HER2 gene activation. Methods: The UPSTREAM trial is a biomarker-driven umbrella trial of targeted therapies and immunotherapy for RM SCCHN (post-platinum, ECOG 0-1, measurable disease). The B1 cohort was a phase II, randomized, open-label substudy evaluating the efficacy of afatinib (orally, 40mg/day) vs physician’s choice (ctrl) (2:1 ratio) in pts with p16 negative RM SCCHN harboring one of the following biomarkers (fresh biopsy): EGFR mutation(mut)/amplification(amp) or HER2 mut/amp or PTEN high by immunohistochemistry (IHC, H-score > 150). Only KRAS/HRAS/NRAS wild type tumors were included. The primary endpoint was progression-free survival rate (PFSR) at 16 weeks after randomization. A 2-stage Simon optimal design was applied to arm A (H1 40%, H0 20%, 1-sided α 10%, power 90%) with 17 and 20 pts enrolled in the first and second stage respectively. Secondary endpoints included ORR, toxicity, PFS, and OS. Results: 59 RM SCCHN pts were included in the B1 cohort (A: n = 40, ctrl: n = 19). Out of the 59 patients, EGFR amp, HER2 amp and HER2 mut were found in 6 pts (10%), 1 pt (2%) and 1 pt (2%), respectively. PTEN (IHC) was high in 97%. 55 pts were evaluable (A: n = 38, ctrl: n = 17) (median age: 62, oral cavity: 29%, oropharynx: 36%, hypopharynx: 24%, larynx: 11%). 69% of pts had received ≥ 2 lines of systemic treatment for RM disease. 80% were pretreated with cetuximab. In arm A, 13/38 patients were alive and free of objective progression at 16 weeks (34.2%, 1-sided 90%CI, 23.9±inf), meeting the predefined criteria of success. The PFSR at 16W in ctrl arm was 29.4% (5/17). 4 objective responses were observed in arm A (3 PR and 1 CR) (ORR 11%, 95%CI 2.9-24.8%) and 1 PR in ctrl arm (6%). The median duration of response under A was 5.7 mo (range: 3.7-25.9 mo). All responding patients had previously been treated with cetuximab, were PTEN high but none had EGFR or HER2 alterations. The median PFS and OS were 2.2/2.4 mo and 7.2/5.0 mo in the A/ctrl arms, respectively. Grade ≥3 drug-related AEs were reported in 31% in arm A and 12% in ctrl arm. The most frequent drug-related AE with A were diarrhea (67%, G3:8%), rash (41%, G3: 3%), fatigue (31%, no G3) and mucositis (23%, G3: 3%). Conclusions: The B1 substudy met its primary endpoint of PFSR at 16 weeks. Responses were seen in cetuximab pre-treated pts. However, the clinical activity of A in this biomarker-selected population remains limited. Translational research is ongoing to better define potential biomarkers. Clinical trial information: NCT03088059.