Background: PSqCC is a rare tumor with poor prognosis. The standard of care for advanced disease (AD) has been palliative platinum-based chemotherapy (CT). Most PSqCC patients (pts) have high levels of programmed death-ligand 1 (PD-L1). ORPHEUS has evaluated the efficacy and safety of Rf –a PD-1 antagonist– in pts with unresectable locally advanced or metastatic PSqCC. Methods: ORPHEUS (NCT04231981) is an international, multicenter, open-label, single-arm phase II trial. The study enrolled pts aged ≥18 years with locally advanced or metastatic PSqCC who had not received any previous treatment with PD-1 or PD-L1/2 agents. Pts received Rf 500 mg intravenously on day 1 of each 28-day cycle until progressive disease, unacceptable toxicity, discontinuation or death. The primary endpoint was investigator-assessed objective response rate (ORR) as per RECIST v.1.1. Secondary endpoints included clinical benefit rate, progression-free survival (PFS), duration of response (DoR), time to response (TTR), disease control rate (DCR), overall survival (OS) and safety evaluated as per NCI-CTCAE 5.0. The design was planned to attain an 80% power at 5% one-sided α level (H0: ORR≤5%; HA: ORR≥25%). Results: Between Jul 7, 2020, and Aug 26, 2021, 18 men were enrolled at 8 sites from Italy and Spain. Median age was 64.2 years (range 42-81),13 (72.2%) pts had an AD at first diagnosis, 15 (83.3%) had a previous surgical procedure for penile cancer (lymphadenectomy in 7 pts), 10 (55.6%) had previously received platinum-based CT (TPF/TIP in 8 pts) and 7 (38.9%) were naïve to AD treatment. At data cutoff (Aug 26, 2022), with a median follow-up of 7.2 months, no pts remained on therapy. ORR (3 partial responses) was 16.7% (95% CI 5.8%-39.2%) with a median DoR and TTR of 3.3 (range 1.8-8.5) and 1.9 (range 1.7-2.4) months, respectively. DCR was (33.3%) (1 stable disease ≥ 6 months). Median PFS was 2.0 months (95% CI 1.6-3.3) and median OS with 17 events was 7.2 months (95% CI 3.0-9.8). Most common non-hematological treatment emergent adverse events (TEAEs) of any grade (G) were fatigue (27.8%; 5.6% G≥3), cellulitis, groin infection and Pseudomonas infection (11.1%; 5.6% G≥3), respectively. Treatment related (TR) TEAEs were fatigue (22.2%; 5.6% G≥3), rash (5.6%; 0% G≥3) and decreased appetite (5.6% G≥3). Anemia (11.1%; 0% G≥3) was the most frequent hematological TEAE. Serious unrelated TEAEs occurred in 5 pts (27.8%). Two (11.1%) pts discontinued treatment due to unrelated non-hematological TEAEs; 1 case of infected lymphocele (G3) and 1 case of skin neoplasm bleeding (G3). No TR deaths were reported. Conclusions: Rf in monotherapy showed signals of clinical activity in advanced or metastatic PSqCC pts. Safety profile was consistent with previous data. These results along with identification of suitable biomarkers could help to develop novel immunotherapy combination strategies in this orphan genitourinary tumor. Clinical trial information: NCT04231981.