4068 Background: The usefulness of combined therapy with immune checkpoint inhibitors (ICI) and chemoradiotherapy (CRT) and its potential biomarker of treatment efficacy for esophageal cancer remains unclear. We investigated the safety, efficacy, and associated biomarkers of combined therapy with ICI and definitive CRT in operable and inoperable esophageal squamous cell carcinoma (ESCC). Methods: This prospective single-arm phase 2 trial was performed at five hospitals in Japan. Patients aged 20–75 years with histologically confirmed ESCC, an ECOG PS score of 0 or 1, and adequate organ and bone marrow functions were eligible. Inoperable patients did not have distant organ metastasis. Patients did not receive any chemotherapy or surgical resection as an initial therapy. Concurrent radiotherapy (50.4 Gy/28 fractions to the primary lesion and 41.4 Gy/23 fractions to the regional LN area) consisted of 4 courses of CF (cisplatin, 75 mg/m2/day, day 1; 5-fluorouracil, 1000 mg/m2/day, days 1–4) plus nivolumab (240 mg/m2/day, every 2 weeks for up to 1 year). The primary endpoint was safety. The incidence rates of nonhematological toxicity grade ≥ 4 and pneumonitis grade ≥ 3 were ≤10% and ≤15%, respectively. The secondary endpoints included overall survival (OS), progression-free survival (PFS), complete response (CR) rate, and time to esophagectomy-free survival (EFS). Sixty patients were to be accrued, but the trial enrollment was terminated because of slow accrual. This trial was registered as jRCT1091220408. Results: Between January 2019 and September 2021, 42 patients were enrolled. Twenty-five patients had operable disease and 16 had inoperable disease. Two patients (4.8%) experienced grade 3 pneumonitis, suggesting that the primary endpoint was met. The median relative dose intensity of nivolumab was 92.4%. One-year OS, PFS, and EFS rates in all cases were 92.7%, 65.4%, and 78.0%, respectively. One-year OS, PFS, and EFS rates in operable and inoperable cases were 100%, 79.6%, and 84.0% and 81.3%, 43.8%, and 68.8%, respectively. The CR and objective response rates were 73.2% (95% confidence interval [CI]: 58.1%–84.3%) and 87.8% (95% CI: 74.5%–94.7%), respectively. The CR rates in operable and inoperable cases were 84% (95% CI: 65.3%–93.6%) and 56.3% (95% CI: 33.2%–76.9%), respectively. Immune high-active intrinsic subtype before treatment (n=4) with an expression score > 80 of 51 immune-related genes showed a high CR rate (100%, n=4), whereas the immune low-active intrinsic subtype (n=37) with an expression score < 80 showed a relatively high CR rate (70%). Conclusions: Nivolumab combined with definitive CRT provided encouraging efficacy and acceptable toxicity in patients with ESCC. Immune high-active intrinsic subtype before treatment has the potential to predict a high CR rate for this combination treatment. Clinical trial information: jRCT1091220408 .