Background: Pembrolizumab is approved for the treatment of adult and pediatric patients (pts) with previously treated MSI-H cancer regardless of tumor type or site. This approval was based in part on data from cohort A of the phase 2 KEYNOTE-164 (NCT02460198) study of pts with MSI-H CRC after ≥2 prior lines of therapy including fluoropyrimidine, oxaliplatin, and irinotecan. In addition, in Cohort B of KEYNOTE-164, we evaluated the activity of pembrolizumab in pts with metastatic MSI-H CRC treated with ≥1 prior line of therapy. Methods: KEYNOTE-164 cohort B enrolled pts with metastatic CRC, MSI-H status confirmed locally by IHC or PCR, and ≥1 prior line of therapy (fluoropyrimidine, oxaliplatin, irinotecan, or anti VEGF/EGFR). Eligible pts received pembrolizumab 200 mg Q3W for 2 years or until progression, unacceptable toxicity, or withdrawal of consent. Tumor response was assessed Q9W per RECIST v1.1 by independent review. The primary endpoint was ORR. Secondary endpoints included DOR, PFS, OS, and safety. Results: Of 63 pts enrolled, median age (range) was 59 years (23-83), and 52% were male. Pts had a median of 2 prior therapies, and 94% had ≥1 prior therapy for advanced disease. As of Sep 12, 2017, median (range) duration of follow-up was 12.6 months (0.1-15.4). ORR was 32% (95% CI, 21-45) with 2 CRs and 18 PRs. Median DOR was not reached (NR); 95% of responses were ongoing with DOR ≥6 mo in 75% of responders. Median PFS was 4.1 month (95% CI, 2.1-NR) with a 12-month PFS rate of 41%. Median OS was NR with a 12-month OS rate of 76%. Forty (64%) pts had any grade treatment-related AEs, most commonly (≥10%) fatigue (18%), hypothyroidism (16%), and hyperthyroidism (11%); 7 (11%) pts had grade 3-4 treatment-related AEs of anemia, thrombocytopenia, diarrhea, pneumatosis intestinalis, arthritis, syncope, pneumonitis, and vasculitis (n = 1 each). There were no treatment-related deaths. Twenty (32%) pts had any grade immune-mediated AEs; 2 (3%) pts had grade 3-4 immune-mediated AEs of colitis and pneumonitis (n = 1 each). Conclusions: Pembrolizumab provided durable antitumor activity with a manageable safety profile in patients with MSI-H CRC that progressed after ≥1 prior line of therapy. Clinical trial information: NCT02460198