PURPOSE National Cancer Institute–Molecular Analysis for Therapy Choice is a multicohort trial that assigns patients with advanced cancers to targeted therapies on the basis of central tumor genomic testing. Arm B evaluated afatinib, an ErbB family tyrosine kinase inhibitor, in patients with ERBB2-activating mutations. METHODS Eligible patients had selected ERBB2 single-nucleotide variants or insertions/deletions detected by the National Cancer Institute–Molecular Analysis for Therapy Choice next-generation sequencing assay. Patients had performance status ≤ 1, left ventricular ejection fraction > 50%, grade ≤ 1 diarrhea, and no prior human epidermal growth factor receptor 2 (HER2) therapy. Patients received afatinib 40 mg once daily in 28-day cycles. The primary end point was objective response rate (ORR). Secondary end points were 6-month progression-free survival, overall survival, toxicity, and molecular correlates. RESULTS A total of 59 patients were assigned and 40 were enrolled. The median age was 62 years, 78% were female, 68% had performance status = 1, and 58% had received > 3 prior therapies. The confirmed ORR was 2.7% (n = 1 of 37; 90% CI, 0.14 to 12.2), and 6-month progression-free survival was 12.0% (90% CI, 5.6 to 25.8). A confirmed partial response occurred in a patient with adenocarcinoma of extra-mammary Paget disease of skin who progressed after cycle 6. Two unconfirmed partial responses were observed (low-grade serous gynecological tract and estrogen receptor–positive/HER2-negative immunohistochemistry breast ductal carcinoma). Of 12 patients with breast cancer, 1 additional patient with lobular carcinoma (estrogen receptor–positive/HER2 fluorescent in situ hybridization) had a 51% reduction in target lesions but progressed because of a new lesion at cycle 6. The most common (> 20%) treatment-related adverse events were diarrhea (68%), mucositis (43%), fatigue (40%), acneiform rash (30%), dehydration (27%), vomiting (27%), nausea (27%), anemia (27%), and anorexia (22%). Four patients (11%) discontinued because of adverse events. CONCLUSION Although afatinib did not meet the prespecified threshold for antitumor activity in this heavily pretreated cohort, the response in a rare tumor type is notable. The safety profile of afatinib was consistent with prior studies.