Background: MTAP-deficiency occurs primarily due to homozygous loss in chromosome 9p21 and is seen in approximately 25% of urothelial cancers. MTAP-deficiency portends aggressive biology with poorer outcomes than in MTAP-proficient tumors; for example, in the IMvigor210 data, MTAP-deficient patients had median overall survival of 8.0 months, compared to 11.3 months in MTAP-proficient patients (p = 0.042). MTAP-deficient tumors lack salvage nucleotide synthesis pathways and thus are uniquely sensitive to anti-folate agents; however, in prior clinical work with pemetrexed in these tumors, responses are relatively short-lived. In-vivo data has suggested that pemetrexed can increase tumor infiltrating T-cells, macrophages, dendritic cells, and PD-L1 expression in MTAP-deficient tumors and decrease myeloid-derived suppressor cells (MDSCs). As such, it was postulated that sequential treatment with anti-folate chemotherapy (pemetrexed) and immune checkpoint inhibition (avelumab) would improve responses in MTAP-deficient urothelial cancer. Methods: A prospective phase II trial of patients with advanced MTAP-deficient urothelial cancer is being conducted under IRB-approved protocol NCT03744793. This study will enroll 25 patients at a single site (The University of TX, MD Anderson Cancer Center). Key eligibility criteria include patients with advanced MTAP-deficient urothelial cancer with measurable disease in second-line or beyond. MTAP-deficiency is confirmed with CLIA-approved IHC or NGS. Patients are stratified as being IO-pretreated versus IO-naïve. All patients receive a lead-in cycle of pemetrexed (500 mg/m2) followed by combination pemetrexed (500 mg/m2) and avelumab (10 mg/kg) IV every three weeks until disease progression, patient withdrawal, or unacceptable toxicity. Three biopsies per patient with tissue collection at trial baseline, on single agent pemetrexed, and on combination pemetrexed-avelumab are performed. The primary objective of this trial is to evaluate response rate with sequential pemetrexed and avelumab in MTAP-deficient urothelial cancer. Imaging response assessment is done via RECIST v1.1. Secondary objectives are to evaluate progression-free survival (PFS) and overall survival (OS), as well as to perform correlative studies evaluating the effect of this therapy on immune cells and tumor microenvironment. This correlative work includes but is not limited to evaluation of peripheral T-cells, tumor-infiltrating T-cells, macrophages, and MDSCs. Interim analyses for futility and safety occur in cohorts of 5 patients and are performed based on Bayesian sequential methods. Fifteen patients have been enrolled thus far. Clinical trial information: NCT03744793.