Background: Infigratinib (BGJ398) is a potent and selective FGFR1–3 tyrosine kinase inhibitor (TKI), previously reported to be effective and well tolerated in patients with locally advanced/unresectable or metastatic urothelial carcinoma (mUC) bearing FGFR3 alterations [Pal et al. Cancer Discovery 2018]. However, this previous study did not examine differences in infigratinib activity based on number of prior lines of treatment (LOT). TKIs studied in other indications (e.g. VEGFRis in renal cell carcinoma) have shown consistent activity in both the first and later LOT. Given the effect seen with other TKIs, we sought to determine if infigratinib showed consistent treatment responses in patients with mUC according to LOT. Methods: Eligible patients had mUC and prior platinum-based chemotherapy unless contraindicated and activating FGFR3 mutations/fusions. Infigratinib was dosed at 125 mg orally daily (3 weeks on/1 week off). The primary endpoint of objective response rate (ORR: partial response [PR] and complete response [CR]) was assessed, as well as disease control rate (DCR: CR + PR + stable disease [SD]). After classification of LOT for each patient, subgroup analysis of response by LOT was performed. Results: 67 patients were enrolled; 81% received ≥1 prior LOT for mUC. 13 patients (19.4%) received infigratinib as 1^st LOT for mUC due to ineligibility to receive platinum-based chemotherapy. ORR for the 67 patients was 25% (95% CI 15.5–37.5) and DCR was 64% (95% CI 51.5–75.5). The ORR with 1^st-line infigratinib was 31% (95% CI 9.1–61.4) compared with 24% (95% CI 13.5–37.6) for 2^nd and later (salvage) LOT. DCR was 46% (95% CI 19.2–74.9) for 1^st-line and 69% (95% CI 54.4–80.5) for ≥2 LOT. 13 of the 59 patients with urothelial bladder carcinoma (UBC) received 1^st-line treatment with an ORR of 31% (95% CI 9.1–61.4) and 46 patients in ≥2 LOT had an ORR of 20% (95% CI 9.4–33.9). All 8 patients with upper tract urothelial carcinoma (UTUC) received salvage therapy, with an ORR of 50% and a DCR of 100%. An analysis of other outcome measures (e.g. PFS, OS) will be presented. Conclusions: These results indicate that infigratinib has activity in patients with mUC regardless of LOT. Additionally, patients with UTUC showed a trend for better ORR and DCR. Taken together, these results support the ongoing adjuvant PROOF 302 study comparing infigratinib with placebo in patients with resected disease, assessing infigratinib in an even earlier setting in a UTUC-enriched population (NCT04197986). Clinical trial information: NCT01004224.