Background: Patients with advanced or metastatic BTC who progress on first-line (1L) gemcitabine-based doublet chemotherapy have few 2L treatment options. Varlitinib is a reversible small molecule pan human epidermal growth factor receptor (HER) inhibitor with low nanomolar potency against HER1 (EGFR), HER2 and HER4 with promising early results in advanced BTC. Methods: TreeTopp (NCT03093870) was a global, multicenter, double blind phase 2 study in which patients with advanced BTC who progressed after 1L therapy that included ≥6 doses of gemcitabine, with radiographically measurable disease based on RECIST v1.1, ECOG PS 0 or 1 and albumin ≥3 g/dL were randomized (1:1) to varlitinib (300 mg BID) plus capecitabine (1000 mg/m² BID 14 days on/ 7 off)(V+C) or placebo plus capecitabine (P+C). The dual primary endpoints were Objective Response Rate (ORR) and Progression Free Survival (PFS) defined as the time from randomization to radiological progression assessed by Independent Central Review. Secondary end points included Overall Survival (OS). Results: Overall, 127 patients were randomized (V+C, n = 64; P+C, n = 63) from May−Dec 2018 and demographics/baseline characteristics were generally well balanced, although the V+C arm had a lower proportion of females vs. P+C (31% vs. 48%). The odds ratio for ORR was numerically higher with V+C vs. P+C was 2.278 (9.4% vs. 4.8%, p = 0.42), the HR for PFS for V+C vs. P+C was 0.90 (median PFS, 2.8 vs. 2.8 months; p = 0.63), and the HR for OS for P+C vs. V+C was 1.11 (median OS, 7.8 vs. 7.5 months; p = 0.66). Although not powered to evaluate sub-group interactions, in sub-group analysis, V+C showed PFS benefit versus P+C in two sub-groups; gallbladder cancer (GBC, HR = 0.55, 95% CI: 0.25, 1.22; median PFS, 2.9 vs. 1.6 months) and females (HR = 0.59, 95% CI: 0.28, 1.23; median PFS, 4.1 vs. 2.8 months). There was no PFS benefit for V+C vs. P+C among males and non-GBC. Toxicities were generally balanced between arms apart from a slightly higher incidence of hyperbilirubinemia, diarrhea and fatigue in the V+C vs. P+C arm. Grade 3/4 toxicities were reported in 66% and 59% of patients in the V+C and P+C arms, respectively. Conclusions: V+C is well tolerated but did not improve ORR, PFS or OS vs. P+C in 2L advanced BTC. Exploratory analyses suggested that patients with GBC and female patients achieved comparatively higher median PFS with V+C vs. P+C. Clinical trial information: NCT03093870.