Background: Metastatic gastroesophageal cancer (GC/GEJC) is an aggressive disease with poor prognosis and a median overall survival (mOS) of around 1 year. While new treatments have emerged, the need for better understanding of the biology and patient profiling is essential given the heterogeneity of the disease. Previously, the TAGS study showed an improved OS with trifluridine/tipiracil (FTD/TPI) vs placebo in heavily pretreated metastatic GC/GEJC patients with a 31% risk reduction in death. This exploratory subgroup analysis of TAGS aims to assess the efficacy, safety, and quality of life (QoL) results observed in patients who received FTD/TPI or placebo in third line (3L) and those who received FTD/TPI beyond the third line (4L+). This analysis will help provide physicians a clearer view of what is expected in the third-line setting in terms of outcomes and to confirm the efficacy of FTD/TPI in later lines. Methods: Patients were divided into two groups: (i) patients who had received FTD/TPI or placebo after two lines of previous systemic therapy (3L) (n = 126 vs 64); and (ii) patients who had received FTD/TPI or placebo (n = 211 vs 106) after three or more lines of previous systemic therapy (4L+). Patient demographics/baseline characteristics, efficacy (OS, progression-free survival [PFS], and time to ECOG deterioration to 2 or more), safety, and QoL were assessed accordingly in these two groups. Results: Patient baseline characteristics in both groups were well balanced between FTD/TPI and placebo. The mOS for FTD/TPI vs placebo in 3L was 6.8 vs 3.2 months (HR, 0.67; 95% CI, 0.47-0.97; P= 0.0318); whereas, in 4L+, it was 5.2 vs 3.7 months (HR, 0.72; 95% CI, 0.55-0.95; P= 0.0192). The mPFS for FTD/TPI vs placebo in 3L was 3.1 vs 1.9 months (HR, 0.54; 95% CI, 0.38-0.77; P= 0.0004); whereas, in 4L+, it was 1.9 vs 1.8 months (HR, 0.57; 95% CI, 0.44-0.74; P< 0.0001). Time to deterioration of ECOG to 2 or more for FTD/TPI vs placebo in 3L was 4.8 vs 2.0 months (HR, 0.60; 95% CI, 0.42-0.86; P= 0.0049); whereas, in 4L+, it was 4.0 vs 2.5 months (HR, 0.75; 95% CI, 0.57-0.98; P= 0.0329). No relevant clinical differences were found in the impact on QoL in the overall study population. The safety profile of FTD/TPI remained consistent through all subgroup analyses. Conclusions: This analysis confirms the efficacy and safety of using FTD/TPI vs placebo in GC/GEJC patients in third and later lines with a survival benefit that seems to be slightly superior in 3L. Results of QoL in both 3L and 4L+ were consistent with previously published subgroup analyses and with the overall TAGS study population. When FTD/TPI is taken in 3L as recommended in the international guidelines, physicians can expect to provide their patients with a mOS of 6.8 and mPFS of 3.1 months. Clinical trial information: NCT02500043