Background: The phase III RELAY study (NCT02411448) showed significantly improved progression-free survival (PFS) for RAM+ERL vs PL+ERL in 449 pts with previously untreated EGFR mutation-positive metastatic NSCLC (median PFS 19.4 vs 12.4 mo, HR 0.591 [95% CI 0.461–0.760], p<.0001). To understand baseline genetic mutations and treatment-emergent (TE) resistance mechanisms, this exploratory liquid biopsy substudy examined biomarkers in ctDNA from participating Japanese pts by next-generation sequencing (NGS) and droplet digital PCR (ddPCR). Methods: Plasma samples were collected at baseline, during treatment (Cycle 4, 13, and every 6 cycles to Cycle 53) and post-study treatment discontinuation (30-day follow-up [30d FU]). Mutations were assessed at baseline and 30d FU by NGS (Ion AmpliSeq Colon and Lung Cancer panel). EGFR mutations and MET and ERBB2 copy number (CN) were assessed at all time points by ddPCR. Baseline markers were analyzed in pts with any detectable baseline mutation (to confirm ctDNA presence; NGS N=84, ddPCR N=74). TE mutations were analyzed in pts with any detectable mutation at baseline and 30d FU (NGS N=26, ddPCR N=28). Among these pts, 81% and 57% for NGS and ddPCR, respectively, had progressed by 30d FU. Results: By plasma NGS, baseline EGFR activating mutations (exon 19 deletion or exon 21 [L858R] mutation) were detected in 83.3% of pts. Common co-occurring baseline mutations were TP53 (42.9%), PTEN (7.1%) and KRAS (6.0%). Baseline TP53 mutation rate was higher in men vs women (p=.02). No difference in PFS was detected by baseline TP53 status (interaction predictive p=.45, prognostic p=.33). TE mutations were detected in EGFR (including T790M), FGFR3, KRAS and TP53. Slightly higher rates of TE KRAS (p=.03) and TP53 (p=.07) mutations were detected in RAM+ERL than in PL+ERL. TE total EGFR mutations (p=.65) or TE T790M (p=.69) did not differ by arm. By ddPCR, baseline EGFR activating mutations were detected in all pts. T790M was detected at baseline in 2/37 pts/arm (5.4%) and was TE in 6/11 (55%) RAM+ERL pts and 7/17 (41%) PL+ERL pts. There was a trend (p=.054) for greater ERBB2 CN in RAM+ERL vs PL+ERL at Cycle 4. MET CN decreased slightly at Cycle 4 in both arms (significant in PL+ERL, p=.003). Biomarker levels by ddPCR across all time points will be presented. Conclusions: Though limited by sample size and likely inconsistent tumor shedding, this exploratory study identified potential differences in TP53, KRAS, ERBB2 and MET by demographics, treatment and/or time. Clinical trial information: NCT02411448.