Background: Pembrolizumab and trastuzumab (P&T) and chemotherapy demonstrated 27 month mOS, 13 month mPFS, and 91% response rate in first-line HER2-positive mEG cancer irrespective of PD-L1 status (Janjigian Lancet Oncology 2020). Biomarkers including ⁸⁹Zr-trastuzumab PET, blood, and tumor analysis were correlated with progression-free survival. Methods: Twenty-five patients received P&T once 3 weeks prior to addition of chemotherapy to P&T. Pre-treatment tumor biopsies, ⁸⁹Zr-trastuzumab PET scans, serial plasma ctDNA (Guardant360, Redwood City, CA) and CT scans were performed. Tumor-matched DNA alterations were identified by correlating ctDNA and tissue-NGS variant calls. Pre-, on-, and post-treatment biopsies were analyzed using WES and IHC (HER2, PD-L1). Biomarkers were correlated with mPFS and 6-month PFS, the primary endpoint. Results: Of patients with tumor-matched mutations ctDNA at baseline, 12 of 16 had a decline in their maxVAF by week 3, corresponding to a mPFS of 14.7 (11.0-NR) vs 5.9 (95% CI 4.1-NR) months (p=0.009) and a mOS of 29.7 (95% CI 27.2-NR) vs 7.71 (95% CI 6.6-NR) months (p=0.006). 9 of 12 (75%) patients with decline in ctDNA at 3 weeks post-P&T achieved the 6-month PFS primary endpoint while the 4 patients with no decline in ctDNA all progressed in under 6-months. Similarly, 7 of 9 (78%) patients who had a decline in CT-measurements in all disease sites achieved the 6 month PFS primary endpoint, versus 10 of 16 (62.5%) of patients who did not respond in all sites (p=0.66), suggesting that ctDNA is superior to CT as an early predictive biomarker of response. Lack of ERBB2 amplification (amp) by NGS in ctDNA and/or tumor was associated with lack of response to P&T alone prior to addition of chemotherapy. Interestingly, no lesions from patients lacking ERBB2 ctDNA amp (n=3) responded to induction P&T by CT, while lesions from 3/9 patients lacking ERBB2 tissue amp responded to P&T by 3-week CT, suggesting intrapatient HER2 heterogeneity. Eight patients also underwent ⁸⁹Zr-Trastuzumab PET scans prior to P&T and up to 5 lesions per disease site were measured on serial CT scans. All 15 lesions with intense uptake (SUVmax>10) responded to P&T, but only 9/24 lesions with SUVmax<10. All 4 patients who had at least 1 intense lesion achieved a post-P&T CT response and later 6+ month PFS. All 3 of 3 evaluable patients with intense uptake had baseline ctDNA ERBB2 amp. Conclusions: Patients with a decline in tumor-matched maxVAF after one dose of P&T were more likely to achieve durable PFS. Pre-treatment ctDNA ERBB2 amp and/or intense ⁸⁹Zr-trastuzumab PET avidity are non-invasive predictive biomarkers of response to HER2-directed therapy. Evaluation of tumor immune environment digital spatial profiling is underway. Clinical trial information: NCT02954536