Purpose: In JACOB trial, pertuzumab added to trastuzumab-chemotherapy did not significantly improve survival of patients with HER2-positive metastatic gastric cancer, despite 3.3 months increase versus placebo. HER2 copy number variation (CNV) and AMNESIA panel encompassing primary resistance alterations (KRAS/PIK3CA/MET mutations, KRAS/EGFR/MET amplifications) may improve patients’ selection for HER2 inhibition. Experimental design: In a post-hoc analysis of JACOB on 327 samples successfully sequenced by NGS (Oncomine Focus DNA), HER2 CNV, HER2 expression by IHC and AMNESIA were correlated with ORR, PFS and OS by uni/multivariable models. Results: Median HER2 CNV was 4.7 (IQR 2.2-16.9). HER2 CNV-high vs low using the median as cut-off was associated with longer median PFS (10.5 vs 6.4 months; HR=0.48, 95%CI: 0.38-0.62; p<.001) and OS (20.3 vs 13.0 months; HR=0.54, 0.42-0.72; p<.001). Combining HER2 CNV and IHC improved discriminative ability, with better outcomes restricted to HER2-high/HER2 3+ subgroup. AMNESIA positivity was found in 51 (16%), with unadjusted HR=1.35 (0.98-1.86) for PFS; 1.43 (1.00-2.03) for OS. In multivariable models, only HER2 CNV status remained significant for PFS (p<.001)/OS (p=.004). Higher ORR was significantly associated with IHC 3+ [61% vs 34% in 2+; odds ratio (OR)=3.11 (1.89-5.17)] and HER2-high [59% vs 43% in HER2-low; OR=1.84 (1.16-2.94)], with highest OR in the top CNV quartile. These biomarkers were not associated with treatment effect of pertuzumab. Conclusions: HER2 CNV-high assessed by NGS may be associated with better ORR, PFS, OS in a JACOB subgroup, especially if combined with HER2 3+. The negative prognostic role of AMNESIA requires further clinical validation.