ObjectiveEndoscopic mucosal biopsies of primary gastric cancers (GCs) are used to guide diagnosis, biomarker testing and treatment. Spatial intratumoural heterogeneity (ITH) may influence biopsy-derived information. We aimed to study ITH of primary GCs and matched lymph node metastasis (LN_met).DesignGC resection samples were annotated to identify primary tumour superficial (PT_sup), primary tumour deep (PT_deep) and LN_met subregions. For each subregion, we determined (1) transcriptomic profiles (NanoString ‘PanCancer Progression Panel’, 770 genes); (2) next-generation sequencing (NGS, 225 gastrointestinal cancer-related genes); (3) DNA copy number profiles by multiplex ligation-dependent probe amplification (MLPA, 16 genes); and (4) histomorphological phenotypes.ResultsNanoString profiling of 64 GCs revealed no differences between PT_sup1 and PT_sup2, while 43% of genes were differentially expressed between PT_sup versus PT_deep and 38% in PT_sup versus LN_met. Only 16% of genes were differently expressed between PT_deep and LN_met. Several genes with therapeutic potential (eg IGF1, PIK3CD and TGFB1) were overexpressed in LN_met and PT_deep compared with PT_sup. NGS data revealed orthogonal support of NanoString results with 40% mutations present in PT_deep and/or LN_met, but not in PT_sup. Conversely, only 6% of mutations were present in PT_sup and were absent in PT_deep and LN_met. MLPA demonstrated significant ITH between subregions and progressive genomic changes from PT_sup to PT_deep/LN_met.ConclusionIn GC, regional lymph node metastases are likely to originate from deeper subregions of the primary tumour. Future clinical trials of novel targeted therapies must consider assessment of deeper subregions of the primary tumour and/or metastases as several therapeutically relevant genes are only mutated, overexpressed or amplified in these regions.