Discrete precancer subtypes and biomarkers of cancer progression identified with spatiotemporal analysis of CDH1-driven diffuse gastric cancer

Introduction: Diffuse gastric cancer (DGC) is a sub-type of gastric adenocarcinoma for which early detection is uncommon. Early-stage gastric signet ring cell (SRC) lesions are ubiquitous in patients with germline CDH1 variants. Though SRC lesions appear to be non-obligate precursors of DGC, the drivers of SRC progression to DGC remain unknown. We sought to derive transcriptomic gene signatures associated with gastric SRC and advanced DGC. Methods: We employed a single-cell spatial transcriptomic analysis (10X Genomics Xenium) of both early SRC (pT1a) and advanced DGC (≥T2) from 18 patients with germline CDH1 variants. Spatially resolved analyses were conducted on 42 fields of view derived from gastric biopsies and total gastrectomy specimens that were analyzed according to depth of cancer cell invasion. Biomarkers of SRC precancer progression were identified within discrete epithelial cell subpopulations according to differentially expressed gene profiles. Immunohistochemistry was performed to correlate protein expression with mRNA markers of interest. Results: Advanced DGC exhibited three distinct transcriptomic clusters based on depth of tumor invasion into the mucosa, submucosa and muscularis propria layers and clustered distinctly from early SRC precursor lesions. SRC phenotype varied based on depth of invasion of the gastric mucosa; SRC1 (superficial) and SRC2 (deep). Multi-modal segmentation of SRC1 and SRC2 subtypes within the SRC precancer cluster demonstrated distinct, spatially resolved transcriptomic profiles. Pathway analysis showed potential functional divergence between SRC1 and SRC2, such that SRC2 possessed a migratory and proliferative phenotype whereas SRC1 displayed loss of cell adhesion and epithelial architecture. We observed three differentially expressed genes with increased expression according to depth of tumor invasion: clusterin (CLU ) , eukaryotic translation initiation factor 3 (EIF3E), and annexin A2 (ANXA2). CLU, a known regulator of epithelial-to-mesenchymal transition and apoptosis, was over-expressed both in advanced SRC lesions (p<0.05) compared to early SRC lesions and within SRC lesions compared to gastric epithelium (p=0.03). Clusterin protein expression was quantitatively greater in SRC lesions compared to unaffected gastric epithelium, with more intense staining associated with depth of tumor cell invasion in advanced DGC. Conclusions: Signet ring cell precancers that originate due to CDH1 loss-of-function mutations have discrete subtypes based on cell phenotype, depth of mucosal invasion, and spatial transcriptomic profile. Clusterin appears to be a biomarker of early SRC lesions and correlates with depth of gastric cancer invasion. These results may support novel strategies for cancer interception and increase our knowledge of diffuse gastric precancers.