Spatial Analysis of Hereditary Diffuse Gastric Cancer Reveals Indolent Phenotype of Signet Ring Cell Precursors

Abstract

Germline CDH1 loss-of-function mutations are causally linked to an increased lifetime risk of diffuse gastric cancer (DGC). Early, multifocal signet ring cell (SRC) lesions are ubiquitous among CDH1 variant carriers, yet only a subset of patients will develop advanced DGC. A multiomic analysis was performed to establish the molecular phenotype of early SRC lesions and how they differ from advanced DGC using 20 samples from human total gastrectomy specimens of germline CDH1 variant carriers. Spatial transcriptomic analysis demonstrated reduced CDH1 gene expression and increased expression of extracellular matrix remodeling in SRC lesions compared with unaffected adjacent gastric epithelium. Single-cell RNA sequencing revealed an SRC-enriched signature with markers REG1A, VIM, AQP5, PRR4, MUC6, and AGR2. Importantly, SRC lesions lacked alterations in known drivers of gastric cancer (TP53, ARID1A, and KRAS) and activation of associated signal transduction pathways. Advanced DGC demonstrated E-cadherin reexpression, somatic TP53 and ERBB3 mutations, and upregulated CTNNA1, MYC, and MET expression when compared with SRC lesions.

Implications: The marked differences in the genomic and transcriptomic profiles of SRC lesions and advanced DGC support the consideration of SRC lesions as precancers in patients with germline CDH1 mutations.

Figure 1

(A) Location of germline CDH1 P/LP variants in four patients with pT1aN0 (Stage IA) signet ring cell (SRC) lesions (top arrows) and four patients with pathologically confirmed advanced diffuse gastric cancer (DGC) (bottom arrows) utilized for GeoMx Nanostring^™ digital spatial profiling (DSP). (B) Multi-omics study overview. Single-cell suspensions of gastric mucosa containing SRC lesions were subjected to single cell RNA sequencing. Co-detection by indexing (CODEX) multiplexed immunofluorescence tissue imaging and GeoMx Nanostring^™ digital spatial profiling was performed on formalin-fixed, paraffin-embedded (FFPE) gastric cancer tissue from total gastrectomy specimens. This figure panel was created with BioRender.com. (C) Occult SRC lesions on upper endoscopy. (D, E) H&E staining of an individual with Stage IA disease at (D) 100x magnification, scale bar measures 100 μm and (E) 200x magnification, scale bar measures 50 μm. Arrows indicate SRC lesions. (F) Masked region of interest (ROI) selection and immunofluorescent imaging of SRC lesions. (G) UMAP demonstrating transcriptomic clustering of adjacent unaffected gastric epithelium (EP) ROIs from pT1aN0 (n=11) gastrectomy specimens with SRC lesion (n=9) ROIs. Advanced DGC (n=61) ROIs form a distinct cluster. (H) Volcano plot demonstrating extracellular matrix (ECM)-related genes with increased expression in EP or SRC (p< 0.05 and |log2 fold-change| ≥1). (I) Differences in expression of select adherens junction genes in SRC ROIs (n = 9) compared to EP ROIs (n = 11). (J) Heatmap demonstrating enrichment of select genes within the ECM proteoglycans gene set in SRC compared to EP from GSEA. (K) Multiplex immunofluorescence imaging of Stage IA disease. Panel 1 H&E, panel 2 pancytokeratin in yellow, beta-catenin in red, SMA in blue, panel 3 nuclei in blue, panel 4 pancytokeratin in yellow, Ki67 in purple, panel 5 β-catenin in orange, and panel 6 E-cadherin in blue. (L) No significant upregulation in expression of classical tumor proliferation markers in SRC compared to EP.

Figure 2

(A, B) Number of genes (A) and transcripts (B) projected onto the UMAP derived from single cell RNA sequencing of Stage IA gastric cancer mucosa. (C) Dot plot depicting the average expression and detection rate (percentage of cells with any expression) levels of cell type specific markers. The color of each circle represents the average expression and the size represents the gene detection rate. (D) UMAP projection of the annotated cell types: chief, parietal, plasma, pit, NK, endothelial, mast, B cell, T cell, signet ring cell (SRC), stem/pericyte/fibroblast, and gastrin. (E) Enriched pathways derived from genes that are differentially expressed between the SRC and non-SRC populations. ECM-related gene sets that are downregulated (DR) in SRC compared to the stem cell/pericyte/fibroblast cluster include ECM-proteoglycans, cell-extracellular matrix interactions, and miRNA targets in ECM and membrane receptors.

Figure 3

(A) Advanced diffuse gastric cancer (DGC) lesion found on endoscopy (arrow). (B, C) H&E of DGC at 200x magnification with 50 μm scale bars. (B) Cancer cells from advanced lesions exhibit high nuclear-cytoplasm ratio with no mucin and highly atypical nuclei with size variation (black arrows). (C) Advanced DGC cells infiltrate singly (black arrows) or in a pile formation (ellipse). Mitoses (red arrows) are frequently seen. (D) Masked region of interest selection and immunofluorescent imaging of advanced DGC. (E) Volcano plot showing representative genes with increased expression in SRC or DGC (p< 0.05 and |log2 fold-change| ≥1). (F) Differences in expression of select adherens junction genes in SRC ROIs compared to DGC ROIs. (G) Multiplex immunofluorescence imaging of advanced DGC lesions (arrows). Panel 1 H&E, panel 2 nuclei in blue, panel 3 pancytokeratin in yellow, Ki67 in purple, panel 4 E-cadherin in blue, panel 5 αSMA in blue, CD4 in yellow and CD8 in red. (H) Heatmap demonstrating enrichment of select genes within the Mitotic G1 phase and G1 S transition gene set in DGC compared to SRC. (I) Heatmap comparison of differentially expressed genes from RNA sequencing analysis between gastric epithelium (EP), SRC, and DGC ROIs.