FOLH1 as a novel biomarker for risk stratification in gastric intestinal metaplasia

Figure 1 Flowchart of the study. GIM: Gastric intestinal metaplasia; N group: Non-progression group; P group: Progression group; PanCK: Pan-cytokeratin; CD: Cluster of differentiation; ROIs: Regions of interest; IHC: Immunohistochemical; IF: Immunofluorescence; ROC: Receiver operating characteristic; AUC: Area under the curve.

Figure 2 Digital spatial profiling of formalin-fixed, paraffin-embedded samples from gastric intestinal metaplasia patients. A: Representative hematoxylin-eosin and immunofluorescence staining of regions of interest (ROIs); B: Principal component analysis for dimensionality reduction, visualizing all ROIs based on overall gene expression profiles, including the start of observation was defined as the time of the first gastroscopic biopsy [non-progression group (N)] N1-intestinal metaplasia (IM) (red color), progression group (P group) P1-IM (purple color), P2-IM (pink color), and P2-cancerous lesions (blue color). N group: Non-progression group; P group: Progression group; IF: Immunofluorescence; HE: Hematoxylin-eosin; PanCK: Pan-cytokeratin; CD: Cluster of differentiation; CTR: Containing normal tissue; IM: Intestinal metaplasia.

Figure 3 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of differentially expressed genes. A: Volcano plot of differentially expressed genes; B: Kyoto Encyclopedia of Genes and Genomes analysis; C: Gene Ontology analysis including biological processes, cellular components, and molecular functions. N group: Non-progression group; P group: Progression group; IM: Intestinal metaplasia; BP: Biological processes; CC: Cellular components; MF: Molecular functions.

Figure 4 A total of 14 genes were identified as enriched in the pan-cytokeratin region. A: Overlap of significantly differentially expressed genes in the pan-cytokeratin region between [non-progression group (N)] N1-intestinal metaplasia (IM) and N1-containing normal tissue, N1-IM and N2-IM, N1-IM and progression group (P group) P1-IM, and N1-IM and P2-IM; B: The protein-protein interaction network from the String Database based on 14 significantly differentially expressed genes and enrichment analysis results of these genes; C: The expression levels of 14 genes are shown by boxplots. P compared to N1-IM. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. N group: Non-progression group; P group: Progression group; IM: Intestinal metaplasia.

Figure 5 Immunohistochemical staining of additional gastric intestinal metaplasia patients. Positive staining for CES2, CRIP1, DHRS11, EPCAM, FOLH1, NPC1 L1, S100A10, and TMEM176B is observed in brown color. N group: Non-progression group; P group: Progression group; IM: Intestinal metaplasia.

Figure 6 Hematoxylin-eosin and immunohistochemical staining of FOLH1 in 60 additional gastric intestinal metaplasia patients. A: Positive staining of FOLH1 (brown color) was significantly higher in the epithelium of [non-progression group (N)] N1-intestinal metaplasia (IM), compared to progression group (P group) P1-IM; B: Integrated optical density (IOD) of FOLH1 expression in different groups: N1-IM, N2-IM, P1-IM, and P2-IM; C: The diagnostic cutoff value for FOLH1 is 0.302, a sensitivity of 1.0, and a specificity of 0.967, respectively. The corresponding IOD value for FOLH1 is 31.46. ^cP < 0.001. HE: Hematoxylin-eosin; IHC: Immunohistochemical; N group: Non-progression group; P group: Progression group; IM: Intestinal metaplasia; IOD: Integrated optical density; AUC: Area under the curve.

Figure 7 Immunofluorescence staining of additional gastric intestinal metaplasia patients. Positive staining for FOLH1 (red color) was significantly increased in the epithelium of the [non-progression group (N)] N1-intestinal metaplasia (IM) group compared to the progression group (P group) P1-IM group. ^cP < 0.001. N group: Non-progression group; P group: Progression group; IM: Intestinal metaplasia; IOD: Integrated optical density; DAPI: 4’,6-diamidino-2-phenylindole.

Figure 8 Prognostic value of FOLH1 for risk stratification in gastric intestinal metaplasia. A: Kaplan-Meier progression-free survival analysis by FOLH1 expression. Cumulative probability of progression-free survival stratified by FOLH1 integrated optical density levels. Patients with low FOLH1 expression had a significantly higher rate of malignant transformation during 5-year follow-up than the high-expression group (Log-rank P < 0.0001); B: Multivariate Cox regression hazard ratios for gastric intestinal metaplasia (GIM) progression. Forest plot illustrating the independent risk of progression. Low FOLH1 expression is a dominant independent risk factor [hazard ratio (HR) = 65.5, P < 0.001] when adjusted for age, gender, and clinical diagnosis; C: Multivariate Cox regression incorporating operative link on GIM (OLGIM) staging. Forest plot evaluating the protective effect of continuous FOLH1 levels (HR = 0.92 per unit increase, P = 0.002) alongside clinical risk factors. Severe GIM remained a significant risk factor (HR = 4.07, P = 0.044), while OLGIM types II and III did not show independent statistical significance in this model. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. F: Female; M: Male; GIM: Gastric intestinal metaplasia; OLGIM: Operative link on gastric intestinal metaplasia.