The tight junction protein claudin 6 is a potential target for patient-individualized treatment in esophageal and gastric adenocarcinoma and is associated with poor prognosis

Abstract

Background: The prognosis of esophageal adenocarcinoma (EAC) and gastric adenocarcinoma (GAC) remains poor, and new therapeutic approaches are urgently needed. Claudin 6 (CLDN6) is an oncofetal antigen that is largely absent in healthy tissues and upregulated in several cancers, making it a promising therapeutical target. In this study, the expression of CLDN6 was assessed in an large Caucasian EAC and GAC cohort.

Methods: RNA-Seq data from 89 EACs and 371 GACs were obtained from The Cancer Genome Atlas project and EAC/GAC cases were stratified by CLDN6 mRNA expression based on a survival-associated cutoff. For groups with CLDN6 expression above or below this cutoff, differential gene expression analyses were performed using DESeq, and dysregulated biological pathways were identified using the Enrichr tool. Additionally, CLDN6 protein expression was assessed in more than 800 EACs and almost 600 GACs using a CLDN6-specific immunohistochemical antibody (clone 58-4B-2) that is currently used in Phase I/II trials to identify patients with CLDN6-positive tumors (NCT05262530; NCT04503278). The expression of CLDN6 was also correlated with histopathological parameters and overall survival (OS).

Results: EACs and GACs with high CLDN6 mRNA levels displayed an overexpression of pathways regulating the cell cycle, DNA replication, and receptor / extracellular matrix interactions. CLDN6 protein expression was associated with shorter OS in EAC and GAC, both in treatment-naïve subgroups and cohorts receiving neoadjuvant therapy. In multivariate analysis, CLDN6 protein expression was an independent adverse prognostic factor in EAC associated with a shorter OS (HR: 1.75; p = 0.01) and GAC (HR: 2.74; p = 0.028).

Conclusions: High expression of CLDN6 mRNA is associated with the dysregulation of distinct biological pathways regulating cell growth, proliferation, and cell-matrix interactions. Clinically, the expression of CLDN6 protein is a valuable adverse prognostic marker in EAC and GAC.

Keywords: Adenocarcinoma; CAR T-cell therapy; CLDN6; Claudin; Esophageal cancer; Gastric cancer; Immunotherapy; Tight junctions.

Fig. 1

Higher CLDN6 mRNA expression is associated with shorter OS and a poor prognosis. A OS for 89 EAC specimens dichotomized by the median CLDN6 expression (TPM, RSEM-normalized). B OS in EACs dichotomized by the best survival-associated cutoff identified by the Cutoff Finder tool; n > best cutoff = 14, n < best cutoff = 75. C OS in GAC specimens (n = 371) stratified by the median CLDN6 expression; D OS differences in GAC patients, dichotomized by the best survival-associated cutoff identified by the Cutoff Finder tool; n > best cutoff = 68, n < best cutoff = 303. CLDN6 = claudin 6, EAC esophageal adenocarcinoma, GAC gastric adenocarcinoma, OS overall survival, TCGA The Cancer Genome Atlas, TPM transcripts per million. A p-value < 0.05 (log-rank test) was considered significant

Fig. 2

EACs with high or low CLDN6 expression have distinct biological profiles. A, B Volcano plots of dysregulated genes in EAC compared to benign tissue dichotomized by CLDN6 expression above and below the best survival-associated cutoff. A log2 fold change (log2FC) ≥ 1.5 was considered for upregulation, and a log2FC ≤ -1.5 for downregulation. An adjusted p-value < 0.05 (Wald test, Bonferroni correction) was considered significant. C Exclusively upregulated pathways in EAC with CLDN6 expression above and below the best survival-associated cutoff and the top 20 exclusively upregulated genes in both subgroups, respectively. D Exclusively downregulated pathways and top 20 downregulated genes in CLDN6-high and -low EAC. CLDN6 Claudin 6, WP WikiPathway Human 2021, GO BP GO Biological Process 2021, KEGG KEGG 2021 Human, GO MF GO Molecular Function 2021.P. P-values and levels of significance: *** p adjusted < 0.001, ** p adj. < 0.01, *p adj. < 0.05 (Enrichr pathway allocation with Bonferroni correction)

Fig. 3

GACs with high or low CLDN6 levels express distinct biological pathways. A, B Volcano plots of dysregulated genes in GAC compared to benign tissue dichotomized by CLDN6 expression above and below the best survival-associated cutoff. A log2 fold change (log2FC) ≥ 1.5 was considered for upregulation, and a log2FC of ≤ -1.5 for downregulation. An adjusted p-value < 0.05 (Wald test, Bonferroni correction) was considered significant. C, D Exclusively upregulated and downregulated pathways in GAC with CLDN6 expression above and below the best survival-associated cutoff and the top 20 exclusively up- and downregulated genes in both subgroups. CLDN6 Claudin 6, GO BP GO Biological Process 2021, KEGG KEGG 2021 Human, GO CC GO Cellular Compartment 2021, BioPlanet BioPlanet 2019; p-values and levels of significance: *** p adjusted < 0.001, ** p adj. < 0.01, *p adj. < 0.05 (Enrichr pathway allocation with Bonferroni correction)

Fig. 4

Adenocarcinomas of the upper gastrointestinal tract, immunohistochemically stained for CLDN6. Red arrows indicate tumor cells and highlight membrane staining. A CLDN6-negative adenocarcinoma. B Adenocarcinoma with only weak focal expression of CLDN6 (score 1 +). C Low-grade CLDN6-positive adenocarcinoma (focally 2 +) D, E Two different carcinomas expressing high levels of CLDN6 (focally 3 +); The blue arrow in D represents peritumoral, CLDN6-negative inflammatory cells; Magnification 200x. CLDN6 claudin 6

Fig. 5

CLDN6 expression is associated with shorter OS in EAC and GAC cohorts. A-C OS in patient cohorts of EAC dichotomized by presence of CLDN6-negative and CLDN6-positive tumors. Primary surgery cohort = patients without neoadjuvant treatment before surgery; Neoadjuvant treatment cohort = patients treated with CROSS and FLOT regime. D–F OS in patient cohorts of GAC dichotomized by CLDN6 expression (positive vs. negative); Neoadjuvant treatment cohort included patients treated with PFL, MAGIC and FLOT regimens; A p-value < 0.05 (log-rank test) was considered significant. CLDN6 claudin 6, CROSS Chemo-radiotherapy for Oesophageal cancer followed by Surgery Study, EAC esophageal adenocarcinoma, FLOT fluorouracil-leucovorin-oxaliplatin-docetaxel, GAC gastric adenocarcinoma, MAGIC Medical Research Council Adjuvant Gastric Infusional Chemotherapy, OS overall survival, PFL Cisplatin, 5-Fluorouracil and Leucovorin