Clinicopathological analysis of claudin 18.2 focusing on intratumoral heterogeneity and survival in patients with metastatic or unresectable gastric cancer

Abstract

Background: This study aimed to investigate the prevalence of claudin 18.2 (CLDN18.2) positivity, with a particular focus on intratumoral heterogeneity, and its association with clinicopathological features in metastatic or unresectable gastric cancer (GC).

Patients and methods: We investigated 400 patients who received systemic chemotherapy for unresectable, metastatic, or recurrent GC. Immunohistochemistry for CLDN18 (43-14A), human epidermal growth factor receptor 2 (HER2), programmed death-ligand 1 (PD-L1), and fibroblast growth factor receptor 2, as well as HER2 silver in situ hybridization (ISH), Epstein-Barr virus (EBV) ISH, and microsatellite instability testing were carried out. CD3+, CD8+, CD4+, and Foxp3-positive immune cell densities were calculated using digital image analysis.

Results: In GC cases with any CLDN18.2 expression, more than half of the cases (61.3%) showed different expression results between four different tissue microarray (TMA) cores. When comparing CLDN18.2 status between whole tissue sections and the combined results from the four TMA cores, discrepancies were observed in only 2 out of 85 GC cases (2.4%), with 1 false positive and 1 false negative. After considering intratumoral heterogeneity, a CLDN18.2 positivity rate of 31.3% was observed among the 400 GC patients. CLDN18.2 positivity was rare in GCs located in the antrum (or lower third) and in HER2-positive cases but was common in EBV-positive GCs (P < 0.05). No differences in overall survival (OS) were observed according to CLDN18.2 positivity (P = 0.116). Additionally, there was no association between OS and CLDN18.2 positivity in patients treated with fluoropyrimidine plus platinum, chemotherapy plus trastuzumab, paclitaxel with or without ramucirumab, and immuno-oncologic agents. CLDN18.2-positive/PD-L1-high GCs showed statistically significantly longer OS than others (P = 0.025) and higher CD8+ T-cell densities in both the tumor center and periphery (P < 0.001).

Conclusions: Characterizing unresectable, metastatic, or recurrent GC with positive CLDN18.2 expression and evaluating intratumoral heterogeneity and prognostic implications of various therapeutics help advance treatment strategies and develop new therapies for patients with GC.

Keywords: claudin 18.2; gastric cancer; immune microenvironment; immunohistochemistry; intratumoral heterogeneity.

Figure 1

Schematic of the study design. TMA construction (four cores in each case) was carried out in 286 surgically resected samples, and whole-section slides were used in 114 biopsy samples. Among 286 surgically resected samples, immunohistochemistry on whole-section slides was additionally done in 85 cases to compare between TMA and whole-section slides. CLDN18.2, claudin 18.2; GC, gastric cancer; TMA, tissue microarray.

Figure 2

CLDN18.2 positivity in individual TMA cores and intratumoral heterogeneity. (A) CLDN18.2 positivity rates in each single TMA core (cores A, B, C, and D), as well as the positivity rate in at least one of these cores (positive in ≥1 core). (B) The results of CLDN18.2 immunohistochemistry in 111 cases where CLDN18.2 was positive in at least one core. Homogeneous positivity in all four TMA cores was observed in 43 cases (38.7%) and heterogeneous positivity (different results between TMA cores) occurred in the remaining 68 cases (61.3%). CLDN18.2, claudin 18.2; TMA, tissue microarray.

Figure 3

OS analysis based on treatment regimen. (A) OS between CLDN18.2-negative and CLDN18.2-positive patients across the entire study population, regardless of treatment. (B) OS analysis of patients receiving first-line fluoropyrimidine plus platinum chemotherapy. (C) Comparison of OS in patients treated with trastuzumab plus chemotherapy. (D) OS in patients treated with second-line paclitaxel with or without ramucirumab. (E) OS in patients who were exposed to IO. (F) OS in patients treated with irinotecan-based third- or later-line treatment. (G-H) Analysis of OS in patients not exposed to IO. (G) Stratified by CLDN18.2 and PD-L1 status. (H) Survival difference between CLDN18.2-positive/PD-L1-high GC and all other groups (PD-L1 high defined as PD-L1 IHC CPS ≥5). CI, confidence interval; CLDN18.2, claudin 18.2; CPS, combined positive score; CTx, chemotherapy; IHC, immunohistochemistry; IO, immuno-oncologic agents; NA, not available; OS, overall survival; PD-L1, programmed death-ligand 1.

Figure 4

Immune cell densities according to CLDN18.2 and PD-L1 status (negative + low, CLDN18.2 negative and PD-L1 CPS <5; positive + low, CLDN18.2 positive and PD-L1 CPS <5; negative + high, CLDN18.2 negative and PD-L1 CPS ≥5; positive + high, CLDN18.2 positive and PD-L1 CPS ≥5). CLDN18.2, claudin 18.2; CPS, combined positive score; PD-L1, programmed death-ligand 1.