Claudin 18.2 testing in gastroesophageal and pancreatobiliary cancers: current status, clinical implications and challenges

Introduction

Claudins (CLDNs) are a family of small transmembrane proteins with a molecular weight of 20 to 27 kDa that are key components of tight junctions between epithelial cells (1,2). They consist of two extracellular loops, four transmembrane domains, and a cytoplasmic domain (2,3). Approximately 27 CLDN isoforms have been identified. Their primary functions include regulating tissue permeability, facilitating paracellular transport, and enabling signal transduction by sealing the spaces between epithelial cells (4). The expression of CLDNs varies by tissue type, with most normal tissues expressing multiple CLDN isoforms (5,6).

CLDN18 comprises two isoforms generated by alternative splicing: CLDN18.1 and CLDN18.2 (7). CLDN18.1 is primarily expressed in alveolar epithelial cells of the lung, whereas CLDN18.2 is exclusively expressed in differentiated epithelial cells of the gastric mucosa (5). The expression of CLDN18.2 is regulated by multiple pathways, including protein kinase C, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), Human epidermal growth factor receptor 2/3 (HER2/HER3) signaling, microRNA, and Cytosine-phosphate-Guanine (CpG) island methylation (8-14). Aberrant surface expression of CLDN18.2 in gastric, esophageal, gastroesophageal junction, pancreaticobiliary, non-small cell lung, and ovarian carcinomas due to the loss of cell polarity make it an ideal target for cancer therapy (15-18). In October of 2024, the US Food and Drug Administration (FDA) approved the anti-CLDN18.2 monoclonal antibody (zolbetuximab) as the first-line treatment for patients with locally advanced unresectable or metastatic HER2-negative, CLDN18.2-positive gastric and gastroesophageal junction (G/GEJ) adenocarcinoma base on the results from two large phase 3 clinical trials: SPOTLIGHT and GLOW trials (19,20). In addition, development and clinical trials of CLDN18.2-targeted therapies, including other monoclonal and bispecific antibodies, antibody-drug conjugates (ADCs), and CLDN18.2-targeted chimeric antigen receptor (CAR) T cells, has prompted widespread testing for CLDN18.2 expression in gastric cancer, pancreatobiliary and other gastrointestinal malignancies. This review provides an up-to-date overview of CLDN18.2 in gastrointestinal and pancreatobiliary cancers, including the patterns of CLDN18.2 expression, therapeutic implications of CLDN18.2-targeted therapies, current testing methodologies, practical recommendations, and challenges associated with standardized assessment and reporting for patient selection in clinical practice.

Expression of CLDN18.2 in G/GEJ cancers

Previous studies reported that the prevalence of CLDN18.2 positivity in G/GEJ cancers ranges from 21.6% to 64.8% (21-23). This variability is largely attributable to differences in primary antibody clones, immunohistochemical staining platforms, positivity thresholds, and scoring systems used in these studies. Using the 43-14A clone, the Ventana Benchmark platform and a cutoff of moderate to strong membranous staining of CLDN18.2 in ≥75% tumor cells, three recent large studies and clinical trials reported the positive rates of CLDN18.2 from 24.0% to 38.4% in G/GEJ cancer (19,20,23-25). In the study by Pellino et al., positive CLDN18.2 correlated with younger age (<70 years), advanced stage at the time of diagnosis, peritoneal metastasis and less frequent liver metastases, but not overall survival (OS) (25). However, other studies reported no significant association between CLDN18.2 positivity and clinicopathologic factors or prognosis in patients with G/GEJ cancer (24,26-29).

The correlation of CLDN18.2 expression with other commonly used biomarkers in G/GEJ cancers, including HER2/neu, programmed death-ligand 1 (PD-L1), Epstein-Barr virus (EBV), and microsatellite instability (MSI) have been investigated and summarized in Table 1 (19,20,23-25). A positive correlation between CLDN18.2 and EBV status was found in two studies by Kwak et al. and Pellino et al., but not in others (24,25). Similarly, a positive correlation between the expression of CLDN18.2 and PD-L1 in G/GEJ cancers was reported only in the study by Kwak et al. (19,20,23-25). Positive correlation between the expression of CLDN18.2 and PD-L1 may suggest synergistic antitumor effects when combining CLDN18.2-targeted therapies with immune checkpoint inhibitors. No significant correlation between CLDN18.2 expression and MSI status was identified in previous studies.

CLDN18.2-targeted therapies for G/GEJ cancers

The efficacy and safety of zolbetuximab have been demonstrated through phase II and III clinical trials. The findings from these trials are summarized in Table 2 and briefly summarized below.

The MONO phase IIa trial was designed to assess the efficacy and safety of zolbetuximab monotherapy in patients with CLDN18.2-positive advanced G/GEJ adenocarcinoma (30). In this study, CLDN18.2 positivity was defined as ≥50% of tumor cells exhibiting moderate to strong (2+ to 3+) membranous staining using the Invitrogen^RM immunohistochemistry (IHC) assay (3J4G7 antibody, ThermoFisher Scientific, Waltham, US). Among 43 patients assessed for response and disease status, the objective response rate (ORR) was 9% and the disease control rate (DCR) was 23%. Of the patients who achieved clinical benefit, 90% demonstrated moderate to strong membranous staining in at least 70% of tumor cells (30).

In the ILUSTRO phase II trial, the eligibility criteria required tumors to exhibit high CLDN18.2 expression (2+ to 3+ membranous staining in ≥75% of tumor cells) for cohorts 1A and 2 (31). The antibody and IHC platform used in this study was not described. Cohort 1A enrolled 30 patients to receive third/later-line zolbetuximab monotherapy. The ORR and DCR were 0% and 44.4%, respectively. The median progression-free survival (PFS) and OS was 1.54 months [95% confidence interval (CI): 1.31–2.56], while the median OS was 5.62 months (95% CI: 2.27–11.53). Cohort 2 enrolled 21 patients with high CLDN18.2 and negative HER2 status, who received mFOLFOX (modified Folinic acid, fluorouracil, and oxaliplatin) plus zolbetuximab as first-line treatment. This cohort demonstrated an ORR of 71.4% and a DCR of 100%. The median PFS was 17.8 months (95% CI: 8.05–25.69). Cohort 3A consisted of only 3 patients with either high or intermediate CLDN18.2 expression (≥50% but <75% of tumor cells), who were treated with third or later-line zolbetxumab plus pembrolizumab. No responses were observed in this cohort, although the DCR was 66.7% (31).

The FAST trial was a randomized phase II study that compared the combination of EOX (epirubicin, oxaliplatin, and capecitabine) with zolbetuximab to EOX alone as a first-line therapy for advanced G/GEJ adenocarcinomas. This study targeted patients whose tumors showed ≥40% of cells with moderate to strong (2+ to 3+) membranous staining, as assessed by the CLAUDETECT RM 18.2 histology kit (CLDN18 43-14A antibody, Ganymed Pharmaceuticals, Mainz, Germany) (32). Among the 686 enrolled patients, 334 (48.7%) were classified as CLDN18.2-positive. The ORR for the combination of EOX with zolbetuximab was 39%, compared to 25% for EOX alone. Patients treated with EOX combined with zolbetuximab experienced a significantly longer PFS of 7.5 months, compared with 5.3 months with EOX alone [hazard ratio (HR) 0.44, 95% CI: 0.29–0.67, P<0.0005]. Furthermore, the OS was also significantly better in the combination group, with 13.0 months compared to 8.3 months for EOX alone (HR 0.55, 95% CI: 0.39–0.77, P<0.0005). Importantly, patients with CLDN18.2 expression in ≥70% of tumor cells demonstrated a significant better PFS of 9.0 months compared to 5.7 months for those receiving EOX alone (HR 0.38, 95% CI: 0.23–0.62, P<0.0005), as well as improved OS of 16.5 months versus 8.9 months (HR 0.44, 95% CI: 0.33–0.74, P<0.0005). In contrast, no survival benefit was observed in patients whose tumors had moderate to strong membranous CLDN18.2 expression in ≥40% but <70% of tumor cells. Based on these results, the eligibility criteria for the phase 3 SPOTLIGHT and GLOW trials established CLDN18.2 positivity as ≥75% of tumor cells exhibiting moderate to strong (2+ to 3+) membranous staining.

The SPOTLIGHT Phase 3 study aimed to assess the efficacy and safety of zolbetuximab in combination with mFOLFOX6 (which includes modified folinic acid or levofolinate, fluorouracil, and oxaliplatin) compared to mFOLFOX6 alone in patients with CLDN18.2-positive, HER2-negative, previously untreated, locally advanced unresectable or metastatic G/GEJ adenocarcinoma (19). CLDN18.2 positivity was defined as ≥75% of tumor cells demonstrating moderate to strong (2+ to 3+) membranous staining, as determined by the VENTANA CLDN18 (43-14A) RXDx Assay [CLDN18 (43-14A) antibody, ROCHE Diagnostic Solutions; Tucson, AZ, USA]. Of the 2,403 patients assessed, 922 (38.4%) tested positive for CLDN18.2. Among these, 565 patients with CLDN18.2-positive and HER2-negative tumors were enrolled in the trial. Those who received zolbetuximab plus mFOLFOX6 demonstrated a significantly longer median PFS of 10.6 months, compared to 8.7 months for those receiving mFOLFOX alone (HR 0.75, 95% CI: 0.6–0.94, P=0.007). Additionally, the median OS was longer in the zolbetuximab plus mFOLFOX6 group at 18.2 months, compared to 15.5 months in the mFOLFOX alone group (HR 0.55, 95% CI: 0.6–0.94, P=0.005). The ORRs were similar between the two treatment groups.

The GLOW phase 3 study was designed to evaluate the efficacy of zolbetuximab combined with capecitabine and oxaliplatin (CAPOX) compared to a placebo plus CAPOX in patients with CLDN18.2-positive and HER2-negative locally advanced unresectable or metastatic G/GEJ adenocarcinoma (20). The antibody, IHC platform, and criteria for determining CLDN18.2 positivity used in this trial were consistent with those employed in the SPOTLIGHT trial. Among 2,104 patients assessed for CLDN18.2 status, 808 (38.4%) were identified as CLDN18.2-positive. Out of these, 729 patients whose tumors were both CLDN18.2 positive and HER2 negative were enrolled in the study. Patients receiving zolbetuximab plus CAPOX demonstrated a median PFS of 8.2 months, compared to 6.8 months in the placebo plus CAPOX group (HR 0.69, 95% CI: 0.54–0.87, P<0.001). The median OS was 14.4 months in the zolbetuximab plus CAPOX group versus 12.2 months in the placebo plus CAPOX group (HR 0.77, 95% CI: 0.62–0.97, P=0.01). The ORR was 52.5% (95% CI: 36.4–48.9%) in the zolbetuximab group compared to 40.3% (95% CI: 34.2–46.6%) in the placebo group.

The most common adverse events of zolbetuximab are vomiting, nausea, and infusion-related reactions. The significant survival benefit and safety profile demonstrated in these clinical trials led to the FDA approval of zolbetuximab as the first-line treatment for patients with locally advanced unresectable or metastatic CLDN18.2-positive, HER2-negative G/GEJ adenocarcinoma. Currently, many new therapeutic strategies targeting CLDN18.2 are in phase I/II trials or development. These include monoclonal antibodies, bispecific T-cell engager antibodies, peptide-antibody conjugates, CAR T-cell (CAR-T) therapies, and messenger RNA (mRNA)-based therapies. Ongoing studies are also evaluating combination therapies with cytotoxic drugs or immune checkpoint inhibitors to assess potential synergistic effects and are expanding the application of these agents to other tumor types, such as pancreatic cancer. In a phase 1 study involving 23 patients with CLDN18.2-positive advanced G/GEJ adenocarcinomas, combination therapy with CAPOX and AB011, a humanized anti-CLDN18.2 immunoglobulin G1 (IgG1) monoclonal antibody, achieved an ORR of 65.2% without an increase in treatment-related adverse effects (33). ASKB589, another humanized anti-CLDN 18.2 IgG1 monoclonal antibody, has shown favorable antitumor activity both as monotherapy and in combination with CAPOX in patients with CLDN18.2-positive gastric cancer (34). As monotherapy, ASKB589 achieved an ORR of 9.5%. However, when combined with CAPOX, the ORR increased significantly to 75%. Osemitamab (TST011), the second-most advanced antibody targeting CLDN18.2, also showed favorable antitumor activity in combination with CAPOX, resulting in an ORR of 66.7% and a PFS of 9.5 months (35). Regarding ADCs, SASA1801 and CMG 901 are CLDN18.2-targeted conjugates that demonstrated promising efficacy in patients with G/GEJ cancer, achieving ORRs of 47.1% and 29%, respectively (36,37). Additionally, CT041, a CAR-T cell therapy targeting CLDN18.2, showed promising efficacy in patients with gastric cancer and other tumor types, including pancreatic cancer, with an ORR of 54.9% in G/GEJ cancers and 22.2% in other cancer types including pancreatic and biliary adenocarcinomas (38). Across these trials, the most common treatment-related adverse events were similar to those observed with zolbetuximab, primarily nausea and vomiting. Overall, new CLDN18.2-targeted therapies have been well-tolerated and exhibit manageable safety profiles.

Three ADCs targeting CLDN18.2, including SHR-A1904 [an anti-CLDN18.2 IgG1 conjugated to a DNA topoisomerase I (TOPO1) inhibitor payload], IBI343 (an Fc-silenced anti-CLDN18.2 IgG1 conjugated to exatecan), and AZD0901 [an IgG1 conjugated to the anti-microtubule agent monomethyl auristatin E (MMAE)], have demonstrated encouraging activity in patients with G/GEJ adenocarcinoma in early phase trials with ORRs ranging from 25% to 40% (37,39,40). Randomized clinical trials evaluating these agents are ongoing.

Expression of CLDN18.2 in pancreatic cancers and ongoing clinical trials

Approximately 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed at an advanced stage (stage III or IV), which contributes to a poor prognosis, with only 10.8% of patients surviving five years (41). For patients with unresectable tumors and metastatic lesions, FOLFIRINOX and gemcitabine (GEM) with nab-paclitaxel (Nab-P) are the first-line treatment (42). Currently, targeted therapies are effective only in a small, molecularly defined subset of patients, such as those with defective DNA repair pathways. This includes the use of PARP inhibitors in patients harboring germline or somatic BRCA1/2 or PALB2 mutations. Immune checkpoint inhibitors are effective only in rare patients with MSI-high (MSI-H) tumors (43-46). Therefore, new targeted therapies are continuously being investigated for patients with PDAC.

Although CLDN 18.2 is expressed exclusively in gastric epithelial cells, aberrant expression of CLDN18.2 has been reported in PDACs (17) (Figure 1A,1B). The reported positive rates of CLDN18.2 expression in PDAC range from 30% to 100% depending on different antibodies and scoring systems (17,47-51). Using the Ventana CLDN18.2 assay and current criteria for CLDN18.2 positivity in G/GEJ adenocarcinomas, the prevalence of CLDN18.2 positivity in PDAC is approximately 30% (17,50,52). Most studies showed a positive correlation between CLDN18.2 positivity using a cut off of moderate to strong membranous staining ≥75% tumor cells and well to moderately differentiated histology and better OS in patients with PDAC (17,50-52). These results suggest that approximately a third of PDAC patients may benefit from CLDN18.2-targeted therapies.

Figure 1 Claudin 18 (CLDN18) immunoperoxidase staining uses a mouse monoclonal anti-claudin 18 antibody (clone 43-14A) and is performed on a Roche Ventana Benchmark Ultra autostainer. This staining enables the detection of CLDN18.1 and CLDN18.2 isoforms in formalin-fixed, paraffin-embedded tissue samples. (A) Aberrant expression of CLDN18.2 in PDAC at 10× magnification. (B) Scattered aberrant expression of CLDN18.2 in intraductal carcinoma of the pancreas at 10× magnification. (C) Intrahepatic cholangiocarcinoma with compact neoplastic glands and focal mucin production on hematoxylin-eosin staining at 20× magnification. (D) Diffuse membranous and cytoplasmic staining of CLDN18.2 in intrahepatic cholangiocarcinoma at 20× magnification. PDAC, pancreatic ductal adenocarcinoma.

A recent study has shown that CLDN18.2-targeted therapy demonstrates promising efficacy in CLDN18.2-positive other types of cancers, including PDACs, without increasing treatment-related adverse effects (38). A phase 2 clinical trial (NCT03816163) is currently underway to investigate the efficacy and safety of zolbetuximab in combination with Nab-P and GEM as a first-line treatment for patients with CLDN18.2-positive, metastatic PDAC (53). Depending on the trial results, there may be potential to expand CLDN18.2 targeted therapy as part of the first-line treatment for patients with advanced PDAC. Furthermore, evidence suggests that CLDN18.2 may regulate T cell infiltration and modulate the tumor immune microenvironment in PDACs (54). These findings suggest that targeting CLDN18.2 may produce a synergistic cytotoxic effect when combined with immunotherapy in PDACs.

In G/GEJ adenocarcinomas, the threshold of 75% of tumor cells with moderate (2+) to strong (3+) membrane staining is the established standard for identifying CLDN18.2-positive tumors. This biological scoring modality was initially revealed by the FAST trial and subsequently supported by the SPOTLIGHT and GLOW trials (19,20,32). In contrast, no clinical trials currently validate the use of a 75% tumor cell threshold for CLDN18.2 positivity in pancreaticobiliary-type adenocarcinomas. Consequently, pathologists report CLDN18.2 expression by indicating both the proportion of positive tumor cells and the intensity of membranous staining, such as CLDN18.2 expression with 1+ to 3+ membranous staining in a specified percentage of tumor cells. The current reporting approach may be revised based on forthcoming results from ongoing clinical trials investigating CLDN18.2-positive pancreaticobiliary-type adenocarcinomas.

Expression of CLDN18.2 in biliary cancers

Recent studies have reported on the prevalence of CLDN18.2 expression in gallbladder carcinomas as well as cholangiocarcinoma (55,56) (Figure 1C,1D). Ni et al. found that 12.3% and 24.6% of 167 surgically resected gallbladder carcinomas were positive for CLDN18.2, using the cutoffs of ≥75% and ≥50% of tumor cells showing 2+ to 3+ membranous staining (55). Their study reported a significant correlation of CLDN18.2 positivity with better tumor differentiation, less frequent lymph node and distant metastasis, and better survival. In addition, they showed that CLDN18.2 positivity correlated with the percentages of Foxp3⁺ Treg cells, CD8⁺ T and CD20⁺ B lymphocytes (P<0.001), indicating potential immune activation associated with CLDN18.2 expression in gallbladder carcinomas. These findings suggest that combining CLDN18.2-targeted therapy with immunotherapy may produce a synergistic cytotoxic effect in patients with gallbladder cancer.

Kinzler et al. reported that approximately 13% of 160 surgically resected cholangiocarcinomas were positive for CLDN18.2 using the cutoff of ≥75% of tumor cells showing 2+ to 3+ membranous staining (56). In their study, 26.5% (9 cases) of 34 perihilar cholangiocarcinomas and 7.4% (7 cases) of 95 intrahepatic cholangiocarcinomas exhibited CLDN18.2 positivity. The frequency of CLDN18.2 positivity in perihilar cholangiocarcinomas was higher than intrahepatic cholangiocarcinoma. In addition, among the intrahepatic cholangiocarcinomas, large duct types showed a higher prevalence of CLDN18.2 positivity compared to small duct types. These findings suggest that patients with perihilar and large duct type cholangiocarcinomas may be more likely to be potential candidates for CLDN18.2-targeted therapy. However, these differences did not reach statistical significance due to the relatively small number of cases in each subgroup.

Testing for CLDN18.2 expression

Antibodies and immunohistochemical platforms

With the increasing clinical demand, assessment of CLDN18.2 expression is routinely performed on formalin fixed paraffin embedded (FFPE) biopsy or resected tumor samples using IHC. Although the Ventana CLDN18 (43-14A) and the Ventana CLDN18.2 RxDx assay is currently FDA-approved as an IHC companion diagnostic for detecting CLDN18.2 protein expression (57), multiple other immunohistochemical assays utilizing different antibody clones and staining platforms have been used in different laboratories and are summarized in Table 3. The recent global ring study compared performance of three CLDN18.2 antibodies (Ventana, LSBio, and Novus) on the staining platforms (Ventana, Dako, and Leica) in gastric cancer in 27 laboratories across 11 countries. This study showed a high concordance for the Ventana CLDN18 (43-14A) on Ventana platform only and LSBio antibodies on both the Dako and Leica platforms and reached a minimum acceptable ≥85% threshold and good-to-excellent levels of concordance in accuracy, precision, sensitivity, and specificity rates. In comparison, the Novus antibody exhibited the highest variability across the same antibody/platform combinations and did not meet the minimum thresholds for accuracy and sensitivity when tested on the Dako or Leica platforms (58). The results from this study demonstrated that different antibodies using different IHC platforms have different accuracy, sensitivity and specificity in detecting CLDN18.2 expression in gastric cancer.

Reporting of CLDN18.2 results

Standardized scoring systems are essential for the accurate evaluation of CLDN18.2 expression for the accurate selection of patients for CLDN18.2-targeted therapies. The pathologist is required to evaluate the intensity of membranous staining (not cytoplasmic staining) in tumor cells and the percentage of tumor cells with moderate to strong membranous staining for CLDN18.2 in the entire tumor area using the criteria defined as follows:

• Negative (0): no membranous staining (Figure 2A);
• Weak (1+): staining appears light brown, with membranous staining that may be partial or circumferential (Figure 2B);
• Moderate (2+): staining exhibits a chocolate brown hue, with membrane staining that may also be partial or circumferential (Figure 2C);
• Strong (3+): staining presents a dark brown to black with an evident chicken wire distribution similar to normal gastric glands at low magnification. The membrane staining is thickened and may be either partial or circumferential (Figure 2D).

Figure 2 Representative micrographs showing different immunohistochemical scores of CLDN18.2 expression at 20× magnification. (A) No membranous or cytoplasmic staining for CLDN18.2 (Score: 0); The strong (3+) staining in the intervening gastric glands served as the internal positive control. (B) Light brown staining with partial or circumferential membranous staining (Score: 1+). (C) Chocolate brown staining with partial or circumferential membranous staining (Score: 2+). (D) Dark brown to black staining with a chicken wire distribution and thickened membranous staining, exhibiting partial or circumferential membranous staining (Score: 3+).

The CLDN18.2 staining results should be reported as the percentage of tumor cells with 2+/3+ membranous staining. Based on the SPOTLIGHT and GLOW trials in G/GEJ cancers, CLDN18.2 positivity was defined as ≥75% of tumor cells exhibiting moderate (2+) to strong (3+) membranous staining, otherwise, the tumor is classified as CLDN18.2 negative (19,20). The same criteria are being used for pancreatobiliary cancers.

However, several questions remained regarding the optimal cut off for CLDN18.2 positivity. First, the CLDN18.2 positivity cut off 75% of tumor cells exhibiting moderate (2+) to strong (3+) membranous staining was established based on G/GEJ cancers and primarily on resected tumor samples. Whether this same cutoff is appropriate for other gastrointestinal tumor types, including pancreatobiliary cancers, remains uncertain. Second, different scoring criteria for HER2 IHC are used in G/GEJ cancers, with HER2 positivity defined as strong complete, basolateral, or lateral membranous reactivity in ≥10% of tumor cells for resection specimens, and as strong complete, basolateral, or lateral membranous reactivity in a cluster of ≥5 tumor cells, irrespective of percentage, for biopsy specimens. Currently, there is no data indicating whether distinct scoring criteria should be applied for CLDN18.2 in resection versus biopsy specimens. This issue is particularly relevant for small biopsy and cytology specimens, where limited sampling, intratumoral heterogeneity, and technical variability may affect the scoring and interpretation (29,59,60). Most patients with PDAC or biliary adenocarcinomas are present with locally advanced or metastatic disease at the time of diagnosis, rendering them ineligible for surgical intervention. Therefore, the majority of specimens from these patients are obtained through fine-needle aspiration cytology or fine-needle/core biopsy of the primary or metastatic tumors. For these types of specimens, it is necessary to evaluate both specimen adequacy and tumor cellularity before assessing CLDN18.2 expression. The current protocol recommends a minimum of 50 viable tumor cells for CLDN18.2 evaluation; however, at least 100 viable tumor cells may be necessary to ensure reliable results in some cases (61).

In a recent clinical trial, Ruan et al. reported the ORR of 23% in patients with G/GEJ adenocarcinoma treated with SHR-A1904 using a CLDN18.2 cut-off value of 2+/3+ membranous staining in ≥50% tumor cells for positivity. No response was observed in patients with low CLDN18.2 expression (>1+ in 1–50% tumor cells) (40). These findings raise the question of whether different cutoff values for CLDN18.2 positivity should be applied for different drugs or classes of CLDN18.2-targeted therapies. Future clinical trials and validation studies are needed to establish optimal thresholds that accurately identify patients most likely to benefit.

Several European countries, including Germany, Austria, and Switzerland, are implementing the proficiency program for CLDN18.2 in gastric cancer tissue samples (62). The preliminary proficiency tests among 52 institutions demonstrated 91% overall accuracy but a relatively high false-negative rate of 12% and indicated that staining quality was the major factor for successful analysis.

In the United States, while the College of American Pathologists requires proficiency testing for all pathologists interpreting HER2 IHC, no such proficiency program currently exists for CLDN18.2 testing, underscoring the need for standardization and quality assurance in reporting.

Potential challenges in CLDN18.2 assessment

There are several cautions to consider when interpreting the immunohistochemical status of CLDN18.2 as follows.

Cytoplasmic staining is frequently observed but should not be considered positive. Tumors with high CLDN18.2 expression may exhibit both diffuse strong membranous and cytoplasmic staining. In tumors containing signet ring cells, signet ring cells may display moderate to strong granular cytoplasmic staining (Figure 3A,3B), which may lead to overestimation of CLDN18.2 expression (63). Only distinct or clearly perceptible linear membranous staining in neoplastic cells should be included in the scoring.

Figure 3 Representative micrographs demonstrating atypical CLDN18.2 staining and insufficient tissue for CLDN18.2 testing. (A) Signet ring cell carcinoma with solid sheets of neoplastic signet ring cells on hematoxylin-eosin staining at 10× magnification. (B) Diffuse granular cytoplasmic staining with 1+ intensity for CLDN18.2 in signet ring cell carcinoma at 20x magnification, corresponding to micrograph in (A). (C) Scattered neoplastic cells in the lamina propria indicate low tumor cellularity in a gastric biopsy specimen after neoadjuvant therapy at 20× magnification. (D) Rare neoplastic cells are positive for CLDN18.2 staining. The tissue is insufficient for CLDN18.2 testing and evaluation, as shown in micrograph (C). (E) Acellular mucin pools without tumor cells at 20× magnification. (F) Acellular mucin pools without neoplastic cells exhibit a non-specific positive staining pattern for CLDN18.2, corresponding to micrograph in (E).

Strong membranous staining is consistently observed in non-neoplastic gastric epithelium, providing a reliable internal positive control. In certain cases, clusters of neoplastic cells are interspersed among non-neoplastic gastric epithelial glands, particularly in diffuse-type gastric adenocarcinoma. Both neoplastic and non-neoplastic cells may exhibit strong membranous staining. In this situation, it is important to identify the neoplastic cells on matched hematoxylin and eosin (H&E)-stained slides. CLDN18.2 expression should be evaluated only in tumor cells, not non-neoplastic cells.

Assessment of CLDN18.2 status should account for sampling limitations and technical variability encountered by pathologists. When inadequate tumor cells are present, the specimen should be considered as insufficient for the interpretation of CLDN18.2 expression (Figure 3C,3D). In addition, cautery artifacts in tumor tissue may result in false-positive staining, whereas under fixation of tumor tissue may lead to false-negative results.

Aberrant staining may occur in stroma mucin (Figure 3E,3F) and non-epithelial cells, including inflammatory and stromal cells (61). These cells should not be misidentified as neoplastic cells expressing CLDN18.2.

Both low- and high-grade dysplastic epithelium in the gastroesophageal junction exhibit similar rates of CLDN18.2 expression to those in invasive adenocarcinomas (64). Evaluation of CLDN18.2 expression should be restricted to the invasive adenocarcinoma component. CLDN18.2 is often expressed in pancreatic precursor lesions. Intestinal metaplasia, dysplasia and adenomas commonly arise in the gastric mucosa adjacent to gastric cancers and typically show minimal or patchy staining. These metaplastic/dysplastic epithelial cells must not be confused with intestinal-type adenocarcinoma cells when interpreting CLDN18.2 staining.

Intra-tumoral heterogeneity and selection of testing samples for CLDN18.2

In G/GEJ adenocarcinomas, intratumoral heterogeneity in CLDN18.2 expression is commonly observed across different regions of tumor sections (29,59) (Figure 4). Heterogeneity of CLDN18.2 expression may contribute to tumor resistance to CLDN18.2-targeted therapies. Coati et al. reported that approximately 40% of CLDN18.2-positive gastric adenocarcinomas and 33.6% of GEJ adenocarcinomas exhibited significant intratumor heterogeneity in CLDN18.2 expression (29). Additionally, in metastatic tumors, around 29% showed intratumoral heterogeneity in CLDN18.2 expression. Recently, Choi et al. described that approximately 38.5% (15/32) CLDN18.2-positive gastric adenocarcinomas also displayed intratumoral heterogeneity and reported a concordance rate of 75% in CLDN18.2 expression between paired primary and metastatic tumors (59). In another study by Son et al., intratumoral heterogeneity was observed in 25.5% of primary G/GEJ cancers and 19.6% of metastatic G/GEJ cancers and the discordance rate between the primary and metastatic G/GEJ cancers was approximately 20% (65). In a study of 27 cases of metastatic G/GEJ adenocarcinomas, there was approximately a 67% concordance in CLDN18.2 expression when comparing matched primary and metastatic tumor samples (25). The concordance rate of CLDN18.2 expression is 70–80% between primary tumors and metastatic lymph nodes (28,66). Given the heterogeneous CLDN18.2 expression and discordant expression of CLDN18.2 between the primary and metastatic tumors, it is essential to select the tumor block that represents different differentiation and phenotypes for CLDN18.2 testing.

Figure 4 Representative micrographs illustrating heterogeneous CLDN18.2 expression in gastroesophageal junctional adenocarcinoma and PDAC at 10× magnification. (A) Transition area of gastroesophageal adenocarcinoma with a moderately differentiated component (left side) and a poorly differentiated component (right side) on hematoxylin-eosin staining. (B) Diffuse and strong membranous staining pattern in the moderately differentiated component (left side) and weak, scattered staining in the poorly differentiated component (right side), corresponding to micrograph (A). (C) Moderately differentiated PDAC with haphazardly arranged small and large neoplastic glands on hematoxylin-eosin staining. (D) Skipped or scattered staining within neoplastic glands, corresponding to micrograph (C). PDAC, pancreatic ductal adenocarcinoma.

Currently, no specific guidelines are available regarding sample selection for CLDN18.2 testing, including the type of specimen (resection vs. biopsy), the use of primary versus metastatic adenocarcinoma samples, or the minimal amount of tumor tissue required. Therefore, the College of American Pathologists guidelines for cancer biomarker testing, including recommendations for specimen handling, fixation, and IHC assay validation should be followed (67). The CLDN18.2 testing should be performed on resection specimens when both biopsy and resection tissue are available. If the assay is performed on a biopsy specimen, an international panel of gastrointestinal pathologists recommends evaluating more than five tissue fragments. When there are fewer fragments available, all tumor tissue specimens should be tested to ensure representative sampling for CLDN18.2 assessment in endoscopic or interventional radiology biopsies (61). This panel also recommended that at least 50 viable malignant cells (preferably 100) be evaluated in effusion cytology when assessing CLDN18.2 expression. If the biopsy or cytology specimens contains fewer than 50 viable tumor cells, the specimen is deemed inadequate for CLDN18.2 testing, and a new specimen should be obtained (57). In addition, if the tumor cell staining pattern is ambiguous or unusual, such as exhibiting only nuclear staining, the specimen should be classified as inadequate for CLDN18.2 scoring, and retesting should be performed with a new or different specimen. The panel also emphasized the importance of optimizing preanalytical variables, such as collection media, fixatives, and preservatives, to ensure accurate staining and interpretation. However, additional studies are needed to validate these recommendations.

Although the international panel of gastrointestinal pathologists suggested that CLDN18.2 expression is highly concordant in matched primary and metastatic lesions and that either primary or metastatic adenocarcinoma sample may be used for CLDN18.2 testing (61). Recent studies reported concordance rates between primary and metastatic G/GEJ cancers ranging from 67% to 80% (25,28,65,66). The discordant expression of CLDN18.2 may result from intratumoral heterogeneity, with different tumor clones in the metastatic lesion, or from sampling bias. In some cases, using metastatic tumor samples may provide a more representative assessment of CLDN18.2 status. Regardless of sample type, it is critical to ensure that tumor specimens are quantitatively adequate to allow accurate evaluation of CLDN18.2 expression.

Currently, no data is available regarding the potential impact of cancer therapies on CLDN18.2 expression. It remains unclear whether therapeutic interventions, including chemotherapy, radiotherapy, or targeted agents, influence the stability or level of CLDN18.2 expression following treatment. The clinical value of re-evaluating CLDN18.2 status in patients receiving neoadjuvant therapy has not been established. Future studies are needed to determine whether repeat testing should be recommended to guide treatment decisions.

Conclusions

The growing use and ongoing clinical trials of CLDN18.2-targeted therapies in G/GEJ cancers, as well as pancreatobiliary cancers, have increased the need for reliable diagnostic testing. Optimizing the clinical utility of CLDN18.2 testing requires rigorous control of pre-analytic, analytic, and post-analytic variables, including the implementation of standardized scoring methods and clearly defined cutoff criteria. CLDN18.2 expression frequently exhibits substantial heterogeneity within these malignancies. Consequently, careful selection and evaluation of tumor samples, especially from small biopsies or cytology specimens, are essential for accurate assessment. Comprehensive standardization of all steps in CLDN18.2 IHC, from specimen preparation to interpretation, is necessary to achieve accurate, reproducible, and clinically meaningful results that guide therapeutic decisions for patients with G/GEJ and pancreatobiliary cancers.

Acknowledgments

None.

Footnote

Peer Review File: Available at https://cco.amegroups.com/article/view/10.21037/cco-2025-1-170/prf

Funding: None.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://cco.amegroups.com/article/view/10.21037/cco-2025-1-170/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of the University of Texas MD Anderson Cancer Center (UT MDACC) (IRB: LAB05-0854). Informed consent was obtained from all participants enrolled in the study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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