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JCO Precis Oncol
JCO Precis Oncol
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JCO Precision Oncology
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Wolters Kluwer Health

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PO.23.00543
10.1200/PO.23.00543
00116
Original Reports
Biomarkers
Retrospective Study of Claudin 18 Isoform 2 Prevalence and Prognostic Association in Gastric and Gastroesophageal Junction Adenocarcinoma
Waters Rebecca DO 1
https://orcid.org/0000-0003-4804-1891
Sewastjanow-Silva Matheus MD 1
Yamashita Kohei MD 1
https://orcid.org/0000-0001-7186-1332
Abdelhakeem Ahmed MD 1
https://orcid.org/0009-0003-5842-2863
Iwata Kenneth K. PhD 2
Moran Diarmuid PhD 2
Elsouda Dina MSIA, MSc 2
Guerrero Abraham PhD 2
Pizzi Melissa MSc 1
https://orcid.org/0009-0004-1272-7650
Vicentini Ernesto Rosa MD 1
Shanbhag Namita MS 1
Ta Anh PhD 1
https://orcid.org/0000-0002-3322-1163
Chatterjee Deyali MD 1
https://orcid.org/0000-0001-9946-0629
Ajani Jaffer A. MD 1
1 MD Anderson Cancer Center, Houston, TX
2 Astellas Pharma, Inc, Northbrook, IL
Jaffer A. Ajani, MD; Twitter: @JafferAjaniMD; e-mail: jajani@mdanderson.org.
2024
23 5 2024
23 5 2024
8 e230054329 9 2023
15 3 2024
11 4 2024
© 2024 by American Society of Clinical Oncology
2024
American Society of Clinical Oncology
https://creativecommons.org/licenses/by-nc-nd/4.0/ Creative Commons Attribution Non-Commercial No Derivatives 4.0 License: http://creativecommons.org/licenses/by-nc-nd/4.0/

PURPOSE

Claudin 18 isoform 2 (CLDN18.2) is an emerging biomarker and therapeutic target in gastric and gastroesophageal junction (G/GEJ) adenocarcinoma. This study aimed to obtain deeper understanding of CLDN18.2 positivity patterns, prognostic implications, and associations with various demographic, clinical, and molecular characteristics in G/GEJ adenocarcinoma.

METHODS

Archived tumor tissue samples from 304 patients with G/GEJ adenocarcinoma in the United States were assessed for CLDN18.2 positivity by immunohistochemistry. CLDN18.2 positivity was defined as ≥50% or ≥75% of tumor cells with CLDN18 staining intensity ≥2+. CLDN18.2 positivity patterns were analyzed for association with prognosis and clinicopathologic/demographic characteristics. Where possible, CLDN18.2 positivity was analyzed for matched tissue samples to assess concordance between primary and metastatic tumors and concordance before and after chemotherapy.

RESULTS

The overall prevalence of CLDN18.2-positive tumors (with ≥75% cutoff) was 44.4% (n = 135 of 304). CLDN18.2-positive tumors had a prevalence of 51.4% (n = 91 of 177) in gastric and 34.6% (n = 44 of 127) in GEJ adenocarcinoma. With a ≥50% cutoff, the prevalence of CLDN18.2-positive tumors was 64.4% (n = 114 of 177) in gastric adenocarcinoma and 44.9% (n = 57 of 127) in GEJ adenocarcinoma. There was no association between overall survival and CLDN18.2 positivity using either threshold. Statistically significant associations were noted between CLDN18.2 positivity and sex, histologic type of G/GEJ adenocarcinoma, and adenocarcinoma subtype (≥75% cutoff), and metastasis site and tumor grade (≥50% cutoff). The overall concordance of CLDN18.2 positivity (≥75% cutoff) was 73% (27 of 37) for matched primary versus metastatic tumor samples and 74% (29 of 39) for matched samples before and after chemotherapy.

CONCLUSION

This study demonstrated that CLDN18.2 positivity did not correlate with survival in G/GEJ adenocarcinoma, consistent with published data. On the basis of matched sample analysis, CLDN18.2 appears to demonstrate >70% concordance as a biomarker. Observed correlations with certain patient/tumor characteristics warrant further study.

OPEN-ACCESSTRUE
==== Body
pmcINTRODUCTION

Gastric and gastroesophageal junction (G/GEJ) adenocarcinoma is a significant health burden. In 2020, there were an estimated 1.7 million new cases of gastric and esophageal cancer globally and an estimated 1.3 million related deaths.1 In the United States, the estimated numbers of new cases and related deaths for the year 2023 were 48,060 and 27,250, respectively.2 Treatment options for G/GEJ adenocarcinoma are limited, and the 5-year relative survival rate in cases with distant spread has remained at approximately 10% since 2007 in the United States.3 Hence, there is a continuous need for better understanding of disease characteristics, biomarkers, and how clinical and tumor-related features might affect diagnosis, management, and prognosis.

CONTEXT

Key Objective

In light of emerging interest in claudin 18 isoform 2 (CLDN18.2) as a biomarker and a therapeutic target in gastric/gastroesophageal junction (G/GEJ) adenocarcinoma, we assessed clinicopathologic/molecular characteristics and potential prognostic correlations associated with CLDN18.2 status using archived tissue samples from patients with G/GEJ adenocarcinoma.

Knowledge Generated

Our data did not demonstrate any correlation between CLDN18.2 positivity and survival, but associations were noted between CLDN18.2 positivity and site of metastasis, Lauren histologic type, and adenocarcinoma subtype. Analysis of matched samples (primary/metastatic tumor tissues; prechemotherapy/postchemotherapy tissues) suggested that CLDN18.2 demonstrates concordance as a biomarker.

Relevance

This study contributes to a growing body of research aimed at a better understanding of the prevalence and potential therapeutic implications of CLDN18.2 positivity in patients with G/GEJ adenocarcinoma. Ongoing interpretation of the accumulating data in this area will help steer the development and clinical role of new therapies targeting CLDN18.2.

The claudin (CLDN) family of transmembrane proteins includes at least 27 unique proteins that serve as primary components of tight junction complexes in cell membranes.4,5 CLDNs, found in normal and cancerous tissues, have unique patterns of expression.4,5 Claudin 18 isoform 2 (CLDN18.2) is present in gastric mucosa. In malignant transformation of gastric mucosal cells, CLDN18.2 is retained and may become exposed and accessible as tight junctions become disrupted.6-8 As a result, CLDN18.2 has gained substantial interest as an emerging biomarker and a therapeutic target in G/GEJ adenocarcinoma.

An antibody against CLDN18.2 (zolbetuximab) has shown considerable promise in randomized, controlled, global clinical studies in patients with CLDN18.2-positive G/GEJ adenocarcinoma. In the phase III SPOTLIGHT trial, zolbetuximab plus modified FOLFOX6 (mFOLFOX6; leucovorin [folinic acid], fluorouracil, and oxaliplatin) was associated with significantly longer median progression-free survival versus mFOLFOX6 plus placebo (10.61 v 8.67 months; hazard ratio [HR], 0.751; P = .0066) and longer median overall survival (18.23 v 15.54 months; HR, 0.75; P = .0053).9 In the phase III GLOW trial, zolbetuximab plus CAPOX (capecitabine and oxaliplatin) significantly prolonged median progression-free survival (8.21 v 6.80 months; HR, 0.687; P = .0007) and median overall survival (14.39 v 12.16 months; HR, 0.771; P = .0118) versus placebo in treatment-naïve patients.10 A randomized phase II study (FAST11) demonstrated significant improvements in median progression-free survival (7.5 v 5.3 months; HR, 0.44; P < .0005) and median overall survival (13 v 8.3 months; HR, 0.55; P < .0005) with zolbetuximab plus EOX (epirubicin, oxaliplatin, and capecitabine) versus EOX alone.

There remains a need for data on the prevalence of CLDN18.2 positivity in G/GEJ adenocarcinoma, its association with disease prognosis, and correlation with demographic, clinical, and molecular characteristics. This study was conducted to determine the prevalence of CLDN18.2 positivity among tumor specimens from patients with advanced G/GEJ adenocarcinoma. This study was also designed to explore CLDN18.2 positivity as a prognostic factor and evaluate clinicopathologic and molecular characteristics associated with CLDN18.2 positivity.

METHODS

Study Design

This was a retrospective translational research study that used tissue samples from the MD Anderson Cancer Center (MDACC) biorepository of patients with G/GEJ adenocarcinoma, along with clinical data extracted from patient medical records. The study was approved by the MDACC Institutional Review Board and conducted according to Good Clinical and Pharmacoepidemiology Practice and in adherence with all applicable federal, state, and local laws, rules, and regulations. All patients provided written informed consent.

Tissue samples were required to be from patients with histologically confirmed G/GEJ adenocarcinoma, must have been formalin-fixed, paraffin-embedded whole tissue specimens available for further analysis, and appropriately consented by source patients. In addition, there must have been additional clinicopathologic information available on the source patient such as treatment history, clinical outcomes, and additional biomarker data. Tissue samples were excluded if older than 10 years and/or if clinical outcome data were not available.

Biomarker status, histologic subtypes, and other clinicopathologic characteristics were obtained from records.

Immunohistochemistry

Immunohistochemistry (IHC) for the CLDN18 antigen was performed on 4-µm paraffin sections of each cancer tissue sample. Tissue sample slide staining was performed by the MDACC pathology laboratory using the on-market VENTANA CLDN18 (43-14A) Assay (08504148001; Ventana Medical Systems, Inc; hereafter VMSI), as per its instructions for use. CLDN18.2 positivity for stained samples was assessed independently by one of two MDACC pathologists. The VENTANA CLDN18 (43-14A) Assay detects total CLDN18 expression (both 18.1 and 18.2 isoforms); positive staining of CLDN18 was interpreted as CLDN18.2 because CLDN18.2 is the dominant isoform observed in G/GEJ adenocarcinoma, whereas the CLDN18.1 isoform is absent/negligible.12

A pilot phase was conducted on the first 60 stained samples to validate the technical quality of slide staining by MDACC personnel as compared with historical VMSI-processed samples and slides. Review of pilot phase data confirmed successful staining by MDACC personnel using the on-market VENTANA CLDN18 (43-14A) Assay (08504148001).

Clinical Characteristics

Tumor stage was defined according to American Joint Committee on Cancer criteria.13 Histologic type was determined using Lauren classification.14 Staining of available pair-matched metastatic and primary tissues and pair-matched pre- and postchemotherapy tissue samples was performed. All tissue sample slides were reviewed by two GI pathologists; in the event of a discrepancy between pathology reports, a third pathologist reviewed the tissue sample slide.

Molecular Characteristics

Molecular characteristics evaluated in the study included human epidermal growth factor receptor 2 (HER2), mismatch repair (MMR) status, Helicobacter pylori, Epstein-Barr virus (EBV), and PD-L1. Assessment for Her2-neu (PATHWAY anti-HER2 [4B5] Rabbit Monoclonal Antibody; VMSI) status was assessed according to the modified scoring system used in the Trastuzumab for Gastric Cancer trial.15 Results were considered negative (score of 0 or 1+), equivocal (score of 2+), or positive (score of 3+). With an equivocal result, fluorescence in situ hybridization (PathVysion HER-2/neu DNA Probe Kit, Abbott, Abbott Park, IL) was performed. For the MMR assessment, immunoperoxidase stains were performed on sections from formalin-fixed, paraffin-embedded invasive carcinoma with antibodies for the DNA MMR enzymes MLH1, MSH2, MSH6, and PMS2. Intact nuclear expression of the four enzymes was considered as microsatellite stable/proficient MMR. Loss of expression of any of the four enzymes was considered a microsatellite instability (MSI)-high/deficient MMR result. H. pylori presence or absence was determined by hematoxylin and eosin stain or IHC stain. EBV-encoded RNA was used to determine the presence or absence of EBV by in situ hybridization. Assessment of PD-L1 (clone 22C3; Dako, Santa Clara, CA) was performed on a subset of formalin-fixed, paraffin-embedded samples, and the combined positive score (CPS) was reported. This scoring method evaluated the number of PD-L1–staining cells (tumor cells, lymphocytes, macrophages) relative to all viable tumor cells. CPS < 1 was considered a negative result, and CPS ≥ 1 was considered a positive result.

Statistical Analysis

A sample size of 300 patients/samples was estimated to provide a 95% margin of error of 0.057 and a 95% CI width of 0.113.

CLDN18.2 positivity was defined and analyzed on the basis of two cutoffs: ≥75% of tumor cells with 2+ and/or 3+ CLDN18 staining intensity and ≥50% of tumor cells with 2+ and/or 3+ CLDN18 staining intensity (hereafter, 2+ and/or 3+ CLDN18 staining intensity above or below a given cutoff is referred to as CLDN18.2-positive or CLDN18.2-negative status, respectively).

Associations between CLDN18.2 status and demographic and clinicopathologic variables were assessed using the chi-square test of independence. The association between CLDN18.2 status and overall survival was assessed using Pearson and Spearman rank correlation coefficients and Kaplan-Meier methods. HRs were calculated using a univariate log-rank model to determine if CLDN18.2 positivity (at ≥50% or ≥75% cutoff) had a prognostic effect on overall survival; these univariate models did not adjust for any potential covariates. Patients with no events were censored at last follow-up date. All statistical comparisons were exploratory in nature, and therefore, no multiplicity adjustments were required. Where possible, CLDN18.2 positivity was analyzed for matched tissue samples to assess concordance between primary and metastatic tumors and concordance before and after chemotherapy. The overall percent agreement rate was determined on the basis of a 75% cutoff. CLDN18.2 positivity patterns within subgroups were summarized descriptively.

P values <.05 were considered statistically significant. All statistical analyses were conducted using SAS version 9.4 (SAS Institute, Cary, NC).

RESULTS

The analysis included a total of 304 G/GEJ adenocarcinoma tissue samples from 304 unique patients, archived between 2011 and 2021. Of these, 177 (58.2%) were gastric and 127 (41.8%) were GEJ adenocarcinoma.

Prevalence of CLDN18.2 Positivity

On the basis of a ≥75% cutoff for 2+ and/or 3+ staining, 44.4% (135 of 304) of all samples were CLDN18.2-positive, 51.4% (91 of 177) of gastric adenocarcinoma samples were CLDN18.2-positive, and 34.6% (44 of 127) of GEJ adenocarcinoma samples were CLDN18.2-positive (Table 1). On the basis of a ≥50% cutoff, the proportions of CLDN18.2-positive samples were 56.3% (171 of 304) overall, 64.4% (114 of 177) in gastric adenocarcinoma samples, and 44.9% (57 of 127) in GEJ adenocarcinoma samples.

TABLE 1. Prevalence of CLDN18.2 Positivity in Study Samples Using ≥50% and ≥75% Cutoffs

CLDN18.2 Positivity Threshold	Gastric Adenocarcinoma (n = 177), No. (%)	GEJ Adenocarcinoma (n = 127), No. (%)	Total (N = 304), No. (%)	
2+ and 3+ staining ≥50%	114 (64.4)	57 (44.9)	171 (56.3)	
2+ and 3+ staining ≥75%	91 (51.4)	44 (34.6)	135 (44.4)	
NOTE. The denominator used for each percentage calculation was the sample n for each column (ie, number of samples of each cancer subtype and total number of samples).

Abbreviations: CLDN18.2, claudin 18 isoform 2; GEJ, gastroesophageal junction.

CLDN18.2 Positivity as a Prognostic Factor in G/GEJ Adenocarcinoma

There was no association between overall survival and CLDN18.2 positivity using either the ≥50% or ≥75% cutoff. The Pearson correlation coefficient and 95% CI for overall survival and CLDN18.2 positivity were 0.04 (–0.12 to 0.20) on the basis of a ≥50% cutoff and –0.04 (–0.21 to 0.14) on the basis of a ≥75% cutoff. The HR (95% CI) for overall survival was 0.92 (0.62 to 1.37) using a ≥50% cutoff, indicating that the overall survival was 8.6% higher for the CLDN18.2-negative group compared with the CLDN18.2-positive group. Similarly, using a ≥75% cutoff, the HR (95% CI) was 0.75 (0.50 to 1.14), indicating that the overall survival was 33.3% higher for the CLDN18.2-negative group compared with the CLDN18.2-positive group (Fig 1). Neither of these results were statistically significant.

FIG 1. Kaplan-Meier curves for overall survival by CLDN18.2 positivity status on the basis of (A) ≥75% or (B) ≥50% cutoff. CLDN18.2, claudin 18 isoform 2; HR, hazard ratio.

Association Between CLDN18.2 and Demographic Characteristics

Among the demographic variables tested for association with CLDN18.2 positivity (sex, ethnicity, race, and age), sex was the only statistically significant characteristic (P ≤ .001). The percentage of males was greater among CLDN18.2-negative samples compared with CLDN18.2-positive samples for both ≥50% (P = .0003) and ≥75% (P = .001) cutoffs (Table 2).

TABLE 2. Demographic, Clinical, and Molecular Characteristics of CLDN18.2-Negative and CLDN18.2-Positive Subgroups

Characteristic	≥50% Cutoff	≥75% Cutoff	
CLDN18.2-Negative (<50%; n = 133)	CLDN18.2-Positive (≥50%; n = 171)	P	CLDN18.2-Negative (<75%; n = 169)	CLDN18.2-Positive (≥75%; n = 135)	P	
Sex, No. (%)	
 Male	108 (81.2)	106 (62)	.0003	132 (78.1)	82 (60.7)	.001	
 Female	25 (18.8)	65 (38)	37 (21.9)	53 (39.3)	
Ethnicity, No. (%)	
 Hispanic/Latino	15 (11.3)	21 (12.3)	.7155	18 (10.7)	18 (13.3)	.3442	
 Not Hispanic/Latino	117 (88)	147 (86)	150 (88.8)	114 (84.4)	
 Unknown	1 (0.8)	3 (1.8)	1 (0.6)	3 (2.2)	
Race, No. (%)	
 White	105 (78.9)	136 (79.5)	.6422	134 (79.3)	107 (79.3)	.4062	
 Black/African American	14 (10.5)	13 (7.6)	16 (9.5)	11 (8.1)	
 Asian	6 (4.5)	6 (3.5)	9 (5.3)	3 (2.2)	
 Other	6 (4.5)	14 (8.2)	8 (4.7)	12 (8.9)	
 Unknown	2 (1.5)	2 (1.2)	2 (1.2)	2 (1.5)	
Age	
 Mean (SD)	64.4 (11.2)	62.4 (12.1)	.4796	64 (11.5)	62.4 (12.1)	.1248	
 Median (min, max)	66 (26, 89)	65 (21, 86)	66 (26, 89)	65 (21, 86)	
Site of metastasis (only those >7% depicted), No. (%)	
 Peritoneum	10 (7.5)	45 (26.3)	.0403	16 (9.5)	39 (28.9)	.0930	
 Unknown	86 (64.7)	94 (55)	109 (64.5)	71 (52.6)	
ECOG PS, No. (%)	
 0	52 (39.1)	61 (35.7)	.1448	64 (37.9)	49 (36.3)	.7076	
 1	73 (54.9)	104 (60.8)	96 (56.8)	81 (60)	
 2	6 (4.5)	2 (1.2)	6 (3.6)	2 (1.5)	
 3	2 (1.5)	1 (0.6)	2 (1.2)	1 (0.7)	
 NS	0	3 (1.8)	1 (0.6)	2 (1.5)	
Disease stage, No. (%)	
 I	6 (4.5)	12 (7)	.4831	10 (5.9)	8 (5.9)	.4928	
 IIA	2 (1.5)	2 (1.2)	2 (1.2)	2 (1.5)	
 IIB	20 (15)	28 (16.4)	29 (17.2)	19 (14.1)	
 III	51 (38.3)	46 (26.9)	61 (36.1)	36 (26.7)	
 IV	33 (24.8)	54 (31.6)	42 (24.9)	45 (33.3)	
 IVA	8 (6)	8 (4.7)	9 (5.3)	7 (5.2)	
 IVB	13 (9.8)	20 (11.7)	16 (9.5)	17 (12.6)	
 NA	0	1 (0.6)	0	1 (0.7)	
Tumor grade, No. (%)	
 G1—well differentiated	1 (0.8)	0	.0399	1 (0.6)	0	.0764	
 G2—moderately differentiated	53 (39.8)	41 (24)	62 (36.7)	32 (23.7)	
 G2-G3—moderately to poorly differentiated	7 (5.3)	13 (7.6)	8 (4.7)	12 (8.9)	
 G3—poorly differentiated	72 (54.1)	115 (67.3)	97 (57.4)	90 (66.7)	
 GX—undetermined grade	0	1 (0.6)	0	1 (0.7)	
 NA	0	1 (0.6)	1 (0.6)	0	
Lauren histologic type, No. (%)	
 Intestinal	21 (15.8)	31 (18.1)	.0222	28 (16.6)	24 (17.8)	.0489	
 Diffuse	30 (22.6)	62 (36.3)	42 (24.9)	50 (37)	
 NS	82 (61.7)	77 (45)	99 (58.6)	60 (44.4)	
 Other	0	1 (0.6)	0	1 (0.7)	
Adenocarcinoma subtype, No. (%)	
 Signet ring carcinoma	39 (29.3)	78 (45.6)	.0038	53 (31.4)	64 (47.4)	.0043	
 NOS	94 (70.7)	93 (54.4)	116 (68.6)	71 (52.6)	
HER2, No. (%)	
 Positive	10 (7.5)	15 (8.8)	.2849	13 (7.7)	12 (8.9)	.4002	
 Negative	80 (60.2)	115 (67.3)	103 (60.9)	92 (68.1)	
 Equivocal (2+)	1 (0.8)	3 (1.8)	2 (1.2)	2 (1.5)	
 NS	42 (31.6)	38 (22.2)	51 (30.2)	29 (21.5)	
MSI, No. (%)	
 MSI-H	6 (4.5)	4 (2.3)	.2167	7 (4.1)	3 (2.2)	.5694	
 MSS	46 (34.6)	74 (43.3)	64 (37.9)	56 (41.5)	
 NS	81 (60.9)	83 (54.4)	98 (58)	76 (56.3)	
Helicobacter pylori, No. (%)	
 Positive	7 (5.3)	16 (9.4)	.0847	10 (5.9)	13 (9.6)	.3749	
 Negative	95 (71.4)	130 (76)	125 (74)	100 (74.1)	
 NS	31 (23.3)	25 (14.6)	34 (20.1)	22 (16.3)	
EBV, No. (%)	
 Positive	0	3 (1.8)	.2671	0	3 (2.2)	.1318	
 Negative	4 (3)	7 (4.1)	7 (4.1)	4 (3)	
 NS	129 (97)	161 (94.2)	162 (95.9)	128 (94.8)	
NOTE. Cutoffs defined according to the percentage of tumor cells with combined 2+ and 3+ staining intensities for CLDN18.2 meeting or exceeding a given cutoff (≥50% or ≥75%).

Abbreviations: CLDN18.2, claudin 18 isoform 2; EBV, Epstein-Barr virus; ECOG PS, Eastern Cooperative Oncology Group performance status; HER2, human epidermal growth factor receptor 2; max, maximum; min, minimum; MSI, microsatellite instability; MSI-H, MSI-high; MSS, microsatellite stable; NA, not applicable; NOS, not otherwise specified; NS, not specified; SD, standard deviation.

Regardless of cutoff (≥50% or ≥75%), CLDN18.2 positivity was higher among females compared with males by approximately 20% (Fig 2; Appendix Table A1). CLDN18.2 positivity rates for samples from White, Black/African American, and Asian patients were generally similar.

FIG 2. CLDN18.2 positivity within demographic, clinical, and molecular patient/disease subgroups on the basis of ≥50% and ≥75% cutoffs. Only data points with a sample n ≥ 10 are shown; as a result, total n for some categories may not total 100. CLDN18.2, claudin 18 isoform 2; ECOG PS, Eastern Cooperative Oncology Group performance status; G2, moderately differentiated; G3, poorly differentiated; HER2, human epidermal growth factor receptor 2; MSI, microsatellite instability; MSI-H, MSI-high; MSS, microsatellite stable; NOS, not otherwise specified; SRC, signet ring carcinoma. aCells staining 2+ and/or 3+ for CLDN18 at or above the designated cutoff.

Association Between CLDN18.2 and Clinical Characteristics

Among the clinical characteristics analyzed (site of metastasis, Eastern Cooperative Oncology Group performance score, disease stage, tumor grade, and histologic type of gastric adenocarcinoma), significant associations were observed between CLDN18.2 and metastasis site and tumor grade at a ≥50% cutoff, whereas the histologic type of gastric adenocarcinoma and adenocarcinoma subtypes (signet ring carcinoma [SRC] and not otherwise specified) was statistically significantly associated with CLDN18.2 positivity at both cutoffs (Table 2).

Site of Metastasis

Location of metastasis was documented only for distant metastasis (M1). Patients classified with unknown metastasis either had no metastasis (N0 M0) or had metastatic disease that only involved regional lymph nodes (LNs) and not a distant site. The only metastasis site reported in >7% of patients was the peritoneum (18.1%; 55 of 304). On the basis of a ≥50% cutoff, a higher percentage of CLDN18.2-positive samples versus CLDN18.2-negative samples was from patients with peritoneal metastases (26.3% [45 of 171] v 7.5% [10 of 133]; P = .0403). There was no significant association between the metastasis site and CLDN18.2 status using a ≥75% cutoff.

Among samples from patients with peritoneum as the known site of metastasis, 81.8% (45 of 55) and 71% (39 of 55) of samples were CLDN18.2-positive on the basis of ≥50% and ≥75% cutoffs, respectively (Fig 2; Appendix Table A1). These percentages were notably higher than those for the remaining 180 samples for which the metastasis site was unknown (52.2% [94 of 180] and 39.4% [71 of 180], respectively).

Tumor Grade

On the basis of a ≥50% cutoff, a larger percentage of CLDN18.2-positive samples versus CLDN18.2-negative samples was G3 tumor grade (67.3% [115 of 171] v 54.1% [72 of 133]) although the percentage of G2 grade samples was smaller among CLDN18.2-positive versus CLDN18.2-negative samples (24% [41 of 171] v 39.8% [53 of 133]; P = .0399 for association between the tumor grade and CLDN18.2). A similar, but not statistically significant, pattern was observed using a ≥75% cutoff.

Tumor grades across all samples were predominantly G3 (poorly differentiated; 61.5% [187 of 304]) or G2 (moderately differentiated; 30.9% [94 of 304]). CLDN18.2 positivity increased with increasing tumor grade. Among the 94 patients with G2 tumor grade, 43.6% (41 of 94) and 34% (32 of 94) of samples were CLDN18.2-positive using cutoffs of ≥50% and ≥75%, respectively (Fig 2; Appendix Table A1). CLDN18.2 positivity was higher among the 187 patients with G3 tumor grade (61.5% [115 of 187] and 48.1% [90 of 187] on the basis of ≥50% and ≥75% cutoffs, respectively).

Histologic Type

According to Lauren classification, intestinal type was observed in 15.8% (21 of 133) of the CLDN18.2-negative cohort and 18.1% (31 of 171) of the CLDN18.2-positive cohort (using a ≥50% cutoff), whereas diffuse type was observed in 22.6% (30 of 133) of the CLDN18.2-negative and 36.3% (62 of 171) of the CLDN18.2-positive samples (P = .0222). Similar patterns were observed using a ≥75% cutoff (P = .0489).

CLDN18.2 positivity was not notably different between samples with diffuse histologic type (67.4%, 62 of 92 [≥50% cutoff]; 54.3%, 50 of 92 [≥75% cutoff]) or intestinal histologic type (59.6%, 31 of 52 [≥50% cutoff]; 46.2%, 24 of 52 [≥75% cutoff]; Fig 2; Appendix Table A1).

Adenocarcinoma Subtype

On the basis of a ≥50% cutoff, signet ring adenocarcinoma subtype was noted in 29.3% (39 of 133) of CLDN18.2-negative samples and 45.6% (78 of 171) of CLDN18.2-positive samples (P = .0038). On the basis of a ≥75% cutoff, signet ring adenocarcinoma subtype was noted in 31.4% (53 of 169) of CLDN18.2-negative samples and 47.4% (64 of 135) of CLDN18.2-positive samples (P = .0043).

CLDN18.2 positivity among samples with a SRC histologic subtype was 66.7% (78 of 117) at the ≥50% cutoff and 54.7% (64 of 117) at the ≥75% cutoff (Fig 2; Appendix Table A1).

Association Between CLDN18.2 and Molecular Characteristics

The molecular characteristics examined included HER2, MSI, H. pylori, and EBV. There were no statistically significant associations between CLDN18.2 positivity and any of these molecular characteristics at either cutoff (≥50% or ≥75%).

CLDN18.2 positivity was not notably different between HER2-positive samples (60%, 15 of 25 [≥50% cutoff]; 48%, 12 of 25 [≥75% cutoff]) and HER2-negative samples (59%, 115 of 195 [≥50% cutoff]; 47.2%, 92 of 195 [≥75% cutoff]; Fig 2; Appendix Table A1).

Among the small number of patients with known EBV status (n = 14), 100% of EBV-positive samples (3 of 3) were CLDN18.2-positive regardless of cutoff, whereas among EBV-negative samples, 63.6% (7 of 11) and 36.4% (4 of 11) were CLDN18.2-positive using ≥50% and ≥75% cutoffs, respectively.

PD-L1 positivity (defined as CPS ≥ 1) was not significantly different between CLDN18.2-positive samples (66.03%, 35 of 53 [≥75% cutoff]) and CLDN18.2-negative samples (72.13%, 44 of 61 [<75% cutoff]; P = .615; Appendix Table A2).

Concordance Analyses Between Matched Tumor Samples

CLDN18.2 positivity was generally similar between matched primary and metastatic samples (Table 3 and Appendix Fig A1) and between tissues taken before and after chemotherapy (Table 4).

TABLE 3. Comparison of CLDN18.2 Positivity (≥75% cutoff) Between Matched Primary and Metastatic Tumors

Characteristic	Metastatic ≥75%	Metastatic <75%	Total	
Primary ≥75%	14	8	22	
Primary <75%	2	13	15	
Total	16	21	37	
NOTE. Overall percentage agreement: 73%.

Abbreviation: CLDN18.2, claudin 18 isoform 2.

TABLE 4. Comparison of CLDN18.2 Positivity (≥75% cutoff) From the Same Tumor Before (untreated) and After (treated) Chemotherapy

Characteristic	Treated ≥75%	Treated <75%	Total	
Untreated ≥75%	13	9	22	
Untreated <75%	1	16	17	
Total	14	25	39	
NOTE. Overall percentage agreement: 74.3%.

Abbreviation: CLDN18.2, claudin 18 isoform 2.

DISCUSSION

This study evaluated the prevalence of CLDN18.2 positivity, prognostic implications, and relationships with clinical characteristics and select biomarkers in G/GEJ adenocarcinoma from a single site in the United States. We found no correlation between CLDN18.2 positivity and overall survival, consistent with other recent studies that demonstrated no significant impact of CLDN18.2 positivity on prognosis in G/GEJ adenocarcinoma.6,16

CLDN18.2 positivity was evaluated using ≥50% or ≥75% cutoffs for tumors demonstrating moderate to strong staining as determined by IHC using the on-market VENTANA CLDN18 (43-14A) assay (08504148001). The overall prevalence of CLDN18.2 positivity at the ≥75% cutoff was 44%, consistent with 38% from the phase III SPOTLIGHT and GLOW studies.9,10

We observed statistically significant associations between CLDN18.2 and sex, histologic type of gastric adenocarcinoma, and adenocarcinoma subtype with a ≥75% cutoff, as well as peritoneal metastasis and tumor grade with a ≥50% cutoff.

Certain previous cohort studies have demonstrated associations between CLDN18.2 or CLDN18 positivity and histologic type of G/GEJ adenocarcinoma,4,17,18 whereas others have not.19,20 Our study found a significant correlation between CLDN18.2 positivity and signet ring cell carcinomas, which aligns with previous research. Xu et al21 studied tissue samples from 105 patients with advanced gastric signet ring cell carcinomas and reported CLDN18.2 expression (≥2+ staining intensity, ≥40% cutoff) in 64.8% (68 of 105) of samples, consistent with 66.7% (78 of 117) CLDN18.2 positivity (≥50% cutoff) in our study. Our data showed no association of disease stage with CLDN18.2 positivity, as noted previously.17

Some previous data have demonstrated lower CLDN18.2 positivity in individuals with peritoneal metastases in contrast to those without peritoneal involvement.22 However, in our study, tissue samples from patients with peritoneal metastases displayed a high prevalence of CLDN18.2 positivity (82%; ≥50% cutoff). Our analysis also found a significant correlation between CLDN18.2 (≥50% cutoff) and site of metastasis (peritoneal or unknown). A study by Pellino et al16 noted CLDN18.2 expression to be significantly correlated with peritoneal metastasis. The exact mechanism underlying the interaction between CLDN18.2 expression and specific metastatic spread remains poorly understood, and additional studies are needed.

No association was identified for the relationship between CLDN18.2 positivity and MMR, HER2, PD-L1, EBV, or H. pylori status. These findings are largely similar to previous studies with the exception of EBV status. Previous studies have demonstrated an association between CLDN18.2 or CLDN18 expression and EBV-positive status.16,18,19 Pellino et al16 found an association between positive EBV status and CLDN18 expression but no association with MMR or HER2. Kubota et al6 had findings similar to ours, with CLDN18.2-positive tumors distributed among MMR-deficient, EBV-positive, HER2-positive, and all negative subtypes without significant associations. The absence of correlation of CLDN18.2 positivity with EBV status in our study contradicts previous findings. All samples that were EBV-positive (n = 3) were also positive for CLDN18.2 (≥50% cutoff); however, these findings are limited by a very small number of samples tested for EBV (n = 14). Nonetheless, these data suggest that CLDN18.2 may be a viable target and emphasize the need for additional research.

In concordance analyses, we found high concordance (73%) in CLDN18.2-positive/negative status between samples collected from matched primary and metastatic sites, in agreement with previous studies.7,16,17 In a study assessing the prevalence of CLDN18.2 positivity in primary tumors and LN metastases of Japanese patients with gastric cancer, CLDN18.2 positivity was noted in 66% of paired primary/LN metastasis samples17; that study evaluated LN metastases only but used a much broader definition of CLDN18.2 positivity (staining ≥1+ intensity in any fraction of tumor cells). In another study, 22 of 27 (81.5%) matched samples showed concordance in CLDN18.2 status (≥75% cutoff), similar to our study.16 Further studies are required to clarify the precise role of CLDN18.2 in gastric cancer progression because some data suggest that reduced CLDN18 expression may be associated with more aggressive tumor characteristics and greater invasive potential.23,24 In addition, our data demonstrated 74% concordance between samples collected from the same tumor pre- and postchemotherapy (≥75% threshold). In another study, the concordance rate of CLDN positivity (≥75% threshold) was reported to be 75% before and after first-line chemotherapy in tissue samples from 17 Japanese patients with advanced G/GEJ adenocarcinoma.6

Despite a sizable number of samples in this analysis, a major limitation was the small numbers for some subgroups, precluding meaningful conclusions. For example, the racial makeup of the patients from which the samples were sourced was largely White and there were small numbers of samples from non-White patients. Furthermore, the samples were obtained from a single institution in the United States, which may limit the generalizability of the data to other regions. Finally, comparisons with other published data are complicated by differences in patient populations or sample sources and nonstandardized definitions of CLDN18.2 positivity. Similar to other biomarkers, CLDN18.2 is heterogeneous and detection of CLDN18.2 is dependent on biopsy location, which can make it challenging to obtain samples. Although more information about intratumoral heterogeneity and the dynamics of CLDN18.2 during different modalities of treatment are needed, we believe that this study provides some insight into the different characteristics that are observed in patients with CLDN18.2-positive G/GEJ adenocarcinoma.

In summary, the prevalence of CLDN18.2 positivity in tissue samples from patients with gastric or GEJ adenocarcinoma in the United States was consistent with observations from previous studies (SPOTLIGHT,9 GLOW10) and did not correlate with survival. Although CLDN18.2 positivity was associated with the site of metastasis, Lauren histologic type, and adenocarcinoma subtype, no statistically significant associations were identified with molecular biomarkers. The overall percentage agreement between matched primary and metastatic samples and primary tumor samples taken before and after receiving chemotherapy was 73% and 74.3%, respectively. This study adds to our understanding of the prevalence and potential clinical implications of CLDN18.2 positivity in patients with G/GEJ adenocarcinoma.

ACKNOWLEDGMENT

We thank the patients and their family members for their participation.

SUPPORT

DATA SHARING STATEMENT

A data sharing statement provided by the authors is available with this article at DOI https://doi.org/10.1200/PO.23.00543.

AUTHOR CONTRIBUTIONS

Conception and design: Matheus Sewastjanow-Silva, Ahmed Abdelhakeem, Kenneth K. Iwata, Diarmuid Moran, Dina Elsouda, Jaffer A. Ajani

Administrative support: Matheus Sewastjanow-Silva, Namita Shanbhag, Anh Ta

Provision of study materials or patients: Matheus Sewastjanow-Silva, Melissa Pizzi, Jaffer A. Ajani

Collection and assembly of data: Rebecca Waters, Matheus Sewastjanow-Silva, Kohei Yamashita, Ahmed Abdelhakeem, Kenneth K. Iwata, Melissa Pizzi, Ernesto Rosa Vicentini, Namita Shanbhag, Anh Ta, Deyali Chatterjee, Jaffer A. Ajani

Data analysis and interpretation: Matheus Sewastjanow-Silva, Kenneth K. Iwata, Diarmuid Moran, Dina Elsouda, Abraham Guerrero, Anh Ta, Deyali Chatterjee, Jaffer A. Ajani

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center.

Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).

APPENDIX

FIG A1. Representative images of matched primary and metastatic samples.

TABLE A1. CLDN18.2 Status Within Demographic, Clinical, and Molecular Characteristic Subgroups

Characteristic	≥50% Cutoff	≥75% Cutoff	
CLDN18.2-Negative (<50%)	CLDN18.2-Positive (≥50%)	CLDN18.2-Negative (<75%)	CLDN18.2-Positive (≥75%)	
Overall (N = 304), No. (%)	133 (43.8)	171 (56.3)	169 (55.6)	135 (44.4)	
Sex, No. (%)	
 Male (n = 214)	108 (50.5)	106 (49.5)	132 (61.7)	82 (38.3)	
 Female (n = 90)	25 (27.8)	65 (72.2)	37 (41.1)	53 (58.9)	
Ethnicity, No. (%)	
 Hispanic/Latino (n = 36)	15 (41.7)	21 (58.3)	18 (50)	18 (50)	
 Not Hispanic/Latino (n = 264)	117 (44.3)	147 (55.7)	150 (56.8)	114 (43.2)	
 Unknown (n = 4)	1 (25)	3 (75)	1 (25)	3 (75)	
Race, No. (%)	
 White (n = 241)	105 (43.6)	136 (56.4)	134 (55.6)	107 (44.4)	
 Black/African American (n = 27)	14 (51.9)	13 (48.1)	16 (59.3)	11 (40.7)	
 Asian (n = 12)	6 (50)	6 (50)	9 (75)	3 (25)	
 Other (n = 20)	6 (30)	14 (70)	8 (40)	12 (60)	
 Unknown (n = 4)	2 (50)	2 (50)	2 (50)	2 (50)	
Site of metastasis (only those >7% depicted), No. (%)	
 Peritoneum (n = 55)	10 (18.2)	45 (81.8)	16 (29.1)	39 (70.9)	
 Unknown (n = 180)	86 (47.8)	94 (52.2)	109 (60.6)	71 (39.4)	
ECOG PS, No. (%)	
 0 (n = 113)	52 (46)	61 (54)	64 (56.6)	49 (43.4)	
 1 (n = 177)	73 (41.2)	104 (58.8)	96 (54.2)	81 (45.8)	
 2 (n = 8)	6 (75)	2 (25)	6 (75)	2 (25)	
 3 (n = 3)	2 (66.7)	1 (33.3)	2 (66.7)	1 (33.3)	
 NS (n = 3)	0	3 (100)	1 (33.3)	2 (33.3)	
Disease stage, No. (%)	
 I (n = 18)	6 (33.3)	12 (66.7)	10 (55.6)	8 (44.4)	
 IIA (n = 4)	2 (50)	2 (50)	2 (50)	2 (50)	
 IIB (n = 48)	20 (41.7)	28 (58.3)	29 (60.4)	19 (39.6)	
 III (n = 97)	51 (52.6)	46 (47.4)	61 (62.9)	36 (37.1)	
 IV (n = 87)	33 (37.9)	54 (62.1)	42 (48.3)	45 (51.7)	
 IVA (n = 16)	8 (50)	8 (50)	9 (56.3)	7 (43.8)	
 IVB (n = 33)	13 (39.4)	20 (60.6)	16 (48.5)	17 (51.5)	
 NA (n = 1)	0	1 (100)	0	1 (100)	
Tumor grade, No. (%)	
 G1—well differentiated (n = 1)	1 (100)	0	1 (100)	0	
 G2—moderately differentiated (n = 94)	53 (56.4)	41 (43.6)	62 (66)	32 (34)	
 G2-G3—moderately to poorly differentiated (n = 20)	7 (35)	13 (65)	8 (40)	12 (60)	
 G3—poorly differentiated (n = 187)	72 (38.5)	115 (61.5)	97 (51.9)	90 (48.1)	
 GX—undetermined grade (n = 1)	0	1 (100)	0	1 (100)	
 NA (n = 1)	0	1 (100)	1 (100)	0	
Lauren histologic type, No. (%)	
 Intestinal (n = 52)	21 (40.4)	31 (59.6)	28 (53.8)	24 (46.2)	
 Diffuse (n = 92)	30 (32.6)	62 (67.4)	42 (45.7)	50 (54.3)	
 No comment (n = 159)	82 (51.6)	77 (48.4)	99 (62.3)	60 (37.7)	
 Other (n = 1)	0	1 (100)	0	1 (100)	
Adenocarcinoma histologic subtype, No. (%)	
 Signet ring carcinoma (n = 117)	39 (33.3)	78 (66.7)	53 (45.3)	64 (54.7)	
 NOS (n = 187)	94 (50.3)	93 (49.7)	116 (62)	71 (38)	
HER2 status, No. (%)	
 Positive (n = 25)	10 (40)	15 (60)	13 (52)	12 (48)	
 Negative (n = 195)	80 (41)	115 (59)	103 (52.8)	92 (47.2)	
 Equivocal (2+; n = 4)	1 (25)	3 (75)	2 (50)	2 (50)	
 NS (n = 80)	42 (52.5)	38 (47.5)	51 (63.8)	29 (36.3)	
MSI, No. (%)	
 MSI-H (n = 10)	6 (60)	4 (40)	7 (70)	3 (30)	
 MSS (n = 120)	46 (38.3)	74 (61.7)	64 (53.3)	56 (46.7)	
 NS (n = 174)	81 (46.6)	93 (53.4)	98 (56.3)	76 (43.7)	
Helicobacter pylori, No. (%)	
 Positive (n = 23)	7 (30.4)	16 (69.6)	10 (43.5)	13 (56.5)	
 Negative (n = 225)	95 (42.2)	130 (57.8)	125 (55.6)	100 (44.4)	
 NS (n = 56)	31 (55.4)	25 (44.6)	34 (60.7)	22 (39.3)	
EBV, No. (%)	
 Positive (n = 3)	0	3 (100)	0	3 (100)	
 Negative (n = 11)	4 (36.4)	7 (63.6)	7 (63.6)	4 (36.4)	
 NS (n = 290)	129 (44.5)	161 (55.5)	162 (55.9)	128 (44.1)	
NOTE. Percentages are row percentages. Cutoffs defined according to the percentage of tumor cells with 2+ and/or 3+ staining intensity for CLDN18 meeting or exceeding a given cutoff (≥50% or ≥75%).

Abbreviations: CLDN18.2, claudin 18 isoform 2; EBV, Epstein-Barr virus; ECOG PS, Eastern Cooperative Oncology Group performance status; HER2, human epidermal growth factor receptor 2; MSI, microsatellite instability; MSI-H, MSI-high; MSS, microsatellite stable; NA, not applicable; NOS, not otherwise specified; NS, not specified.

TABLE A2. Summary of CPS ≥ 1 Prevalence in CLDN18.2-Positive and CLDN18.2-Negative Tumors

Characteristic	Total No. of CPS Results (n = 117)a	
Validated CLDN18.2 results with CPS results, No.	114	
CLDN18.2-positive (≥75%) with CPS results, No.	53	
 Prevalence of CPS ≥ 1 in CLDN18.2-positive samples, No./No. (%)	35/53 (66.03)	
CLDN18.2-negative (<75%) with CPS results, No.	61	
 Prevalence of CPS ≥ 1 in CLDN18.2-negative samples, No./No. (%)	44/61 (72.13)	
NOTE. P = .615.

Abbreviations: CLDN18.2, claudin 18 isoform 2; CPS, combined positive score.

a CPS of three samples was unknown.

Supported by Astellas Pharma, Inc. Medical writing/editorial support was provided by Sandra Westra, PharmD, Pamela Barendt, PhD, and Cheryl Casterline, MA, from Peloton Advantage, LLC, an OPEN Health company, Parsippany, NJ, and funded by the study sponsor.

Rebecca Waters

Consulting or Advisory Role: Astellas Pharma

Kenneth K. Iwata

Employment: Astellas Pharma

Research Funding: Astellas Pharma

Patents, Royalties, Other Intellectual Property: Patent Number 11833140

Travel, Accommodations, Expenses: Astellas Pharma

Diarmuid Moran

Employment: Astellas Pharma

Dina Elsouda

Employment: Astellas Pharma

Research Funding: Astellas Pharma

Abraham Guerrero

Employment: Astellas Pharma

Research Funding: Astellas Pharma

Ernesto Rosa Vicentini

Employment: MD Anderson Cancer Center

Jaffer A. Ajani

Honoraria: Lilly, Bristol Myers Squibb, Merck, Aduro Biotech, DAVA Pharmaceuticals, AstraZeneca, Acrotech Biopharma, Zymeworks, Astellas Pharma, Amgen, Oncotherics, Daiichi Sankyo, Novartis, SERVIER, Gilead Sciences, BeiGene, Fresenius Kabi, Boehringer Ingelheim, GRAIL

Consulting or Advisory Role: American Cancer Society, BeiGene, Vaccinogen, Insys Therapeutics, Merck, Bristol Myers Squibb, Novartis, Astellas Pharma, Gilead Sciences, Amgen, SERVIER, Geneos, Arcus Biosciences

Research Funding: Novartis, Bristol Myers Squibb, Taiho Pharmaceutical, Roche/Genentech, Amgen, Lilly/ImClone, Merck, Delta-Fly Pharma, Gilead Sciences, Takeda, ProLynx, Zymeworks, Daiichi Sankyo, Astellas Pharma (Inst)

Patents, Royalties, Other Intellectual Property: I have research funding from: Genentech, Roche, BMS, Taiho, MedImmune, Merck, Amgen, Lilly

No other potential conflicts of interest were reported.
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