CLDN 18.2

Preparing and testing for CLDN18.2

Learn more about testing for CLDN18.2, including the impact of preanalytical factors on staining, the importance of antibody selection, and the significance of reflexive CLDN18.2 testing upon diagnosis.1, 2

Laboratory technician handling tissue cassettes at an embedding workstation.
Laboratory technician handling tissue cassettes at an embedding workstation.
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IHC testing is the only clinically validated method to detect CLDN18.2 expression in G/GEJ adenocarcinoma.1-3 Other methods, including whole-transcriptome sequencing and liquid biopsy, while useful in other contexts, cannot directly evaluate protein expression or localization and are not suitable for assessing CLDN18.2 positivity in clinical practice.4,5

IHC testing: Using CLDN18 antibodies to detect CLDN18.2 in G/GEJ tumor samples

CLDN18 antibodies can identify both CLDN18 isoforms—CLDN18.1 and CLDN18.2; however, when evaluating G/GEJ tumor tissue, staining can be attributed to the presence of CLDN18.2 only, because3:

  • CLDN18.1 is the dominant isoform expressed in normal and malignant lung tissue, and its expression is negligible or absent in G/GEJ cancers3
  • CLDN18.2 is normally expressed in gastric epithelium, and expression is often retained in malignant gastric tissue3

Sample preparation and preanalytics

Appropriate specimen handling and preparation are essential to ensure the accuracy of biomarker results.1

Sample preparation

Guidelines recommend daily tissue processor maintenance per the manufacturer recommendations and rigorous quality maintenance of processor fluids, including formalin pH/purity and water contamination of alcohols.1

Cold ischemic time

Cold ischemic time should be limited to ≤60 minutes according to current guidelines.1

Fixation

Guidelines provide recommendations regarding dimensions and duration involved in sample fixation.1

Key dimensions1

  • Tissue should be completely submerged in fixative
  • Ensure a fixative volume-to-tissue mass ratio of no less than 4:1, with an optimal ratio of 10:1

Key durations1

  • As part of stabilization, tissue should be fixed in 10% neutral phosphate-buffered formalin (pH 7.0) for at least 6 hours and no longer than 24 to 36 hours
  • If the tissue has high fat content, fixation may require up to 48 hours

Specimen preparation1,2

  • Routinely processed, formalin-fixed, paraffin-embedded (FFPE) tissues are suitable for use with IHC testing
    • Low-melt paraffin should be melted at <60 °C
    • Specimen should contain sufficient tumor tissue for analysis
  • Specimens that are fine-needle aspirate (FNA), cytology specimens, or metastatic bone lesion do not qualify for CLDN18 staining
  • Tissue sections should be cut at ~4 μm thick with a range from 3 μm to 5 μm*
  • It is recommended that slides are stained immediately, as antigenicity of cut tissue sections may diminish over time

*CLDN18-specific.

Storage conditions2,6

Cut slides/FFPE tissue should be:

  • Desiccated
  • Stored at room temperature
  • Stored out of direct sunlight
  • Kept away from an open flame or direct heat source

Environmental factors affect antigen stability, and antigenicity decreases over time; therefore:

  • The VENTANA method sheet (package insert) recommends that laboratories validate cut slide stability within their own environment beyond 45 days
  • Discovery Life Sciences recommends using slides within one calendar month from the date of sectioning

Suboptimal tissue fixation1

It is important to optimize preanalytical variables to minimize staining artifacts.

Cytoplasmic blushing due to suboptimal fixation

Suboptimal tissue fixation can cause cytoplasmic blushing and interfere with accurate membranous scoring.

Testing

Antibody options for evaluating CLDN18.2 expression via IHC2,7,8

A number of assays and antibodies are available to assess CLDN18.2 expression. The list below is not exhaustive. An appropriate, validated IHC antibody/assay with high performance should be used when guiding clinical decision-making.

Select appropriate controls

Appropriate controls are essential for the detection of CLDN18.2 in G/GEJ tumor samples. Here are some key points on their selection and use.2,9

Validation controls

Guidelines recommend that laboratories validate and/or verify immunohistochemical tests before placing them into clinical service. These tests should include positive, negative, and borderline tissue, reflecting the intended use of the assay.10

Tissue controls are commercially available through various providers.

Run controls2

  • It is recommended to use optimal run controls, including positives and negatives
  • Gastric intestinal metaplasia tissue is an example of an appropriate positive and negative control as it demonstrates
    • Strong membranous staining in normal gastric epithelial cells
    • Weak-to-moderate membranous staining of epithelial cells in areas of metaplasia
    • Absence of staining in lamina propria, lymphocytes, smooth muscle, blood vessels, and peripheral nerve
  • If the positive controls fail to demonstrate staining, results of the test specimen should be considered invalid
  • Known positive tissue controls should be utilized only for monitoring performance of reagents and instruments, not as an aid in determining the specific diagnosis of test samples

Considerations for laboratory-developed tests (LDTs)11

Companion diagnostic (CDx) assays are clinically validated and approved for use with specific therapies and indications;
however, clinical laboratories may opt to use LDTs instead of a CDx assay due to limited resources or lack of access.

CAP Guidelines for the use of LDTs include the following:

  • Laboratories must analytically validate all LDTs before reporting results on patient tissues
  • For initial analytic validation or verification of every assay used clinically, laboratories should achieve at least 90% overall concordance
between the new assay and the comparator assay or expected results
  • For initial analytic validation of all predictive LDTs, laboratories should test a minimum of 20 positive and 20 negative tissues
  • Laboratories should use validation tissues that have been processed using the same fixative and processing methods as cases that will be tested clinically, when possible

CAP Guidelines provide direction on IHC assay validation to help ensure accuracy, reproducibility, and consistency of results11

CLDN18.2 expression demonstrates intratumoral heterogeneity

As with other biomarkers (such as HER2), CLDN18.2 expression in G/GEJ cancer exhibits intratumoral heterogeneity, which should be taken into account when sampling tumor tissue.12,13

In a study showing high concordance between primary and metastatic tumor samples, intratumoral heterogeneity in CLDN18.2 expression was observed in12*:

*Data are from a single-institution study that assessed 523 archival cases of surgically treated gastric (n=408) and gastroesophageal (n=115) cancers. Intratumoral heterogeneity of CLDN18.2 was determined by assessing different samples from the same tumor for concomitant high and low CLDN18 expression.12

Navigating intratumoral heterogeneity14

A single-institution study in Italian patients with G/GEJ adenocarcinoma evaluated the sensitivity and specificity of CLDN18 testing according to the number of virtual biopsies scored on 93 surgically treated cases (77 GC and 16 GEJ), evaluated by two blinded pathologists.

Using the CLDN18.2-positive cutoff of ≥75%:

  • Sensitivity increased steadily upon evaluating additional biopsies
    • From 92.9% with 2 biopsies to 100% with 9 biopsies
  • After 6 biopsies, the increase was minimal
    • 97.7% with 6 biopsies compared with 98.2% with 7 biopsies: 0.5% increase
    • 98.2% with 7 biopsies compared with 98.7% with 8 biopsies: 0.5% increase
    • 98.7% with 8 biopsies compared with 100% with 9 biopsies: 1.3% increase
  • Similar results have been demonstrated for HER2 testing in G/GEJ adenocarcinoma
CLDN18.2-Positive Cutoff of ≥75%
CLDN18.2-Positive Cutoff of ≥75%

Multiple biopsies (ideally, 6+, if tissue allows) should be obtained to more accurately evaluate CLDN18.2 expression.14,15

Proficiency evaluations have identified variability in antibody and platform performance

Global concordance study9

In the global concordance study, which assessed the reproducibility and comparability of three CLDN18 antibodies—VENTANA (43-14A), LSBio (LS-B16145), and Novus (NBP2-32002)—and IHC staining platforms across a cohort of 27 global laboratories, sensitivity, specificity, accuracy, and precision were analyzed.*,†

  • Analytical performance of the VENTANA assay was >95% and reproducible across all of the 27 laboratories vs consensus reference results
  • Analytical performance was equivalent across the VENTANA (43-14A) IVD assay and the LSBio antibody when stained on the Dako or Leica platform; staining was also reproducible for the LSBio antibody
  • Performance was less consistent for the Novus antibody compared to the VENTANA assay

*Antibodies in the study comprised the VENTANA CLDN18 (43-14A) IVD Assay from Roche Tissue Diagnostics, the PathPlus™ CLDN18 Antibody from LSBio, and the Claudin-18 Antibody from Novus Biologicals. Platforms comprised BenchMark ULTRA, Dako Autostainer, and Leica Bond.

†Consensus reference scores from all antibodies for each sample were determined by central pathology review. CLDN18.2 positivity was defined with a threshold of ≥75% of tumor cells expressing membranous CLDN18 with moderate-to-strong (≥2+) staining intensity. Accordingly, participating pathologists were required to submit a binary positive/negative call as well as an estimation of the percent of cells stained. Laboratory-submitted IHC scores were compared to the reference consensus score and considered discordant if the positive/negative binary result differed. Statistical analysis was performed for comparison, and an acceptance criteria of 85% (≥0.85) was applied.

NordiQC external quality assessment results from Run C17*

The objective of the NordiQC EQA was to evaluate the analytical accuracy of IHC assays for detecting CLDN18.2. 75 laboratories participated in this first assessment with ~87% (65/75) achieving a sufficient mark.16

  • “Sufficient” stains were defined as “optimal” +  “good”
  • 100% of IHC stains submitted using the CE-IVD VENTANA CLDN18 (43-14A) Assay were marked as “sufficient,” of which ~89% (8/9) were “optimal”
  • For the assessment of 6 concentrated antibodies
    • Only ~46% (6/13) of IHC stains submitted were marked as “sufficient”
    • The monoclonal antibody clone 43-14A was the most successful compared with other clones, with 80% (4/5) marked as “sufficient”
    • Insufficient results were mainly caused by too weak or false negative staining reaction in gastric carcinomas expected to have ≥75% positivity
IHC Stains from VENTANA CLDN18.2(43-14A) Assay and Concentrated Antibodies
IHC Stains from VENTANA CLDN18.2(43-14A) Assay and Concentrated Antibodies

NordiQC is a professional scientific organization that conducts EQAs for immunohistochemistry proficiency.17

*NordiQC EQA sponsored by Astellas.

The G/GEJ adenocarcinoma landscape continues to evolve, with emerging considerations for testing approaches

Reflexive testing may improve patient care through timely availability of biomarker results and initiation of optimal systemic therapy18

Rationale for Reflex Testing18

Expert consensus supports reflex biomarker testing in G/GEJ adenocarcinoma to ensure that clinically relevant biomarker results are available at the time of initial oncology consultation, helping to facilitate timely treatment decision‑making and improve overall patient care.

Initiation of Reflex Testing18

Consensus recommendations favor a pathologist‑initiated reflex testing approach, activated once diagnostic histology is confirmed, rather than waiting for a separate clinician request. This model is intended to streamline the diagnostic pathway and reduce avoidable delays in care.

Biomarker Testing Approach18

Experts agreed that reflex testing should be considered based on tumor histology in patients with G/GEJ adenocarcinoma, with biomarker assessment coordinated as part of the initial diagnostic work‑up.

Operational Principles for Reflex Testing18

Best‑practice implementation of reflex testing requires:

  • Coordination across the full tissue pathway, from biopsy acquisition through testing and reporting
  • Use of standardized, quality‑assured laboratory processes to ensure accuracy and validity of results
  • Pathology reports that provide clear, sufficiently detailed biomarker results to support downstream clinical decision‑making

"It’s critical that pathologists advocate for testing at diagnosis for all prevalent, actionable biomarkers."

Profile photo of Dr. Matteo Fassan

 

Matteo Fassan, MD, PhD

Learn more about interpreting CLDN18.2 expression

CAP, College of American Pathologists; CLDN18, claudin 18; CLDN18.1, claudin 18 isoform 1; CLDN18.2, claudin 18 isoform 2; EQA, external quality assessment; GC, gastric cancer; G/GEJ, gastric/gastroesophageal junction; HER2, human epidermal growth factor receptor-2; IHC, immunohistochemistry; IVD, in vitro diagnostic; RUO, research use only.

References:

  • 1.Compton CC, Robb JA, Anderson MW, et al. Preanalytics and precision pathology: pathology practices to ensure molecular integrity of cancer patient biospecimens for precision medicine. Arch Pathol Lab Med. 2019;143(11):1346-1363.
  • 2.Ventana CLDN18 (43-14A) RxDx assay. Package insert. Tucson, AZ: Ventana Medical Systems, Inc; 2024.
  • 3.Sahin U, Koslowski M, Dhaene K, et al. Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development. Clin Cancer Res. 2008;14(23):7624-7634.
  • 4.Hashimoto T, Iida N, Nakamura Y, et al. Landscape analysis of CLDN18 expression and isoform distribution in solid tumors: insights from MONSTAR-SCREEN-2 study. Cancer Sci. 2025;116(8):2218-2231.
  • 5.Sun Y, Puspanathan P, Lim T, Lin D. Advances and challenges in gastric cancer testing: the role of biomarkers. Cancer Biol Med. 2025;22(3):212-230.
  • 6.Discovery Life Sciences. Recommended handling and storage for biological specimens. Published August 2023. Accessed June 30, 2026. https://dls.com/wp-content/uploads/2023/08/Recommended_Handling_and_Storage_for_Biological_Specimens_2020-2.pdf
  • 7.Abcam. Anti-claudin18 antibody [43-14A]. Accessed July 8, 2026. https://doc.abcam.com/datasheets/active/ab314690/en-us/claudin18-antibody-43-14a-ab314690.pdf
  • 8.LSBio. PathPlus CLDN18 Monoclonal Antibody Mouse IHC. Accessed July 15, 2026. https://www.lsbio.com/pathplus-antibodies/pathplus-cldn18-antibody-claudin-18-antibody-ihc-ls-b16145/789855#speci%EF%AC%81cations-section
  • 9.Jasani B, Taniere P, Schildhaus HU, et al. Global Ring Study to investigate the comparability of total assay performance of commercial claudin18 antibodies for evaluation in gastric cancer [published online November 8, 2023]. Lab Invest. 2024. Accessed June 19, 2026. https://www.laboratoryinvestigation.org/action/showPdf?pii=S0023-6837%2823%2900227-1
  • 10.Fitzgibbons PL, Bradley LA, Fatheree LA, et al. Principles of analytic validation of immunohistochemical assays: guideline from the College of American Pathologists Pathology and Laboratory Quality Center. Arch Pathol Lab Med. 2014;138(11):1432-1443.
  • 11.Goldsmith JD, Troxell ML, Roy-Chowdhuri S, et al. Principles of analytic validation of immunohistochemical assays: guideline update. Arch Pathol Lab Med. 2024;148(6):e111-e153.
  • 12.Coati I, Lotz G, Fanelli GN, et al. Claudin-18 expression in oesophagogastric adenocarcinomas: a tissue microarray study of 523 molecularly profiled cases. Br J Cancer. 2019;121(3):257-263.
  • 13.Grillo F, Fassan M, Sarocchi F, et al. HER2 heterogeneity in gastric/gastroesophageal cancers: from benchside to practice. World J Gastroenterol. 2016;22(26):5879-5887.
  • 14.Pellino A, Brignola S, Riello E, et al. Association of CLDN18 protein expression with clinicopathological features and prognosis in advanced gastric and gastroesophageal junction adenocarcinomas [published online October 26, 2021]. J Pers Med. 2021. Accessed April 3, 2026. https://www.mdpi.com/2075-4426/11/11/1095
  • 15.Pak S, Simon AG, Lyu SI, et al. The expression of the tight junction protein and therapeutical target Claudin 18.2 is heterogeneously distributed within esophageal and gastric adenocarcinoma [published online August 7, 2025]. Sci Rep. 2025. Accessed June 19, 2026. https://www.nature.com/articles/s41598-025-12337-4
  • 16.NordiQC. Assessment run C17 2025: Claudin 18.2 (CLDN18.2). Published April 7, 2025. Accessed June 19, 2026. https://www.nordiqc.org/downloads/assessments/206_123.pdf
  • 17.NordiQC. About NordiQC. Accessed August 4, 2025. https://www.nordiqc.org/about.php
  • 18.West NP, Mansoor W, Taniere P, et al. Best-practice biomarker testing of oesophago-GC in the UK: expert consensus recommendations developed using a modified Delphi. Clin Oncol (R Coll Radiol). 2024;36(11):701-709.