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.^1-3

More more_horiz

Jump to:

Open anchor link menu

Close anchor link menu

IHC testing is the only clinically validated method to detect CLDN18.2 expression in G/GEJ.^1-4 Other methods, including whole-transcriptome sequencing and liquid biopsy, while useful in other contexts, cannot directly evaluate protein expression or localisation and are not suitable for assessing CLDN18.2 positivity in clinical practice.^5,6

IHC testing: using CLDN18 antibodies to detect CLDN18.2 in G/GEJ tumour samples

CLDN18 antibodies can identify both CLDN18 isoforms—CLDN18.1 and CLDN18.2; however, when evaluating G/GEJ tumour tissue, staining can be attributed to the presence of CLDN18.2 only, because^3,4:

CLDN18.1 is the dominant isoform expressed in normal and malignant lung tissue, and its expression is negligible or absent in G/GEJ cancers^3,4
CLDN18.2 is normally expressed in gastric epithelium and expression is often retained in malignant gastric tissue^3,4

CLDN18 antibodies (such as the 43-14A clone) are appropriate to measure CLDN18.2 expression in G/GEJ.^3,7

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

How to Prepare CLDN18.2 Samples for Testing

Takeshi Kuwata, MD, PhD

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 stabilisation, 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 preparation²

Routinely processed, formalin-fixed, paraffin-embedded (FFPE) tissues are suitable for use with IHC testing
- Paraffin should be melted at <60°C
- Specimen should contain sufficient tumour 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*
Before staining, the cut slides should be dried completely either at room temperature (air dried) or by offline baking (baked in oven) at 60°C for 60 minutes*

*CLDN18-specific.

Storage conditions^2,8

It is recommended that slides are stained immediately, as antigenicity of cut tissue sections may diminish over time

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 PI 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.

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

Testing

Antibody options for evaluating CLDN18.2 expression via IHC

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.

How to Test for CLDN18.2

Christoph Röcken, MD

This video provides an overview of performing CLDN18.2 testing with immunohistochemistry, highlighting key elements of the assay workflow. It discusses what to look for when running and reviewing a CLDN18.2 stain, including the importance of focusing on membranous staining patterns and ensuring appropriate controls. The intent is to support consistent laboratory practice and improve comparability of results across sites.

Select appropriate controls

Appropriate controls are essential for the detection of CLDN18.2 in G/GEJ tumour samples. Here are some key points on their selection and use.^2,7

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.⁹

Tissue controls are commercially available through various sources such as Discovery Life Sciences.

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 utilised only for monitoring performance of reagents and instruments, not as an aid in determining the specific diagnosis of test samples

CLDN18.1=claudin 18 isoform 1. CLDN18.2=claudin 18 isoform 2. G/GEJ=gastric/gastroesophageal junction. IHC=immunohistochemistry. IVD=in vitro diagnostic. RUO=research use only.

Considerations for laboratory-developed tests (LDTs)¹⁰

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 results¹⁰

Navigating intratumoural heterogeneity¹¹

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

Navigating intratumoural heterogeneity11

To more accurately evaluate CLDN18.2 expression from a primary tumour, at least 6 biopsies should be obtained if possible.^3,11,12

Proficiency evaluations have identified variability in antibody and platform performance

Global concordance study⁷

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 analysed.*^,†

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 least 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 tumour 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.

2024 QuIP independent proficiency testing study¹³*

Results from the 2024 Quality in Pathology (QuIP) independent proficiency testing study highlight the importance of antibody selection for accurate results.

Fifty-three^† pathology labs participated to assess CLDN18.2 testing proficiency in GC using 10 consensus-scored, validated cases

Performance Overview

A total of 42 participating institutes (~79.2%) successfully passed the independent proficiency test^‡
Among the CLDN18-specific antibodies, the CLDN18 (43-14A) clone was the most frequently utilized and demonstrated consistent performance, with no significant technical or interpretative issues reported
In the evaluation of CLDN18.2-specific antibodies, significant issues were identified with staining quality; two antibody clones were found to account for 75% of the unsuccessful submissions:
- EPR19202 clone: Achieved a 0% success rate (0/5) due to low sensitivity and weak staining intensity
- ZR451 clone: Achieved a 56% success rate (5/9) but exhibited low staining specificity

Key Considerations

Accurate determination of CLDN18.2 expression relies on the use of well-optimized staining protocols and high-performing antibody clones
To ensure reliable patient test results, it is essential to maintain ongoing quality control through external quality assessments (EQAs), regardless of the antibody clone employed

QuIP is an accredited and experienced independent institution that specialises in conducting EQAs for IHC and molecular biomarker testing on tissue samples.

*QuIP independent proficiency testing study sponsored by Astellas. ^†Fifty-four institutes registered for the open proficiency test and one resigned. ^‡Successful passing was defined as ≥ 90% of cases evaluated correctly.

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. Seventy-five laboratories participated in this first assessment with ~87% (65/75) achieving a sufficient mark.¹⁴

“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

NordiQC is a professional scientific organization that conducts EQAs for immunohistochemistry proficiency.¹⁵

*NordiQC EQA sponsored by Astellas.

Validation of AmoyDx CLDN18 (43-14A) Assay and Concordance With VENTANA® CLDN18 (43-14A) RxDx¹⁶

This study evaluated the consistency between two CLDN18 IHC testing approaches in 68 gastric cancer samples using AmoyDx CLDN18 (43-14A) and VENTANA® CLDN18 (43-14A) RxDx Assays.

This study suggests that these two testing methods may be reliable in clinical practice
When the positivity threshold was exploratively adjusted to “≥50% of tumour cells with moderate to strong (2+/3+) membranous CLDN18 staining,” high concordance was maintained with positive percent agreement at 100%, negative percent agreement at ~94%, and overall percent agreement at ~97%

Reflexive testing at diagnosis may improve patient outcomes

Reflexive testing may improve patient care through timely availability of biomarker results and initiation of optimal systemic therapy.^17,18

Reflexive testing is where the pathologist initiates and controls testing for a set of prespecified biomarkers, agreed upon within the context of the multidisciplinary team¹⁸

Delaying biomarker testing may worsen clinical outcomes

Challenges associated with nonreflexive testing include¹⁸

Delays in the initiation of complex and often lengthy testing procedures
Delays in the availability of the information necessary to inform therapy
Risk of clinical deterioration while waiting for the results of biomarker tests
Delays in test reporting, which can lead to the initiation of suboptimal therapy before determining complete biomarker status
Long test turnaround times, which may also deter clinicians from ordering biomarker tests

ESMO guidelines recommend testing for all available, actionable biomarkers at the time of diagnosis if metastatic GC is documented or suspected¹⁹; to allow for timely reporting of results, biomarker assessment should be performed concurrently^17,19

Reflexive testing allows targeted therapy to begin sooner, improving clinical outcomes

In a retrospective real-world study of 1,423 patients with advanced cancer—including NSCLC (22%), colorectal cancer (16%), and breast cancer (12%) as the most frequently tested tumour types—a pathologist-initiated reflex testing protocol at diagnosis was associated with improved OS¹⁷

OS at 12 months

Pathologists and oncologists recognise the positive impact of reflexive testing on improved patient care

A recent study utilizing an online survey of UK pathologists, oncologists and gastroenterologists involved in treating G/GEJ cancers explored the importance of biomarker testing immediately following G/GEJ adenocarcinoma diagnosis (reflexive testing); this work predated routine CLDN18.2 biomarker testing and underscores the importance of establishing robust pathways for adding new biomarkers as they emerge²⁰

Participants agreed that reflexive testing has the potential to improve the speed of treatment decisions and improve patient care
This study emphasises the importance of testing all patients for key biomarkers as soon as G/GEJ adenocarcinoma is confirmed²⁰

"We’ve seen evidence that reflexive testing improves patient outcomes. It’s critical that pathologists advocate for testing at diagnosis for all prevalent, actionable biomarkers."

Matteo Fassan, MD, PhD

Learn more about interpreting CLDN18.2 expression

Stain Interpretation

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) assay [package insert]. Mannheim, Germany: Roche Diagnostics GmbH.
3.Fassan M, Kuwata T, Matkowskyj KA, et al. Claudin-18.2 immunohistochemical evaluation in gastric and gastroesophageal junction adenocarcinomas to direct targeted therapy: a practical approach. Mod Pathol. 2024;37(11):100589.
4.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.
5.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.
6.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.
7.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. Lab Invest. 2024;104(1):100284.
8.Discovery Life Sciences. Recommended handling and storage for biological specimens. Published July 10, 2020. Accessed February 12, 2026. https://f.hubspotusercontent00.net/hubfs/207850/Protocols/Recommended%20Handling%20and%20Storage%20for%20Biological%20Specimens_2020.pdf
9.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.
10.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.
11.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. J Pers Med. 2021;11(11):1095.
12.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. Sci Rep. 2025;15(1):28958.
13.Röcken C, Höhn AK, Neumann J, et al. Concordance of laboratory assays for claudin 18.2 in gastric cancer tissue samples: independent proficiency testing and a descriptive non-interventional study. Virchows Arch. 2025;487(5):1117-1125.
14.NordiQC. Assessment Run C17 2025 Claudin 18.2 (CLDN18.2). Accessed June 16, 2025. https://w.nordiqc.org/downloads/assessments/206_123.pdf
15.NordiQC. About NordiQC. Accessed July 21, 2025. https://www.nordiqc.org/about.php
16.Tang Z, Cheng P, Liu X, et al. A novel approach of Claudin 18 immunohistochemistry testing demonstrated comparable performance and high concordance compared to Ventana 43-14A in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res. 2025;85(8_Suppl_1):Abstract nr 681.
17.Piening B, Bapat B, Weerasinghe RK, et al. Improved outcomes from reflex comprehensive genomic profiling-guided precision therapeutic selection across a major US healthcare system [Abstract 6622]. J Clin Oncol. 2023;41(Suppl 16):6622.
18.Gosney JR, Paz-Ares L, Jänne P, et al. Pathologist-initiated reflex testing for biomarkers in non-small-cell lung cancer: expert consensus on the rationale and considerations for implementation. ESMO Open. 2023;8(4):101587.
19.ESMO GC Living Guidelines. Version 1.4. September 2024. Accessed June 16, 2025. https://www.esmo.org/guidelines/living-guidelines/esmo-living-guideline-gastriccancer/diagnosis-pathology-and-molecular-biology
20.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.