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Why use CLDN18 antibodies for detecting CLDN18.2 in G/GEJ tumor samples?
CLDN18 antibodies can identify both CLDN18 isoforms—CLDN18.1 and CLDN18.2. But when evaluating G/GEJ tumor tissue, staining can be attributed to the presence of CLDN18.2 because2,3:
- CLDN18.1 is primarily expressed in lung adenocarcinoma tissue and its expression is negligible or absent in G/GEJ cancers3
- CLDN18.2 is normally present in gastric epithelium and 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
How to Prepare CLDN18.2 Samples for Testing
Takeshi Kuwata, MD, PhD
Sample preparation
College of American Pathologists (CAP) 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
CAP 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
Paraffin should be melted at <60°C
Specimen containing sufficient tumor tissue for analysis
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
Suboptimal tissue fixation1
It is important to optimize pre-analytical variables to minimize staining artifacts, which can interfere with accurate scoring.
Cytoplasmic blushing due to suboptimal fixation, which can interfere with accurate membranous scoring.
Specimen preparation2
Routinely processed, formalin-fixed, paraffin-embedded (FFPE) tissues are suitable for use with IHC testing
Specimens that are fine-needle aspirate (FNA), cytology specimens or metastatic bone lesions do not qualify for CLDN18 staining
Tissue sections can be cut at 3 μm-6 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 conditions1
To ensure integrity of specimens, storage areas should be:
Dry
Pest-free
Room temperature (18°C to 25°C)
Testing
Options to evaluate for the presence of CLDN18.2 through IHC
A number of assays and antibodies are available to assess CLDN18.2 expression. The list below is not exhaustive. The appropriate test should be used when guiding clinical decision-making.2,4-6
| Name | Company | Clone Name | Clonality | Host | Isotype | Use |
|---|---|---|---|---|---|---|
| VENTANA® CLDN18 (43-14A) IVD Assay | Ventana Medical Systems a member of the Roche Group | 43-14A | Monoclonal | Mouse | IgG2b | IVD |
| PathPlusTM CLDN18/Claudin 18 Antibody | LSBio an Absolute Biotech Company | LS-B16145 | Monoclonal | Mouse | IgG | RUO |
| Recombinant Anti-Claudin 18 antibody (43- 14A) | Abcam | 43-14A | Monoclonal | Mouse | IgG2b | RUO |
| Claudin-18 Antibody | Novus Biologicals | NBP2-32002 | Polyclonal | Rabbit | IgG | RUO |
IVD=in vitro diagnostic; RUO=research use only.
How to Test for CLDN18.2
Christoph Röcken, MD
Platforms and antibody performance characteristics
In the global RING study, assessing the reproducibility and comparability of three CLDN18 antibodies and IHC staining platforms across a cohort of 27 global laboratories where sensitivity, specificity, accuracy, and precision were analyzed7*†:
- Analytical performance of the VENTANA CLDN18 (43-14A) IVD Assay was >95% and reproducible across the 27 laboratories vs consensus reference results7
- Analytical performance was equivalent to the VENTANA (43-14A) IVD Assay for the LSBio antibody when stained on the Dako or Leica platform.7 Staining was also reproducible for the LSBio antibody.
- Performance was least consistent for the Novus antibody compared to the VENTANA CLDN18 (43-14A) Class I IVD Assay7
*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.7
†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.7
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.2,8
Validation controls
Guidelines recommend that laboratories validate and/or verify immunohistochemical tests before placing them into clinical service and should include positive, negative, and borderline tissue, reflecting the intended use of the assay.8
Tissue controls are commercially available through various providers.
Run controls
- It is recommended to use optimal run controls, including positives and negatives2
- Normal gastric epithelium that includes areas of gastric intestinal metaplasia is an example of an appropriate positive and negative control as it demonstrates2:
- 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 invalid2
- Known positive tissue controls should be utilized only for monitoring performance of reagents and instruments, not as an aid in determining specific diagnosis of test samples2
CAP PPMPT, College of American Pathologists Preanalytics for Precision Medicine Project Team; CLDN, claudin; CLDN18.1, claudin 18 isoform 1; CLDN18.2, claudin 18 isoform 2; FDA, US Food and Drug Administration; G/GEJ, gastric/gastroesophageal junction; IHC, immunohistochemistry; IVD, in vitro diagnostic.
Reflexive testing may contribute to improved patient care9
Biomarker testing is a critical step in the patient’s journey
ESMO Guidelines recommend testing for all available, actionable biomarkers at the time of diagnosis if advanced or metastatic gastric cancer is documented or suspected.10
Any new biomarker should be tested concurrently with other biomarkers to allow for timely reporting of results.9,10
Click below to find out what is recommended in the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Gastric Cancer and Esophageal and Esophagogastric Junction Cancers.
It’s critical that pathologists advocate for testing at diagnosis for all prevalent, actionable biomarkers.
Matteo Fassan, MD, PhD
The changing landscape of actionable biomarkers in G/GEJ cancer may require consideration of a new testing strategy
Lessons learned in lung cancer: A case study for reflex testing11
Implementation of a reflex ordered testing strategy at the time of pathologic diagnosis:
- Significantly decreased biomarker test result turnaround times
- Increased the number of informative mutations detected
- Ensured timely delivery of key molecular information to inform the prognosis and personalized treatment
In a lung cancer analysis, establishing reflex testing resulted in:
Additionally, reflex testing can12:
- Expedite testing and thus reducing the time from diagnosis to personalized treatment
- Facilitate optimized tissue stewardship
- Reduce disparities in care by establishing consistent biomarker testing protocols
- Improve patient testing rates
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):1346-63. 2. Ventana CLDN18 (43-14A) assay [package insert]. Mannheim, Germany: Roche Diagnostics GmbH. 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):7624-34. 4. LSBio. PathPlus CLDN18 Claudin 18 Monoclonal Antibody Mouse IHC. Accessed May 15, 2024. https://www.lsbio.com/pathplus-antibodies/pathplus-cldn18-antibody-claudin-18-antibody-ihc-ls-b16145/789855. 5. Abcam. Product datasheet. Anti-Claudin18 antibody [43-14A] ab314690. Accessed May 15, 2024. https://www.abcam.com/products/primary-antibodies/claudin18-antibody-43-14a-ab314690.html. 6. Novusbio. Product datasheet. Claudin-18 Antibody NBP2-32002. Accessed May 15, 2024. https://www.novusbio.com/products/claudin-18-antibody_nbp2-32002. 7. Jasani B, Taniere P, Schildhaus HU, et al. Global ring study to investigate the comparability of total assay performance of commercial claudin 18 antibodies for evaluation in gastric cancer [published ahead online November 8, 2023]. Lab Invest 2024. https://www.sciencedirect.com/science/article/pii/S0023683723002271?via%3Dihub. 8. 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. 9. 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). 10. ESMO Gastric Cancer Living Guidelines (10-2023). https://www.esmo.org/living-guidelines/esmo-gastric-cancer-living-guideline/diagnosis-pathology-and-molecular-biology/article/diagnosis-pathology-and-molecular-biology. Accessed 01-08-2024. 11. Anand K, Phung TL, Bernicker EH, Cagle PT, Olsen RJ, Thomas JS. Clinical utility of reflex ordered testing for molecular biomarkers in lung adenocarcinoma. Clin Lung Cancer 2020;21(5):437-42. 12. Gosney JR, Paz-Ares L, Janne 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);1-8.