CLDN 18.2

Interpretation of CLDN18 staining

Accurate CLDN18 staining is critical to guide treatment decision-making.1-5

IHC interpretation of CLDN18.2 expression banner
IHC interpretation of CLDN18.2 expression
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CLDN18.2-positivity is defined as ≥75% of tumour cells demonstrating moderate-to-strong (2+/3+) membranous CLDN18 staining, based on IHC assessments in two recently published global Phase 3 studies.1-3

In G/GEJ tumours, assessment for CLDN18.2 expression is determined by the percentage of tumour cells with moderate-to-strong membrane staining1,4

How to Interpret CLDN18.2

Josef H. Rüschoff, MD

Example-CLDN
  • CLDN18-stained tissue sections may exhibit a range of membrane staining intensities, from an absence of staining to strong, well-defined staining1
  • Staining intensity is scored between 0 and 3+ (absent, 0; weak, 1+; moderate, 2+; and strong, 3+)1

Practical guidance on CLDN18.2 expression interpretation1,6

Evaluation guidelines

Staining definition and intensity scoring1

  • A cell is defined as CLDN18.2 positive in the presence of crisp membranous staining (complete, basolateral, or lateral)
  • Granular cytoplasmic staining is considered negative
  • Account for tumour necrosis, cytoplasmic staining, and edge effect during interpretation
  • Staining intensity is scored between 0 and 3+ (absent, 0; weak, 1+; moderate, 2+; and strong, 3+)
  • A 3+ intensity is defined as a strong brown immunoreactivity with an evident chicken wire distribution at low magnification (×5 objective), when 3,3ʹ-diaminobenzidine is used as a chromogen

Heterogeneity and special scenarios1

  • CLDN18.2 staining shows a high degree of intratumoral heterogeneity
  • A low-magnification inspection of the stained slide should be completed to determine an overall impression of the staining pattern and heterogeneity of expression
  • In poorly cohesive carcinomas, joint evaluation of CLDN18 staining and its matched haematoxylin and eosin-stained slide is strongly suggested
  • In cases with mixed histology, the most representative tumour sample/block exhibiting morphologic tumour heterogeneity should be selected for testing
    • CLDN18.2 expression may be different in the two neoplastic constituents; therefore, the report should represent a comprehensive evaluation of both components

Controls, inclusion, cutoff, and review1,6

  • Normal gastric glands are CLDN18.2 positive and show the maximal staining intensity in a given sample
  • False-negative staining should be considered if a sample is devoid of any staining and in cases with unstained adenocarcinomas that lack internal nonneoplastic positive controls; repeat staining or testing of an alternative tissue block (if available) is advised
  • Ensure preinvasive lesions are distinguished from invasive carcinomas, especially in biopsy specimens
    • Only evaluate CLDN18.2 expression in invasive carcinomas, not in preinvasive lesions
  • Precursor lesions (both intestinal metaplasia and dysplasia) may express CLDN18.2 but should not be included in the scoring
  • Normal gastric epithelium and intestinal metaplasia may be used as appropriate controls in routine practice
  • Moderate to strong (2+/3+) positive membrane staining in ≥75% of tumour cells is the proposed cutoff required for the clinical use of monoclonal antibody anti-CLDN18.2 zolbetuximab
  • Consider evaluation by a second pathologist, when possible, for difficult/borderline cases
  • Cases initially considered 2+/3+ positive in the 60%-80% of cancer cells by a single pathologist should be reviewed by a second pathologist
  • Ambiguous staining should be interpreted as inadequate for CLDN18.2 scoring, and a separate sample should be obtained for retesting and scoring purposes

Staining considerations

Signet ring cells and dysplastic lesions

signet-ring-cell-and-dysplastic-lesions
signet-ring-cell-and-dysplastic-lesions
  • Exclude tumour cells with nonspecific staining in signet ring cells or dysplastic lesions1,6
  • Signet ring cells may be misinterpreted because of nonspecific staining in the marginated cytoplasm similar to that noted for HER21
  • Although dysplastic lesions show CLDN18.2 expression similar to that observed in invasive adenocarcinoma, there are no data available on the concordance of CLDN18.2 expression in dysplastic lesions and their matched invasive counterparts1

Cytoplasmic vs nuclear staining

cytoplasmic-vs-nuclear-staining
cytoplasmic-vs-nuclear-staining
  • Exclude tumour cells with only cytoplasmic staining1,6
  • In cases with high CLDN18.2 expression, membrane expression may be obscured by strong cytoplasmic staining1
    • A chicken wire pattern can usually still be observed to indicate membrane staining
    • Examining staining at 40X may be required to determine true positivity

Aberrant positivity

Aberrant-positivity
Aberrant-positivity
  • Ignore aberrant positivity in inflammatory cells, nonneoplastic cells, or acellular mucus of mucinous adenocarcinoma; CLDN18.2 staining should be repeated on another sample when available1,6
  • Rare cases showing aberrant, faint cytoplasmic positivity in inflammatory cells or other nonneoplastic cells have been characterized by obvious preanalytical artifacts, and should not be scored

Preanalytical artifacts

preanalytical-artifacts
  • Thermal artifacts: Tissue subjected to electrocautery, especially from mucosal/submucosal dissections, may appear histologically torn and coagulated; in these samples, antigen preservation may be impaired, and areas with thermal artifacts should be excluded from CLDN18.2 evaluation1
  • Underfixation issues: Underfixation can be detrimental for antigen maintenance, potentially causing false-negative staining with edge effects and nonspecific cytoplasmic staining1

Impact of necrosis on CLDN18.2 interpretation

impact-of-necrosis-on-cldn18.2-interpretation
impact-of-necrosis-on-cldn18.2-interpretation

To evaluate CLDN18.2 expression, while a minimum of 50 viable neoplastic cells should be required, 100 cells is a more effective benchmark, similar to other predictive biomarker tests.1

Magnification rule1,7

  • For CLDN18.2 assessment, the “magnification rule” can be easily translated into clinical practice to aid in the interpretation of IHC scores1:
    • 3+ intensity is defined as a strong brown immunoreactivity with an evident chicken wire distribution at low power (ie, 2.5X–5X objective/25X–50X magnification) when 3,3’-diaminobenzidine is used as a chromogen; if a higher magnification is required to confirm membrane expression, then the evaluation is usually 1+ or 2+ for membrane staining intensity
    • 2+ membrane staining is visible using a 10X objective and 20X may be needed for confirmation of specific membrane staining
    • 1+ staining can be visible at 20X but needs a 40X objective for confirmation and should be interpreted as negative
  • The magnification rule is a useful tool for reproducibly subdividing the continuous spectrum of IHC staining intensities into distinct categories to help overcome scoring subjectivity for membrane-bound IHC staining1

Click to view stain gallery or test your knowledge via the stain quiz

CLDN=claudin. CLDN18.2=claudin 18 isoform 2. G/GEJ=gastric/gastroesophageal junction. H&E=hematoxylin and eosin. HER2=human epidermal growth factor receptor 2. IHC=immunohistochemistry. PD-L1=programmed death-ligand 1.

References:

  • 1.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.
  • 2.Shitara K, Xu RH, Ajani JA, et al. Global prevalence of claudin 18 isoform 2 in tumours of patients with locally advanced unresectable or metastatic gastric or gastroesophageal junction adenocarcinoma. Gastric Cancer. 2024;27(5):1058-1068.
  • 3.Shah MA, Shitara K, Ajani JA, et al. Zolbetuximab plus CAPOX in CLDN18.2-positive gastric or gastroesophageal junction adenocarcinoma: the randomized, phase 3 GLOW trial. Nat Med. 2023;29(8):2133-2141.
  • 4.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.
  • 5.Rohde C, Yamaguchi R, Mukhina S, et al. Comparison of claudin 18.2 expression in primary tumours and lymph node metastases in Japanese patients with gastric adenocarcinoma. Jpn J Clin Oncol. 2019;49(9):870-876.
  • 6.Angerilli V, Callegarin M, Govoni I, et al. Heterogeneity of predictive biomarker expression in gastric and esophago-gastric junction carcinoma with peritoneal dissemination. Gastric Cancer. 2025;28(4):569-578.
  • 7.Scheel AH, Penault-Llorca F, Hanna W, et al. Physical basis of the 'magnification rule' for standardized immunohistochemical scoring of HER2 in breast and gastric cancer. Diagn Pathol. 2018;13(1):19.