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Investigating Various Thresholds as Immunohistochemistry Cutoffs for Observer Agreement
Asif Ali1, Sarah Bell, Alan Bilsland
1*Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow §Institute of Cardiovascular and Medical Sciences, University of Glasgow, Western Infirmary ¶Academic Unit of Surgery, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow Royal Infirmary ‡Department of Pathology, Laboratory Medicine Building, Queen Elizabeth University Hospital, Greater Glasgow & Clyde NHS ∥West of Scotland Pancreatic Unit and Glasgow Royal Infirmary, Alexandra Parade, Glasgow #Pathology Laboratory, Forth Valley Royal Hospital, Larbert, UK †Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan.
Insights
Three common immunohistochemistry (IHC) cutoffs for biomarker interpretation show reasonable observer agreement, enhancing reproducibility in cancer pathology. These thresholds help reduce variability in staining interpretation among pathologists.
Area of Science:
- Pathology
- Biomarker Discovery
- Cancer Research
Background:
- Clinical translation of immunohistochemistry (IHC) biomarkers necessitates reliable interpretation cutoffs.
- Current research often prioritizes biomarker clinical utility over observer agreement.
- Three common IHC cutoffs (10% positive cells, 20% positive cells, moderate-to-strong intensity) were selected for analysis.
Purpose of the Study:
- To assess interobserver and intraobserver agreement for commonly used IHC interpretation cutoffs.
- To evaluate the reproducibility of IHC biomarker interpretation among pathologists.
- To determine if specific staining patterns influence agreement.
Main Methods:
- A set of 36 IHC-stained microarray core images with variable staining was scored by seven pathologists.
- Three predefined cutoffs (10% positive cells, 20% positive cells, +2/+3 intensity) were used for scoring.
- Kappa (κ) statistic was employed to quantify interobserver and intraobserver agreement.
Main Results:
- Reasonably good interobserver agreement was observed across all three cutoffs (mean κ: 0.59–0.64).
- Experienced pathologists showed better agreement with the 10% cutoff compared to junior pathologists.
- Good intraobserver agreement was also achieved (mean κ: 0.60–0.73), with higher agreement for cytoplasmic-only staining.
Conclusions:
- The investigated IHC cutoffs demonstrate reasonable agreement, reducing interpretation variability.
- These established cutoffs are reproducible among practicing pathologists, supporting their clinical utility.
- The findings provide evidence for the reliability of these thresholds in cancer pathology.
Background:
Clinical translation of immunohistochemistry (IHC) biomarkers requires reliable and reproducible cutoffs or thresholds for interpretation of immunostaining. Most IHC biomarker research focuses on the clinical relevance (diagnostic, prognostic, or predictive utility) of cutoffs, with less emphasis on observer agreement using these cutoffs. From the literature, we identified 3 commonly used cutoffs of 10% positive epithelial cells, 20% positive epithelial cells, and moderate to strong staining intensity (+2/+3 hereafter) to use for investigating observer agreement.
Materials And Methods:
A series of 36 images of microarray cores stained for 4 different IHC biomarkers, with variable staining intensity and percentage of positive cells, was used for investigating interobserver and intraobserver agreement. Seven pathologists scored the immunostaining in each image using the 3 cutoffs for positive and negative staining. Kappa (κ) statistic was used to assess the strength of agreement for each cutoff.
Results:
The interobserver agreement between all 7 pathologists using the 3 cutoffs was reasonably good, with mean κ scores of 0.64, 0.59, and 0.62, respectively, for 10%, 20%, and +2/+3 cutoffs. A good agreement was observed for experienced pathologists using the 10% cutoff, and their agreement was statistically higher than for junior pathologists (P=0.02). In addition, the mean intraobserver agreement for all 7 pathologists using the 3 cutoffs was reasonably good, with mean κ scores of 0.71, 0.60, and 0.73, respectively, for 10%, 20%, and +2/+3 cutoffs. For all 3 cutoffs, a positive correlation was observed with perceived ease of interpretation (P<0.003). Finally, cytoplasmic-only staining achieved higher agreement using all 3 cutoffs than mixed staining patterns.
Conclusions:
All 3 cutoffs investigated achieve reasonable strength of agreement, modestly decreasing interobserver and intraobserver variability in IHC interpretation. These cutoffs have previously been used in cancer pathology, and this study provides evidence that these cutoffs can be reproducible between practicing pathologists.
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