Related Experiment Video
Updated: Jul 11, 2025
Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers
Published on: December 5, 2017
Canadian Multicentric Pan-TRK (CANTRK) Immunohistochemistry Harmonization Study
Martin D Hyrcza1, Sebastiao N Martins-Filho2, Alan Spatz3
1Department of Pathology and Laboratory Medicine, University of Calgary, Arnie Charbonneau Cancer Institute, Calgary, Alberta, Canada.
Abstract:
Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.
Insights
A Canadian study successfully harmonized pan-TRK immunohistochemistry (IHC) screening across 18 labs for TRK inhibitor therapy. The assay achieved high diagnostic sensitivity, enabling broader patient identification for targeted treatments.
Area of Science:
- Oncology
- Pathology
- Molecular Diagnostics
Background:
- Tumor-agnostic testing for NTRK1-3 gene rearrangements is crucial for identifying patients eligible for TRK inhibitor therapies.
- Standardized and high-quality immunohistochemistry (IHC) screening assays are needed for widespread clinical implementation.
Purpose of the Study:
- To establish and validate a high-quality pan-TRK IHC screening assay across multiple Canadian pathology laboratories.
- To assess the diagnostic sensitivity and specificity of the assay using a standardized protocol and reference materials.
Main Methods:
- A multicenter ring study involving 18 regional pathology laboratories in Canada.
- Utilized pan-TRK monoclonal antibody clone EPR17341 and centrally developed reference standards (TRK calibrators and iCAPCs).
- Laboratories optimized IHC protocols, stained 30 TRK-fusion positive/negative tumor samples, and reported staining patterns.
Main Results:
- Fifteen of 18 laboratories achieved 100% diagnostic sensitivity compared to next-generation sequencing.
- Diagnostic specificity ranged from 40% to 90%.
- Median limit of detection was 76,000 molecules/cell, with observed staining in cytoplasmic, nuclear, and membranous patterns.
Conclusions:
- The Canadian multicentric pan-TRK study successfully harmonized IHC screening, demonstrating a viable strategy for multicenter implementation.
- The developed assay achieves high diagnostic sensitivity, facilitating broader patient identification for TRK inhibitor therapies.
- Centrally developed reference materials and analytical sensitivity measurements using TRK calibrators were key to assay performance.
