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Overview

IHC has been widely used for several decades [1]. This technique uses antibodies, usually linked to an enzyme or a fluorescent dye, to visualise cell markers (i.e., antigens) in tissue samples [1, 2].

IHC results and reproducibility are influenced by many factors, including tissue handling and storage, epitope retrieval, choice of antibody, detection method, and interpretation [3].

Currently the most frequently used pan-TRK monoclonal antibody in IHC is a rabbit monoclonal antibody, clone EPR17341, which is available as a diagnostic or by itself from Abcam( www.abcam.com) and Ventana/ Roche Diagnostics (https://diagnostics.roche.com/)]. Another pan-TRK rabbit monoclonal antibody, clone A7H6R, is also available by itself. These antibodies can detect TRKA, TRKB, and TRKC expression which is physiologically expressed in certain tissues and in most tumours with NTRK gene fusions. It should be noted that the staining patterns of the two clones are not strictly identical [4].

IHC using VENTANA pan-TRK (EPR17341) Assay - Roche Diagnostics

Image: Secretory breast cancer tissue section showing typical strong nuclear staining (fusion NTRK3-ETV6)

IHC detects TRK protein expression and can be used to detect or screen for NTRK fusions in tumours without neural, neuroendocrine, or smooth muscle differentiation (in these particular tumours there could be positivity not related to NTRK translocations)[5-7]. TRK is physiologically expressed in smooth muscle, neural, and neoplastic tissue with this differentiation occurring regardless of fusion status, as well as being expressed in tumours with NTRK gene fusions [5, 6, 8]. Hence, care should be taken when endogenous expression occurs in the sample, in which case RNA-based NGS analysis is needed [10].  

A target enrichment model can be applied for detecting NTRK gene fusion in a two-step method, whereby IHC as a screening tool is used initially, followed by NGS to confirm fusions when IHC is positive (see figure below) [5, 8, 9]. Samples that are negative by IHC for any of these markers are then removed to enrich for tumours that potentially have NTRK gene fusions. This approach ensures that pathologists are more likely to detect NTRK gene fusions. Samples are then further tested for NTRK gene fusions using NGS.

Use of IHC to Detect NTRK gene fusionsa

Figure 10: Use of IHC to Detect NTRK gene fusions

IHC, immunohistochemistry; NGS, next-generation sequencing.

IHC, immunohistochemistry; NGS, next-generation sequencing.

aAnother or a complementary strategy to make sure that pathologists are more likely to detect NTRK gene fusion is to focus on high prevalence tumours, as shown in the Algorithm for NTRK gene fusion testing section later in this module.

References

  1. Matos LLd, Trufelli DC, de Matos MGL, da Silva Pinhal MA. Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomark insights 2010; 5: 9-20.
  2. Dixon AR, Bathany C, Tsuei M et al. Recent developments in multiplexing techniques for immunohistochemistry. Expert Rev of Mol Diagn 2015; 15: 1171-1186.
  3. O'Hurley G, Sjöstedt E, Rahman A et al. Garbage in, garbage out: A critical evaluation of strategies used for validation of immunohistochemical biomarkers. Mol Oncol 2014; 8: 783-798.
  4. Guibourg B, Cloarec E, Conan-Charlet V et al. EPR17341 and A7H6R pan-TRK Immunohistochemistry Result in Highly Different Staining Patterns in a Series of Salivary Gland Tumors. Appl Immunohistochem Mol Morphol. 2020 Oct;28(9):719-724.
  5. Albert CM, Davis JL, Federman N et al. TRK Fusion Cancers in Children: A Clinical Review and Recommendations for Screening. J Clin Oncol 2019; 37: 513-524.
  6. Hechtman JF, Benayed R, Hyman DM et al. Pan-Trk Immunohistochemistry Is an Efficient and Reliable Screen for the Detection of NTRK Fusions. Am J Surg Pathol 2017; 41: 1547-1551.
  7. Cocco E, Scaltriti M, Drilon A. NTRK fusion-positive cancers and TRK inhibitor therapy. Nat Rev Clin Oncol 2018; 15: 731-747.
  8. Murphy DA, Ely HA, Shoemaker R et al. Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing. Appl Immunohistochem Mol Morphol 2017; 25: 513-523.
  9. Marchio C, Scaltriti M, Ladanyi M et al. ESMO recommendations on the standard methods to detect NTRK fusions in daily practice and clinical research. ESMO 2018 Congress 2019; 30(9):1417-1427.
  10. De Winne K, Sorber L, Lambin S et al. . Immunohistochemistry as a screening tool for NTRK gene fusions: results of a first Belgian ring trial. Virchows Arch. 2021; 478(2):283-291.

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