IHC has been widely used for several decades . 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 .
A pan-TRK monoclonal antibody for use in IHC is commercially available [clone EPR17341 available from Abcam, Cambridge, MA, USA( www.abcam.com) and Ventana Medical Systems Inc., Tucson, AZ, USA also based on the same EPR17341 clone ( diagnostics.roche.com)]. These antibodies can detect TRKA, TRKB, and TRKC expression which is physiologically expressed in certain tissues and also in most tumours with NTRK gene fusions.
Figure 9: IHC Using Pan-TRK Monoclonal Antibody (EPR17341; Abcam)
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)[4-6]. 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 [4, 5, 7]. One suggested model uses a two-step method for detecting NTRK gene fusion, using IHC as a screening tool followed by NGS to confirm fusions when IHC is positive (see figure below) [4, 7, 8]. 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.
Figure 10: Use of IHC to Detect NTRK gene fusionsa
aAnother 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.
- 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.
- Dixon AR, Bathany C, Tsuei M et al. Recent developments in multiplexing techniques for immunohistochemistry. Expert Rev of Mol Diagn 2015; 15: 1171-1186.
- 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.
- 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.
- 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.
- Cocco E, Scaltriti M, Drilon A. NTRK fusion-positive cancers and TRK inhibitor therapy. Nat Rev Clin Oncol 2018; 15: 731-747.
- 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.
- Marchio C, Scaltriti M, Ladanyi M et al. ESMO recommendations on the standard methods to detect NTRK fusions in daily practice and clinical research. Ann Oncol. 2019 Jul 3. pii: mdz204. doi: 10.1093/annonc/mdz204. [Epub ahead of print].