Disadvantages

The specificity of IHC is limited if tumour tissue has physiological TRK protein expression, particularly in the nervous system and smooth muscle, as well as when there is variable basal expression in other tissues [1].

While two studies reported 98%–100% specificity for IHC [2, 3], studies with a larger number of negative controls have reported higher false positive rates if NGS was not performed for confirmation. For instance, diffuse pan-TRK staining was found to be a sensitive diagnostic IHC marker for infantile fibrosarcoma (NTRK3 fusion; 14/15, 93% sensitivity) and lipofibromatosis-like neural tumour (NTRK1 fusion; 5/5, 100% sensitivity), but non-specific staining was noted in 8% (16/190) of other paediatric spindle-cell tumours [4]. In addition, specificity of IHC was 96% in adults with common solid tumours (4,136 cases analysed, including 28 with confirmed NTRK gene fusion) [5]. However, in uterine leiomyosarcoma, TRKA and/or pan-TRK staining did not correlate with NTRK gene rearrangement. Specifically, TRK staining by IHC was observed in 6 of 97 leiomyosarcomas (which are tumours with smooth muscle differentiation), but these tumours lacked NTRK gene fusions as determined by FISH or whole transcriptome sequencing [6]. Furthermore, differences have been observed with different antibody clones and fixation conditions, and while sensitivity close to 100% for NTRK1 and NTRK2 has been described, much lower sensitivity (79%) was found for NTRK3 [7, 8]. In some of these cases, staining was found to be weak and focal, increasing the chance of false negative result [7-9]. This illustrates the importance of a confirmatory second analysis following IHC screening.

The disadvantages of IHC are summarised in the following table.

Disadvantages of IHC for Testing for NTRK Gene Fusions

Disadvantages
Confirmation of fusion by a second assay is recommended, particularly when cytoplasmic staining is present without nuclear, peri-nuclear, or membranous staining Similarly, confirmation of fusion by a second assay is recommended if there is a possibility of smooth muscle or neurologic differentiation Only detects the TRK protein expression without distinction between wild-type TRK and TRK fusion protein. As a result, a confirmatory test is needed to confirm the presence of a NTRK gene fusion if IHC used

Disadvantages

Confirmation of fusion by a second assay is recommended, particularly when cytoplasmic staining is present without nuclear, peri-nuclear, or membranous staining
Similarly, confirmation of fusion by a second assay is recommended if there is a possibility of smooth muscle or neurologic differentiation
Only detects the TRK protein expression without distinction between wild-type TRK and TRK fusion protein. As a result, a confirmatory test is needed to confirm the presence of a NTRK gene fusion if IHC used

References

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.
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.
Rudzinski ER, Lockwood CM, Stohr BA et al. Pan-Trk Immunohistochemistry Identifies NTRK Rearrangements in Pediatric Mesenchymal Tumors. Am J Surg Pathol 2018; 42: 927-935.
Hung YP, Fletcher CDM, Hornick JL. Evaluation of pan-TRK immunohistochemistry in infantile fibrosarcoma, lipofibromatosis-like neural tumour and histological mimics. Histopathology 2018; 73: 634-644.
Gatalica Z, Xiu J, Swensen J, Vranic S. Molecular characterization of cancers with NTRK gene fusions. Mod Pathol 2019; 32.
Chiang S, Cotzia P, Hyman DM et al. NTRK Fusions Define a Novel Uterine Sarcoma Subtype With Features of Fibrosarcoma. The American Journal of Surgical Pathology 2018; 42: 791-798.
Conde E, Hernandez S, Sanchez E et al. Pan-TRK Immunohistochemistry: An Example-Based Practical Approach to Efficiently Identify Patients With NTRK Fusion Cancer. Arch Pathol Lab Med. 2021 Aug 1;145(8):1031-1040.
Solomon JP, Linkov I, Rosado A et al. NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls. Mod Pathol. 2020; 33(1):38-46.
Strohmeier S, Brcic I, Popper H et al. Applicability of pan-TRK immunohistochemistry for identification of NTRK fusionsin lung carcinoma. Sci Rep. 2021; 11(1):9785.