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Review
. 2020 Jul;26(3):1385-1399.
doi: 10.1007/s12253-019-00685-2. Epub 2019 Jun 29.

Methods for Identifying Patients with Tropomyosin Receptor Kinase (TRK) Fusion Cancer

Derek Wong  1 , Stephen Yip  1 , Poul H Sorensen  2   3   4
Affiliations
Review

Methods for Identifying Patients with Tropomyosin Receptor Kinase (TRK) Fusion Cancer

Derek Wong et al. Pathol Oncol Res. 2020 Jul.

Abstract

NTRK gene fusions affecting the tropomyosin receptor kinase (TRK) protein family have been found to be oncogenic drivers in a broad range of cancers. Small molecule inhibitors targeting TRK activity, such as the recently Food and Drug Administration-approved agent larotrectinib (Vitrakvi®), have shown promising efficacy and safety data in the treatment of patients with TRK fusion cancers. NTRK gene fusions can be detected using several different approaches, including fluorescent in situ hybridization, reverse transcription polymerase chain reaction, immunohistochemistry, next-generation sequencing, and ribonucleic acid-based multiplexed assays. Identifying patients with cancers that harbor NTRK gene fusions will optimize treatment outcomes by providing targeted precision therapy.

Keywords: NGS; NTRK gene fusions; Next-generation sequencing; TRK fusions; TRK inhibitors.

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Conflict of interest statement

Dr. Wong declares no conflicts of interest. Dr. Yip declares consultation fees from Bayer and Pfizer for his participation in advisory boards and travel expense reimbursement from Roche/Foundation Medicine. Dr. Sorensen declares that he is an advisor for Bayer Pharmaceuticals but holds no financial interest in the company.

Figures

Fig. 1
Fig. 1
Tropomyosin receptor kinase (TRK) receptor signaling [5]. AKT, v-akt murine thymoma viral oncogene homolog; BDGF, brain-derived growth factor; DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; GAB1, GRB2-associated-binding protein 1; GRB2, growth factor receptor-bound protein 2; IP3, inositol trisphosphate; MEK, mitogen-activated protein kinase; NGF, nerve growth factor; NTF-3, neurotrophin 3; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; RAF, rapidly accelerated fibrosarcoma kinase; RAS, rat sarcoma kinase; SHC, Src homology 2 domain containing. Reproduced with permission from Amatu A, Sartore-Bianchi A, Siena S. ESMO Open 2016;1(2):e000023
Fig. 2
Fig. 2
NTRK gene fusions. (a) Mechanism of NTRK1/2/3 gene fusions; (b) ETV6-NTRK3 gene fusion [9]. DNA, deoxyribonucleic acid; LBD, ligand-binding domain; PTK, tyrosine kinase; TRK, tropomyosin receptor kinase; TM, transmembrane; SAM, sterile alpha motif. Figure 2b reproduced with permission from Triche TJ, Hicks MJ, Sorensen PH. Diagnostic Pathology of Pediatric Malignancies. In: Pizzo PA, Poplack DG, editors. Principles and Practice of Pediatric Oncology, 7th Edition: Wolters Kluwer Health; 2015
Fig. 3
Fig. 3
Break-apart fluorescent in situ hybridization (FISH). (a) The wildtype pattern shows two pairs of closely situated or fused signals. (b) In break-apart FISH, a set of probes specific for the target gene is used. When translocation occurs involving a breakpoint between the two probe sites, the loci split apart. (c) An example of break-apart FISH testing results in a patient with soft-tissue sarcoma and an LMNA-NTRK1 gene fusion [76]. NTRK1 break-apart FISH demonstrates both paired green (5′ NTRK1) and red (3′ NTRK1) signals corresponding to the normal NTRK1 gene (yellow arrow). Isolated red signals (red arrows) are observed in tumor nuclei (stained blue with DAPI) indicative of a chromosomal deletion leading to an NTRK1 gene fusion. DAPI, 4′,6-diamidino-2-phenylindole. Figures 3a and b reproduced with permission from Cheng L, Zhang S, Wang L, MacLennan GT, Davidson DD. J Pathol Clin Res 2017;3(2):73–99. Figure 3c reproduced with permission from Doebele RC, Davis LE, Vaishnavi A, Le AT, Estrada-Bernal A, Keysar S, et al. Cancer Discov 2015;5(10):1049–57
Fig. 4
Fig. 4
Reverse transcription polymerase chain reaction (RT-PCR) and next-generation sequencing (NGS). (a) Example results for reverse transcription polymerase chain reaction (RT-PCR) testing [80]. RT-PCR for ETV6-NTRK3 fusion transcripts in mammary analogue secretory carcinoma (MASC) tumors. ACTB, ß-actin, MASC 1, MASC 2, MASC 3 and MASC 4, tumor samples from Case 1, Case 2, Case 3, and Case 4, respectively. (b–d) Summary of NGS [79]. (B) RNA is extracted from formalin-fixed, paraffin-embedded (FFPE) tumor specimens and reverse transcribed into complementary DNA (cDNA). The cDNA is amplified with a panel of primers targeting fusion and native control transcripts. The resulting libraries are sequenced on Ion Torrent instruments and the sequence reads are then enumerated using a custom pipeline. Identified fusion transcripts are confirmed in the Integrative Genomics Viewer (IGV) to check that sequence reads span both fusion partners. (C) Fused genes are detected by PCR amplicons that span a known fusion breakpoint. (D) Novel fusions may also be detected based on overexpression of the kinase domain of selected targets. Figure 4a reproduced with permission from Fehr A, Loning T, Stenman G. Am J Surg Pathol 2011;35(10):1600–2. URL: https://journals.lww.com/ajsp/Citation/2011/10000/Mammary_Analogue_Secretory_Carcinoma_of_the.20.aspx. Figures 4b–d reproduced with permission from Beadling C, et al. J Mol Diagnostics. 2016;18(2):165–175
Fig. 5
Fig. 5
Example pan-tropomyosin receptor kinase (TRK) immunohistochemistry (IHC) staining pattern in a patient with colorectal carcinoma with an LMNA-NTRK1 fusion [28]. A moderately differentiated colorectal carcinoma with conventional histology (hematoxylin and eosin) and an LMNA exon 12-NTRK1 exon 12 fusion (A) displays diffuse cytoplasmic and nuclear membrane staining for pan-TRK IHC (pan-TRK IHC clone EPR17341, Abcam, Cambridge, MA) (B, C). Reproduced with permission from Hechtman JF, Benayed R, Hyman DM, Drilon A, Zehir A, Frosina D, et al. Am J Surg Pathol 2017;41(11):1547–51. URL: https://journals.lww.com/ajsp/Abstract/2017/11000/Pan_Trk_Immunohistochemistry_Is_an_Efficient_and.13.aspx
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