Oncogenic events affecting members of the neurotrophin receptor kinase (NTRK) gene family have been recognized across many human tumors in both adults and children. These events occur as in-frame fusions between a 5′ partner gene that facilitates protein dimerization and an NTRK1/NTRK2/NTRK3 gene kinase domain, leading to ligand-independent constitutive activation of the so-called tropomyosin receptor kinase (TRK) kinase proteins encoded by the NTRK genes. These events are recognized as oncogenic drivers that lead to addiction to TRK-driven signaling through downstream growth and proliferation pathways, including mitogen-activated protein kinase, phosphatidyl inositol 3 kinase, and protein kinase C pathways. Several inhibitors of TRK proteins have demonstrated response and survival benefits in clinical trials, leading to regulatory approval specifically for entrectinib, a multikinase inhibitor, and larotrectinib, a selective TRK inhibitor. Any patient with an advanced solid tumor containing an NTRK fusion detected using molecular methods (including fluorescence in situ hybridization, RT-PCR, and DNA- or RNA-based next-generation sequencing) may be eligible for TRK inhibitor therapy. Therefore, NTRK was featured in the Association for Molecular Pathology webinar series entitled “Emerging and Evolving Biomarkers: Recent Findings, Laboratory Considerations, and Clinical Implications” (, last accessed October 25, 2021). The TRK proteins and their neurotrophin ligands have been recognized as essential to central nervous system development and homeostasis via work performed in animal models spanning >70 years. NTRK1, which encodes for TRKA, was formally confirmed as a proto-oncogene in a colon cancer cell line in 1986, with cloning and characterization of NTRK2 (TRKB) and NTRK3 (TRKC) in the ensuing decade. TRK fusions are reported in >90% of several uncommon tumor types, including infantile fibrosarcoma (enriched for ETV6-NTRK3), breast secretory carcinoma and mammary analogue secretory carcinoma, and cellular mixed congenital mesoblastic nephroma. There is a relative enrichment in TRK fusions (estimated prevalence of 5% to 25%) in thyroid carcinomas, KIT/PDGFRA/SDH gene wild-type gastrointestinal stromal tumor, and spitzoid tumors. However, for most solid tumors, TRK fusions occur in <5% of cases, and are typically identified in ≤1% of common adult cancers. Other NTRK gene alterations, including amplification events and single-nucleotide variants, are also reported in a small percentage of tumors, typically independent of fusions. Neither amplification nor de novo mutation of NTRK gene family members has been clearly associated with response to TRK inhibitor therapy. Most reported mutations in NTRK genes are likely passenger events, particularly in the context of tumors with otherwise high mutational burdens. However, some data from myeloid neoplasms have suggested an oncogenic role for select extracellular and transmembrane domain mutations in NTRK2 and NTRK3, with as yet unclear implications for therapeutic targeting. Trials of entrectinib in adult populations with TRK fusion–positive tumors reported a 57% overall response rate with median duration of response of 10.4 months; trials of larotrectinib in combined adult and pediatric cohorts reported 79% objective response rates and median duration of response of 35 months. The expression of NTRK transcripts/TRK proteins is relatively limited outside of the central nervous system; thus, most of the adverse effects associated with specific TRK inhibitor therapy relate to inhibition of the normal function of the TRK proteins as they relate to appetite modulation (TRKB), autonomic control (TRKB/TRKC), and pain control following drug withdrawal (TRKA), and can typically be managed via dose adjustment. However, entrectinib in particular is associated with a broader spectrum of adverse events, including congestive heart failure, likely attributable to its affinity for multiple tyrosine kinase receptors. As with other targeted therapies for cancer, TRK inhibition ultimately loses efficacy as a result of resistance mutations that arise either within the NTRK kinase domains or as off-target mechanisms of overcoming TRK signaling blockade, such as through downstream or parallel activation of mitogen-activated protein kinase pathway members. Despite the rarity of these tumors, the impressive results from trials of TRK inhibitors have led to substantial enthusiasm for detecting and treating patients with NRTK gene fusion events. For laboratorians, this may require reconsidering testing approaches to enhance the likelihood of detection of fusions at the DNA, RNA, or protein level. NTRK2 and NTRK3 genes contain several hundred megabases of noncoding regions; in particular, the introns containing common rearrangement breakpoints are large and contain a high percentage of repetitive sequence, leading to substantial barriers to detection using DNA-based hybrid capture sequencing technologies. RNA-based fusion detection, including via anchored multiplex PCR technologies, has been shown to increase the yield for NTRK fusion detection over DNA sequencing alone. Fluorescence in situ hybridization testing for NTRK gene rearrangement (or ETV6 in the case of infantile fibrosarcoma) is an acceptable modality for identification of patients for TRK inhibitor therapy; however, it may be limited by cost and risk of detecting nonfunctional structural variants. Immunohistochemistry for pan-Trk protein is an appealing, low-cost strategy that can detect abnormal Trk overexpression; however, protein overexpression can result from mechanisms other than NRTK fusion, thus necessitating molecular confirmation. Strategies that may enable more reliable detection of NTRK fusions include a priori comprehensive DNA- and RNA-based sequencing (such as via DNA panel sequencing and transcriptome analysis or targeted fusion detection). Given cost constraints, sequential testing using a comprehensive DNA panel followed by reflexive testing to RNA testing has been proposed in certain clinical contexts where NTRK fusion detection may be enriched, such as in pan–wild-type non–small-cell lung carcinomas or BRAF–wild-type mismatch repair deficient colorectal adenocarcinomas. The detection of an NTRK fusion in an individual patient with an advanced solid tumor may have profound implications for disease management, quality of life, and survival. For laboratorians, recognition of techniques that enhance NTRK fusion detection and an understanding of the strengths and weaknesses of protein versus DNA- versus RNA-based tests can inform operational strategies that ultimately lead to improved clinical outcomes for cancer patients. Learn more at: . Cocco E, Scaltriti M, Drilon A: NTRK fusion-positive cancers and TRK inhibitor therapy. Nat Rev Clin Oncol 2018, 15:731–747 Hsiao SJ, Zehir A, Sireci AN, Aisner DL: Detection of tumor NTRK gene fusions to identify patients who may benefit from tyrosine kinase (TRK) inhibitor therapy. J Mol Diagn 2019, 21:553–571 Solomon JP, Benayed R, Hechtman JF, Ladanyi M: Identifying patients with NTRK fusion cancer. Ann Oncol 2019, 30(Suppl_8):viii16–viii22 The Association for Molecular Pathology (AMP) Practice Guidelines and Reports are developed to be of assistance to laboratory and other health care professionals by providing guidance and recommendations for particular areas of practice. The Guidelines or Reports should not be considered inclusive of all proper approaches or methods, or exclusive of others. The Guidelines or Reports cannot guarantee any specific outcome, nor do they establish a standard of care. The Guidelines or Reports are not intended to dictate the treatment of a particular patient. Treatment decisions must be made on the basis of the independent judgment of health care providers and each patient's individual circumstances. The AMP makes no warranty, express or implied, regarding the Guidelines or Reports and specifically excludes any warranties of merchantability and fitness for a particular use or purpose. The AMP shall not be liable for direct, indirect, special, incidental, or consequential damages related to the use of the information contained herein. Molecular Pathology Education: An Association for Molecular Pathology Webinar Series on Emerging and Evolving Biomarkers Structured for Specific AudiencesThe Journal of Molecular DiagnosticsVol. 24Issue 2PreviewOne of the greatest challenges in molecular diagnostics education is the conveyance of scientific and laboratory principles within a constant stream of advances in research, new testing paradigms, and clinical relevance in the growing field of molecular pathology. This is particularly true with the changing roles of specific biomarkers as prognostic, predictive, and/or diagnostic signals. These biomarkers, often used to augment other pathologic testing, have expanded in both number and importance in recent years, a reality reflected in the laboratory and in the clinical management of patients. Full-Text PDF