Beyond Clinical Trials: Understanding Neurotrophic Tropomyosin Receptor Kinase Inhibitor Challenges and Efficacy in Real-World Pediatric Oncology.

PURPOSE: Our study aimed to explore real-world treatment scenarios for children and adolescents with neurotrophic tropomyosin receptor kinase (NTRK)-fused tumors, emphasizing access, responses, side effects, and outcomes. PATIENTS AND METHODS: Pooled clinical data from 17 pediatric cases (11 soft-tissue sarcomas, five brain tumors, and one neuroblastoma) treated with larotrectinib and radiologic images for 14 patients were centrally reviewed. Testing for gene fusions was prompted by poor response to treatment, tumor progression, or aggressiveness. RESULTS: Six different NTRK fusion subtypes were detected, and various payment sources for testing and medication were reported. Radiologic review revealed objective tumor responses (OR) in 11 of 14 patients: Complete responses: two; partial responses: nine; and stable disease: three cases. Grades 1 or 2 Common Terminology Criteria for Adverse Events adverse effects were reported in five patients. Regarding the entire cohort's clinical information, 15 of 17 patients remain alive (median observation time: 25 months): four with no evidence of disease and 11 alive with disease (10 without progression). One patient developed resistance to the NTRK inhibitor and died from disease progression while another patient died due to an unrelated cause. CONCLUSION: This real-world study confirms favorable agnostic tumor OR rates to larotrectinib in children with NTRK-fused tumors. Better coordination to facilitate access to medication remains a challenge, particularly in middle-income countries like Brazil.

NTRK3 exhibits a pro-oncogenic function in upper tract urothelial carcinomas.

Neurotrophic receptor tyrosine kinase 3 (NTRK3) has pleiotropic functions: it acts not only as an oncogene in breast and gastric cancers but also as a dependence receptor in tumor suppressor genes in colon cancer and neuroblastomas. However, the role of NTRK3 in upper tract urothelial carcinoma (UTUC) is not well documented. This study investigated the association between NTRK3 expression and outcomes in UTUC patients and validated the results in tests on UTUC cell lines. A total of 118 UTUC cancer tissue samples were examined to evaluate the expression of NTRK3. Survival curves were generated using Kaplan-Meier estimates, and Cox regression models were used for investigating survival outcomes. Higher NTRK3 expression was correlated with worse progression-free survival, cancer-specific survival, and overall survival. Moreover, the results of an Ingenuity Pathway Analysis suggested that NTRK3 may interact with the PI3K-AKT-mTOR signaling pathway to promote cancer. NTRK3 downregulation in BFTC909 cells through shRNA reduced cellular migration, invasion, and activity in the AKT-mTOR pathway. Furthermore, the overexpression of NTRK3 in UM-UC-14 cells promoted AKT-mTOR pathway activity, cellular migration, and cell invasion. From these observations, we concluded that NTRK3 may contribute to aggressive behaviors in UTUC by facilitating cell migration and invasion through its interaction with the AKT-mTOR pathway and the expression of NTRK3 is a potential predictor of clinical outcomes in cases of UTUC.

Estimating the Prognostic Value of the NTRK Fusion Biomarker for Comparative Effectiveness Research in The Netherlands.

OBJECTIVES: We evaluated the prognostic value of the neurotrophic tyrosine receptor kinase (NTRK) gene fusions by comparing the survival of patients with NTRK+ tumours with patients without NTRK+ tumours. METHODS: We used genomic and clinical registry data from the Center for Personalized Cancer Treatment (CPCT-02) study containing a cohort of cancer patients who were treated in Dutch clinical practice between 2012 and 2020. We performed a propensity score matching analysis, where NTRK+ patients were matched to NTRK- patients in a 1:4 ratio. We subsequently analysed the survival of the matched sample of NTRK+ and NTRK- patients using the Kaplan-Meier method and Cox regression, and performed an analysis of credibility to evaluate the plausibility of our result. RESULTS: Among 3556 patients from the CPCT-02 study with known tumour location, 24 NTRK+ patients were identified. NTRK+ patients were distributed across nine different tumour types: bone/soft tissue, breast, colorectal, head and neck, lung, pancreas, prostate, skin and urinary tract. NTRK fusions involving the NTRK3 gene (46%) and NTRK1 gene (33%) were most common. The survival analysis rendered a hazard ratio (HR) of 1.44 (95% CI 0.81-2.55) for NTRK+ patients. Using the point estimates of three prior studies on the prognostic value of NTRK fusions, our finding that the HR is > 1 was deemed plausible. CONCLUSIONS: NTRK+ patients may have an increased risk of death compared with NTRK- patients. When using historic control data to assess the comparative effectiveness of TRK inhibitors, the prognostic value of the NTRK fusion biomarker should therefore be accounted for.

Predictors of radioiodine (RAI)-avidity restoration for NTRK fusion-positive RAI-resistant metastatic thyroid cancers.

Context: Two-thirds of metastatic differentiated thyroid cancer (DTC) patients have radioiodine (RAI)-resistant disease, resulting in poor prognosis and high mortality. For rare NTRK and RET fusion-positive metastatic, RAI-resistant thyroid cancers, variable success of re-induction of RAI avidity during treatment with NTRK or RET inhibitors has been reported. Case presentation and results: We report two cases with RAI-resistant lung metastases treated with larotrectinib: an 83-year-old male presenting with an ETV6::NTRK3 fusion-positive tumor with the TERT promoter mutation c.-124C>T, and a 31-year-old female presenting with a TPR::NTRK1 fusion-positive tumor (and negative for TERT promoter mutation). Post larotrectinib treatment, diagnostic I-123 whole body scan revealed unsuccessful RAI-uptake re-induction in the TERT-positive tumor, with a thyroid differentiation score (TDS) of -0.287. In contrast, the TERT-negative tumor exhibited successful I-131 reuptake with a TDS of -0.060. Conclusion: As observed for RAI-resistance associated with concurrent TERT and BRAF mutations, the co-occurrence of TERT mutations and NTRK fusions may also contribute to re-sensitization failure.

NT-3 contributes to chemotherapy-induced neuropathic pain through TrkC-mediated CCL2 elevation in DRG neurons.

Cancer patients undergoing treatment with antineoplastic drugs often experience chemotherapy-induced neuropathic pain (CINP), and the therapeutic options for managing CINP are limited. Here, we show that systemic paclitaxel administration upregulates the expression of neurotrophin-3 (Nt3) mRNA and NT3 protein in the neurons of dorsal root ganglia (DRG), but not in the spinal cord. Blocking NT3 upregulation attenuates paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities and spontaneous pain without altering acute pain and locomotor activity in male and female mice. Conversely, mimicking this increase produces enhanced responses to mechanical, heat, and cold stimuli and spontaneous pain in naive male and female mice. Mechanistically, NT3 triggers tropomyosin receptor kinase C (TrkC) activation and participates in the paclitaxel-induced increases of C-C chemokine ligand 2 (Ccl2) mRNA and CCL2 protein in the DRG. Given that CCL2 is an endogenous initiator of CINP and that Nt3 mRNA co-expresses with TrkC and Ccl2 mRNAs in DRG neurons, NT3 likely contributes to CINP through TrkC-mediated activation of the Ccl2 gene in DRG neurons. NT3 may be thus a potential target for CINP treatment.

Evaluation of NTRK expression and fusions in a large cohort of early-stage lung cancer.

Tropomyosin receptor kinases (TRK) are attractive targets for cancer therapy. As TRK-inhibitors are approved for all solid cancers with detectable fusions involving the Neurotrophic tyrosine receptor kinase (NTRK)-genes, there has been an increased interest in optimizing testing regimes. In this project, we wanted to find the prevalence of NTRK fusions in a cohort of various histopathological types of early-stage lung cancer in Norway and to investigate the association between TRK protein expression and specific histopathological types, including their molecular and epidemiological characteristics. We used immunohistochemistry (IHC) as a screening tool for TRK expression, and next-generation sequencing (NGS) and fluorescence in situ hybridization (FISH) as confirmatory tests for underlying NTRK-fusion. Among 940 cases, 43 (4.6%) had positive TRK IHC, but in none of these could a NTRK fusion be confirmed by NGS or FISH. IHC-positive cases showed various staining intensities and patterns including cytoplasmatic or nuclear staining. IHC-positivity was more common in squamous cell carcinoma (LUSC) (10.3%) and adenoid cystic carcinoma (40.0%), where the majority showed heterogeneous staining intensity. In comparison, only 1.1% of the adenocarcinomas were positive. IHC-positivity was also more common in men, but this association could be explained by the dominance of LUSC in TRK IHC-positive cases. Protein expression was not associated with differences in time to relapse or overall survival. Our study indicates that NTRK fusion is rare in early-stage lung cancer. Due to the high level of false positive cases with IHC, Pan-TRK IHC is less suited as a screening tool for NTRK-fusions in LUSC and adenoid cystic carcinoma.

Srsf1 and Elavl1 act antagonistically on neuronal fate choice in the developing neocortex by controlling TrkC receptor isoform expression.

The seat of higher-order cognitive abilities in mammals, the neocortex, is a complex structure, organized in several layers. The different subtypes of principal neurons are distributed in precise ratios and at specific positions in these layers and are generated by the same neural progenitor cells (NPCs), steered by a spatially and temporally specified combination of molecular cues that are incompletely understood. Recently, we discovered that an alternatively spliced isoform of the TrkC receptor lacking the kinase domain, TrkC-T1, is a determinant of the corticofugal projection neuron (CFuPN) fate. Here, we show that the finely tuned balance between TrkC-T1 and the better known, kinase domain-containing isoform, TrkC-TK+, is cell type-specific in the developing cortex and established through the antagonistic actions of two RNA-binding proteins, Srsf1 and Elavl1. Moreover, our data show that Srsf1 promotes the CFuPN fate and Elavl1 promotes the callosal projection neuron (CPN) fate in vivo via regulating the distinct ratios of TrkC-T1 to TrkC-TK+. Taken together, we connect spatio-temporal expression of Srsf1 and Elavl1 in the developing neocortex with the regulation of TrkC alternative splicing and transcript stability and neuronal fate choice, thus adding to the mechanistic and functional understanding of alternative splicing in vivo.

Elaboration of NTRK-rearranged colorectal cancer: Integration of immunoreactivity pattern, cytogenetic identity, and rearrangement variant.

Fused information from protein status, DNA breakage, and transcripts are still limited because of the low rate of activated-NTRK in colorectal cancer (CRC). In total, 104 archived CRC tissue samples with dMMR were analyzed using immunohistochemistry (IHC), polymerase chain reaction (PCR), and pyrosequencing to mine the NTRK-enriched CRC group, and then subjected to NTRK fusion detection using pan-tyrosine kinase IHC, fluorescence in situ hybridization (FISH), and DNA-/RNA-based next generation sequencing (NGS) assays. Of the 15 NTRK-enriched CRCs, eight NTRK fusions (53.3%, 8/15), including two TPM3(e7)-NTRK1(e10), one TPM3(e5)-NTRK1(e11), one LMNA(e10)-NTRK1(e10), two EML4(e2)-NTRK3(e14), and two ETV6(e5)-NTRK3(e15) fusions, were identified. There was no immunoreactivity for ETV6-NTRK3 fusion. In addition to cytoplasmic staining found in six specimens, membrane positive (TPM3-NTRK1 fusion) and nuclear positive (LMNA-NTRK1 fusion) were also observed in two of them. Atypical FISH-positive types were observed in four cases. Unlike IHC, NTRK-rearranged tumors appeared homogeneous on FISH. ETV6-NTRK3 may be missed in pan-TRK IHC screening for CRC. Regarding break-apart FISH, NTRK detection is difficult because of the diversity of signal patterns. Further research is warranted to identify the characteristics of NTRK-fusion CRCs.

Head and Neck Mesenchymal Tumors with Kinase Fusions: A Report of 15 Cases With Emphasis on Wide Anatomic Distribution and Diverse Histologic Appearance.

Mesenchymal tumors harboring various kinase fusions were recently recognized as emerging entities mainly in the soft tissues. We herein investigate the clinicopathologic and molecular characteristics of head and neck mesenchymal tumors harboring kinase fusions. The study cohort included 15 patients with a median age of 13 years (ranging from congenital to 63 y). The kinase genes involved in descending order were NTRK1 (n=6), NTRK3 (n=5), BRAF (n=2), and 1 each with MET, and RET. The anatomic locations were broad involving all tissue planes, including skin (n=4), intraosseous (n=4), major salivary glands (n=2), sinonasal tract (n=2), soft tissue of face or neck (n=2), and oral cavity (n=1). The histologic spectrum ranged from benign to high grade, in descending order including tumors resembling malignant peripheral nerve sheath tumor (MPNST)-like, fibrosarcoma (infantile or adult-type), lipofibromatosis-like neural tumor (LPFNT), inflammatory myofibroblastic tumor-like, and a novel phenotype resembling myxoma. Perivascular hyalinization/stromal keloid-like collagen bands and staghorn vasculature were common features in MPNST-like and LPFNT-like tumors. Two tumors (1 each with NTRK1 or BRAF rearrangement) were classified as high grade. By immunohistochemistry, S100 and CD34 positivity was noted in 71% and 60%, frequently in MPNST-like and LPFNT-like phenotypes. Pan-TRK was a sensitive marker for NTRK-translocated tumors but was negative in tumor with other kinase fusions. One patient with a high-grade tumor developed distant metastasis. Molecular testing for various kinase fusions should be considered for S100+/CD34+ spindle cell neoplasms with perivascular hyalinization and staghorn vessels, as pan-TRK positivity is seen only in NTRK fusions.

NTRK-rearranged spindle cell neoplasms: a clinicopathological and molecular study of 13 cases with peculiar characteristics at one of the largest institutions in China.

NTRK-rearranged spindle cell neoplasms (NTRK-RSCNs) represent an emerging group of rare tumours defined using molecular means. To the best of our knowledge, there have been no large series of reports about this tumour in the Chinese population in English full-text articles. Herein, we present 13 NTRK-RSCNs with peculiar characteristics. Ten of the 13 (77%) patients were children without sex differences. The tumour locations included six trunks, four extremities, two recta, and one small bowel. The histological morphology included four lipofibromatosis-like neural tumour (LPF-NT)-like, eight malignant peripheral nerve sheath tumours (MPNST)/fibrosarcoma-like, and one extremely rare myxofibrosarcoma-like pattern. Immunohistochemically, all cases were CD34, pan-TRK and TRK-A positive, SOX-10 negative, and H3K27me3 intact. S-100 protein expression was identified in 11 of 13 (85%) cases. Genetically, NTRK1 rearrangements were considered positive (7/13, 54%) or suspicious for positivity (6/13, 46%) by fluorescence in situ hybridisation. Next-generation sequencing and Sanger sequencing confirmed NTRK1 fusions with a variety of partner genes, including five LMNA, three TPM3, one SQSTM1, three novel CPSF6, IGR (downstream PMVK), and GAS2L1 genes. Interestingly, the last tumour concurrently harboured a second EWSR1-PBX1 fusion, which has never been reported. Four patients developed local recurrence and two of them suffered metastasis. In our study, NTRK-RSCNs had peculiar fusions that displayed unusual or complicated clinicopathological features. Histological clues and IHC helped streamline a small subset of potential candidates. Although FISH is a powerful technology for identifying NTRK rearrangements, RNA-/DNA-based NGS is recommended for highly suspected cases in which FISH signal patterns are not discernible as classic positive patterns, particularly if targeted therapy is considered.

Tropomyosin receptor kinase B (TrkB) signalling: targeted therapy in neurogenic tumours.

Tropomyosin receptor kinase B (TrkB), a transmembrane receptor protein, has been found to play a pivotal role in neural development. This protein is encoded by the neurotrophic receptor tyrosine kinase 2 (NTRK2) gene, and its abnormal activation caused by NTRK2 overexpression or fusion can contribute to tumour initiation, progression, and resistance to therapeutics in multiple types of neurogenic tumours. Targeted therapies for this mechanism have been designed and developed in preclinical and clinical studies, including selective TrkB inhibitors and pan-TRK inhibitors. This review describes the gene structure, biological function, abnormal TrkB activation mechanism, and current-related targeted therapies in neurogenic tumours.

Getting Your Laboratory on Track With Neurotrophic Receptor Tyrosine Kinase.

CONTEXT.­: Neurotrophic receptor tyrosine kinase (NTRK) fusion testing has both diagnostic and therapeutic implications for patient care. With 2 tumor-agnostic US Food and Drug Administration-approved tropomyosin receptor kinase (TRK) inhibitors, testing is increasingly used for therapeutic decision making. However, the testing landscape for NTRK fusions is complex, and optimal testing depends on the clinicopathologic scenario. OBJECTIVE.­: To compare different NTRK testing methods to help pathologists understand test features and performance characteristics and make appropriate selections for NTRK fusion detection for their laboratory and individual patient specimens. DATA SOURCES.­: A literature search for NTRK gene fusions and TRK protein was performed, including papers that discussed treatment, testing methodology, and detection or prevalence of fusion-positive cases. CONCLUSIONS.­: As standard of care in some tumor types, next-generation sequencing (NGS) panel testing is a cost effective and reliable way to detect a broad range of NTRK fusions. The design of the panel and use of DNA or RNA will affect performance characteristics. Pan-TRK immunohistochemistry may be used as a rapid, less expensive screen in cases that will not undergo routine NGS testing, or on specimens unsuitable for NGS testing. Fluorescence in situ hybridization may be appropriate for low-tumor-content specimens that are unsuitable for NGS testing. Quantitative reverse transcription polymerase chain reaction is best suited for monitoring low-level disease of a specific, previously identified target. This information should help laboratories develop a laboratory-specific NTRK testing algorithm that best suits their practice setting and patients' needs.

TRK Protein Expression in Merkel Cell Carcinoma Is Not Caused by NTRK Fusions.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignant tumor with neuroendocrine differentiation, with a rapidly growing incidence rate, high risk of recurrence, and aggressive behavior. The available therapeutic options for advanced disease are limited and there is a pressing need for new treatments. Tumors harboring fusions involving one of the neurotrophin receptor tyrosine kinase (NTRK) genes are now actionable with targeted inhibitors. NTRK-fused genes have been identified in neuroendocrine tumors of other sites; thus, a series of 76 MCCs were firstly analyzed with pan-TRK immunohistochemistry and the positive ones with real-time RT-PCR, RNA-based NGS, and FISH to detect the eventual underlying gene fusion. Despite 34 MCCs showing pan-TRK expression, NTRK fusions were not found in any cases. As in other tumors with neural differentiation, TRK expression seems to be physiological and not caused by gene fusions.

Expert consensus on the diagnosis and treatment of NTRK gene fusion solid tumors in China.

Gene fusions can drive tumor development for multiple types of cancer. Currently, many drugs targeting gene fusions are being approved for clinical application. At present, tyrosine receptor kinase (TRK) inhibitors targeting neurotrophic tyrosine receptor kinase (NTRK) gene fusions are among the first "tumor agnostic" drugs approved for pan-cancer use. Representative TRK inhibitors, including larotrectinib and entrectinib, have shown high efficacy for many types of cancer. At the same time, several second-generation drugs designed to overcome first-generation drug resistance are undergoing clinical development. Due to the rarity of NTRK gene fusions in common cancer types and technical issues regarding the complexity of fusion patterns, effectively screening patients for TRK inhibitor treatment in routine clinical practice is challenging. Different detection methods including immunohistochemistry, fluorescence in situ hybridization, reverse transcription-polymerase chain reaction, and (DNA and/or RNA-based) next-generation sequencing have pros and cons. As such, recommending suitable tests for individual patients and ensuring the quality of tests is essential. Moreover, at present, there is a lack of systematic review for the clinical efficacy and development status of first- and second-generation TRK inhibitors. To resolve the above issues, our expert group has reached a consensus regarding the diagnosis and treatment of NTRK gene fusion solid tumors, aiming to standardize clinical practice with the goal of benefiting patients with NTRK gene fusions treated with TRK inhibitors.

A novel TMTC2-NTRK3 fusion in undifferentiated high-grade pleomorphic sarcoma.

Undifferentiated high-grade pleomorphic sarcoma (UHPS) is a rare soft tissue sarcoma (STS) originated from mesenchyme. UHPS is mostly advanced, aggressive and has poor prognosis. Patients with UHPS tend to have a lower 5-year survival rate than patients with other types of STS. NTRK fusions are commonly found in rare histological tumor types. Among sarcomas, 90% of infantile fibrosarcomas have NTRK fusions. Many other types of sarcomas have also been studied for NTRK fusions. Targeted therapy with NTRK inhibitors, such as Larotrectinib and Entrectinib, leads to response in most patients with NTRK1/2/3 gene fusion-positive tumors. Herein, we present a 68-years old man with UHPS by pathological diagnosis. Next-generation sequencing (NGS) revealed a novel TMTC2-NTRK3 fusion, which was also detected by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). This report broadens the spectrum of NTRK fusions in UHPS and highlights a new target for treatment.

Therapeutic targeting of RBPJ, an upstream regulator of ETV6 gene, abrogates ETV6-NTRK3 fusion gene transformations in glioblastoma.

Fusion genes are abnormal genes resulting from chromosomal translocation, insertion, deletion, inversion, etc. ETV6, a rather promiscuous partner forms fusions with several other genes, most commonly, the NTRK3 gene. This fusion leads to the formation of a constitutively activated tyrosine kinase which activates the Ras-Raf-MEK and PI3K/AKT/MAPK pathways, leading the cells through cycles of uncontrolled division and ultimately resulting in cancer. Targeted therapies against this ETV6-NTRK3 fusion protein are much needed. Therefore, to find a targeted approach, a transcription factor RBPJ regulating the ETV6 gene was established and since the ETV6-NTRK3 fusion gene is downstream of the ETV6 promoter/enhancer, this fusion protein is also regulated. The regulation of the ETV6 gene via RBPJ was validated by ChIP analysis in human glioblastoma (GBM) cell lines and patient tissue samples. This study was further followed by the identification of an inhibitor, Furamidine, against transcription factor RBPJ. It was found to be binding with the DNA binding domain of RBPJ with antitumorigenic properties and minimal organ toxicity. Hence, a new target RBPJ, regulating the production of ETV6 and ETV6-NTRK3 fusion protein was found along with a potent RBPJ inhibitor Furamidine.

A novel ETV6-NTRK3 gene fusion in primary renal fibrosarcoma.

INTRODUCTION: Primary renal fibrosarcoma is a relatively uncommon tumor in the urinary system of adults, in fact only 6 cases have been reported in the English literature so far worldwide. The etiology of renal fibrosarcoma is incompletely understood. It is still lacking in simple and specific tissue-based biomarkers to assist the diagnosis of renal fibrosarcoma. Among the previously reported cases in the literature, the ETV6-NTRK3 gene fusion could be detected in the congenital (or infantile) fibrosarcoma, and this rearrangement may play a vital role in initiation of congenital fibrosarcoma. However, the ETV6-NTRK3 expression has not been reported in adult-type fibrosarcoma in the literature so far. CASE PRESENTATION: A 66-year-old male patient admitted to our hospital because of chills, fever, and a right indwelling percutaneous nephrostomy catheter. Compared to normal kidney, the right renal had a thinner cortex and no function. After a week of anti-infective treatment, the patient underwent retroperitoneal laparoscopic right nephrectomy. The postoperative pathological result was fibrosarcoma of the right kidney. CONCLUSIONS: Aberrant expression of EVT6-NTRK3 may contribute to the development of renal fibrosarcoma.