Neurotrophic receptor kinase (NTRK) fusions are actionable oncogenic drivers of multiple pediatric and adult solid tumors, and tropomyosin receptor kinase (TRK) has been considered as an attractive therapeutic target for \"pan-cancer\" harboring these fusions. Currently, two generations TRK inhibitors have been developed. The representative second-generation inhibitors selitrectinib and repotrectinib were designed to overcome clinic acquired resistance of the first-generation inhibitors larotrectinib or entrectinib resulted from solvent-front and gatekeeper on-target mutations. However, xDFG (TRKAG667C/A/S, homologous TRKCG696C/A/S) and some double mutations still confer resistance to selitrectinib and repotrectinib, and overcoming these resistances represents a major unmet clinical need. In this review, we summarize the acquired resistance mechanism of the first- and second-generation TRK inhibitors, and firstly put forward the emerging selective type II TRK inhibitors to overcome xDFG mutations mediated resistance. Additionally, we concluded our perspectives on new challenges and future directions in this field.

BACKGROUND: Tumor genotyping is becoming crucial to optimize the clinical management of patients with advanced differentiated thyroid cancer (DTC); however, its implementation in clinical practice remains undefined. We herein report our single-center experience on molecular advanced DTC testing by next-generation sequencing approach, to better define how and when tumor genotyping can assist clinical decision making.
METHODS: We retrospectively collected data on all adult patients with advanced DTC who received molecular profiling at the IRCSS Sant\'Orsola-Malpighi Hospital from 2008 to 2022. The genetic alterations were correlated with radioactive iodide refractory (RAI-R), RAI uptake/disease status, and time to RAI resistance (TTRR) development.
RESULTS: A significant correlation was found between RAI-R development and genetic alterations (P = 0.0001). About 48.7% of RAI-R cases were positive for TERT/TP53 mutations (as both a single event and comutations with other driver gene alterations, such as BRAF mutations, RAS mutations, or gene fusions), while the great majority of RAI-sensitive cases carried gene fusions (41.9%) or were wild type (WT; 41.9%). RAI uptake/disease status and time to TTRR were significantly associated with genetic alterations (P = 0.0001). In particular, DTC with TERT/TP53 mutations as a single event or as comutations displayed a shorter median TTRR of 35.4 months (range 15.0-55.8 months), in comparison to the other molecular subgroups. TERT/TP53 mutations as a single event or as comutations remained independently associated with RAI-R after Cox multivariate analysis (hazard ratio 4.14, 95% CI 1.51-11.32; P = 0.006).
CONCLUSIONS: Routine testing for genetic alterations should be included as part of the clinical workup, for identifying both the subset of more aggressive tumors and the subset of tumors harboring actionable gene fusions, thus ensuring the appropriate management for all patients with advanced DTC.

Triple-negative breast cancer (TNBC) is usually an aggressive disease with a poor prognosis and limited treatment options. The neurotrophic tyrosine receptor kinase (NTRK) gene fusions are cancer type-agnostic emerging biomarkers approved by the Food and Drug Administration (FDA), USA, for the selection of patients for targeted therapy. The main aim of our study was to investigate the frequency of NTRK aberrations, i.e. fusions, gene copy number gain, and amplification, in a series of TNBC using different methods. A total of 83 TNBCs were analyzed using pan-TRK immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (RT-PCR), and RNA-based next-generation sequencing (NGS). Of 83 cases, 16 showed pan-TRK positivity although no cases had NTRK-fusions. Indeed, FISH showed four cases carrying an atypical NTRK1 pattern consisting of one fusion signal and one/more single green signals, but all cases were negative for fusion by NGS and RT-PCR testing. In addition, FISH analysis showed six cases with NTRK1 amplification, one case with NTRK2 copy number gain, and five cases with NTRK3 copy number gain, all negative for pan-TRK IHC. Our data demonstrate that IHC has a high false-positive rate for the detection of fusions and molecular testing is mandatory; there is no need to perform additional molecular tests in cases negativity for NTRK by IHC. In conclusion, the NTRK genes are not involved in fusions in TNBC, but both copy number gain and amplification are frequent events, suggesting a possible predictive role for other NTRK aberrations.

BACKGROUND: NTRK (neurotrophic tyrosine receptor kinase)-rearranged spindle cell neoplasms are a new group of tumors included in the new 5th edition of the World Health Organization (WHO) classification of soft Tissue and Bone Sarcomas. These tumors are characterized by NTRK gene fusions and show a wide spectrum of histologies and clinical behavior. Several targeted therapies have recently been approved for tumors harboring NTRK fusions, including STS.
METHODS: A 26-year-old male with advanced, pretreated NTRK rearranged spindle cell neoplasm and liver, lung and bone metastases was treated with larotrectinib on a continuous 28-day schedule, at a dose of 100 mg twice daily. An 18FDG-PET/CT scan performed after 7 days of treatment showed tumor shrinkage in both visceral and bone lesions. There was no drug-related toxicity. Subsequent evaluations confirmed continued tumor regression in disease sites. The patient is well and continues treatment.
CONCLUSIONS: The clinical and radiological response of our patient with an uncommon TPM4 (exon 7)-NTRK1 (exon 12) gene fusion tumor treated with a first-generation TRK inhibitor could contribute to a better understanding of the biology of this new STS entity and help to improve patient management.

The aim of the study was to identify patients with NTRK fusion-positive or RET fusion/mutation-positive thyroid cancers, who could benefit from neurotrophic tyrosine kinase receptor (NTRK) or receptor tyrosine kinase (RET) inhibitors.
Patients were identified in the Calgary prospective thyroid cancer database (N= 482). Patients were \'pre-screened\' with clinically available MassARRAY® BRAF test, Colon Panel, Melanoma Panel, or ThyroSPEC™. Mutation-negative tumors were \'screened\' for NTRK fusions and RET fusions/mutations with the Oncomine™ Comprehensive Assay v3 (OCAv3).
A total of 86 patients were included in 1 of 2 separate analyses. Analysis A included 42 patients with radioactive iodine (RAI)-resistant distant metastases. After pre-screening, 20 BRAF and RAS mutation-negative patients underwent OCAv3 screening, resulting in the detection of 4 patients with NTRKfusions and 4 patients with RET fusions (8/20, 40% of analyzed patients). Analysis B included 44 patients, 42 with American Thyroid Association (ATA) high and intermediate risk of recurrence and 2 with medullary thyroid carcinoma. During pre-screening, 1 patient with an NTRK fusion, 1 patient with a RET fusion, and 30 patients with BRAF mutations were identified. The remaining 9 patients received OCAv3 screening, resulting in detection of 1 patient with an NTRKfusion and 1 with a RET fusion (4/11, 36% of analyzed patients).
Our findings indicate a higher rate of NTRK fusions and RETfusions in patients with thyroid cancer with RAI-resistant distant metastases and ATA high or intermediate risk of recurrence. This highlights the importance of early screening to enable intervention with a NTRK or RET inhibitor.

Fusions involving TRK protein tyrosine kinases are oncogenic drivers in a variety of tumors in children and adults, with a prevalence of ∼0.2% in non-small cell lung cancer. Diagnosis can be challenging due to structural features such as NTRK intron length, but next-generation sequencing (NGS), including RNA-based NGS, increases detection. The first-generation TRK inhibitors, larotrectinib and entrectinib, have demonstrated clinically meaningful antitumor activity in TRK fusion-positive cancers in a tumor-agnostic fashion and should be considered first-line therapeutic options for TRK fusion-positive lung cancers. Furthermore, the first-generation TRK inhibitors are well tolerated. Care should be taken, however, to monitor on-target adverse events, such as dizziness, weight gain, paresthesias, and withdrawal pain. On-target and off-target mechanisms mediating TRK inhibitor resistance may occur. Next-generation TRK inhibitors, such as selitrectinib, repotrectinib, and taletrectinib, are available on ongoing clinical trials and address on-target resistance. This review will focus on NTRK fusions and TRK-directed targeted therapy specifically in the context of lung cancer.

Lung cancer is the most common cause of cancer-related death worldwide, and the prognosis for stage IV remains poor. The presence of genetic alterations in tumor cells, such as EGFR and BRAF gene mutations, as well as ALK and ROS1 gene rearrangements, are indications for targeted therapies. Many such treatments are already registered and used on a wide scale. In comparison to standard chemotherapy, they can prolong not only progression-free survival but also overall survival. Moreover, they are able to provide excellent quality of life and rapid improvement of cancer-related symptoms such as dyspnea, cough and pain. Recent years have witnessed great advances in both molecular diagnostics and new molecular therapies for non-small-cell lung cancer. This review presents new therapeutic targets in NSCLC, as well as drugs of which the activity against NTRK, RET, MET or HER2 gene alterations (including EGFR exon 20 insertions) has either been confirmed or is currently being evaluated. Although these particular genetic alterations in NSCLC are generally rare, each accounting for 1-2% of patients, in total about half of all patients have molecular alterations and may ultimately receive targeted therapies.

Introduction: For patients with advanced/unresectable biliary tract cancers, cisplatin-gemcitabine combination is the standard first-line treatment. Beyond the first line, the therapeutic arsenal is limited with minimal benefit. Biliary tract cancers exhibit one of the highest frequencies of targetable molecular alterations across cancer types, and several targeted therapies are emerging as treatment options.Areas covered:We discuss neurotrophic tyrosine kinase receptor gene (NTRK) fusions in biliary tract cancers and the use of NTRK inhibitors (now approved in a \'cancer-agnostic\' way), mechanisms of resistance, and emerging second-generation NTRK inhibitors.Expert opinion: Despite their rarity in biliary tract cancers, NTRK fusions are promising molecular targets because i) NTRK inhibitors have proven highly effective in NTRK-rearranged cancers and are now approved in a \'cancer-agnostic\' way; ii) emerging second-generation NTRK inhibitors may overcome secondary resistance; iii) NTRK rearrangements will be readily detectable with the generalization of next-generation-sequencing in biliary tract cancers, including the detection of other frequent gene rearrangements, such as those involving the fibroblast growth factor receptor 2 gene (FGFR2). However, more data are necessary regarding the prevalence and characteristics of NTRK fusions in biliary tract cancers and the efficacy of NTRK inhibitors in these patients.

Pediatric papillary thyroid carcinoma (PTC) is clinically and biologically distinct from adult PTC. We sequenced a cohort of clinically annotated pediatric PTC cases enriched for high-risk tumors to identify genetic alterations of relevance for diagnosis and therapy.
Tumor DNA and RNA were extracted from FFPE tissue and subjected to next-generation sequencing (NGS) library preparation using a custom 124-gene hybridization capture panel and the 75-gene Archer Oncology Research Panel, respectively. NGS libraries were sequenced on an Illumina MiSeq.
Thirty-six pediatric PTC cases were analyzed. Metastases were frequently observed to cervical lymph nodes (29/36, 81%), with pulmonary metastases less commonly found (10/36, 28%). Relapsed or refractory disease occurred in 18 patients (18/36, 50%). DNA sequencing revealed targetable mutations in 8 of 31 tumors tested (26%), most commonly BRAF p.V600E (n = 6). RNA sequencing identified targetable fusions in 13 of 25 tumors tested (52%): RET (n = 8), NTRK3 (n = 4), and BRAF. Mutually exclusive targetable alterations were discovered in 15 of the 20 tumors (75%) with both DNA and RNA analyzed. Fusion-positive PTC was associated with multifocal disease, higher tumor staging, and higher American Thyroid Association risk levels. Both BRAF V600E mutations and gene fusions were correlated with the presence of cervical metastases.
Targetable alterations were identified in 75% of pediatric PTC cases with both DNA and RNA evaluated. Inclusion of RNA sequencing for detection of fusion genes is critical for evaluation of these tumors. Patients with fusion-positive tumors were more likely to have features of high-risk disease.

A Belgian ring trial for pan-TRK immunohistochemistry (IHC) staining was organised to harmonise pan-TRK IHC staining protocols and interpretation. As a reference method, the VENTANA pan-TRK Assay (clone EPR17341) on the Benchmark Ultra platform was selected. Six samples were selected: 2 negative, 2 fusion positive and 2 samples with wild-type endogenous TRK expression. Each participating laboratory stained the slides using their routine pan-TRK IHC and reported their results. In addition, they were asked to return one TRK-stained slide from each case. The coordinating lab evaluated these slides, compared them with the reference method and scored them. Two clones were used during the ring trial: A7H6R (Cell Signaling) and EPR17341 (Abcam/Ventana). Seven protocols achieved a sufficient performance mark, and three labs were advised to further optimise the protocol. Interpretation of pan-TRK IHC proved to be challenging in cases with physiological TRK expression. In addition, depending on the NTRK fusion partner, the staining can vary strongly in both intensity and staining pattern. Labs using the Ventana ready-to-use system based on the EPR17341 clone and using the recommended protocol settings scored best. However, given some small optimisation, all labs scored well on the technical staining and the succeeding evaluation.