Acquired NF2 mutation confers resistance to TRK inhibition in an ex vivo LMNA::NTRK1-rearranged soft-tissue sarcoma cell model.

Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A Misleading Case of NTRK-Rearranged Papillary Thyroid Carcinoma.

Herein, we present a misleading case of advanced papillary thyroid carcinoma with lung, node, and pleural metastases, initially diagnosed as metastatic lung adenocarcinoma with papillary features, based on the histological and immunohistochemical analysis of a pleural biopsy. Between August 2019 and August 2020, the patient received 2 ineffective lines of systemic therapy, including a first line of chemotherapy with cisplatin and pemetrexed, and a second line of immunotherapy with atezolizumab. Comprehensive genomic profiling by next-generation sequencing on the archival pleural biopsy revealed an NTRK1-TMP3 fusion and comutation of the TERT promoter, commonly found in papillary thyroid carcinoma. After palliative partial thyroidectomy that confirmed the diagnosis of papillary thyroid carcinoma, in February 2021, the patient was enrolled in the STARTRK-2 GO40782 basket trial and received entrectinib, an oral pan-TRK inhibitor specifically targeting NTRK-rearranged tumors. After initially experiencing drug-related grade 2 anorexia, dysgeusia, and neurotoxicity and grade 3 asthenia, the dose was reduced, and an excellent and durable objective response was observed.

DNA hypermethylation of tumor suppressor genes TWIST1, GATA4, MUS81 and NTRK1 in endometrial hyperplasia.

OBJECTIVE: To investigate DNA methylation of specific tumor suppressor genes in endometrial hyperplasia compared to normal endometrial tissue. File and methodology: To search for epigenetic events, methylation-specific multiplex ligation-dependent probe amplification was employed to compare the methylation status of 40 tissue samples with atypical endometrial hyperplasia, 40 tissue samples with endometrial hyperplasia without atypia, and 40 control tissue samples with a normal endometrium. RESULTS AND CONCLUSION: Differences in DNA methylation among the groups were found in TWIST1, GATA4, MUS81, and NTRK1 genes (TWIST1: atypical hyperplasia 67.5%, benign hyperplasia 2.5%, normal endometrium 22.5%; P < 0.00001; GATA4: atypical hyperplasia 95%, benign hyperplasia 65%, normal endometrium 22.5%; P < 0.00001; MUS81: atypical hyperplasia 57.5%, benign hyperplasia 22.5%, normal endometrium 5%; P < 0.00001; NTRK1: atypical hyperplasia 65%, benign hyperplasia 27.5%, normal endometrium 10%; P < 0.00001). Higher methylation rates were observed for the tumor suppressor genes of TWIST1, GATA4, MUS81, and NTRK1 in samples with atypical endometrial hyperplasia compared to samples with normal endometrial tissue, and higher methylation rates were found in samples with atypical endometrial hyperplasia compared to samples of benign endometrial hyperplasia. DNA methylation of TWIST1, GATA4, MUS81, and NTRK1 is involved in the pathogenesis of atypical endometrial hyperplasia.

Clinical, Morphological and Molecular Features of Spitz tumors.

Spitz tumors represent a heterogeneous group of challenging melanocytic neoplasms, displaying a range of biological behaviors, spanning from benign lesions, Spitz nevi (SN) to Spitz melanomas (SM), with intermediate lesions in between known as atypical Spitz tumors (AST). They are histologically characterized by large epithelioid and/or spindled melanocytes arranged in fascicles or nests, often associated with characteristic epidermal hyperplasia and fibrovascular stromal changes. In the last decade, the detection of mutually exclusive structural rearrangements involving receptor tyrosine kinases ROS1, ALK, NTRK1, NTRK2, NTRK3, RET, MET, serine threonine kinases BRAF and MAP3K8, or HRAS mutation, led to a clinical, morphological and molecular based classification of Spitz tumors. The recognition of some reproducible histological features can help dermatopathologist in assessing these lesions and can provide clues to predict the underlying molecular driver. In this review, we will focus on clinical and morphological findings in molecular Spitz tumor subgroups.

Immunosuppressive CD10+ALPL+ neutrophils promote resistance to anti-PD-1 therapy in HCC by mediating irreversible exhaustion of T cells.

BACKGROUND & AIMS: Remodeling the tumor microenvironment is a critical strategy for treating advanced hepatocellular carcinoma (HCC). Yet, how distinct cell populations in the microenvironment mediate tumor resistance to immunotherapies, such as anti-PD-1, remains poorly understood. METHODS: We analyzed the transcriptomic profile, at a single-cell resolution, of tumor tissues from patients with HCC scheduled to receive anti-PD-1-based immunotherapy. Our comparative analysis and experimental validation using flow cytometry and histopathological analysis uncovered a discrete subpopulation of cells associated with resistance to anti-PD-1 treatment in patients and a rat model. A TurboID-based proximity labeling approach was deployed to gain mechanistic insights into the reprogramming of the HCC microenvironment. RESULTS: We identified CD10+ALPL+ neutrophils as being associated with resistance to anti-PD-1 treatment. These neutrophils exhibited a strong immunosuppressive activity by inducing an apparent "irreversible" exhaustion of T cells in terms of cell number, frequency, and gene profile. Mechanistically, CD10+ALPL+ neutrophils were induced by tumor cells, i.e., tumor-secreted NAMPT reprogrammed CD10+ALPL+ neutrophils through NTRK1, maintaining them in an immature state and inhibiting their maturation and activation. CONCLUSIONS: Collectively, our results reveal a fundamental mechanism by which CD10+ALPL+ neutrophils contribute to tumor immune escape from durable anti-PD-1 treatment. These data also provide further insights into novel immunotherapy targets and possible synergistic treatment regimens. IMPACT AND IMPLICATIONS: Herein, we discovered that tumor cells reprogrammed CD10+ALPL+ neutrophils to induce the "irreversible" exhaustion of T cells and hence allow tumors to escape from the intended effects of anti-PD-1 treatment. Our data provided a new theoretical basis for the elucidation of special cell populations and revealed a molecular mechanism underpinning resistance to immunotherapy. Targeting these cells alongside existing immunotherapy could be looked at as a potentially more effective therapeutic approach.

Cost-Efficient Detection of NTRK1/2/3 Gene Fusions: Single-Center Analysis of 8075 Tumor Samples.

The majority of NTRK1, NTRK2, and NTRK3 rearrangements result in increased expression of the kinase portion of the involved gene due to its fusion to an actively transcribed gene partner. Consequently, the analysis of 5'/3'-end expression imbalances is potentially capable of detecting the entire spectrum of NTRK gene fusions. Archival tumor specimens obtained from 8075 patients were subjected to manual dissection of tumor cells, DNA/RNA isolation, and cDNA synthesis. The 5'/3'-end expression imbalances in NTRK genes were analyzed by real-time PCR. Further identification of gene rearrangements was performed by variant-specific PCR for 44 common NTRK fusions, and, whenever necessary, by RNA-based next-generation sequencing (NGS). cDNA of sufficient quality was obtained in 7424/8075 (91.9%) tumors. NTRK rearrangements were detected in 7/6436 (0.1%) lung carcinomas, 11/137 (8.0%) pediatric tumors, and 13/851 (1.5%) adult non-lung malignancies. The highest incidence of NTRK translocations was observed in pediatric sarcomas (7/39, 17.9%). Increased frequency of NTRK fusions was seen in microsatellite-unstable colorectal tumors (6/48, 12.5%), salivary gland carcinomas (5/93, 5.4%), and sarcomas (7/143, 4.9%). None of the 1293 lung carcinomas with driver alterations in EGFR/ALK/ROS1/RET/MET oncogenes had NTRK 5'/3'-end expression imbalances. Variant-specific PCR was performed for 744 tumors with a normal 5'/3'-end expression ratio: there were no rearrangements in 172 EGFR/ALK/ROS1/RET/MET-negative lung cancers and 125 pediatric tumors, while NTRK3 fusions were detected in 2/447 (0.5%) non-lung adult malignancies. In conclusion, this study describes a diagnostic pipeline that can be used as a cost-efficient alternative to conventional methods of NTRK1-3 analysis.

ALK, ROS1, RET and NTRK1-3 Gene Fusions in Colorectal and Non-Colorectal Microsatellite-Unstable Cancers.

This study aimed to conduct a comprehensive analysis of actionable gene rearrangements in tumors with microsatellite instability (MSI). The detection of translocations involved tests for 5'/3'-end expression imbalance, variant-specific PCR and RNA-based next generation sequencing (NGS). Gene fusions were detected in 58/471 (12.3%) colorectal carcinomas (CRCs), 4/69 (5.8%) gastric cancers (GCs) and 3/65 (4.6%) endometrial cancers (ECs) (ALK: 8; RET: 12; NTRK1: 24; NTRK2: 2; NTRK3: 19), while none of these alterations were observed in five cervical carcinomas (CCs), four pancreatic cancers (PanCs), three cholangiocarcinomas (ChCs) and two ovarian cancers (OCs). The highest frequency of gene rearrangements was seen in KRAS/NRAS/BRAF wild-type colorectal carcinomas (53/204 (26%)). Surprisingly, as many as 5/267 (1.9%) KRAS/NRAS/BRAF-mutated CRCs also carried tyrosine kinase fusions. Droplet digital PCR (ddPCR) analysis of the fraction of KRAS/NRAS/BRAF mutated gene copies in kinase-rearranged tumors indicated that there was simultaneous co-occurrence of two activating events in cancer cells, but not genetic mosaicism. CRC patients aged above 50 years had a strikingly higher frequency of translocations as compared to younger subjects (56/365 (15.3%) vs. 2/106 (1.9%), p = 0.002), and this difference was particularly pronounced for tumors with normal KRAS/NRAS/BRAF status (52/150 (34.7%) vs. 1/54 (1.9%), p = 0.001). There were no instances of MSI in 56 non-colorectal tumors carrying ALK, ROS1, RET or NTRK1 rearrangements. An analysis of tyrosine kinase gene translocations is particularly feasible in KRAS/NRAS/BRAF wild-type microsatellite-unstable CRCs, although other categories of tumors with MSI also demonstrate moderate occurrence of these events.

Analysis of CD74 Occurrence in Oncogenic Fusion Proteins.

CD74 is a type II cell surface receptor found to be highly expressed in several hematological and solid cancers, due to its ability to activate pathways associated with tumor cell survival and proliferation. Over the past 16 years, CD74 has emerged as a commonly detected fusion partner in multiple oncogenic fusion proteins. Studies have found CD74 fusion proteins in a range of cancers, including lung adenocarcinoma, inflammatory breast cancer, and pediatric acute lymphoblastic leukemia. To date, there are five known CD74 fusion proteins, CD74-ROS1, CD74-NTRK1, CD74-NRG1, CD74-NRG2α, and CD74-PDGFRB, with a total of 16 different variants, each with unique genetic signatures. Importantly, the occurrence of CD74 in the formation of fusion proteins has not been well explored despite the fact that ROS1 and NRG1 families utilize CD74 as the primary partner for the formation of oncogenic fusions. Fusion proteins known to be oncogenic drivers, including those of CD74, are typically detected and targeted after standard chemotherapeutic plans fail and the disease relapses. The analysis reported herein provides insights into the early intervention of CD74 fusions and highlights the need for improved routine assessment methods so that targeted therapies can be applied while they are most effective.

Infant-Type Hemispheric Glioma in a Chinese Girl: A Newly Defined Entity.

BACKGROUND: Infant-type hemispheric glioma is a newly defined entity in the updated 2021 WHO classification of tumors of the central nervous system. This lesion occurs in the cerebral hemispheres of newborns and infants and harbors molecular alterations in the NTRK family, ALK, ROS, or MET. Case report: A four-month-old female infant presented with a large space occupying lesion of the left cerebral hemisphere, whose histological manifestation was high-grade hemispheric infantile glioma. Tumor expressed panTRK, indicative of rearranged NTRK1, which was validated by next generation sequencing (NGS) as TPM3-NTRK1 fusion. There was homozygous deletion of CDKN2A/B, and there were ROS1, TLX3, FAT1, ABL1, MSH2, and PALB2 mutations. Conclusion: The additional genetic alterations in this case may expand the genotypic spectrum of this distinct cohort.

Inhibition of EGFR and MEK surmounts entrectinib resistance in a brain metastasis model of NTRK1-rearranged tumor cells.

Tropomyosin receptor kinase (TRK) inhibitors have demonstrated histology-agnostic efficacy in patients with neurotrophic receptor tyrosine kinase (NTRK) gene fusion. Although responses to TRK inhibitors can be dramatic and durable, duration of response may eventually be limited by acquired resistance via several mechanisms, including resistance mutations such as NTRK1-G595R. Repotrectinib is a second-generation TRK inhibitor, which is active against NTRK1-G595R. However, its efficacy against entrectinib-resistant tumors has not been fully elucidated. In the present study, we established entrectinib-resistant tumor cells (M3B) in a brain metastasis model inoculated with NTRK1-rearranged KM12SM cells and examined the sensitivity of M3B cells to repotrectinib. While M3B cells harbored the NTRK1-G595R mutation, they were unexpectedly resistant to repotrectinib. The resistance was due to extracellular signal-regulated kinase (ERK) reactivation partially mediated by epidermal growth factor receptor (EGFR) activation. We further demonstrate that the triplet combination of repotrectinib, EGFR inhibitor, and MEK inhibitor could sensitize M3B cells in vitro as well as in a brain metastasis model. These results indicate that resistant mutations, such as NTRK1-G595R, and alternative pathway activation, such as ERK activation, could simultaneously occur in entrectinib-resistant tumors, thereby causing resistance to second-generation inhibitor repotrectinib. These findings highlight the importance of intensive examinations to identify resistance mechanisms and application of the appropriate combination treatment to circumvent the resistance.

Ntrk1 promotes mesangial cell proliferation and inflammation in rat glomerulonephritis model by activating the STAT3 and p38/ERK MAPK signaling pathways.

BACKGROUND: Mesangial proliferative glomerulonephritis (MsPGN) accounts for a main cause of chronic kidney disease (CKD), chronic renal failure and uremia. This paper aimed to examine the effect of Ntrk1 on MsPGN development, so as to identify a novel therapeutic target for MsPGN. METHODS: The MsPGN rat model was constructed by single injection of Thy1.1 monoclonal antibody via the tail vein. Additionally, the Ntrk1 knockdown rat model was established by injection of Ntrk1-RNAi lentivirus via the tail vein. Periodic acid-schiff staining and immunohistochemistry (IHC) were performed on kidney tissues. Moreover, the rat urinary protein was detected. Mesangial cells were transfected and treated with p38 inhibitor (SB202190) and ERK inhibitor (PD98059). Meanwhile, the viability and proliferation of mesangial cells were analyzed by cell counting kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine assays. Gene expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western-blot (WB) assays. RESULTS: The proliferation of mesangial cells was enhanced in glomerulus and Ki67 expression was up-regulated in renal tubule of MsPGN rats. The urine protein level increased in MsPGN rats. Pro-inflammatory factors and Ntrk1 expression were up-regulated in glomerulus of MsPGN rats. Ntrk1 up-regulation promoted the viability, proliferation, expression of pro-inflammatory factors and activation of the STAT3, p38 and ERK signaling pathways in mesangial cells. Ntrk1 knockdown reduced mesangial cell proliferation, urine protein, pro-inflammatory factors, activation of STAT3, p38 and ERK signaling pathways in glomerulus, and decreased Ki67 expression in renal tubule of MsPGN rats. Treatment with SB202190 and PD98059 reversed the effect of Ntrk1 on promoting the viability, proliferation and inflammatory response of mesangial cells. CONCLUSION: Ntrk1 promoted mesangial cell proliferation and inflammation in MsPGN rats by activating the STAT3 and p38/ERK MAPK signaling pathways.

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.

The Morpho-Molecular Landscape of Spitz Neoplasms.

Spitz neoplasms are a heterogeneous group of melanocytic proliferations with a great variability in the histological characteristics and in the biological behavior. Thanks to recent discoveries, the morpho-molecular landscape of Spitz lineage is becoming clearer, with the identification of subtypes with recurrent features thus providing the basis for a more solid and precise tumor classification. Indeed, specific mutually exclusive driver molecular events, namely HRAS or MAP2K1 mutations, copy number gains of 11p, and fusions involving ALK, ROS, NTRK1, NTRK2, NTRK3, MET, RET, MAP3K8, and BRAF genes, correlate with distinctive histological features. The accumulation of further molecular aberrations, instead, promotes the increasing malignant transformation of Spitz neoplasms. Thus, the detection of a driver genetic alteration can be achieved using the appropriate diagnostic tests chosen according to the histological characteristics of the lesion. This allows the recognition of subtypes with aggressive behavior requiring further molecular investigations. This review provides an update on the morpho-molecular correlations in Spitz neoplasms.

Paediatric-type diffuse high-grade gliomas in the 5th CNS WHO Classification.

As a relevant element of novelty, the fifth CNS WHO Classification highlights the distinctive pathobiology underlying gliomas arising primarily in children by recognizing for the first time the families of paediatric-type diffuse gliomas, both high-grade and low-grade. This review will focus on the family of paediatric-type diffuse high-grade gliomas, which includes four tumour types: 1) Diffuse midline glioma H3 K27-altered; 2) Diffuse hemispheric glioma H3 G34-mutant; 3) Diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype; and 4) Infant-type hemispheric glioma. The essential and desirable diagnostic criteria as well as the entities entering in the differential will be discussed for each tumour type. A special focus will be given on the issues encountered in the daily practice, especially regarding the diagnosis of the diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype. The advantages and the limits of the multiple molecular tests which may be utilised to define the entities of this tumour family will be evaluated in each diagnostic context.

NTRK1 gene-related congenital insensitivity to pain with anhidrosis: a nationwide multicenter retrospective study.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disease resulting from mutations in the NTRK1 gene encoding the neurotrophic tyrosine kinase-1 receptor. In this multicenter observational retrospective study, we investigated CIPA patients identified from French laboratories sequencing the NTRK1 gene, and seven patients were identified. Patients originated from France (2), Suriname (2), Mali (1), Kazakhstan (1), and Algeria (1). Mean age of patients was 9.8 years (4-20), four patients were female (57%), infant developmental milestones were delayed in four cases (57%), and four patients had a family history of consanguinity (57%). Mean age at diagnosis was 4.8 months (3-6), and all patients presented with pain insensitivity, anhidrosis, intellectual disability, self-mutilation, febrile episodes, impaired temperature perception, and autonomous nervous system impairment. Patients also showed an assortment of associated findings, including hyperactivity (86%), emotional lability (86%), joint deformities (71%), bone fractures (57%), abnormal sense of touch, vibration and position (50%), skin, hair and nails abnormalities (28%), and hypothermia episodes (28%). Two patients died at age 9 and 12 years from infection. In three cases, nerve conduction studies showed absent lower limbs sensory nerve action potentials. In one case, sensory nerve biopsy showed complete absence of unmyelinated fibers. Nine NTRK1 pathogenic variants were found, including three newly described mutations. This nationwide study confirms that NTRK1 gene-related CIPA is an extremely rare disorder and expands the genotypic spectrum of NTRK1 mutations.

p53 immunohistochemical analysis of fusion-positive uterine sarcomas.

AIMS: Uterine sarcomas can be grouped into tumours with pathognomonic genetic fusions such as low-grade endometrial stromal sarcoma (LGESS), high-grade endometrial stromal sarcoma (HGESS), and inflammatory myofibroblastic tumour (IMT), and tumours lacking genetic fusions such as leiomyosarcoma (LMS) and undifferentiated uterine sarcoma (UUS). Members of the latter group frequently harbour TP53 mutations. The aim of this study was to evaluate TP53 mutations by the use of immunohistochemistry in fusion-positive uterine sarcomas. METHODS AND RESULTS: We performed p53 immunohistochemical staining on 124 uterine sarcomas harbouring genetic fusions and 38 fusion-negative LMSs and UUSs. These included 41 HGESSs with YWHAE, BCOR and BCORL1 fusions/rearrangements, 13 IMTs with ALK fusion, 12 sarcomas with NTRK1/3 fusion, three sarcomas with PDGFB fusion, and 55 LGESSs with JAZF1, SUZ12 and PHF1 fusions/rearrangements. All HGESSs, LGESSs, IMTs and sarcomas with PDGFB fusion showed wild-type p53 expression. Among NTRK1/3-positive sarcomas, a TPR-NTRK1-positive sarcoma with nuclear pleomorphism showed mutation-type p53 expression. The remaining 11 NTRK1/3-positive sarcomas showed wild-type p53 expression, except for the subclonal p53 mutation-type staining in a minor pleomorphic focus of an NTRK3-positive sarcoma. Twenty-one of 27 (78%) LMSs and six of nine (67%) UUSs showed mutation-type p53 expression. CONCLUSION: p53 immunohistochemistry may be considered in the initial work-up of a uterine sarcoma, as mutation-type staining would make a fusion-positive sarcoma very unlikely. Mutation-type p53 expression, however, can be seen in a small subset of NTRK1/3-positive sarcomas showing pleomorphic round/ovoid cell histology, which may represent a mechanism of progression in these tumours.

LMNA-NTRK1 rearranged mesenchymal tumor (lipofibromatosis-like neural tumor) mimicking pigmented dermatofibrosarcoma protuberans.

We present the case of a 31-year-old female with a 1.5 cm pigmented nodule on the scalp. Histopathological examination revealed a proliferation of relatively bland spindle cells and pigmented dendritic cells, with interspersed lymphoid follicles diffusely infiltrating the adipose tissue. The microscopic differential diagnosis included pigmented dermatofibrosarcoma protuberans (DFSP). The spindle cells showed S-100 and CD34 labeling but were negative for SOX-10. Immunohistochemical stain for pan-TRK was positive, while fluorescence in-situ hybridization for PDGFB gene rearrangement was negative. Targeted RNA sequencing revealed an LMNA-NTRK1 (exon2/exon10) fusion. This molecular result coupled with the histopathological findings and immunohistochemical profile supported the diagnosis of the recently characterized NTRK-rearranged spindle cell neoplasm termed "lipofibromatosis-like neural tumor (LPF-NT)." These neoplasms typically occur in superficial soft tissue and are characterized by a distinctive immunoprofile (CD34+, S-100+, SOX10-). Histopathological differential diagnosis for LPF-NT tumors includes lipofibromatosis, DFSP, low-grade malignant peripheral nerve sheath tumor, and spindle cell/desmoplastic melanoma. The pigmented dendritic cells reminiscent of pigmented DFSP and lymphoid follicles noted in our case have not been previously reported in LPF-NT, thus expanding the morphological spectrum of this entity. LMNA-NTRK1 fusion serves both as a diagnostic and therapeutic biomarker, as cases with advanced disease may be amenable to targeted therapy using tyrosine kinase inhibitors.

Unclassified mesenchymal sarcoma with NTRK1-KHDRBS1 gene fusion: a case report of long-term tumor-free survival with crizotinib treatment.

BACKGROUND: Mesenchymal sarcomas are tumors that originate from mesenchymal tissue. Most mesenchymal sarcomas can be accurately classified, but some are unclassifiable in clinical practice. Molecular detection methods enable patients to benefit from molecular-targeted therapies for many cancers, including lung, breast, and bowel cancers. Further, even unclassified tumors can have therapeutic targets. NTRK gene fusions are sporadic genetic alterations that occur across tumor entities. If NTRK gene fusions are detected, TRK inhibitors can be used regardless of the tumor entity. CASE PRESENTATION: This report describes a case with an unclassifiable mesenchymal sarcoma carrying a neurotrophic tyrosine receptor kinase NTRK1-KHDRBS1 gene fusion that was diagnosed and treated at multiple hospitals. Diagnostic work-up included pathological and immunohistochemical analysis, which excluded angiosarcoma, dendritic cell sarcoma, and pseudomyogenic hemangioendothelioma. The patient achieved a long-term survival without tumor relapse after treatment with crizotinib. CONCLUSIONS: This case will be of significant interest to pathologists because, despite the tumor being unclassified, a molecular target was identified. Although the FDA does not currently approve crizotinib for treatment of patients harboring NTRK gene fusions, this case provides new insights for diagnosis and treatment of mesenchymal sarcomas with NTRK1 gene translocations. Similar to ALKomas, which can be successfully treated using NTRK molecular-targeted therapy, tumors with NTRK gene translocations can be classified as NTRKomas, even when they occur at different organ sites, and with varying histological morphologies, and immunophenotypes.

NTRK Gene Fusion Detection in Atypical Spitz Tumors.

Atypical Spitz tumors (AST) deviate from stereotypical Spitz nevi for one or more atypical features and are now regarded as an intermediate category of melanocytic tumors with uncertain malignant potential. Activating NTRK1/NTRK3 fusions elicit oncogenic events in Spitz lesions and are targetable with kinase inhibitors. However, their prevalence among ASTs and the optimal approach for their detection is yet to be determined. A series of 180 ASTs were screened with pan-TRK immunohistochemistry and the presence of NTRK fusions was confirmed using FISH, two different RNA-based NGS panels for solid tumors, and a specific real time RT-PCR panel. Overall, 26 ASTs showed pan-TRK immunostaining. NTRK1 fusions were detected in 15 of these cases showing cytoplasmic immunoreaction, whereas NTRK3 was detected in one case showing nuclear immunoreaction. Molecular tests resulted all positive in only two ASTs (included the NTRK3 translocated), RNA-based NGS and real time RT-PCR were both positive in three cases, and FISH and real time RT-PCR in another two cases. In seven ASTs NTRK1 fusions were detected only by FISH and in two cases only by real time RT-PCR. The frequency of NTRK fusions in ASTs is 9%, with a clear prevalence of NTRK1 compared to NTRK3 alterations. Pan-TRK immunohistochemistry is an excellent screening test. Confirmation of NTRK fusions may require the use of different molecular techniques.

TrkA overexpression in non-tumorigenic human breast cell lines confers oncogenic and metastatic properties.

BACKGROUND/PURPOSE: TrkA overexpression occurs in over 20% of breast cancers, including triple-negative breast cancers (TNBC), and has recently been recognized as a potential driver of carcinogenesis. Recent clinical trials of pan-Trk inhibitors have demonstrated targeted activity against tumors harboring NTRK fusions, a relatively rare alteration across human cancers. Despite this success, current clinical trials have not investigated TrkA overexpression as an additional therapeutic target for pan-Trk inhibitors. Here, we evaluate the cancerous phenotypes of TrkA overexpression relative to NTRK1 fusions in human cells and assess response to pharmacologic Trk inhibition. EXPERIMENTAL DESIGN/METHODS: To evaluate the clinical utility of TrkA overexpression, a panel of TrkA overexpressing cells were developed via stable transfection of an NTRK1 vector into the non-tumorigenic breast cell lines, MCF10A and hTERT-IMEC. A panel of positive controls was generated via stable transfection with a CD74-NTRK1 fusion vector into MCF10A cells. Cells were assessed via various in vitro and in vivo analyses to determine the transformative potential and targetability of TrkA overexpression. RESULTS: TrkA overexpressing cells demonstrated transformative phenotypes similar to Trk fusions, indicating increased oncogenic potential. TrkA overexpressing cells demonstrated growth factor-independent proliferation, increased PI3Kinase and MAPKinase pathway activation, anchorage-independent growth, and increased migratory capacity. These phenotypes were abrogated by the addition of the pan-Trk inhibitor, larotrectinib. In vivo analysis demonstrated increased tumorgenicity and metastatic potential of TrkA overexpressing breast cancer cells. CONCLUSIONS: Herein, we demonstrate TrkA overexpressing cells show increased tumorgenicity and are sensitive to pan-Trk inhibitors. These data suggest that TrkA overexpression may be an additional target for pan-Trk inhibitors and provide a targeted therapy for breast cancer patients.