Molecular and Cytogenetic Features of NTRK Fusions Enriched in BRAF and RET Double-Negative Papillary Thyroid Cancer.

Rare NTRK-driven malignant neoplasms can be effectively inhibited by anti-TRK agents. The discovery of NTRK1/2/3-rich tumors in papillary thyroid cancer (PTC) patients is a precondition for the rapid identification of NTRK fusion tumors. Knowledge of NTRK gene activation is critical to accurately detect NTRK status. A total of 229 BRAF V600E-negative samples from PTC patients were analyzed in this study. Break-apart fluorescence in situ hybridization (FISH) was performed to detect RET fusion. NTRK status was analyzed using FISH, DNA- and RNA-based next-generation sequencing, and quantitative reverse transcription PCR. In 128 BRAF and RET double-negative cases, 56 (43.8%, 56/128) NTRK rearrangement tumors were found, including 1 NTRK2, 16 NTRK1, and 39 NTRK3 fusions. Two novel NTRK fusions, EZR::NTRK1 and EML4::NTRK2, were found in the NTRK rearrangement tumors. Dominant break-apart and extra 3' signal patterns accounted for 89.3% (50/56) and 5.4% (3/56) of all NTRK-positive cases, respectively, as determined by FISH. In this study's cohort, there were 2.3% (3/128) FISH false-negative and 3.1% (4/128) FISH false-positive cases identified. NTRK fusions are highly recurrent in BRAF and RET double-negative PTCs. FISH- or RNA-based next-generation sequencing is a reliable detection approach. NTRK rearrangement can be precisely, rapidly, and economically detected based on the developed optimal algorithm.

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.

NTRK Fusion in a Cohort of BRAF p. V600E Wild-Type Papillary Thyroid Carcinomas.

Owing to the availability of a potent tropomyosin receptor kinase (TRK) inhibitor, it is necessary to develop an effective strategy to identify an enriched population of NTRK fusions in papillary thyroid carcinoma (PTC) in routine diagnostic practice. The reported prevalence of NTRK fusion in a large cohort of PTC is ∼3%. We performed an analysis to refine the characteristic histologic features of PTCs harboring NTRK fusions and further validate the diagnostic utility of pan-TRK immunohistochemistry as a screening tool. In this study, 450 PTCs known to harbor no BRAF p. V600E mutations were screened by pan-TRK immunohistochemistry, and the cases with TRK expression were confirmed by RNA-based next-generation sequencing assay. Eleven NTRK fusion cases were detected (2.4%), and all PTCs were classical subtypes. NTRK1 and NTRK3 were involved in the fusion with 9 different partner genes. Most cases showed similar characteristic histologic findings. Nodular permeative border, multinodular growth with a predominantly follicular pattern, extensive lymphatic invasion, and prominent internodular and intratumoral fibrosis were the characteristic histologic features of NTRK-rearranged PTCs. The ill-defined margins in the ultrasonography findings, which could not be clearly distinguished from the adjacent nontumorous thyroid tissue, were nodular permeative margins in histologic findings. Therefore, preoperative ultrasonographic findings in nodule margins were consistent with the final histologic findings. NTRK1/3 fusion in PTCs showed an overall sensitivity of 100% (95% CI, 71.51%-100%) and specificity of 100% (95% CI, 71.51%-100%) in the 22 cases examined, as confirmed with next-generation sequencing. Our study provides an integrative report of the preoperative ultrasonographic, histologic, immunohistochemical, and molecular features of NTRK-rearranged PTCs. Based on these findings, we propose an algorithmic approach for the stepwise assessment of NTRK fusions in PTCs.

CANTRK: A Canadian Ring Study to Optimize Detection of NTRK Gene Fusions by Next-Generation RNA Sequencing.

The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.

The sensitivity of pan-TRK immunohistochemistry in solid tumours: A meta-analysis.

INTRODUCTION: Since the approval of neurotrophic tropomyosin receptor kinase (NTRK) tyrosine kinase inhibitors for fist-line advanced stage pan-cancer therapy, pathologists and molecular biologists have been facing a complex question: how should the large volume of specimens be screened for NTRK fusions? Immunohistochemistry is fast and cheap, but the sensitivity compared to RNA NGS is unclear. METHODS: We performed RNA-based next-generation sequencing on 1,329 cases and stained 24 NTRK-rearranged cases immunohistochemically with pan-TRK (ERP17341). Additionally, we performed a meta-analysis of the literature. After screening 580 studies, 200 additional NTRK-rearranged cases from 13 studies, analysed with sensitive molecular diagnostics as well as pan-TRK IHC, were included. RESULTS: In the included 224 NTRK-rearranged solid tumours, the sensitivity for pan-TRK IHC was 82% and the false-negative rate was 18%. NTRK3 fusions had more false negatives (27%) compared to NTRK1 (6%) and NTRK2 (14%) (p = 0.0006). Membranous, nuclear and peri-nuclear staining patterns strongly correlated with different fusion products, with membranous staining being more prevalent in NTRK1 and NTRK2, nuclear in NTRK3, and perinuclear in NTRK1. CONCLUSION: Despite a reduction in the number of molecular analysis, using pan-TRK immunohistochemistry as a prescreening method to detect NTRK fusions in solid tumours will miss 18% of all NTRK-fused cases (especially involving NTRK3). Therefore, the most comprehensive and optimal option to detect NTRK fusions is to perform molecular testing on all eligible cases. However, in case of financial or logistical limitations, an immunohistochemistry-first approach is defensible in tumours with a low prevalence of NTRK fusions.

NTRK Gene Fusion Detection in a Pan-Cancer Setting Using the Idylla GeneFusion Assay.

Recently, approval of tyrosine receptor kinase (TRK) inhibitors by Food and Drug Administration and European Medicines Agency in NTRK fusion-positive cancer types has led to a variety of proposed testing algorithms. In this study, performance of the fully automated Idylla GeneFusion Assay was assessed in a set of clinically relevant cancer types, including glioblastoma, non-small-cell lung cancer, microsatellite instability-positive colorectal cancer, and thyroid carcinoma. Analysis with the Idylla GeneFusion Assay revealed significant differences in baseline RNA expression profile between the different cancer types, which corresponded to both literature and pan-TRK immunohistochemical staining. Compared with the RNA-based Oncomine Focus Assay, the Idylla GeneFusion Assay demonstrated an overall percentage agreement, positive percentage agreement, and negative percentage agreement of 92.7%, 81.8%, and 93.8%, respectively; and the pan-TRK immunohistochemistry demonstrated an overall percentage agreement, positive percentage agreement, and negative percentage agreement of 82.1%, 45.5%, and 85.7%, respectively. These findings highlighted the importance of tailoring NTRK testing algorithms per cancer type. In a small subset, data from the RNA-based Archer FusionPlex Assay were also available. NTRK fusion detection efficiency was compared between the four NTRK testing modalities, with a high concordance between the PCR-based methods. Last, RNA degradation was observed when using the Idylla GeneFusion Assay on snap frozen tissue samples as these are nonfixated. This might be countered by increasing the amount of sample input. To conclude, the Idylla GeneFusion Assay has shown a clear potential in identifying NTRK fusions.

Adult NTRK-rearranged spindle cell neoplasms of the viscera: with an emphasis on rare locations and heterologous elements.

NTRK-rearranged mesenchymal neoplasms mostly affect the soft tissues of pediatric patients. Given the responsiveness to selective NTRK inhibitors, it remains critical to identify those ultra-rare cases occurring in the viscera of adults. In five females and two males aged 18-53 years, we characterized visceral mesenchymal tumors harboring TPM3-NTRK1 [uterine cervix (N = 2), pleura, prostate], LMNA-NTRK1 (lung), SQSTM1-NTRK3 (heart), and NTRK3 rearrangement with unknown fusion partner (colon/mesocolon) with RNA sequencing, FISH, RT-PCR, and immunohistochemistry. The tumors exhibited spindled to ovoid/epithelioid or pleomorphic cells, often arranged in fascicles, and were low-to-intermediate-grade and high-grade in three and four cases, respectively. Keloid-like stromal collagen and perivascular hyalinization was noted in five. Adenosarcoma-like appearances were observed in two, manifesting frond-like protrusions in one cervical tumor and phyllodes-like architecture in the prostatic tumor. Abrupt high-grade transformation into pleomorphic liposarcoma was found in another cervical tumor, while the pleural tumor contained intermixed rhabdomyoblasts. Pan-TRK immunostaining was positive in all cases. All cases expressed CD34, while five were S100-positive. CDKN2A homozygous deletion with concomitant p16 loss occurred in 4/7. Whole-exome sequencing identified TP53 mutation (c.672+2T>C, involving a splice site, with concomitant protein loss) in a cervical sarcoma, limited to its heterologous liposarcomatous component. At least moderate pan-TRK immunoreactivity was present in varying proportions of potential pathologic mimics, with BCOR-positive sarcoma (56%, 5/9), undifferentiated uterine sarcoma (50%, 3/6), and spindle cell/sclerosing rhabdomyosarcoma (33%, 2/6) being among the most frequent. This underscored the unsatisfactory specificity of pan-TRK immunohistochemistry and warranted molecular confirmation in the diagnosis of adult NTRK-rearranged visceral mesenchymal neoplasms. The current report highlights the ever-expanding clinicopathologic and genetic spectrum of this entity by describing the unprecedented cardiac and pleural locations and heterologous differentiation, as well as the second NTRK-rearranged "prostatic stromal sarcoma," while substantiating CDKN2A deletion as a frequent occurrence.

Detection of NTRK fusions in glioblastoma: fluorescent in situ hybridisation is more useful than pan-TRK immunohistochemistry as a screening tool prior to RNA sequencing.

Glioblastomas are frequent malignant brain tumours with a very poor prognosis and a need for new and efficient therapeutic strategies. With the approval of anti-TRK targeted therapies to treat patients with advanced NTRK-rearranged cancers, independent of the type of cancer, potential new treatment opportunities are available for the 0.5-5% of patients with NTRK-rearranged glioblastomas. Identification of these rare NTRK-rearranged glioblastomas requires efficient diagnostic tools and strategies which are evaluated in this study. We compared the results of NTRK1, NTRK2 and NTRK3 fluorescent in situ hybridisation (FISH) assays to those of pan-TRK immunohistochemistry (IHC) using two EPR17341 and A7H6R clones in a set of 196 patients with glioblastomas. Cases with at least 15% of positive nuclei using FISH analyses were further analysed using RNA sequencing. Above the 15% threshold, seven positive glioblastomas (3.57%) were identified by FISH assays (4 NTRK1, 3 NTRK2, no NTRK3). NTRK rearrangements were confirmed by RNA sequencing analyses in four cases [1 LMNA-NTRK1, 1 PRKAR2A-NTRK2, 1 SPECC1L-NTRK2 and 1 NACC2-NTRK2 fusions, i.e., 4/196 (2%) of NTRK-rearranged tumours in our series] but no rearrangement was detected in three samples with less than 30% of positive tumour nuclei as determined by NTRK1 FISH. Pan-TRK immunostaining showed major discrepancies when using either the EPR17341 or the A7H6R clones for the following criteria: main intensity, H-Score based scoring and homogeneity/heterogeneity of staining (Kappa values <0.2). This led to defining adequate criteria to identify NTRK-rearranged gliomas exhibiting strong and diffuse immunostaining contrasting to the variable and heterogeneous staining in non-NTRK-rearranged gliomas (p<0.0001). As assessing NTRK rearrangements has become crucial for glioma therapy, FISH seems to be a valuable tool to maximise access to TRK testing in patients with glioblastomas. In contrast to other cancers, pan-TRK IHC appears of limited interest in this field because there is no 'on/off' IHC positivity criterion to distinguish between NTRK-rearranged and non-NTRK-rearranged gliomas. RNA sequencing analyses are necessary in FISH positive cases with less than 30% positive nuclei, to avoid false positivity when scoring is close to the detection threshold.

NTRK fusion-positive colorectal cancer in Japanese population.

ALK, ROS1 and NTRK fusions are involved in the tumorigenesis of various organs, including colorectal cancer. This study aims to clarify the prevalence of these fusions in colorectal cancer in the Japanese population. Immunohistochemical analysis of 1012 specimens of colorectal cancer revealed two NTRK-positive cases (0.2%) whereas no ALK- or ROS1-positive cases were identified. Reverse transcription polymerase chain reaction (RT-PCR) detected an LMNA-NTRK1 fusion in a case of adenosquamous carcinoma and a TPM3-NTRK1 fusion in a case of tubular adenocarcinoma. Both NTRK1 fusion-positive cases lacked activating mutations in KRAS and BRAF and were mismatch repair-deficient with loss of MLH1 and PMS2 expression and MLH1 promoter methylation. Our results show that receptor tyrosine kinase fusions are rare but present in colorectal cancers in Japanese patients, with a prevalence similar to that reported in other countries.

Ultrasonic Imaging Combined with Hysteroscopy in Diagnosis of Endometrial Polyps Based on Multioperator Algorithm and Analysis of Nerve Growth Factor Receptor Transmembrane Protein Expression.

The paper aims to understand the expression of nerve growth factor receptor (TrkA) in the ectopic and entopic endometrium of patients with adenomyosis and dysmenorrhea and, at the same time, explore and analyze the combination of hysteroscopy and ultrasound imaging based on multioperator algorithms. The clinical treatment value of color Doppler ultrasound in the diagnosis and treatment of endometrial polyps. The immunohistochemical avidin-peroxidase staining method (SP method) was used to detect 20 cases of ectopic endometrium and entopic endometrium in patients with dysmenorrhea of adenomyosis (study group) and 22 cases of uterine fibroids in the control group. The expression of nerve growth factor receptor in the endometrium of patients with dysmenorrhea and comparative analysis. At the same time, all patients were examined by transvaginal color Doppler ultrasound and hysteroscopy. The scores of endometrial polyps, diagnostic efficiency of the methods, and receiver operating characteristic curve analysis of various diagnostic methods were observed and recorded in the 2 groups. There was no significant difference in the expression of TrkA in the ectopic endometrium and the entopic endometrium in the study group (P > 0.05), but the expression intensity of TrkA in the 2 groups was significantly higher than that in the control group (P = 0.05). In the diagnosis of endometrial polyps, the area of receiver operating characteristic curve in the parallel combined diagnosis of ultrasound and hysteroscopy is significantly larger than that under the combined diagnosis of series. TrkA is widely expressed in the ectopic and entopic endometrium of patients with adenomyosis and dysmenorrhea and may play an important role in the onset of adenomyosis and dysmenorrhea. Hysteroscopy combined with color Doppler ultrasound can effectively improve the diagnostic accuracy of patients with endometrial polyps.

Incorporating traditional and emerging biomarkers in the clinical management of metastatic colorectal cancer: an update.

INTRODUCTION: Molecular profiling has led to significantly longer survival in metastatic colorectal cancer (mCRC) patients. Clinical guidelines recommend testing for KRAS/NRAS, BRAF and MSI status, and new biomarkers such as HER2 amplification and NTRK fusions have emerged more recently in refractory CRC, supported by overwhelming clinical relevance. These biomarkers can guide treatment management to improve clinical outcomes in these patients. AREAS COVERED: Preclinical and clinical data over the last decade were reviewed for known and novel biomarkers with clinical implications in refractory CRC. Molecular alterations are described for classic and novel biomarkers, and data for completed and ongoing studies with targeted and immunotherapies are presented. EXPERT OPINION: Use of targeted therapies based on biomarker testing in CRC has enabled impressive improvements in clinical outcomes in refractory patients. BRAF, MSI, NRAS and KRAS should be tested upfront in all patients given their indisputable therapeutic implications. Other molecular alterations such as HER2 and NTRK are emerging. Testing for these alterations may further improve outcomes for refractory CRC patients. Nonetheless, many key aspects remain to be defined including the optimal timing and technique for testing, the most adequate panel, and whether all patients should be tested for all alterations.

NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls.

With the FDA approval of larotrectinib, NTRK fusion assessment has recently become a standard part of management for patients with locally advanced or metastatic cancers. Unlike somatic mutation assessment, the detection of NTRK fusions is not straightforward, and various assays exist at the DNA, RNA, and protein level. Here, we investigate the performance of immunohistochemistry and DNA-based next-generation sequencing to indirectly or directly detect NTRK fusions relative to an RNA-based next-generation sequencing approach in the largest cohort of NTRK fusion positive solid tumors to date. A retrospective analysis of 38,095 samples from 33,997 patients sequenced by a targeted DNA-based next-generation sequencing panel (MSK-IMPACT), 2189 of which were also examined by an RNA-based sequencing assay (MSK-Fusion), identified 87 patients with oncogenic NTRK1-3 fusions. All available institutional NTRK fusion positive cases were assessed by pan-Trk immunohistochemistry along with a cohort of control cases negative for NTRK fusions by next-generation sequencing. DNA-based sequencing showed an overall sensitivity and specificity of 81.1% and 99.9%, respectively, for the detection of NTRK fusions when compared to RNA-based sequencing. False negatives occurred when fusions involved breakpoints not covered by the assay. Immunohistochemistry showed overall sensitivity of 87.9% and specificity of 81.1%, with high sensitivity for NTRK1 (96%) and NTRK2 (100%) fusions and lower sensitivity for NTRK3 fusions (79%). Specificity was 100% for carcinomas of the colon, lung, thyroid, pancreas, and biliary tract. Decreased specificity was seen in breast and salivary gland carcinomas (82% and 52%, respectively), and positive staining was often seen in tumors with neural differentiation. Both sensitivity and specificity were poor in sarcomas. Selection of the appropriate assay for NTRK fusion detection therefore depends on tumor type and genes involved, as well as consideration of other factors such as available material, accessibility of various clinical assays, and whether comprehensive genomic testing is needed concurrently.

Expression of neurotrophins and their receptors in primary osteosarcoma. / Expressão de neurotrofinas e de seus receptores no osteossarcoma primário.

OBJECTIVE: to determine the expression of neurotrophins and their tyrosine-kinase receptors in patients with osteosarcoma (OS) and their correlation with clinical outcomes. METHODS: we applied immunohistochemistry to biopsy specimens of patients consecutively treated for primary OS at a single institution between 2002 and 2015, analyzing them for expression receptors of tyrosine kinase A and B (TrKA and TrKB), neural growth factor (NGF) and brain derived neurotrophic factor (BDNF). Independently, two pathologists classified the immunohistochemical markers as negative (negative or weak focal) or positive (moderate focal/diffuse or strong focal/diffuse). RESULTS: we analyzed data from 19 patients (10 females and 9 males), with median age of 12 years (5 to 17.3). Tumors' location were 83.3% in the lower limbs, and 63.2% of patients had metastases at diagnosis. Five-year overall survival was 55.3%. BDNF was positive in 16 patients (84%) and NGF in 14 (73%). TrKA and TrKB presented positive staining in four (21,1%) and eight (42,1%) patients, respectively. Survival analysis showed no significant difference between TrK receptors and neurotrophins. CONCLUSION: primary OS samples express neurotrophins and TrK receptors by immunohistochemistry. Future studies should explore their role in OS pathogenesis and determine their prognostic significance in larger cohorts.

OBJETIVO: determinar a expressão de neurotrofinas e seus receptores tirosina quinases em pacientes com osteossarcoma (OS) e sua correlação com desfechos clínicos. MÉTODOS: biópsias de tumores primários de pacientes com OS tratados em uma única instituição, consecutivamente, entre 2002 e 2015, foram analisados através de imuno-histoquímica para expressão de receptores de tirosina quinase A e B (TrKA e TrKB), fator de crescimento neural (NGF) e fator neurotrófico derivado do cérebro (BDNF). De forma independente, dois patologistas classificaram os marcadores de imuno-histoquímica como negativos (negativos e focais fracos) ou positivos (moderado focal/difuso ou forte focal/difuso). RESULTADOS: foram analisados dados de 19 pacientes (10 do sexo feminino e 9 do masculino) com mediana de idade de 12 anos (5 a 17,3 anos). Dos tumores, 83,3% estavam localizados em membros inferiores e 63,2% dos pacientes eram metastáticos ao diagnóstico. A sobrevida global em cinco anos foi de 55,3%. BDNF foi positivo em 16 pacientes (84%) e NGF em 14 pacientes (73%). TrKA e TrKB apresentaram coloração positiva em quatro (21,1%) e oito (42,1%) pacientes, respectivamente. A análise de sobrevida não demonstrou diferença significativa entre receptores TrK e neurotrofinas. CONCLUSÃO: amostras de OS primário expressam neurotrofinas e receptores TrK através de imuno-histoquímica. Estudos futuros podem auxiliar na identificação do papel das mesmas na patogênese do OS e determinar se há possível correlação prognóstica.

First-in-Human Brain Imaging of [18F]TRACK, a PET tracer for Tropomyosin Receptor Kinases.

The tropomyosin receptor kinase TrkA/B/C family is responsible for human neuronal growth, survival, and differentiation from early nervous system development stages onward. Downregulation of TrkA/B/C receptors characterizes numerous neurological disorders including Alzheimer's disease (AD). Abnormally expressed Trk receptors or chimeric Trk fusion proteins are also well-characterized oncogenic drivers in a variety of neurogenic and non-neurogenic human neoplasms and are currently the focus of intensive clinical research. Previously, we have described the clinical translation of a highly selective and potent carbon-11-labeled pan-Trk radioligand and the preclinical characterization of the optimized fluorine-18-labeled analogue, [18F]TRACK, for in vivo Trk positron emission tomography (PET) imaging. We describe herein central nervous system selectivity assessment and first-in-human study of [18F]TRACK.

Expressão de neurotrofinas e de seus receptores no osteossarcoma primário / Expression of neurotrophins and their receptors in primary osteosarcoma

RESUMO Objetivo: determinar a expressão de neurotrofinas e seus receptores tirosina quinases em pacientes com osteossarcoma (OS) e sua correlação com desfechos clínicos. Métodos: biópsias de tumores primários de pacientes com OS tratados em uma única instituição, consecutivamente, entre 2002 e 2015, foram analisados através de imuno-histoquímica para expressão de receptores de tirosina quinase A e B (TrKA e TrKB), fator de crescimento neural (NGF) e fator neurotrófico derivado do cérebro (BDNF). De forma independente, dois patologistas classificaram os marcadores de imuno-histoquímica como negativos (negativos e focais fracos) ou positivos (moderado focal/difuso ou forte focal/difuso). Resultados: foram analisados dados de 19 pacientes (10 do sexo feminino e 9 do masculino) com mediana de idade de 12 anos (5 a 17,3 anos). Dos tumores, 83,3% estavam localizados em membros inferiores e 63,2% dos pacientes eram metastáticos ao diagnóstico. A sobrevida global em cinco anos foi de 55,3%. BDNF foi positivo em 16 pacientes (84%) e NGF em 14 pacientes (73%). TrKA e TrKB apresentaram coloração positiva em quatro (21,1%) e oito (42,1%) pacientes, respectivamente. A análise de sobrevida não demonstrou diferença significativa entre receptores TrK e neurotrofinas. Conclusão: amostras de OS primário expressam neurotrofinas e receptores TrK através de imuno-histoquímica. Estudos futuros podem auxiliar na identificação do papel das mesmas na patogênese do OS e determinar se há possível correlação prognóstica.

ABSTRACT Objective: to determine the expression of neurotrophins and their tyrosine-kinase receptors in patients with osteosarcoma (OS) and their correlation with clinical outcomes. Methods: we applied immunohistochemistry to biopsy specimens of patients consecutively treated for primary OS at a single institution between 2002 and 2015, analyzing them for expression receptors of tyrosine kinase A and B (TrKA and TrKB), neural growth factor (NGF) and brain derived neurotrophic factor (BDNF). Independently, two pathologists classified the immunohistochemical markers as negative (negative or weak focal) or positive (moderate focal/diffuse or strong focal/diffuse). Results: we analyzed data from 19 patients (10 females and 9 males), with median age of 12 years (5 to 17.3). Tumors' location were 83.3% in the lower limbs, and 63.2% of patients had metastases at diagnosis. Five-year overall survival was 55.3%. BDNF was positive in 16 patients (84%) and NGF in 14 (73%). TrKA and TrKB presented positive staining in four (21,1%) and eight (42,1%) patients, respectively. Survival analysis showed no significant difference between TrK receptors and neurotrophins. Conclusion: primary OS samples express neurotrophins and TrK receptors by immunohistochemistry. Future studies should explore their role in OS pathogenesis and determine their prognostic significance in larger cohorts.

Clinical perineural invasion of cutaneous head and neck cancer: Impact of radiotherapy, imaging, and nerve growth factor receptors on symptom control and prognosis.

OBJECTIVES: Clinical perineural invasion (CPNI) of cutaneous head and neck cancer is associated with poor prognosis and presents a therapeutic dilemma. The purpose of this study was to determine the relationship between CPNI and nerve growth factor receptors (NGFR), and the impact of radiotherapy (RT), imaging, and NGFR on symptom control and disease-related outcomes. MATERIALS AND METHODS: We retrospectively reviewed patients with CPNI of cutaneous head and neck cancer who were treated with RT between 2010 and 2015 at our institution. Exact chi-square and Wilcoxon rank-sum tests compared patients with positive versus negative staining for TrkA and/or CD271. Gray's test determined differences in cumulative incidences of 1- and 2-year locoregional recurrence (LRR) and cancer-specific mortality (CSM). RESULTS: Twenty-three patients had a median overall follow-up of 31.4 months from initial clinical symptoms and 19.7 months from pathological confirmation of PNI. The most prevalent symptoms were numbness (70%) and pain (57%). Sixteen patients (70%) experienced symptom improvement or control, especially decreased pain (85%), within a median of 2.6 months from starting RT. The 1- and 2-year rates of overall LRR were 37% and 71%, while those of overall CSM were 11% and 25%, respectively. Patients who stained positively for TrkA and/or CD271 had significantly worse LRR compared to patients who stained negatively for both markers (p = 0.046). CONCLUSION: Positive TrkA and/or CD271 staining predicts worse outcomes. Patients may benefit from aggressive RT for local control and symptom improvement. Future research is needed to identify the potential for anti-nerve growth factor therapies in CPNI.

4-Methylcatechol prevents streptozotocin-induced acute kidney injury through modulating NGF/TrkA and ROS-related Akt/GSK3ß/ß-catenin pathways.

Nerve growth factor (NGF) has been shown to protect the viability of kidney cells in acute phase of renal damage. However, since the half-life of NGF is very short, it is too large to pass the blood-brain barrier and rapidly transported to the liver for catabolizing its use in therapy is limited. 4-Methylcatechol (4MC) is a substance that increases NGF synthesis in many tissues. This study aimed to investigate the protective effects of 4MC against acute renal injury induced by streptozotocin (STZ). We have investigated the profibrotic, proinflammatory, oxidative changes in STZ-induced acute renal damage and the possible role of the NGF/TrkA system and Akt/GSK3ß/ß-catenin pathway in this mechanism. Experiment was designed as to be started with injection of 4MC for 10 days as a single dose (10 µg/kg) per day and to be terminated after 4 h of a single dose (75 mg/kg) STZ injection. As the result, 4MC pre-treatment decreased kidney damage, ROS production, the renal levels of TGFß1, CD68, tumor necrosis factor-α and interleukin 1ß. Moreover, 4MC pre-treatment increased levels of NGF and its receptor TrkA, p-Akt (Thr308), p-GSK3ß (Ser9) and nuclear ß-catenin. These data suggest that 4MC prevents the development of STZ-induced renal damage by suppressing ROS production and inflammation via Akt/GSK3ß/ß-catenin pathway which may be stimulated by NGF/TrkA signaling. Therefore, 4MC can be suggested as a potential agent for the prevention of acute renal injury.

Evaluation of pan-TRK immunohistochemistry in infantile fibrosarcoma, lipofibromatosis-like neural tumour and histological mimics.

AIMS: Infantile fibrosarcoma is characterised by intersecting fascicles of spindle cells and ETV6-NTRK3 gene fusion in most cases. Given histological overlap with other spindle-cell tumours, the diagnosis can be challenging and often requires molecular confirmation. A recently developed pan-TRK antibody shows promise for identifying tumours with NTRK fusions. The purpose of this study was to evaluate the potential diagnostic utility of pan-TRK immunohistochemistry for infantile fibrosarcoma. METHODS AND RESULTS: We evaluated whole-tissue sections from 210 cases, including 15 infantile fibrosarcomas; five each lipofibromatosis-like neural tumour and lipofibromatosis; 10 each primitive myxoid mesenchymal tumour of infancy (PMMTI) and low-grade myofibroblastic sarcoma; 15 each fibrous hamartoma of infancy (FHI), myofibroma/myofibromatosis and desmoid-type fibromatosis; and 20 each low-grade fibromyxoid sarcoma, synovial sarcoma, spindle-cell rhabdomyosarcoma, malignant peripheral nerve sheath tumour, fibrosarcomatous dermatofibrosarcoma protuberans (F-DFSP) and nodular fasciitis. Immunohistochemistry was performed using a rabbit monoclonal pan-TRK antibody. Immunoreactivity for pan-TRK was observed in all 15 (100%) infantile fibrosarcomas, including diffuse immunoreactivity (>50% of cells) in 14 (93%) cases. Pan-TRK was positive in all five (100%) lipofibromatosis-like neural tumours. Of the 190 histological mimics, diffuse pan-TRK immunoreactivity was noted in 16 (8%) cases, including five PMMTI, five FHI (highlighting predominantly the primitive myxoid spindle-cell components), three F-DFSP, one low-grade myofibroblastic sarcoma, one myofibroma and one spindle-cell rhabdomyosarcoma. CONCLUSIONS: Diffuse pan-TRK immunoreactivity is a highly sensitive but not entirely specific diagnostic marker for infantile fibrosarcoma, and may be helpful in selecting patients for TRK-targeted therapy. As expected, lipofibromatosis-like neural tumours, which harbour NTRK1 fusions, also show diffuse pan-TRK immunoreactivity.

Neurotrophins and Trk receptors in the developing and adult ovary of Coturnix coturnix japonica.

NGF, BDNF, NT-3 and their specific receptors TrkA, TrkB and TrkC are known to be involved in the development and maintenance of vertebrates' nervous system. However, these molecules play a role also in non-neuronal tissue, such as in the reproductive system. In this study we investigated the presence and localization of neurotrophins and Trk receptors to unravel their potential role in the developing and adult ovary of Japanese quail, a model species well suited for reproduction studies. Western blotting analysis on ovaries of three month old quails in the period of egg laying showed the presence of pro and mature forms of neurotrophins and splice variants of Trk receptors. Immunohistochemical investigation reported that in embryonic ovaries from the 9th day of incubation to the hatching NGF and NT-3 were observed in the cortical and medullar areas respectively, whereas Trk receptors were observed in both areas. In adult ovary, all NTs were detected in glandular stromal cells, NGF and NT-3 also in the nervous component. Regarding follicle components, NGF and BDNF were observed in oocytes and follicular cells. All TrK receptors were present in nervous components and only TrkA in glandular stromal cells. In follicles, TrkA was present in oocyte cytoplasm and TrkB in theca cells. The results suggest an involvement of the neurotrophin system in the quail ovary physiology, promoting the oocyte development and follicular organization in the embryo, as well as oocyte and follicular maturation in adults.

The implications of TrkA and MET aberrations in de novo salivary duct carcinoma.

Salivary duct carcinoma (SDC) is an aggressive carcinoma with poor prognosis. Although anti-HER2 therapy is a potential treatment option for HER2-positive SDC, other potential therapeutic targets are not known, in particular for HER2-negative cases. In this study, the recently identified receptors tyrosine kinases MET and tropomyosin-receptor kinase (Trk) were investigated as potential therapeutic targets. A total of 28 consecutive, surgically resected, de novo SDC cases were selected after evaluating histology and immunohistochemical expression of androgen receptor. Immunohistochemical expression of c-erb2, TrkA, TrkB, TrkC, and c-MET was analyzed, and the genetic status of the HER2 and MET genes was investigated through dual-color silver in situ hybridization. High expression of c-MET or Trk was defined as that above the median value. Among the 28 SDC cases, 64.3% (18/28) were HER2-positive. c-MET expression varied, with a median H-score of 65 (range, 0 to 200). Copy number gain and amplification of MET were noted in 57.1% (16/28) and 10.7% (3/28) of cases, respectively. TrkA was variably expressed, with a median H-score of 100 (range, 0 to250). High TrkA expression was significantly related to an inferior overall survival rate in HER2-negative SDC. High expression of TrkA and c-MET and MET copy number gain/amplification were frequent events in SDC, and high expression of TrkA revealed the tendency to be related to poor prognosis in HER2-negative SDC. TrkA and MET may be possible therapeutic targets in SDC, especially in HER2-negative SDC.