ABSTRACT
Introduction: ALK rearrangement is the only druggable oncogenic driver detectable by immunohistochemistry (IHC) not requiring further confirmation of positivity in accessing first-line specific inhibitors. ALK-positive patients experience clinical benefit from pemetrexed-based chemotherapy possibly due to lower thymidylate synthase (TS) levels. This study assesses agreement with three different ALK IHC clones in 37 FISH-positive NSCLC. TS expression by real time (RT)-PCR was compared with ALK FISH-negative cases. Materials and methods: 37 ALK FISH-positive NSCLC cases diagnosed between 2010 and 2015 in 7 Italian centres were investigated with ICH using three different anti-ALK antibodies (ALK1, 5A4 and D5F3). Staining for ALK1 and 5A4 was graded as 0+,1+,2+, and 3+, while the scoring for D5F3 was recorded as negative or positive. Proportion agreement analysis was done using Cohen's unweighted kappa (k). TS and ß-actin expression levels were analysed by quantitative RT-PCR. Comparison between TS expression in ALK FISH-positive specimens and a control cohort of ALK FISH-negative ones was performed with the Mann-Whitney and Kruskal-Wallis tests. Results: Considering 2+ and 3+ as positive, the proportion of IHC agreement was 0.1691 (95% CI 0-0.4595) for ALK1/5A4, 0.1691 (95% CI 0-0.4595) for ALK1/D5F3, and 1 for D5F3/5A4. Considering 3+ as positive, it was 0.1543 (95% CI 0-0.4665) for ALK1/ 5A4, 0.0212 (95% CI 0-0.1736) for ALK1/D5F3, and 0.2269 (95% CI 0-0.5462) for 5A4/D5F3. Median TS expression was 6.07 (1.28-14.94) and ALK-positive cases had a significant lower TS expression than ALK-negative tumours (p = 0.002). Conclusions: IHC proved to be a reliable tool for the diagnosis of ALK-rearranged NSCLC. D5F3 and 5A4 clones have the highest percentage of agreement. TS levels are significantly lower in FISH-positive patients.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Actins , Anaplastic Lymphoma Kinase/genetics , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Clone Cells/chemistry , Clone Cells/metabolism , Clone Cells/pathology , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Pemetrexed , Receptor Protein-Tyrosine Kinases/analysis , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Retrospective Studies , Thymidylate Synthase/geneticsABSTRACT
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.
Subject(s)
Glioblastoma , Immunohistochemistry , In Situ Hybridization, Fluorescence , Receptor Protein-Tyrosine Kinases , Sequence Analysis, RNA , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Brain Neoplasms/therapy , Female , Gene Rearrangement , Glioblastoma/genetics , Glioblastoma/pathology , Glioblastoma/therapy , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Molecular Targeted Therapy , Oncogene Proteins, Fusion/analysis , Oncogene Proteins, Fusion/genetics , Receptor Protein-Tyrosine Kinases/analysis , Receptor Protein-Tyrosine Kinases/genetics , Receptor, trkA/analysis , Receptor, trkA/genetics , Receptor, trkC/analysis , Receptor, trkC/genetics , Young AdultABSTRACT
AIMS: Tropomyosin receptor kinase (TRK)-targeted therapies represent an important therapeutic option for patients with advanced solid tumours harbouring neurotrophin receptor kinase (NTRK) gene fusions. However, NTRK fusions are rare in common adult carcinomas, and systematic approaches to screening for these alterations are lacking. Pan-TRK immunohistochemistry (IHC) has been proposed as one method to screen for NTRK fusion-positive tumours. Reflexive testing strategies have been endorsed for several IHC-based biomarkers, and thus offer a convenient and low-cost entry point to incorporate pan-TRK screening. METHODS AND RESULTS: In this study, 447 consecutive cases of adult solid tumours undergoing mismatch repair (MMR), human epidermal growth factor receptor 2 (HER2) and/or programmed cell death ligand 1 (PD-L1) testing were prospectively stained with pan-TRK IHC. Four cases (0.9%) were pan-TRK positive, including three (1.3% of 223) colonic adenocarcinomas, two of which were MMR-deficient and one (1.4% of 71) was gastroesophageal carcinoma. None of 108 non-small cell lung carcinomas showed pan-TRK expression. NTRK gene fusion was confirmed by DNA sequencing in one MMR-deficient colonic adenocarcinoma. In one MMR-deficient tumour, an alternative mitogen-activated protein kinase (MAPK) driver was identified. In the oesophageal (squamous cell) carcinoma, RNA sequencing identified relative NTRK2 transcript overexpression in the absence of a fusion. In one MMR-proficient colonic adenocarcinoma, no MAPK drivers were identified; therefore, a falsely negative sequencing result was favored. None of the patients met clinical criteria for TRK-targeted therapy. CONCLUSION: The clinical impact of pan-TRK IHC 'piggybacking' on existing reflexive testing strategies in surgical pathology appears negligible. Carcinomas may rarely show high-level pan-TRK expression in the absence of an underlying NTRK fusion event.
Subject(s)
Biomarkers, Tumor/analysis , Early Detection of Cancer/methods , Gene Fusion , Immunohistochemistry/methods , Neoplasms/genetics , Neoplasms/pathology , Receptor Protein-Tyrosine Kinases/analysis , Aged , Aged, 80 and over , Female , Genetic Testing , Humans , Male , Middle Aged , Receptor Protein-Tyrosine Kinases/genetics , Sequence Analysis, RNAABSTRACT
The current coronavirus disease 2019 (COVID-19) pandemic presents a global public health challenge. The viral pathogen responsible, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to the host receptor ACE2 through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. Although the role of ACE2 as a receptor for SARS-CoV-2 is clear, studies have shown that ACE2 expression is extremely low in various human tissues, especially in the respiratory tract. Thus, other host receptors and/or co-receptors that promote the entry of SARS-CoV-2 into cells of the respiratory system may exist. In this study, we found that the tyrosine-protein kinase receptor UFO (AXL) specifically interacts with the N-terminal domain of SARS-CoV-2 S. Using both a SARS-CoV-2 virus pseudotype and authentic SARS-CoV-2, we found that overexpression of AXL in HEK293T cells promotes SARS-CoV-2 entry as efficiently as overexpression of ACE2, while knocking out AXL significantly reduces SARS-CoV-2 infection in H1299 pulmonary cells and in human primary lung epithelial cells. Soluble human recombinant AXL blocks SARS-CoV-2 infection in cells expressing high levels of AXL. The AXL expression level is well correlated with SARS-CoV-2 S level in bronchoalveolar lavage fluid cells from COVID-19 patients. Taken together, our findings suggest that AXL is a novel candidate receptor for SARS-CoV-2 which may play an important role in promoting viral infection of the human respiratory system and indicate that it is a potential target for future clinical intervention strategies.
Subject(s)
COVID-19/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Respiratory Mucosa/cytology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Bronchi/cytology , Bronchi/metabolism , Cell Line , Humans , Lung/cytology , Lung/metabolism , Models, Molecular , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins/analysis , Receptor Protein-Tyrosine Kinases/analysis , Respiratory Mucosa/metabolism , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Virus Internalization , Axl Receptor Tyrosine KinaseABSTRACT
BACKGROUND: Crizotinib is a first-generation tyrosine kinase inhibitor used for anaplastic lymphoma kinase (ALK) positive cancers. Simple and complex renal cyst formation is a rare complication of crizotinib use that has been reported previously in the adult population. CASE: We report a case of a right renal mass in a 17-year-old with ALK-positive epithelioid inflammatory myofibroblastic sarcoma treated with Crizotinib. After cessation of Crizotinib and initiating Alectenib, a second generation ALK inhibitor, the mass decreased in size and the patient remained asymptomatic without evidence of recurrence at three months of follow-up.
Subject(s)
Antineoplastic Agents/adverse effects , Crizotinib/adverse effects , Kidney Diseases, Cystic/chemically induced , Adolescent , Antineoplastic Agents/therapeutic use , Crizotinib/therapeutic use , Humans , Male , Neoplasms, Muscle Tissue/chemistry , Neoplasms, Muscle Tissue/drug therapy , Receptor Protein-Tyrosine Kinases/analysis , Sarcoma/chemistry , Sarcoma/drug therapyABSTRACT
Protein tyrosine kinase-7 (PTK7), as an important membrane receptor, regulates various cellular activities, including cell polarity, movement, migration, and invasion. Although lots of research studies focused on revealing its functions from the aspect of the expression of the gene and protein are present, the relationship between the spatial distribution at the single-molecule level and the function remains unclear. Through combining aptamer probe labeling and super-resolution imaging technology, after verifying the specificity and superiority of the aptamer probe, a more significant clustering distribution of PTK7 is found on the MCF10A cell basal membrane than on the apical membrane, which is thought to be related to their specific functions on different membranes. By exploring the relationship between the assembly of PTK7 and lipid rafts, actin cytoskeleton, and carbohydrate chains on the membrane, the unique distribution of PTK7 on disparate membranes is revealed to be probably because of the varied dominant position of these three factors. These findings present the detailed spatial information of PTK7 and the related potential organization mechanism on the cell membrane, which will facilitate a better understanding of the relationship between the molecular assembly and its function, as well as the overall structure of the cell membrane.
Subject(s)
Aptamers, Nucleotide/chemistry , Cell Adhesion Molecules/analysis , Cell Membrane/chemistry , Receptor Protein-Tyrosine Kinases/analysis , Cells, Cultured , Humans , Microscopy, Fluorescence , Stochastic ProcessesABSTRACT
Nanoparticle-based probes have great potential for imaging specific biomolecules in signal distinguishing and amplification via Förster resonance energy transfer (FRET). Protein-specific sialylation plays key roles in the regulation of protein structure and function, as well as in various pathophysiological processes. Here, we developed a fluorescent polymeric nanoparticle with a biocompatible hydrophilic thin shell loaded with plentiful dye and used it as the donor to enhance the FRET imaging of cell surface protein-specific sialylation. The hydrophobic core decreased the self-quenching of loaded fluorescent molecules, while the hydrophilic thin shell ensured that the nanoparticles remained on the extracellular surface and guaranteed the FRET effect. Thus, the thin-shell polymeric nanoparticles enhanced the FRET imaging of protein tyrosine kinase-7-specific sialylation on the CCRF-CEM cell surface and showed high sensitivity under drug treatment. This nanoparticle has great potential for elucidating the relationship between dynamic specific glycosylation states and disease processes, as well as for the study of new cell surface imaging methodologies.
Subject(s)
Cell Adhesion Molecules/analysis , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Nanoparticles/chemistry , Polymers/chemistry , Receptor Protein-Tyrosine Kinases/analysis , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Cell Membrane/chemistry , Humans , Receptor Protein-Tyrosine Kinases/metabolism , Surface PropertiesABSTRACT
BACKGROUND: RAS mutations are the only validated biomarkers in metastatic colorectal cancer (mCRC) for anti-epidermal growth factor receptor (EGFR) therapy. Limited clinical information is available on AXL expression, marker of epithelial to mesenchymal transition, in mCRC. METHODS: AXL was retrospectively assessed by immunohistochemistry in 307 patients. RAS wild-type (WT) patients (N = 136) received first-line anti-EGFR-based therapy; RAS mutant patients (N = 171) received anti-angiogenic-based regimens. Preclinical experiments were performed using human RAS WT CRC cell lines and xenograft models. AXL RNA levels were assessed in a cohort of patients with available samples at baseline and at progression to anti-EGFR treatment and in the GSE5851 dataset. RESULTS: AXL was expressed in 55/307 tumour tissues, correlating with worse survival in the overall population (AXL-positive, 23.7 months; AXL-negative, 30.8 months; HR, 1.455, P = 0.032) and in RAS WT patients (AXL-positive, 23.0 months; AXL-negative, 35.8 months; HR,1.780, P = 0.032). Progression-free survival (PFS) in the RAS WT cohort was shorter in the AXL-positive cohort (6.2 months versus 12.1 months; HR, 1.796, P = 0.013). Three-dimensional cultures obtained from a patient following anti-EGFR therapy resulted AXL-positive, showing resistance to anti-EGFR drugs and sensitivity to AXL inhibition. AXL transfection in CRC cell lines induced AXL overexpression and resistance to the EGFR blockade. At progression to cetuximab, 2/10 SW48-tumour xenograft mice showed AXL expression. Consistently, AXL RNA levels increased in 5/7 patients following anti-EGFR therapy. Moreover, in the GSE5851 dataset higher AXL RNA levels correlated with worse PFS with cetuximab in KRAS-exon2 WT chemorefractory patients. CONCLUSIONS: AXL is a marker of poor prognosis in mCRC with consistent clinical and preclinical evidences of involvement in primary and acquired resistance to anti-EGFR drugs in RAS WT patients.
Subject(s)
Colorectal Neoplasms/drug therapy , ErbB Receptors/antagonists & inhibitors , Genes, ras , Proto-Oncogene Proteins/analysis , Receptor Protein-Tyrosine Kinases/analysis , Animals , Biomarkers , Cell Line, Tumor , Colorectal Neoplasms/chemistry , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Drug Resistance, Neoplasm , Humans , Mice , Neoplasm Metastasis , Xenograft Model Antitumor Assays , Axl Receptor Tyrosine KinaseABSTRACT
Integration of multi-functional diagnosis and treatment competencies can improve effect of the drug and its visual distribution to the location of tumor focus site, and play a pivotal role by visualizing the tumor size in the assessment of chemotherapy. With the objective of developing integrated multi-therapeutic and diagnostic agent that could target the kinase receptor with high expression in tumor cells, herein, a biologically releasable drug-drug conjugate compound 9 with dual therapeutic and diagnostic effects was designed, synthesized and evaluated for pharmacodynamics and diagnostic functions in vitro and in vivo. The results of antitumor effects evaluations and compound 9 visual imaging indicated that compound 9 not only improved the anti-proliferative activity of chemotherapy and photodynamic treatment (PDT) in vitro and in vivo compared with those of compound 8 and PpIX but also allowed the photodynamic diagnosis (PDD). The present study verified a facile and effective strategy using a drug-drug conjugate to integrate diagnosis and multi-therapies, showing its potential a candidate clinical drug.
Subject(s)
Neoplasms/diagnostic imaging , Neoplasms/drug therapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Design , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , Fluorescent Dyes/therapeutic use , Humans , Mice, Inbred BALB C , Molecular Docking Simulation , Molecular Targeted Therapy , Neoplasms/metabolism , Optical Imaging , Photochemotherapy , Photosensitizing Agents/therapeutic use , Protoporphyrins/chemistry , Protoporphyrins/pharmacology , Receptor Protein-Tyrosine Kinases/analysis , Receptor Protein-Tyrosine Kinases/metabolismABSTRACT
Dynamic changes of protein-glycosylation on cell surface act as an important indicator that reflects cellular physiological states and disease developments. The enhanced visualization of protein-specific glycosylation is of great value to interpret its functions and mechanisms. Hence, we present an intramolecular trigger remodeling-induced hybridization chain reaction (HCR) for imaging protein-specific glycosylation. This strategy relies on designing two DNA probes, protein and glycan probes, labeled respectively on protein by aptamer recognition and glycan through metabolic oligosaccharide engineering (MOE). Upon the same glycoprotein was labeled, the complementary domain of two probes induces hybridization and thus to remodel an intact trigger, followed by initiating HCR assembly. Applying this strategy, we successfully achieved imaging of specific protein-glycosylation on CEM cell surface and monitored dynamic changes of the glycosylation after treating with drugs. It provides a powerful tool with high flexibility, specificity and sensitivity in the research field of protein-specific glycosylation on living cells.
Subject(s)
Cell Adhesion Molecules/analysis , DNA Probes/chemistry , Nucleic Acid Hybridization , Receptor Protein-Tyrosine Kinases/analysis , Aptamers, Nucleotide/chemistry , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/metabolism , Cells, Cultured , DNA Probes/chemical synthesis , Glycosylation/drug effects , Humans , Particle Size , Polysaccharides/analysis , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/metabolism , Surface Properties , Tunicamycin/pharmacologySubject(s)
Biomarkers, Tumor/adverse effects , Immunohistochemistry/methods , Pancreatic Neoplasms/genetics , Receptor Protein-Tyrosine Kinases/analysis , Receptor Protein-Tyrosine Kinases/genetics , Adult , Aged , Aged, 80 and over , Female , Gene Rearrangement , Humans , Male , Middle Aged , Pancreatic NeoplasmsABSTRACT
Noninvasive diagnostics using circulating tumor cells (CTCs) are expected to be useful for decision making in precision cancer therapy. AXL, a receptor tyrosine kinase is associated with tumor progression, epithelial-to-mesenchymal transition (EMT), and drug resistance, and is a potential therapeutic target. However, the epithelial markers generally used for CTC detection may be not enough to detect AXL-expressing CTCs due to EMT. Here, we evaluated the detection of AXL-expressing CTCs using the mesenchymal marker vimentin with a microcavity array system. To evaluate the recovery of cancer cells, spike-in experiments were performed using cell lines with varying cytokeratin (CK) or vimentin (VM) expression levels. With high CK and low VM-expressing cell lines, PC-9 and HCC827, the recovery rate of AXL-expressing cancer cells was 1%-17% using either CK or VM as markers. Whereas, with low CK and high VM-expressing cell lines, MDA-MB231 and H1299, it was 52%-75% using CK and 72%-88% using VM as a marker. For clinical evaluation, peripheral blood was collected from 20 non-small cell lung cancer patients and CTCs were detected using CK or VM as markers in parallel. Significantly more AXL-expressing single CTCs were detected in VM-positive than CK-positive CTCs (P < .001). Furthermore, CTC clusters were identified only among VM-positive CTCs in 20% of patients. Patients with one or more prior treatments harbored significantly more VM-positive AXL-expressing CTCs, suggesting the involvement of these CTCs in drug resistance. These results indicate the necessity of integrating mesenchymal markers with CTC detection and this should be further evaluated clinically.
Subject(s)
Carcinoma, Non-Small-Cell Lung/diagnosis , Cell Separation/instrumentation , Lung Neoplasms/diagnosis , Neoplastic Cells, Circulating/metabolism , Proto-Oncogene Proteins/analysis , Receptor Protein-Tyrosine Kinases/analysis , Aged , Aged, 80 and over , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , Drug Resistance, Neoplasm , Feasibility Studies , Female , Humans , Lung Neoplasms/blood , Lung Neoplasms/drug therapy , Male , Middle Aged , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Vimentin/analysis , Vimentin/metabolism , Axl Receptor Tyrosine KinaseABSTRACT
Gastric cancer (GC) is the most prevalent human cancer around the globe. In GC, Wnt signaling is deregulated, and receptor-like tyrosine kinase (RYK) coreceptors have been identified to interact with noncanonical Wnt ligand Wnt5a. We, therefore, aimed to evaluate the role of RYK in GC development and metastasis. GC tumor samples were collected from 250 GC patients. Expressions of RYK, as well as markers for the epithelial-mesenchymal transition (EMT), such as N-cadherin and E-cadherin, were subjected to correlation analysis with clinicopathological features. Endogenous RYK expression levels were compared in GC cell lines with ascending metastatic potentials followed by stable RYK knockdown. Effect of RYK knockdown on GC cell migration, invasion, and EMT phenotype were assessed in vitro, and on GC tumor growth in vivo in a xenograft rodent model. Particularly, liver metastasis potential of tail vein-injected GC cells was also analyzed following RYK knockdown. RYK was highly correlated with liver metastasis of GC tumors and the expression profiles of EMT markers toward the mesenchymal tendency. RYK expression was also positively correlated with the metastasis potential of GC cells. RYK knockdown not only inhibited migration, invasion, and EMT of GC cells in vitro, but also suppressed tumorigenesis and liver metastasis of GC cells in vivo using the mouse xenograft model. RYK is highly correlated with GC tumorigenesis and potential of liver metastasis, suggesting it may be a novel oncogenic factor of the noncanonical Wnt signaling pathway contributing to GC.NEW & NOTEWORTHY RYK is highly correlated with gastric cancer tumorigenesis and the potential of liver metastasis, suggesting it may be a novel oncogenic factor of the noncanonical Wnt signaling pathway contributing to gastric cancer.
Subject(s)
Liver Neoplasms/secondary , Receptor Protein-Tyrosine Kinases/metabolism , Stomach Neoplasms/metabolism , Wnt-5a Protein/metabolism , Animals , Biomarkers, Tumor/analysis , Cadherins/analysis , Cell Line, Tumor , Cell Movement/genetics , Epithelial-Mesenchymal Transition , Female , Gene Knockdown Techniques , Humans , Male , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness/genetics , Receptor Protein-Tyrosine Kinases/analysis , Wnt-5a Protein/analysis , Xenograft Model Antitumor AssaysABSTRACT
Background: Protein tyrosine kinase-7, a regulatory protein in the Wnt signaling pathway, was highly overexpressed in various cancer types and assumed to be related to prognosis. Aims: The purpose of this study is to assess whether protein tyrosine kinase-7 expression status in curatively resected gastric carcinoma would independently identify patients with a high risk of recurrence and death. Study Design: Retrospective cohort study. Methods: We included patients who were at least 18 years of age and diagnosed with gastric cancer. The exclusion criterion was a metastatic disease at the time of diagnosis or operation. Data on clinicopathological prognostic determinants and clinical courses, including the date of disease relapse and survival status, were collected with the use of medical records. Surgically removed tumor tissue specimens were examined by two independent pathologists at the pathology department of our institution. Protein tyrosine kinase-7 expression status was assessed with immunohistochemical processing and stratified on a scale ranging from 0 to +3 according to the extent of stained tumor cells. It was then further categorized into two groups, one being + (positive), including +1, +2, and +3 scores, another was-(negative), including-and +/− scores. Results: A total of 114 patients were analyzed. Protein tyrosine kinase-7 expression was present in 66.7% of the surgical tumor specimens. There was no statistically significant difference in almost all relevant parameters between the protein tyrosine kinase-7 positive and negative groups. The estimated median survival in the protein tyrosine kinase-7 positive group was significantly better than the protein tyrosine kinase-7 negative group (60 vs 22 months, p<0.001). Disease-free survival was found to be 55 months in the protein tyrosine kinase-7 positive group, whereas it was 21 months in the negative group (p=0.015). In the multivariate analysis, along with negative protein tyrosine kinase-7 expression, poor performance status, and advanced stage were significantly associated with the risk of death (p<0.001 for each). Conclusion: Compared to patients with negative PTK-7 expression, patients with positive PTK-7 expression have better disease-free survival and overall survival rates. Efforts should be made to enhance this finding and translate it into clinical practice.
Subject(s)
Cell Adhesion Molecules/analysis , Gene Expression , Receptor Protein-Tyrosine Kinases/analysis , Stomach Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/analysis , Biomarkers, Tumor/blood , Cell Adhesion Molecules/blood , Chi-Square Distribution , Cohort Studies , Female , Follow-Up Studies , Humans , Male , Middle Aged , Prognosis , Receptor Protein-Tyrosine Kinases/blood , Retrospective Studies , Stomach Neoplasms/blood , Stomach Neoplasms/physiopathology , Survival RateABSTRACT
AIMS: Inflammatory myofibroblastic tumour (IMT) is a spindle cell neoplasm of intermediate malignancy, and the diagnosis is often challenging due to the morphological overlap with other spindle cell neoplasms and reactive lesions. More than half of IMTs have the ALK gene rearrangement, and a minor subset have ROS1, NTRK3 or RET gene rearrangements. We sought to determine the potential diagnostic utility of pan-Trk immunohistochemistry for IMTs. METHODS AND RESULTS: We retrospectively examined 40 cases of IMT using immunohistochemistry with a rabbit monoclonal pan-Trk antibody. Gene rearrangement was confirmed by fluorescence in-situ hybridisation and/or reverse transcription-polymerase chain reaction. The IMTs were classified as the ALK (n = 29), ROS1 (n = 2), NTRK3 (n = 2), RET (n = 0) and 'quadruple-negative' (n = 7) genotypes by molecular analyses. Both of the ETV6-NTRK3 fusion-positive cases showed nuclear and cytoplasmic staining for pan-Trk in the majority of tumour cells. None of the ALK, ROS1 or quadruple-negative-type IMTs showed nuclear staining for pan-Trk, but approximately one-third of these IMTs showed focal and weak cytoplasmic staining. One exceptional case of a RANBP2-ALK-positive epithelioid inflammatory myofibroblastic sarcoma (an aggressive variant of IMT) showed moderate cytoplasmic staining for pan-Trk. CONCLUSIONS: These results suggest that pan-Trk immunoreactivity with a nuclear and cytoplasmic staining pattern may be useful to identify ETV6-NTRK3-positive IMTs and may be helpful in selecting patients for Trk-targeted therapy.
Subject(s)
Biomarkers, Tumor/analysis , Myofibroma/diagnosis , Receptor Protein-Tyrosine Kinases/analysis , Female , Humans , Immunohistochemistry , Male , Retrospective StudiesABSTRACT
Small molecule inhibitors such as Larotrectinib have been recently approved in the treatment of patients with fusions of the neurotrophic-tropomyosin receptor tyrosine kinase (NTRK) genes 1-3. These genomic rearrangements have been reported across different tumor subtypes with a high prevalence in rare tumors. However, in gastric and esophageal adenocarcinoma (AGE) NTRK fusions have also been described in a subset of Asian patients. In order to study the prevalence of this alteration in Caucasian patients with AGE we performed immunohistochemistry for pan-NTRK in 438 formalin-fixed paraffin embedded (FFPE) tumor samples. While we found NTRK expression in gastric glands and tumor adjacent nerve tissue, we did not detect this marker in the tumor compartment. Based on our findings NTRK fusions do not seem to play a role in the molecularpathology of Caucasian AEG patients, so that other treatment options are required.
Subject(s)
Adenocarcinoma/genetics , Esophageal Neoplasms/genetics , Receptor Protein-Tyrosine Kinases/genetics , Stomach Neoplasms/genetics , Adenocarcinoma/enzymology , Adult , Aged , Esophageal Neoplasms/enzymology , Female , Humans , Immunohistochemistry , Male , Middle Aged , Oncogene Fusion , Receptor Protein-Tyrosine Kinases/analysis , Receptor Protein-Tyrosine Kinases/biosynthesis , Stomach Neoplasms/enzymologySubject(s)
Myasthenia Gravis/diagnosis , Myasthenia Gravis/therapy , Antibodies/analysis , Antibodies/blood , Biomarkers/analysis , Biomarkers/blood , Humans , Myasthenia Gravis/physiopathology , Quality of Life/psychology , Receptor Protein-Tyrosine Kinases/analysis , Receptor Protein-Tyrosine Kinases/blood , Receptors, Cholinergic/analysis , Receptors, Cholinergic/blood , Receptors, Nicotinic/analysis , Receptors, Nicotinic/bloodABSTRACT
The leading proteomic method for identifying N-glycosylated peptides is liquid chromatography coupled with tandem fragmentation mass spectrometry (LCMS/MS) followed by spectral matching of MS/MS fragment masses to a database of possible glycan and peptide combinations. Such database-dependent approaches come with challenges such as needing high-quality informative MS/MS spectra, ignoring unexpected glycan or peptide sequences, and making incorrect assignments because some glycan combinations are equivalent in mass to amino acids. To address these challenges, we present GlycopeptideGraphMS, a graph theoretical bioinformatic approach complementary to the database-dependent method. Using the AXL receptor tyrosine kinase (AXL) as a model glycoprotein with multiple N-glycosylation sites, we show that those LCMS features that could be grouped into graph networks on the basis of glycan mass and retention time differences were actually N-glycopeptides with the same peptide backbone but different N-glycan compositions. Conversely, unglycosylated peptides did not exhibit this grouping behavior. Furthermore, MS/MS sequencing of the glycan and peptide composition of just one N-glycopeptide in the graph was sufficient to identify the rest of the N-glycopeptides in the graph. By validating the identifications with exoglycosidase cocktails and MS/MS fragmentation, we determined the experimental false discovery rate of identifications to be 2.21%. GlycopeptideGraphMS detected more than 500 unique N-glycopeptides from AXL, triple the number found by a database search with Byonic software, and detected incorrect assignments due to a nonspecific protease cleavage. This method overcomes some limitations of the database approach and is a step closer to comprehensive automated glycoproteomics.
Subject(s)
Proto-Oncogene Proteins/analysis , Receptor Protein-Tyrosine Kinases/analysis , Software , Chromatography, Liquid , Databases, Protein , Humans , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Tandem Mass Spectrometry , Time Factors , Axl Receptor Tyrosine KinaseABSTRACT
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.
Subject(s)
Brain/metabolism , Fluorine Radioisotopes/pharmacokinetics , Neuroimaging/methods , Positron-Emission Tomography/methods , Radiopharmaceuticals/pharmacokinetics , Receptor Protein-Tyrosine Kinases/analysis , Animals , Humans , Membrane Glycoproteins/analysis , Membrane Glycoproteins/metabolism , Mice , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, trkA/analysis , Receptor, trkA/metabolism , Receptor, trkB/analysis , Receptor, trkB/metabolism , Receptor, trkC/analysis , Receptor, trkC/metabolismABSTRACT
We propose a signal amplification method that enables visualization analysis of membrane proteins on living cells at a single-molecule level. Using the proposed method, we achieved imaging of PTK7 membrane proteins on HeLa cells and analyzed the down-regulated expression of EpCAM on the MCF-7 cell surface during epithelial-mesenchymal transition (EMT).