crizotinib and Pancreatic-Neoplasms

Inflammatory myofibroblastic tumor (IMT) is a rare, mesenchymal tumor that has an increased incidence in childhood. Tumors are usually isolated to the chest, abdomen, and retroperitoneum, but metastatic presentations can be seen. Presenting symptoms are nonspecific and include fever, weight loss, pain, shortness of breath, and cough. Approximately 85% of IMTs harbor actionable kinase fusions. The diagnosis can be delayed because of overlapping features with inflammatory disorders, such as elevated inflammatory markers, increased immunoglobin G levels, fever, weight loss, and morphologic similarity with nonmalignant conditions. We present a girl aged 11 years with a TFG-ROS1 fusion-positive tumor of the lung that was initially diagnosed as an immunoglobin G4-related inflammatory pseudotumor. She underwent complete left-sided pneumonectomy and later recurred with widely metastatic disease. We then report the case of a boy aged 9 years with widely metastatic TFG-ROS1 fusion-positive IMT with rapid molecular diagnosis. In both children, there was an excellent response to oral targeted therapy. These cases reveal that rapid molecular testing of inflammatory tumors is not only important for diagnosis but also reveals therapeutic opportunities. Targeted inhibitors produce significant radiologic responses, enabling potentially curative treatment approaches for metastatic ROS1 fusion IMT with previously limited treatment options. Primary care pediatricians and pediatric subspecialists have a crucial role in the early consultation of a pediatric oncology center experienced in molecular diagnostics to facilitate a comprehensive evaluation for children with inflammatory tumors.

Topics: Antineoplastic Agents, Immunological; Child; Crizotinib; Diagnosis, Differential; Female; Glucocorticoids; Humans; Immunoglobulin G4-Related Disease; Inflammation; Lung Neoplasms; Male; Molecular Targeted Therapy; Neoplasm Recurrence, Local; Neoplasms, Muscle Tissue; Oncogene Proteins, Fusion; Pancreatic Neoplasms; Plasma Cell Granuloma, Pulmonary; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins; Rare Diseases; Rituximab

Peritoneal dissemination is a frequent occurrence in pancreatic cancer, which is associated with a poor prognosis. MET is associated with the progression of pancreatic cancer; therefore, we evaluated the effect of a MET inhibitor, crizotinib, on peritoneal dissemination of pancreatic cancer. Crizotinib inhibited the growth of 8 pancreatic cancer cell lines with the IC50 ranging from 1.4 to 4.3 µM. Invasion of the pancreatic cancer cell line Suit-2, was suppressed in vitro at a concentration of 1.0 µM, which is sufficient for the inhibition of MET phosphorylation. This effect on cell invasion was also recapitulated by the reduction of MET expression in Suit-2 with siRNA. Crizotinib also inhibited RhoA activation in addition to MET phosphorylation. We further evaluated the effect of crizotinib on peritoneal dissemination of pancreatic cancer in vivo. Crizotinib reduced tumor burden and ascites accumulation due to development of peritoneal dissemination after inoculation of Suit-2. Taken together, crizotinib may be a potent drug for treating peritoneal dissemination of pancreatic cancer by inhibiting cancer cell proliferation and invasion, at least in part through the suppression of HGF/MET signaling and RhoA activation.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Cell Proliferation; Crizotinib; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Pancreatic Neoplasms; Peritoneal Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Signal Transduction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p < 0.01). These results were associated with reduced expression of miR-21 and increased expression of miR-155. Our study provides the first evidence that transduced primary PDAC cells can form tumors on the CAM, retaining several histopathological and (epi)genetic characteristics of original tumors. Moreover, our results support the use of these models for drug testing, providing insights on molecular mechanisms underlying antitumor activity of new drugs/combinations.

Topics: Adenocarcinoma; Animals; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Proliferation; Chickens; Chorioallantoic Membrane; Crizotinib; Deoxycytidine; DNA Mutational Analysis; Drug Evaluation, Preclinical; Gemcitabine; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 6; Luminescent Measurements; MicroRNAs; Models, Biological; Pancreatic Neoplasms; Sequence Analysis, DNA; SOX9 Transcription Factor; Tumor Cells, Cultured

Cancer cells with high metastatic potential and stem cell-like characteristics express the cell surface marker CD44. CD44 isoforms that include the v6 exon are co-receptors for the receptor tyrosine kinases MET and Vascular Endothelial Growth factor Receptor-2 (VEGFR-2). We studied CD44v6 signaling in several pancreatic cancer cell lines, and its role in tumor growth and metastasis in several models of pancreatic cancer.. We analyzed the effects of v6 peptides that interfere with the co-receptor functions of CD44v6 for MET and VEGFR-2 in tumors and metastases grown from cells that express different CD44 isoforms, including CD44v6. The peptides were injected into rats with syngeneic tumors and mice with orthotopic or xenograft tumors. We also tested the effects of the peptides in mice with xenograft tumors grown from patient tumor samples and mice that express an oncogenic form of RAS and develop spontaneous pancreatic cancer (KPC mice). We measured levels of CD44v6 messenger RNA (mRNA) in pancreatic cancer tissues from 136 patients.. Xenograft tumors grown from human cancer cells injected with v6 peptides were smaller and formed fewer metastases in mice. The v6 peptide was more efficient than the MET inhibitor crizotinib and/or the VEGFR-2 inhibitor pazopanib in reducing xenograft tumor growth and metastasis. Injection of KPC mice with the v6 peptide increased their survival time. Injection of mice and rats bearing metastases with the v6 peptide induced regression of metastases. Higher levels of CD44v6 mRNA in human pancreatic tumor tissues were associated with increased expression of MET, tumor metastasis, and shorter patient survival times.. Peptide inhibitors of CD44v6 isoforms block tumor growth and metastasis in several independent models of pancreatic cancer. The v6 peptides induced regression of metastases. Levels of CD44v6 mRNA are increased, along with those of MET mRNA, in patients with metastatic pancreatic tumors, compared with nonmetastatic tumors; the increased levels correlated with shorter patient survival time.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Crizotinib; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Hyaluronan Receptors; Indazoles; Kaplan-Meier Estimate; Lung Neoplasms; Male; Mice, Nude; Mice, Transgenic; Mutation; Neoplasm Metastasis; Pancreatic Neoplasms; Peptides; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Pyrimidines; Rats; RNA, Messenger; Signal Transduction; Sulfonamides; Time Factors; Transfection; Tumor Burden; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

Drug-resistance is a major contributing factor for the poor prognosis in patients with pancreatic cancer. We have shown previously that the irreversible ErbB family blocker afatinib, is more effective than the reversible EGFR tyrosine kinase inhibitor erlotinib in inhibiting the growth of human pancreatic cancer cells. The aim of this study was to develop human pancreatic cancer cell (BxPc3) variants with acquired resistance to treatment with gemcitabine, afatinib, or erlotinib, and to investigate the molecular changes that accompany the acquisition of a drug-resistant phenotype. We also investigated the therapeutic potential of various agents in the treatment of such drug-resistant variants. Three variant forms of BxPc3 cells with acquired resistance to gemcitabine (BxPc3GEM), afatinib (BxPc3AFR) or erlotinib (BxPc3OSIR) were developed following treatment with increasing doses of such drugs. The expression level, mutational and phosphorylation status of various growth factor receptors and downstream cell signaling molecules were determined by FACS, human phopsho-RTK array, and western blot analysis while the sulforhodamine B assay was used for determining the effect of various agents on the growth of such tumours. We found that all three BxPc3 variants with acquired resistance to gemcitabine (BxPc3GEM), afatinib (BxPc3AFR) or erlotinib (BxPc3OSIR) also become less sensitive to treatment with the two other agents. Acquisition of resistance to these agents was accompanied by upregulation of p-c-MET, p-STAT3, CD44, increased autocrine production of EGFR ligand amphiregulin and differential activation status of EGFR tyrosine residues as well as downregulation of total and p-SRC. Of all therapeutic interventions examined, including the addition of an anti-EGFR antibody ICR62, an anti-CD44 monoclonal antibody, and of STAT3 or c-MET inhibitors, only treatment with the STAT3 inhibitor Stattic produced a higher growth inhibitory effect in all three drug-resistant variants. In addition, treatment with a combination of afatinib with either c-MET inhibitor Crizotinib or Stattic resulted in an additive or synergistic growth inhibition in all three variants. Our results suggest that activation of STAT3 may play an important role in the acquisition of resistance to gemcitabine and HER inhibitors in pancreatic cancer and warrant further studies on the therapeutic potential of STAT3 inhibitors in such a setting.

Topics: Afatinib; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Crizotinib; Cyclic S-Oxides; Deoxycytidine; DNA Mutational Analysis; Drug Resistance, Neoplasm; Gemcitabine; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Ligands; MAP Kinase Signaling System; Mutation; Pancreatic Neoplasms; Phenotype; Phosphorylation; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Quinazolines; Receptor, ErbB-2; Signal Transduction; STAT3 Transcription Factor

Crizotinib, a c-MET/ALK inhibitor, has exhibited antitumor efficacy in different types of cancers. However, studies regarding Crizotinib in pancreatic cancer have been limited. Thus, we investigated the effect of Crizotinib on pancreatic cancer and its mechanism of action. Crizotinib strongly suppressed the growth and proliferation of pancreatic cancer cells in a dose-dependent manner. Also, it induced apoptosis by modulating its related factors. In the study, with regard to the mechanism of action, Crizotinib did not inhibit c-MET expression on pancreatic cancer cells; instead, it specifically inhibited the activity of ALK, which was identified to be highly expressed on various pancreatic cancer cells and tissues in our study. In 42 different receptor tyrosine kinase (RTKs) array, Crizotinib also strongly inhibited the expression of activated ALK in pancreatic cancer cells, modulating its downstream mediators such as STAT3, AKT, and ERK. Furthermore, Crizotinib inhibited angiogenesis in a mouse Matrigel plug assay as well as the progression of tumor growth in a mouse xenograft model. Taken together, our investigation shows that Crizotinib inhibits the ALK signaling pathway in pancreatic cancer, resulting in cell growth/angiogenesis inhibition and apoptosis induction. We suggest that Crizotinib might be used as a novel therapeutic drug for treating pancreatic cancer.

Topics: Anaplastic Lymphoma Kinase; Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Crizotinib; Extracellular Signal-Regulated MAP Kinases; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Pancreatic Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Receptor Protein-Tyrosine Kinases; STAT3 Transcription Factor; Transplantation, Heterologous

Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem. Most drugs that pass preclinical tests fail in these patients, emphasizing the need of improved preclinical models to test novel anticancer strategies. Here, we developed four orthotopic mouse models using primary human PDAC cells genetically engineered to express firefly- and Gaussia luciferase, simplifying the ability to monitor tumor growth and metastasis longitudinally in individual animals with MRI and high-frequency ultrasound. In these models, we conducted detailed histopathologic and immunohistochemical analyses on paraffin-embedded pancreatic tissues and metastatic lesions in liver, lungs, and lymph nodes. Genetic characteristics were compared with the originator tumor and primary tumor cells using array-based comparative genomic hybridization, using frozen specimens obtained by laser microdissection. Notably, the orthotopic human xenografts in these models recapitulated the phenotype of human PDACs, including hypovascular and hypoxic areas. Pursuing genomic and immunohistochemical evidence revealed an increased copy number and overexpression of c-Met in one of the models; we examined the preclinical efficacy of c-Met inhibitors in vitro and in vivo. In particular, we found that crizotinib decreased tumor dimension, prolonged survival, and increased blood and tissue concentrations of gemcitabine, synergizing with a cytidine deaminase-mediated mechanism of action. Together, these more readily imaged orthotopic PDAC models displayed genetic, histopathologic, and metastatic features similar to their human tumors of origin. Moreover, their use pointed to c-Met as a candidate therapeutic target in PDAC and highlighted crizotinib and gemcitabine as a synergistic combination of drugs warranting clinical evaluation for PDAC treatment.

Topics: Animals; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Crizotinib; Deoxycytidine; Female; Gemcitabine; Humans; Inactivation, Metabolic; Mice; Mice, Nude; Pancreatic Neoplasms; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Pancreatic-ductal-adenocarcinoma (PDAC) is amongst the most lethal malignancies, mainly because of its metastatic spread and multifactorial chemoresistance. Since c-Met is a marker of pancreatic-cancer-stem-cells (CSC), playing a key role in metastasis and chemoresistance, this study evaluated the therapeutic potential of the novel c-Met/ALK inhibitor crizotinib against PDAC cells, including the Capan-1-gemcitabine-resistant cells (Capan-1-R). Crizotinib inhibited PDAC cell-growth with IC50 of 1.5 μM in Capan-1-R, and synergistically enhanced the antiproliferative and proapoptotic activity of gemcitabine, as detected by sulforhodamine-B-assay, flow cytometry and combination-index method. Capan-1-R had higher expression of the CSC markers CD44+/CD133+/CD326+, but their combined expression was significantly reduced by crizotinib, as detected by quantitative-RT-PCR and FACS-analysis. Similarly, Capan-1-R cells had significantly higher protein-expression of c-Met (≈2-fold), and increased migratory activity, which was reduced by crizotinib (e.g., > 50% reduction of cell-migration in Capan-1-R after 8-hour exposure, compared to untreated-cells), in association with reduced vimentin expression. Capan-1-R had also significantly higher mRNA expression of the gemcitabine catabolism-enzyme CDA, potentially explaining the higher CDA activity and statistically significant lower levels of gemcitabine-nucleotides in Capan-1-R compared to Capan-1, as detected by Liquid-chromatography-massspectrometry. Conversely, crizotinib significantly reduced CDA expression in both Capan-1 and Capan-1-R cells. In aggregate, these data show the ability of crizotinib to specifically target CSC-like-subpopulations, interfere with cell-proliferation, induce apoptosis, reduce migration and synergistically interact with gemcitabine, supporting further studies on this novel therapeutic approach for PDAC.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromatography, Liquid; Crizotinib; Deoxycytidine; Drug Resistance, Neoplasm; Drug Synergism; Gemcitabine; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Mass Spectrometry; Neoplastic Stem Cells; Pancreatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Pyrazoles; Pyridines