Serial xenotransplantation of sorted cancer cells in immunodeficient mice remains the most complex test of cancer stem cell (CSC) phenotype. However, we have demonstrated in various sarcomas that putative CSC surface markers fail to identify CSCs, thereby impeding the isolation of CSCs for subsequent analyses. Here, we utilized serial xenotransplantation of unsorted rhabdomyosarcoma cells in NOD/SCID gamma (NSG) mice as a proof-of-principle platform to investigate the molecular signature of CSCs. Indeed, serial xenotransplantation steadily enriched for rhabdomyosarcoma stem-like cells characterized by enhanced aldehyde dehydrogenase activity and increased colony and sphere formation capacity in vitro. Although the expression of core pluripotency factors (SOX2, OCT4, NANOG) and common CSC markers (CD133, ABCG2, nestin) was maintained over the passages in mice, gene expression profiling revealed gradual changes in several stemness regulators and genes linked with undifferentiated myogenic precursors, e.g., SOX4, PAX3, MIR145, and CDH15. Moreover, we identified the induction of a hybrid epithelial/mesenchymal gene expression signature that was associated with the increase in CSC number. In total, 60 genes related to epithelial or mesenchymal traits were significantly altered upon serial xenotransplantation. In silico survival analysis based on the identified potential stemness-associated genes demonstrated that serial xenotransplantation of unsorted rhabdomyosarcoma cells in NSG mice might be a useful tool for the unbiased enrichment of CSCs and the identification of novel CSC-specific targets. Using this approach, we provide evidence for a recently proposed link between the hybrid epithelial/mesenchymal phenotype and cancer stemness.

• Keywords
• cancer stem cells, epithelial/mesenchymal phenotype, in vivo tumorigenicity assay, rhabdomyosarcoma, serial xenotransplantation, stem-like state, stemness,
• Publication type
• Journal Article MeSH

The specific targeting of signal transduction by low-molecular-weight inhibitors or monoclonal antibodies represents a very promising personalized treatment strategy in pediatric oncology. In this study, we present the successful and clinically relevant use of commercially available phospho-protein arrays for analyses of the phosphorylation profiles of a broad spectrum of receptor tyrosine kinases and their downstream signaling proteins in tumor tissue samples. Although these arrays were made for research purposes on human biological samples, they have already been used by several authors to profile various tumor types. Our study performed a systematic analysis of the advantages and pitfalls of the use of this method for personalized clinical medicine. In certain clinical cases and their series, we demonstrated the important aspects of data processing and evaluation, the use of phospho-protein arrays for single sample and serial sample analyses, and the validation of obtained results by immunohistochemistry, as well as the possibilities of this method for the hierarchical clustering of pediatric solid tumors. Our results clearly show that phospho-protein arrays are apparently useful for the clinical consideration of druggable molecular targets within a specific tumor. Thus, their potential validation for diagnostic purposes may substantially improve the personalized approach in the treatment of relapsed or refractory solid tumors.

• Keywords
• low-molecular-weight inhibitors, pediatric solid tumors, phospho-protein arrays, phosphorylation profiling, receptor tyrosin kinases, signal transduction,
• Publication type
• Journal Article MeSH

UNLABELLED: In order to identify reasons for treatment failures when using targeted therapies, we have analyzed the comprehensive molecular profiles of three relapsed, poor-prognosis Burkitt lymphoma cases. All three cases had resembling clinical presentation and histology and all three patients relapsed, but their outcomes differed significantly. The samples of their tumor tissue were analyzed using whole-exome sequencing, gene expression profiling, phosphoproteomic assays, and single-cell phosphoflow cytometry. These results explain different treatment responses of the three histologically identical but molecularly different tumors. Our findings support a personalized approach for patient with high risk, refractory, and rare diseases and may contribute to personalized and customized treatment efforts for patients with limited treatment options like relapsed/refractory Burkitt lymphoma. SUMMARY: The main aim of this study is to analyze three relapsed Burkitt lymphoma patients using a comprehensive molecular profiling, in order to explain their different outcomes and to propose a biomarker-based targeted treatment. In cases 1 and 3, the tumor tissue and the host were analyzed prospectively and appropriate target for the treatment was successfully implemented; however, in case 2, analyses become available only retrospectively and his empirically based rescue treatment did not hit the right target of his disease.

• Keywords
• Burkitt lymphoma, pediatric oncology, precision medicine, targeted therapy, theranostics,
• Publication type
• Journal Article MeSH

Infantile myofibromatosis represents one of the most common proliferative fibrous tumors of infancy and childhood. More effective treatment is needed for drug-resistant patients, and targeted therapy using specific protein kinase inhibitors could be a promising strategy. To date, several studies have confirmed a connection between the p.R561C mutation in gene encoding platelet-derived growth factor receptor beta (PDGFR-beta) and the development of infantile myofibromatosis. This study aimed to analyze the phosphorylation of important kinases in the NSTS-47 cell line derived from a tumor of a boy with infantile myofibromatosis who harbored the p.R561C mutation in PDGFR-beta. The second aim of this study was to investigate the effects of selected protein kinase inhibitors on cell signaling and the proliferative activity of NSTS-47 cells. We confirmed that this tumor cell line showed very high phosphorylation levels of PDGFR-beta, extracellular signal-regulated kinases (ERK) 1/2 and several other protein kinases. We also observed that PDGFR-beta phosphorylation in tumor cells is reduced by the receptor tyrosine kinase inhibitor sunitinib. In contrast, MAPK/ERK kinases (MEK) 1/2 and ERK1/2 kinases remained constitutively phosphorylated after treatment with sunitinib and other relevant protein kinase inhibitors. Our study showed that sunitinib is a very promising agent that affects the proliferation of tumor cells with a p.R561C mutation in PDGFR-beta.

• Keywords
• FR180204, U0126, erlotinib, infantile myofibromatosis, platelet-derived growth factor receptor, protein kinase inhibitors, receptor tyrosine kinases, sunitinib, targeted therapy,
• MeSH
• Butadienes administration & dosage   therapeutic use   MeSH
• Child MeSH
• Erlotinib Hydrochloride administration & dosage   therapeutic use   MeSH
• Phosphorylation drug effects   MeSH
• Protein Kinase Inhibitors administration & dosage   therapeutic use   MeSH
• Infant MeSH
• Humans MeSH
• Mutation * MeSH
• Myofibromatosis congenital   drug therapy   genetics   MeSH
• Cell Line, Tumor MeSH
• Nitriles administration & dosage   therapeutic use   MeSH
• Cell Proliferation drug effects   MeSH
• Pyrazoles administration & dosage   therapeutic use   MeSH
• Pyridazines administration & dosage   therapeutic use   MeSH
• Receptor, Platelet-Derived Growth Factor beta * genetics   metabolism   MeSH
• Sunitinib administration & dosage   therapeutic use   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Butadienes MeSH
• Erlotinib Hydrochloride MeSH
• FR 180204 MeSH Browser
• Protein Kinase Inhibitors MeSH
• Nitriles MeSH
• PDGFRB protein, human MeSH Browser
• Pyrazoles MeSH
• Pyridazines MeSH
• Receptor, Platelet-Derived Growth Factor beta * MeSH
• Sunitinib MeSH
• U 0126 MeSH Browser

BACKGROUND: Infantile myofibromatosis belongs to a family of soft tissue tumors. The majority of these tumors have benign behavior but resistant and malignant courses are known, namely in tumors with visceral involvement. The standard of care is surgical resection. Observations suggest that low dose chemotherapy is beneficial. The treatment of resistant or relapsed patients with multifocal disease remains challenging. Patients that harbor an actionable mutation in the kinase domain are potential subjects for targeted tyrosine kinase inhibitor therapy. CASE PRESENTATION: An infant boy with inborn generalized infantile myofibromatosis that included bone, intracranial, soft tissue and visceral involvement was treated according to recent recommendations with low dose chemotherapy. The presence of a partial but temporary response led to a second line of treatment with six cycles of chemotherapy, which achieved a partial response again but was followed by severe toxicity. The generalized progression of the disease was observed later. Genetic analyses were performed and revealed a PDGFRB gene c.1681C>A missense heterozygous germline mutation, high PDGFRβ phosphokinase activity within the tumor and the heterozygous germline Slavic Nijmegen breakage syndrome 657del5 mutation in the NBN gene. Targeted treatment with sunitinib, the PDGFRβ inhibitor, plus low dose vinblastine led to an unexpected and durable response without toxicities or limitations to daily life activities. The presence of the Slavic NBN gene mutation limited standard chemotherapy dosing due to severe toxicities. Sister of the patient suffred from skull base tumor with same genotype and histology. The same targeted therapy led to similar quick and durable response. CONCLUSION: Progressive and resistant incurable infantile myofibromatosis can be successfully treated with the new approach described herein. Detailed insights into the biology of the patient's tumor and genome are necessary to understand the mechanisms of activity of less toxic and effective drugs except for up to date population-based chemotherapy regimens.

• Keywords
• Case report, Chemotherapy, Infantile myofibromatosis, PDGFR, Theranostics, Tyrosine kinase inhibitor,
• MeSH
• Drug Resistance, Neoplasm drug effects   genetics   MeSH
• Molecular Targeted Therapy methods   MeSH
• Heterozygote MeSH
• Indoles administration & dosage   MeSH
• Humans MeSH
• Myofibromatosis congenital   drug therapy   genetics   metabolism   MeSH
• Infant, Newborn MeSH
• Antineoplastic Combined Chemotherapy Protocols therapeutic use   MeSH
• Pyrroles administration & dosage   MeSH
• Receptor, Platelet-Derived Growth Factor beta antagonists & inhibitors   genetics   metabolism   MeSH
• Sunitinib MeSH
• Vinblastine administration & dosage   MeSH
• Treatment Outcome MeSH
• Germ-Line Mutation * MeSH
• Family Health MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Names of Substances
• Indoles MeSH
• Pyrroles MeSH
• Receptor, Platelet-Derived Growth Factor beta MeSH
• Sunitinib MeSH
• Vinblastine MeSH

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. Its dismal prognosis is often attributed to the presence of cancer stem cells (CSCs) that have been identified in PDAC using various markers. However, the co-expression of all of these markers has not yet been evaluated. Furthermore, studies that compare the expression levels of CSC markers in PDAC tumor samples and in cell lines derived directly from those tumors are lacking. Here, we analyzed the expression of putative CSC markers-CD24, CD44, epithelial cell adhesion molecule (EpCAM), CD133, and nestin-by immunofluorescence, flow cytometry and quantitative PCR in 3 PDAC-derived cell lines and by immunohistochemistry in 3 corresponding tumor samples. We showed high expression of the examined CSC markers among all of the cell lines and tumor samples, with the exception of CD24 and CD44, which were enriched under in vitro conditions compared with tumor tissues. The proportions of cells positive for the remaining markers were comparable to those detected in the corresponding tumors. Co-expression analysis using flow cytometry revealed that CD24+/CD44+/EpCAM+/CD133+ cells represented a significant population of the cells (range, 43 to 72%) among the cell lines. The highest proportion of CD24+/CD44+/EpCAM+/CD133+ cells was detected in the cell line derived from the tumor of a patient with the shortest survival. Using gene expression profiling, we further identified the specific pro-tumorigenic expression profile of this cell line compared with the profiles of the other two cell lines. Together, CD24+/CD44+/EpCAM+/CD133+ cells are present in PDAC cell lines derived from primary tumors, and their increased proportion corresponds with a pro-tumorigenic gene expression profile.

• MeSH
• Adenocarcinoma diagnosis   metabolism   MeSH
• Epithelial Cell Adhesion Molecule metabolism   MeSH
• AC133 Antigen metabolism   MeSH
• CD24 Antigen metabolism   MeSH
• Hyaluronan Receptors metabolism   MeSH
• Stem Cells metabolism   MeSH
• Cells, Cultured MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Middle Aged MeSH
• Humans MeSH
• Biomarkers, Tumor MeSH
• Pancreatic Neoplasms diagnosis   metabolism   MeSH
• Nestin metabolism   MeSH
• Prognosis MeSH
• Flow Cytometry MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Aged MeSH
• Transcriptome MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Epithelial Cell Adhesion Molecule MeSH
• AC133 Antigen MeSH
• CD24 Antigen MeSH
• Hyaluronan Receptors MeSH
• EPCAM protein, human MeSH Browser
• Biomarkers, Tumor MeSH
• NES protein, human MeSH Browser
• Nestin MeSH
• PROM1 protein, human MeSH Browser