PredictONCO: a web tool supporting decision-making in precision oncology by extending the bioinformatics predictions with advanced computing and machine learning.

PredictONCO 1.0 is a unique web server that analyzes effects of mutations on proteins frequently altered in various cancer types. The server can assess the impact of mutations on the protein sequential and structural properties and apply a virtual screening to identify potential inhibitors that could be used as a highly individualized therapeutic approach, possibly based on the drug repurposing. PredictONCO integrates predictive algorithms and state-of-the-art computational tools combined with information from established databases. The user interface was carefully designed for the target specialists in precision oncology, molecular pathology, clinical genetics and clinical sciences. The tool summarizes the effect of the mutation on protein stability and function and currently covers 44 common oncological targets. The binding affinities of Food and Drug Administration/ European Medicines Agency -approved drugs with the wild-type and mutant proteins are calculated to facilitate treatment decisions. The reliability of predictions was confirmed against 108 clinically validated mutations. The server provides a fast and compact output, ideal for the often time-sensitive decision-making process in oncology. Three use cases of missense mutations, (i) K22A in cyclin-dependent kinase 4 identified in melanoma, (ii) E1197K mutation in anaplastic lymphoma kinase 4 identified in lung carcinoma and (iii) V765A mutation in epidermal growth factor receptor in a patient with congenital mismatch repair deficiency highlight how the tool can increase levels of confidence regarding the pathogenicity of the variants and identify the most effective inhibitors. The server is available at https://loschmidt.chemi.muni.cz/predictonco.

Clinical and biological landscape of constitutional mismatch-repair deficiency syndrome: an International Replication Repair Deficiency Consortium cohort study.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) syndrome is a rare and aggressive cancer predisposition syndrome. Because a scarcity of data on this condition contributes to management challenges and poor outcomes, we aimed to describe the clinical spectrum, cancer biology, and impact of genetics on patient survival in CMMRD. METHODS: In this cohort study, we collected cross-sectional and longitudinal data on all patients with CMMRD, with no age limits, registered with the International Replication Repair Deficiency Consortium (IRRDC) across more than 50 countries. Clinical data were extracted from the IRRDC database, medical records, and physician-completed case record forms. The primary objective was to describe the clinical features, cancer spectrum, and biology of the condition. Secondary objectives included estimations of cancer incidence and of the impact of the specific mismatch-repair gene and genotype on cancer onset and survival, including after cancer surveillance and immunotherapy interventions. FINDINGS: We analysed data from 201 patients (103 males, 98 females) enrolled between June 5, 2007 and Sept 9, 2022. Median age at diagnosis of CMMRD or a related cancer was 8·9 years (IQR 5·9-12·6), and median follow-up from diagnosis was 7·2 years (3·6-14·8). Endogamy among minorities and closed communities contributed to high homozygosity within countries with low consanguinity. Frequent dermatological manifestations (117 [93%] of 126 patients with complete data) led to a clinical overlap with neurofibromatosis type 1 (35 [28%] of 126). 339 cancers were reported in 194 (97%) of 201 patients. The cumulative cancer incidence by age 18 years was 90% (95% CI 80-99). Median time between cancer diagnoses for patients with more than one cancer was 1·9 years (IQR 0·8-3·9). Neoplasms developed in 15 organs and included early-onset adult cancers. CNS tumours were the most frequent (173 [51%] cancers), followed by gastrointestinal (75 [22%]), haematological (61 [18%]), and other cancer types (30 [9%]). Patients with CNS tumours had the poorest overall survival rates (39% [95% CI 30-52] at 10 years from diagnosis; log-rank p<0·0001 across four cancer types), followed by those with haematological cancers (67% [55-82]), gastrointestinal cancers (89% [81-97]), and other solid tumours (96% [88-100]). All cancers showed high mutation and microsatellite indel burdens, and pathognomonic mutational signatures. MLH1 or MSH2 variants caused earlier cancer onset than PMS2 or MSH6 variants, and inferior survival (overall survival at age 15 years 63% [95% CI 55-73] for PMS2, 49% [35-68] for MSH6, 19% [6-66] for MLH1, and 0% for MSH2; p<0·0001). Frameshift or truncating variants within the same gene caused earlier cancers and inferior outcomes compared with missense variants (p<0·0001). The greater deleterious effects of MLH1 and MSH2 variants as compared with PMS2 and MSH6 variants persisted despite overall improvements in survival after surveillance or immune checkpoint inhibitor interventions. INTERPRETATION: The very high cancer burden and unique genomic landscape of CMMRD highlight the benefit of comprehensive assays in timely diagnosis and precision approaches toward surveillance and immunotherapy. These data will guide the clinical management of children and patients who survive into adulthood with CMMRD. FUNDING: The Canadian Institutes for Health Research, Stand Up to Cancer, Children's Oncology Group National Cancer Institute Community Oncology Research Program, Canadian Cancer Society, Brain Canada, The V Foundation for Cancer Research, BioCanRx, Harry and Agnieszka Hall, Meagan's Walk, BRAINchild Canada, The LivWise Foundation, St Baldrick Foundation, Hold'em for Life, and Garron Family Cancer Center.

Personalized dendritic cell vaccine in multimodal individualized combination therapy improves survival in high-risk pediatric cancer patients.

A lot of hope for high-risk cancers is being pinned on immunotherapy but the evidence in children is lacking due to the rarity and limited efficacy of single-agent approaches. Here, we aim to assess the effectiveness of multimodal therapy comprising a personalized dendritic cell (DC) vaccine in children with relapsed and/or high-risk solid tumors using the N-of-1 approach in real-world scenario. A total of 160 evaluable events occurred in 48 patients during the 4-year follow-up. Overall survival of the cohort was 7.03 years. Disease control after vaccination was achieved in 53.8% patients. Comparative survival analysis showed the beneficial effect of DC vaccine beyond 2 years from initial diagnosis (HR = 0.53, P = .048) or in patients with disease control (HR = 0.16, P = .00053). A trend for synergistic effect with metronomic cyclophosphamide and/or vinblastine was indicated (HR = 0.60 P = .225). A strong synergistic effect was found for immune check-point inhibitors (ICIs) after priming with the DC vaccine (HR = 0.40, P = .0047). In conclusion, the personalized DC vaccine was an effective component in the multimodal individualized treatment. Personalized DC vaccine was effective in less burdened or more indolent diseases with a favorable safety profile and synergized with metronomic and/or immunomodulating agents.

Final results of the Choroid Plexus Tumor study CPT-SIOP-2000.

INTRODUCTION: Standards for chemotherapy against choroid plexus tumors (CPT) have not yet been established. METHODS: CPT-SIOP-2000 (NCT00500890) was an international registry for all CPT nesting a chemotherapy randomization for high-risk CPT with Carboplatin/Etoposide/Vincristine (CarbEV) versus Cyclophosphamide/Etoposide/Vincristine (CycEV). Patients older than three years were recommended to receive irradiation: focal fields for non-metastatic CPC, incompletely resected atypical choroid plexus papilloma (APP) or metastatic choroid plexus papilloma (CPP); craniospinal fields for metastatic CPC/APP and non-responsive CPC. High risk was defined as choroid plexus carcinoma (CPC), incompletely resected APP, and all metastatic CPT. From 2000 until 2010, 158 CPT patients from 23 countries were enrolled. RESULTS: For randomized CPC, the 5/10 year progression free survival (PFS) of patients on CarbEV (n = 20) were 62%/47%, respectively, compared to 27%/18%, on CycEV (n = 15), (intention-to-treat, HR 2.6, p = 0.032). Within the registry, histological grading was the most influential prognostic factor: for CPP (n = 55) the 5/10 year overall survival (OS) and the event free survival (EFS) probabilities were 100%/97% and 92%/92%, respectively; for APP (n = 49) 96%/96% and 76%/76%, respectively; and for CPC (n = 54) 65%/51% and 41%/39%, respectively. Without irradiation, 12 out of 33 patients with CPC younger than three years were alive for a median of 8.52 years. Extent of surgery and metastases were not independent prognosticators. CONCLUSIONS: Chemotherapy for Choroid Plexus Carcinoma is feasible and effective. CarbEV is superior to CycEV. A subset of CPC can be cured without irradiation.

Childhood survivors of high-risk neuroblastoma show signs of immune recovery and not immunosenescence.

Neuroblastoma survivors show signs of immunosenescence early after therapy in CD8+ T cell compartment and elevated plasma TNF-α but in later follow-up immune recovery comes into play. Whether the recovery phenotype is long lasting or transient remains to be elucidated, however, late adverse effects often occur in childhood cancer survivors.

Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma.

Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.

Enhanced Antiproliferative Effect of Combined Treatment with Calcitriol and All-Trans Retinoic Acid in Relation to Vitamin D Receptor and Retinoic Acid Receptor α Expression in Osteosarcoma Cell Lines.

The main objective of this study was to analyze changes in the antiproliferative effect of vitamin D3, in the form of calcitriol and calcidiol, via its combined application with all-trans retinoic acid (ATRA) in osteosarcoma cell lines. The response to treatment with calcitriol and calcidiol alone was specific for each cell line. Nevertheless, we observed an enhanced effect of combined treatment with ATRA and calcitriol in the majority of the cell lines. Although the levels of respective nuclear receptors did not correlate with the sensitivity of cells to these drugs, vitamin D receptor (VDR) upregulation induced by ATRA was found in cell lines that were the most sensitive to the combined treatment. In addition, all these cell lines showed high endogenous levels of retinoic acid receptor α (RARα). Our study confirmed that the combination of calcitriol and ATRA can achieve enhanced antiproliferative effects in human osteosarcoma cell lines in vitro. Moreover, we provide the first evidence that ATRA is able to upregulate VDR expression in human osteosarcoma cells. According to our results, the endogenous levels of RARα and VDR could be used as a predictor of possible synergy between ATRA and calcitriol in osteosarcoma cells.

Traffic lights for retinoids in oncology: molecular markers of retinoid resistance and sensitivity and their use in the management of cancer differentiation therapy.

For decades, retinoids and their synthetic derivatives have been well established anticancer treatments due to their ability to regulate cell growth and induce cell differentiation and apoptosis. Many studies have reported the promising role of retinoids in attaining better outcomes for adult or pediatric patients suffering from several types of cancer, especially acute myeloid leukemia and neuroblastoma. However, even this promising differentiation therapy has some limitations: retinoid toxicity and intrinsic or acquired resistance have been observed in many patients. Therefore, the identification of molecular markers that predict the therapeutic response to retinoid treatment is undoubtedly important for retinoid use in clinical practice. The purpose of this review is to summarize the current knowledge on candidate markers, including both genetic alterations and protein markers, for retinoid resistance and sensitivity in human malignancies.

Why Differentiation Therapy Sometimes Fails: Molecular Mechanisms of Resistance to Retinoids.

Retinoids represent a popular group of differentiation inducers that are successfully used in oncology for treatment of acute promyelocytic leukemia in adults and of neuroblastoma in children. The therapeutic potential of retinoids is based on their key role in the regulation of cell differentiation, growth, and apoptosis, which provides a basis for their use both in cancer therapy and chemoprevention. Nevertheless, patients treated with retinoids often exhibit or develop resistance to this therapy. Although resistance to retinoids is commonly categorized as either acquired or intrinsic, resistance as a single phenotypic feature is usually based on the same mechanisms that are closely related or combined in both of these types. In this review, we summarize the most common changes in retinoid metabolism and action that may affect the sensitivity of a tumor cell to treatment with retinoids. The availability of retinoids can be regulated by alterations in retinol metabolism or in retinoid intracellular transport, by degradation of retinoids or by their efflux from the cell. Retinoid effects on gene expression can be regulated via retinoid receptors or via other molecules in the transcriptional complex. Finally, the role of small-molecular-weight inhibitors of altered cell signaling pathways in overcoming the resistance to retinoids is also suggested.

Effects of Sunitinib and Other Kinase Inhibitors on Cells Harboring a PDGFRB Mutation Associated with Infantile Myofibromatosis.

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.

Case report: rapid and durable response to PDGFR targeted therapy in a child with refractory multiple infantile myofibromatosis and a heterozygous germline mutation of the PDGFRB gene.

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 PDGFRß phosphokinase activity within the tumor and the heterozygous germline Slavic Nijmegen breakage syndrome 657del5 mutation in the NBN gene. Targeted treatment with sunitinib, the PDGFRß 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.

Metronomic therapy has low toxicity and is as effective as current standard treatment for recurrent high-risk neuroblastoma.

The metronomic therapy concept uses low doses of continuously applied chemotherapeutic, anti-angiogenetic, and immunomodulating drugs. Twenty patients with recurrent and 3 with refractory high-risk neuroblastoma were treated by the metronomic concept using celecoxib, cyclophosphamide, vinblastine, and etoposide for up to 24 months. The outcome was compared to 274 matched patients with a first recurrence from stage 4 neuroblastoma using the variables time from diagnosis to first recurrence, number of organs involved, and MYCN amplification. All were treated with dose-intensive conventional chemotherapy. The study patients experienced 1-3 recurrences and had 1-3 sites involved (osteomedullary, primary tumor, central nervous system, lymph nodes, liver, lungs) before the metronomic therapy started. Two patients in complete remission and three with active refractory disease following recurrence treatment were excluded from the outcome analysis. The curves for secondary event-free and overall survival demonstrated no significant differences. The toxicity was minimal except for ≥3 grade thrombocytopenia and leukopenia (all heavily pretreated). The treatment was realized in an outpatient setting. The metronomic approach is similarly effective as standard treatment in recurrent high-risk neuroblastoma, has low toxicity, and is applicable in an outpatient setting. A prospective study including propranolol as a fifth drug is underway.

Prognostic value of medulloblastoma extent of resection after accounting for molecular subgroup: a retrospective integrated clinical and molecular analysis.

BACKGROUND: Patients with incomplete surgical resection of medulloblastoma are controversially regarded as having a marker of high-risk disease, which leads to patients undergoing aggressive surgical resections, so-called second-look surgeries, and intensified chemoradiotherapy. All previous studies assessing the clinical importance of extent of resection have not accounted for molecular subgroup. We analysed the prognostic value of extent of resection in a subgroup-specific manner. METHODS: We retrospectively identified patients who had a histological diagnosis of medulloblastoma and complete data about extent of resection and survival from centres participating in the Medulloblastoma Advanced Genomics International Consortium. We collected from resections done between April, 1997, and February, 2013, at 35 international institutions. We established medulloblastoma subgroup affiliation by gene expression profiling on frozen or formalin-fixed paraffin-embedded tissues. We classified extent of resection on the basis of postoperative imaging as gross total resection (no residual tumour), near-total resection (<1·5 cm(2) tumour remaining), or sub-total resection (≥1·5 cm(2) tumour remaining). We did multivariable analyses of overall survival and progression-free survival using the variables molecular subgroup (WNT, SHH, group 4, and group 3), age (<3 vs ≥3 years old), metastatic status (metastases vs no metastases), geographical location of therapy (North America/Australia vs rest of the world), receipt of chemotherapy (yes vs no) and receipt of craniospinal irradiation (<30 Gy or >30 Gy vs no craniospinal irradiation). The primary analysis outcome was the effect of extent of resection by molecular subgroup and the effects of other clinical variables on overall and progression-free survival. FINDINGS: We included 787 patients with medulloblastoma (86 with WNT tumours, 242 with SHH tumours, 163 with group 3 tumours, and 296 with group 4 tumours) in our multivariable Cox models of progression-free and overall survival. We found that the prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. We identified a progression-free survival benefit for gross total resection over sub-total resection (hazard ratio [HR] 1·45, 95% CI 1·07-1·96, p=0·16) but no overall survival benefit (HR 1·23, 0·87-1·72, p=0·24). We saw no progression-free survival or overall survival benefit for gross total resection compared with near-total resection (HR 1·05, 0·71-1·53, p=0·8158 for progression-free survival and HR 1·14, 0·75-1·72, p=0·55 for overall survival). No significant survival benefit existed for greater extent of resection for patients with WNT, SHH, or group 3 tumours (HR 1·03, 0·67-1·58, p=0·89 for sub-total resection vs gross total resection). For patients with group 4 tumours, gross total resection conferred a benefit to progression-free survival compared with sub-total resection (HR 1·97, 1·22-3·17, p=0·0056), especially for those with metastatic disease (HR 2·22, 1·00-4·93, p=0·050). However, gross total resection had no effect on overall survival compared with sub-total resection in patients with group 4 tumours (HR 1·67, 0·93-2·99, p=0·084). INTERPRETATION: The prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. Although maximum safe surgical resection should remain the standard of care, surgical removal of small residual portions of medulloblastoma is not recommended when the likelihood of neurological morbidity is high because there is no definitive benefit to gross total resection compared with near-total resection. FUNDING: Canadian Cancer Society Research Institute, Terry Fox Research Institute, Canadian Institutes of Health Research, National Institutes of Health, Pediatric Brain Tumor Foundation, and the Garron Family Chair in Childhood Cancer Research.

Pre-treatment VD levels and VDR receptors as potential predictors of occurrence and overall survival in paediatric patients with solid tumours-a single institution pilot study.

Recently, vitamin D has been recognized as an important player in the immune system, and multiple studies suggested its involvement in cancer, too. The aims of this study were to investigate selected single nucleotide polymorphisms (SNPs) in the VDR gene, BsmI (rs1544410; A > G), FokI (rs 2228570; C > T), TaqI (rs731236; T > C), ApaI (rs 7975232; C > T) and Cdx-2 (rs11568820; A > G), and to evaluate their possible predictive role for outcomes in patients with paediatric solid tumours. A total of 111 children with paediatric solid tumours were enrolled at the Department of Paediatric Oncology, University Hospital Brno (Brno, Czech Republic) along with a control population of 787 adults; all study subjects were available for genotyping of selected SNPs, and the prediagnostic levels of 25-hydroxycholecalciferol (25(OH)D3) and 1,25-dihydroxycholecalciferol (1,25(OH)2D3) were measured in the cases, too. In FokI, the heterozygote CT genotype was weakly associated with a decreased risk of paediatric solid cancer occurrence 0.82 (0.53-1.28), while the CC genotype was associated with a decreased risk of 0.58 (0.30-1.09), p = 0.09. The 1,25(OH)2D3 prediagnostic levels were indicative of the overall survival in the cases (ß = -0.012, HR 0.988, 95 % CI (0.978-0.998), while higher prediagnostic levels of 1,25(OH)2D3 were associated with a statistically significant increase in overall mortality. We observed multiple effects of the alleles of the investigated polymorphisms and of 1,25(OH)2D3 on overall survival, regardless of the underlying disease.

Cancer stem cell markers in pediatric sarcomas: Sox2 is associated with tumorigenicity in immunodeficient mice.

The three most frequent pediatric sarcomas, i.e., Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma, were examined in this study: three cell lines derived from three primary tumor samples were analyzed from each of these tumor types. Detailed comparative analysis of the expression of three putative cancer stem cell markers related to sarcomas-ABCG2, CD133, and nestin-was performed on both primary tumor tissues and corresponding cell lines. The obtained results showed that the frequency of ABCG2-positive and CD133-positive cells was predominantly increased in the respective cell lines but that the high levels of nestin expression were reduced in both osteosarcomas and rhabdomyosarcomas under in vitro conditions. These findings suggest the selection advantage of cells expressing ABCG2 or CD133, but the functional tests in NOD/SCID gamma mice did not confirm the tumorigenic potential of cells harboring this phenotype. Subsequent analysis of the expression of common stem cell markers revealed an evident relationship between the expression of the transcription factor Sox2 and the tumorigenicity of the cell lines in immunodeficient mice: the Sox2 levels were highest in the two cell lines that were demonstrated as tumorigenic. Furthermore, Sox2-positive cells were found in the respective primary tumors and all xenograft tumors showed apparent accumulation of these cells. All of these findings support our conclusion that regardless of the expression of ABCG2, CD133 and nestin, only cells displaying increased Sox2 expression are directly involved in tumor initiation and growth; therefore, these cells fit the definition of the cancer stem cell phenotype.

Non-DHFR-mediated effects of methotrexate in osteosarcoma cell lines: epigenetic alterations and enhanced cell differentiation.

BACKGROUND: Methotrexate is an important chemotherapeutic drug widely known as an inhibitor of dihydrofolate reductase (DHFR) which inhibits the reduction of folic acid. DHFR-mediated effects are apparently responsible for its primary antineoplastic action. However, other non-DHFR-mediated effects of methotrexate have been recently discovered, which might be very useful in the development of new strategies for the treatment of pediatric malignancies. The principal goal of this study was to analyze the possible impact of clinically achievable methotrexate levels on cell proliferation, mechanisms of epigenetic regulation (DNA methylation and histone acetylation), induced differentiation and the expression of differentiation-related genes in six osteosarcoma cell lines. METHODS: The Saos-2 reference cell line and five other patient-derived osteosarcoma cell lines were chosen for this study. The MTT assay was used to assess cell proliferation, DNA methylation and histone acetylation were detected using ELISA, and western blotting was used for a detailed analysis of histone acetylation. The expression of differentiation-related genes was quantified using RT-qPCR and the course of cell differentiation was evaluated using Alizarin Red S staining, which detects the level of extracellular matrix mineralization. RESULTS: Methotrexate significantly decreased the proliferation of Saos-2 cells exclusively, suggesting that this reference cell line was sensitive to the DHFR-mediated effects of methotrexate. In contrast, other results indicated non-DHFR-mediated effects in patient-derived cell lines. Methotrexate-induced DNA demethylation was detected in almost all of them; methotrexate was able to lower the level of 5-methylcytosine in treated cells, and this effect was similar to the effect of 5-aza-2'-deoxycytidine. Furthermore, methotrexate increased the level of acetylated histone H3 in the OSA-06 cell line. Methotrexate also enhanced all-trans retinoic acid-induced cell differentiation in three patient-derived osteosarcoma cell lines, and the modulation of expression of the differentiation-related genes was also shown. CONCLUSIONS: Overall non-DHFR-mediated effects of methotrexate were detected in the patient-derived osteosarcoma cell lines. Methotrexate acts as an epigenetic modifier and has a potential impact on cell differentiation and the expression of related genes. Furthermore, the combination of methotrexate and all-trans retinoic acid can be effective as a differentiation therapy for osteosarcoma.

Overexpression of the ∆Np73 isoform is associated with centrosome amplification in brain tumor cell lines.

The p73 protein is a member of the p53 family, and this protein is known to be essential for the maintenance of genomic stability, DNA repair, and apoptosis regulation. Transcription from two promoters leads to two main N-terminal isoforms: the TAp73 isoform is reported to have tumor suppressor function, whereas the ΔNp73 isoform likely has oncogenic potential. The present study is focused on the investigation of a possible role of both these p73 N-terminal isoforms in the process of centrosome amplification. HGG-02 and GM7 glioblastoma cell lines and the Daoy medulloblastoma cell line were used in this study. The cells were analyzed using indirect immunofluorescence to determine TAp73 and ΔNp73 expression patterns and possible co-localization with the BubR1 protein, as well as the number of centrosomes. A transiently transfected GM7 cell line was used to verify the results concerning the N-terminal isoforms in relation to centrosome amplification. We found that increased immunoreactivity for the ΔNp73 isoform is associated with the occurrence of an abnormal number of centrosomes in particular cells. Using the transiently transfected GM7 cell line, we confirmed that centrosome amplification is present in cells with overexpression of the ΔNp73 isoform. In contrast, the immunoreactivity for the TAp73 isoform was weak or medium in most of the cells with an aberrant number of centrosomes. To determine the putative counterpart of the p73 N-terminal isoforms among spindle assembly checkpoint (SAC) proteins, we also evaluated possible interactions between the N-terminal isoforms and BubR1 protein, but no co-localization of these proteins was observed.

Inflammatory myofibroblastic tumor of the esophagus in childhood: a case report and a review of the literature.

Inflammatory myofibroblastic tumor (IMT) is a rare mesenchymal disease of low malignancy consisting of inflammatory cells inside a mesenchymal stroma comprising myofibroblasts. Biological behavior is variable, recurrence is uncommon, metastatic disease is rare. Treatment strategy is based on the status of low-grade malignant tumor. Radical surgery is considered to be the principal treatment modality, except if it requires a mutilating procedure. Only 6 cases of pediatric patients treated for IMT of the esophagus have been reported in English-language literature. Herein, a case of a 13-year-old boy with an IMT of the esophagus is presented.

[The prospects for children with acute lymphoblastic leukemia of being cured has increased in the Czech Republic in the 21st century to 90% - outcome of the ALL-IC BFM 2002 trial]. / Sance na vylécení detí s akutní lymfoblastickou leukémií stoupla v Ceské republicev 21. století na 90 % - výsledky studie ALL IC-BFM 2002.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most frequent childhood malignancy. Treatment has been unified in the middle of 1980 in the Czech Republic. In 2002-2007 children and adolescents with acute lymphoblastic leukemia were treated in an international randomized trial ALL-IC BFM 2002 in the Czech Republic. 291 patients aged 1-18 years were enrolled; infants below 1 year entered a separate trial. METHODS AND RESULTS: Patients were stratified into three risk groups according to their age, initial leukocyte count, prednisone response, presence of fusion genes BCR/ABL or MLL/AF4, bone marrow D+15 and remission status D+33. The whole therapy took 24 months. Randomized late intensification compared standard BFM therapy with extended, usually more intensive experimental treatment. The median follow-up was 8.7 years. Complete remission was achieved in 97.9% patients, 1% died in remission. 11% of children relapsed, 1.7% with CNS involvement. Six children (2.1%) developed secondary malignancy. Event free survival (EFS) 8 years from diagnosis was 83.5%, overall survival (OS) 91.4%. EFS and OS of the risk groups were: standard risk: 89.4%; 98.1%; intermediate risk: 82.6%; 89.6%; high risk: 68.8%; 78.1%. Male sex and age above 10 years were adverse prognostic factors. CONCLUSIONS: In comparison with the previous trial ALL-BFM 95, significant improvement was achieved.

LOX/COX inhibitors enhance the antineoplastic effects of all-trans retinoic acid in osteosarcoma cell lines.

The induced differentiation of tumor cells into mature phenotypes is a promising strategy in cancer therapy. In this study, the effects of combined treatment with all-trans retinoic acid (ATRA) and lipoxygenase/cyclooxygenase inhibitors were examined in two osteosarcoma cell lines, Saos-2 and OSA-01. Caffeic acid and celecoxib were used as inhibitors of 5-lipoxygenase and of cyclooxygenase-2, respectively. Changes in the cell proliferation, matrix mineralization, and occurrence of differentiation markers were evaluated in treated cell populations at intervals. The results confirmed the capability of caffeic acid to enhance the antiproliferative effect of ATRA in both cell lines. In contrast, celecoxib showed the same effect in Saos-2 cells only. Furthermore, the extension of matrix mineralization was observed after combined treatment with ATRA and celecoxib or caffeic acid. The increased expression of osteogenic differentiation markers was observed in both cell lines after the combined application of ATRA and inhibitors. The obtained results clearly demonstrate the capability of lipoxygenase/cyclooxygenase inhibitors to enhance the antiproliferative and differentiating effect of ATRA in osteosarcoma cells, although some of these effects are specific and depend on the biological features of the respective tumor or cell line.