Jaroslav Sterba

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.

Intertumoral Heterogeneity within Medulloblastoma Subgroups

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

Combined biodifferentiating and antiangiogenic oral metronomic therapy is feasible and effective in relapsed solid tumors in children: single-center pilot study.

To outline an outpatient-based treatment for children with relapsed solid tumors, who already have been extensively pretreated, we defined a 4-drug protocol named COMBAT (combined oral maintenance biodifferentiating and antiangiogenic therapy). Using this protocol, we performed a pilot study to determine its feasibility in children with relapsed and/or high-risk pediatric solid tumors. Patients and Methods: 22 children received the COMBAT protocol. Treatment consisted of daily celecoxib administration along with daily 13-cisretinoic acid (2 weeks on / 2 weeks off) and cycles of metronomic temozolomide (90 mg/m2 for 42 days) and low-dose etoposide (21 days). The treatment was scheduled for a period of 1 year. Results: 9 of the 14 patients assessable for response demonstrated evidence of treatment benefit manifested as prolonged disease stabilization or response. The protocol medication was well tolerated with very good compliance. Only minimal side effects where observed which responded to dose modification or local therapy. Conclusions: The COMBAT regimen is well tolerated by patients with intensive prior therapy including myeloablative regimens. Favorable responses observed in this cohort of patients support the further exploration of this and/or similar strategies in the treatment of pediatric solid tumors.

Efficacy of pregabalin in neuropathic pain in paediatric oncological patients

OBJECTIVE To evaluate the safety and efficacy of pregabalin in the management of chemotherapy-induced neuropathic pain in patients with childhood solid tumors and leukaemia. MATERIALS AND METHODS In an open-label study, 30 children (11 boys and 19 girls; mean age 13.5 years) who were treated for solid tumors and leukaemia, and developed a painful peripheral neuropathy, were medicated with pregabalin in the daily dose of 150-300 mg for 8 weeks. RESULTS Twenty-eight patients completed the 8-week follow-up. A significant and long-lasting pain relief was noted in 86% of these patients. Median VAS score decreased by 59% at the 8th week from baseline. Adverse effects were infrequent and transient. CONCLUSION The treatment with pregabalin resulted in a significant improvement in pain symptoms. The use of pregabalin in children is off-label so far. However, this drug seems to be a safe and effective remedy, which could significantly broaden the therapeutic spectrum in paediatric oncological patients suffering from neuropathic pain.

Expression of immune-modulatory molecules HLA-G and HLA-E by tumor cells in glioblastomas: An unexpected prognostic significance?

The role of nonclassical human leukocyte antigens G and E (HLA‐G and HLA‐E) was originally thought to be restricted to the protection of the fetus from a maternal allorecognition. Now it is known that HLA‐G and HLA‐E exert multiple immunoregulatory functions. A prognostic significance of the expression of HLA‐G and HLA‐E by neoplastic cells in glioblastoma is not well characterized. In this study, we evaluated the expression of HLA‐G and HLA‐E by neoplastic cells in 39 cases of glioblastoma. We found the production of HLA‐G and HLA in a majority of cases. There was an unexpected positive correlation between the expression of HLA‐E and length of survival. We speculate that the expression of this molecule by neoplastic cells may represent a coincidental selective pro‐host advantage related to better response to subsequent therapeutic modalities. Mechanisms of glioblastoma cell pathophysiology and mechanisms of responses to therapeutic interventions in respect to the expression of these molecules deserves further study.

Nestin expression in osteosarcomas and derivation of nestin/CD133 positive osteosarcoma cell lines

BackgroundNestin was originally identified as a class VI intermediate filament protein that is expressed in stem cells and progenitor cells in the mammalian CNS during development. This protein is replaced in the adult organism by other intermediate filament proteins; however, nestin may be re-expressed under certain pathological conditions such as ischemia, inflammation, brain injury, and neoplastic transformation. Nestin has been detected in many kinds of tumors, especially in tumors derived from the CNS. Co-expression of nestin and the CD133 surface molecule is considered to be a marker for cancer stem cells in neurogenic tumors. Our work was aimed at a detailed study of nestin expression in osteosarcomas and osteosarcoma-derived cell lines.MethodsUsing immunodetection methods, we examined nestin in tumor tissue samples from 18 patients with osteosarcomas. We also successfully established permanent cell lines from the tumor tissue of 4 patients and immunodetection of nestin and CD133 was performed on these cell lines.ResultsNestin-positive tumor cells were immunohistochemically detected in all of the examined osteosarcomas, but the proportion of these cells that were positively stained as well as the intensity of staining varied. Nestin-positive cells were rarely observed in 2 tumor samples, and the remaining 16 tumor samples showed various nestin expression patterns ranging from very sporadic occurrence to an overwhelming proportion of cells with strong positive staining. Three of the established osteosarcoma cell lines were demonstrated to be nestin-positive, and only one cell line showed no expression of nestin; this finding corresponds with the rare occurrence of nestin-positive cells in the respective tumor sample. Moreover, three of these osteosarcoma cell lines were undoubtedly proven to be Nes+/CD133+.ConclusionOur results represent the first evidence of nestin expression in osteosarcomas and suggest the possible occurrence of cells with a stem-like phenotype in these tumors.

Metronomic Chemotherapy with the COMBAT Regimen in Advanced Pediatric Malignancies: A Multicenter Experience

Background: The outcome of children with refractory/relapsed malignancies remains poor and novel therapies are urgently required. One of the promising approaches is metronomic chemotherapy. We present the clinical results of 74 children with advanced solid tumors treated according to treatment recommendation with data registry in three European pediatric centers. Methods: COMBAT (Combined Oral Metronomic Biodifferentiating Antiangiogenic Treatment) included low-dose daily temozolomide, etoposide, celecoxib, vitamin D, fenofibrate and retinoic acid. From 2004 to 2010, 74 children were enrolled. Results: The 2-year overall survival (OS) was 43.1% (median 15.4, range 1.3–69.9 months). Of the 74 patients, 50 patients (68%) died and 24 are alive: 6 (8%) with progressive disease, 7 (9%) with stable disease/partial response and 11 (15%) in complete response. Median time to response was 6 months. Of 62 patients with initially measurable disease, 25 (40%) had radiological response or stable disease. Fourteen of 25 showing clinical benefit responded within the first 6 months. The treatment was well tolerated on an outpatient basis. Regarding non-hematological toxicity of grade ≥2, hepatotoxicity of grade 3 occurred in 8 children and grade 3 cheilitis in 16 children. Conclusion: COMBAT is a feasible and effective treatment option for patients with relapsing/refractory malignancies. The treatment is well tolerated with a low acute toxicity profile.

Analysis of nuclear nestin localization in cell lines derived from neurogenic tumors

Nestin is a class VI intermediate filament protein expressed in the cytoplasm of stem and progenitor cells in the mammalian CNS during development. In adults, nestin is present only in a small subset of cells and tissues, including the subventricular zone of the adult mammalian brain, where neurogenesis occurs. Nestin expression has also been detected under such pathological conditions as ischemia, inflammation, and brain injury, as well as in various types of human solid tumors and their corresponding cell lines. Furthermore, nestin was recently found in the nuclei of glioblastoma, neuroblastoma, and angiosarcoma cells and it was proved to interact directly with the nuclear DNA in neuroblastoma cells. Here, we perform the first study of the intracellular distribution of nestin in cell lines derived from neurogenic tumors. Using immunodetection methods, we examined nestin expression in tumor-derived cell lines obtained from 11 patients with neuroblastoma, medulloblastoma, or glioblastoma multiforme. Besides its standard cytoplasmic localization, nestin was present in the nuclei of two neuroblastoma cell lines and one medulloblastoma cell line. Nestin was only present in the nuclei of cells with diffuse cytoplasmic staining for this protein, and the proportion of cells positive for nestin in nuclei, as well as the intensity of staining, varied. The presence of nestin in the nuclei was confirmed by both transmission electron microscopy and Western blotting. Our results indicate that the presence of nestin in the nuclei of tumor cells is not very rare, especially under in vitro conditions.

CD133 Expression and Identification of CD133/nestin Positive Cells in Rhabdomyosarcomas and Rhabdomyosarcoma Cell Lines

Background: Co-expression of CD133, cell surface glycoprotein, and nestin, an intermediate filament protein, was determined to be a marker of neural stem cells and of cancer stem cells in neurogenic tumors. Methods: We examined the expression of CD133 and nestin in ten tumor tissue samples taken from patients with rhabdomyosarcomas and in five rhabdomyosarcoma cell lines. Immunohistochemistry and immunofluorescence were used to examine FFPE tumor tissue samples. Cell lines were analyzed by immunofluorescence, immunoblotting, flow cytometry, and RT-PCR. Functional assays (clonogenic in vitro assay and tumorigenic in vivo assay) were also performed using these cell lines. Results: CD133 and nestin were detected in all 10 tumor tissue samples and in all 5 cell lines; however, the frequency of CD133+, Nes+, and CD133+/Nes+ cells, as well as the intensity of fluorescence varied in individual samples or cell lines. The expression of CD133 and nestin was subsequently confirmed in all cell lines by immunoblotting. Furthermore, we observed an increasing expression of CD133 in relation to the cultivation. All cell lines were positive for Oct3/4 and nucleostemin; NSTS-11 cells were also able to form xenograft tumors in mice. Conclusion: Our results represent the first evidence of CD133 expression in rhabdomyosarcoma tissue and in rhabdomyosarcoma cell lines. In addition, the co-expression of CD133 and nestin as well as results of the functional assays suggest a possible presence of cancer cells with a stem-like phenotype in these tumors.

Second complete remission of relapsed medulloblastoma induced by metronomic chemotherapy.

Prognosis for children with relapsed medulloblastoma remains poor. Metronomic chemotherapy may offer some benefit to patients treated initially with intensive regimens. However, dosing and duration of such palliative treatment have not been systematically studied. Here we describe a child with medulloblastoma relapsing after initial high‐dose chemotherapy and standard radiotherapy. The patient was then treated with metronomic chemotherapy and achieved second complete remission after 21 months of treatment. Three months off therapy he relapsed again and died from progressive disease. This case illustrates the potential benefit of metronomic chemotherapy but also shows the uncertainty of when to stop metronomic chemotherapy while balancing toxicity. Pediatr Blood Cancer 2010;54:616–617.