Molecular Classification of Medullobastomas by Whole Genome Expression Profiling.

BACKGROUND: Medulloblastoma (MB) is the most common malignant tumour of the central nervous system in children. MB is considered to be high risk tumour propensity to metastasize. In the Czech Republic, approximately 10-12 children are affected annually by this tumour. Recent progress in molecular diagnostics helps to refine the diagnosis and estimate clinical prognosis of the disease. Currently, MBs are subclassified into WNT-activated, SHH-activated, group 3, and 4 based on molecular pathways that drive their tumorigenesis. Each subtype differs in its histopathology, clinical features, genomic changes and gene expressions. The aim of our study is to classify patients MBs into four basic molecular groups and compare our results with published data. MATERIAL AND METHODS: In our study we analysed expression profiles using Affymetrix GeneChip Human Gene 1.0. ST Array (Thermo Fisher Scientific, MA, USA). As input material RNA extracted from the fresh frozen tissue was used. Molecular classification based on the method established by P. Northcott in 2011 was performed. RESULTS: From April 2015 to February 2019, 21 patients with MBs were included in our study. Median age of the patients at the time of diagnosis was 6 years, 14 boys and 7 girls were enrolled. Gene expression profiling and molecular classification of MBs was performed. Based on this methodology, we found the most frequently represented subgroup of MB was group 4 (9 patients, 43%), followed by group 3 (5 patients, 24%), SHH-activated MB (4 patients, 19%) and the least represented subgroup was WNT-activated MB (3 patients, 14%). Results of molecular subgroup classification of MBs were successfully correlated with histopathological findings and other molecular-genetic examinations. CONCLUSION: Molecular classification of MBs has been established in our institution allowing better understanding of this heterogeneous disease and helping clinicians in therapeutic planning in affected patients. This work was supported by the Czech Ministry of Health grant No. 16-33209A. All rights reserved.  he authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 4. 3. 2019.

Effective Immunotherapy of Glioblastoma in an Adolescent with Constitutional Mismatch Repair-Deficiency Syndrome.

BACKGROUND: Individuals with constitutional mismatch repair-deficiency syndrome (CMMR-D) are characterised by early occurrence of colon cancer, haematological malignancies, and brain tumors (malignant gliomas, high-grade gliomas) in childhood, adolescence, and early adulthood. High mutational tumor burden is typical of glioblastoma in CMMR-D patients and could be a reason why this type of glioblastoma responds well to immunotherapies, including those that employ checkpoint inhibitors. OBSERVATION: We describe a case of an adolescent with CMMR-D that had been genetically proven by whole exome sequencing (c.2T>A/p.M1K and c.2521delT/p.W841fs PMS2 gene mutation). The patient presented successively with colon cancer and glioblastoma with a high mutational burden. The individualized glioblastoma therapy was based on the biological tumor profile and included immunotherapy with a combination of vaccination with autologous dendritic cells producing IL-12 and nivolumab, in addition to radiotherapy with metronomic temozolomide. The patient is still alive 21 months after the initial glioblastoma diagnosis and shows a complete therapeutic response documented by repeated magnetic resonance examinations. CONCLUSION: Individuals with CMMR-D should be regularly examined using established algorithms. Whole body magnetic resonance imaging can play a key role, because it enables the early diagnosis of malignancy during the asymptomatic period. Malignancies in CMMR-D patients usually exhibit a hypermutated genotype and respond to immunotherapy. Conventional glioblastoma therapy is only palliative. Patients can benefit from an individualized therapeutic plan based on the tumor biological profile. Extensive molecular analysis of the tumor tissue is necessary. Key words hereditary cancer predisposition syndromes - glioblastoma - whole exome sequencing - immunotherapy - vaccines - checkpoint inhibitors This study was supported by the research project of the Czech Ministry of Health AZV 16-33209A (Next generation sequencing and express profiling as diagnostic tools for personalized therapeutic plans in children with solid tumors). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 26. 9. 2018 Accepted: 18. 11. 2018.

NEUROFIBROMATOSIS TYPE 1 AND OPTIC PATHWAY GLIOMA.

PURPOSE: Evaluate the effectiveness of treatment of patients with optic pathway glioma. MATERIALS AND METHODS: Comparison of literature research on neurofibromatosis and optic pathway glioma with a cohort of pediatric patients treated at the Childrens Ophthalmology Clinic of the University Hospital in Brno from January 2013 until June 2018. DISCUSSION: The main challenge of this and other retrospective studies is variable intervals between ophthalmologic examinations. In some pediatric patients it is also difficult to objectively assess visual functions. The main risk factors are age at the time of treatment and tumor localization. Tumor progression itself does not always correlate with worse visual acuity outcomes, and it remains to be evaluated whether some patients would be better off without treatment. As of now, there are no clinical biomarkers able to predict impending visual acuity loss. CONCLUSION: The cohort outcome agrees with literature. Chemotherapy remains a treatment of choice and its most likely outcome is visual acuity stabilization. In order to properly evaluate the treatments effectiveness, better collaboration between medical specialists and regular standardized ophthalmology examinations are required.

[Identification of a Family with SUFU Germline Deletion Based on a Case of Desmoplastic Medulloblastoma in an Infant]. / Identifikace rodiny s nosicstvím germinální delece genu SUFU na podklade dia-gnózy desmoplastického meduloblastomu u batolete.

BACKGROUND: Medulloblastoma, an embryonal neuroectodermal tumor of the cerebellum, is the most common malignant brain tumor in children. There are approximately 15 cases diagnosed in the Czech Republic each year. The recent World Health Organization classification recognizes several histopathological subtypes of medulloblastoma: classical, desmoplastic/ nodular with its extensive-nodularity variant, and anaplastic/ large-cell variant. Further molecular analysis identified four basic subgroups of medulloblastoma: WNT, SHH, Group 3, and Group 4. The subgroup of SHH meduloblastoma is associated with somatic mutations of SHH, PTCH1, SUFU, SMO and TP53, while the most common mutations found in infants up to three years of age were PTCH1 and SUFU. The majority of medulloblastomas are sporadic diseases, whereas only about 5- 10% of all cases occur in connection with hereditary genetic syndromes. CASE: We present a case of a 21-months old girl diagnosed with a localized posterior fossa tumor. The histopathological examination revealed a desmoplastic/ nodular medulloblastoma. The treatment comprised a radical exstirpation of the tumor followed by adjuvant chemotherapy. With the use of array-CGH, a partial biallelic deletion of the SUFU gene (locus 10q24.32) was detected in the tumor DNA, whereas a monoallelic deletion was found in the peripheral lymphocyte DNA of the patient. These findings were confirmed by an independent qPCR method. Monoallelic germline deletion of SUFU was also identified in the patients mother, who was a healthy carrier. Pedigree of the family suggested a transition of the germline deletion of SUFU, since another brain tumors (including one case diagnosed before the age of three years) were identified in previous generations. CONCLUSION: Germline mutations in SUFU gene are believed to predispose to infant desmoplastic/ nodular medulloblastomas, basal cell carcinomas and meningiomas. The susceptibility gene shows autosomal dominant inheritance with an incomplete penetrance. There is no evidence-based surveillance strategy suggested for the carriers of germline SUFU mutations/ deletions so far. Our recommendation is based both on a family history of our patient and similar cases described in the literature. Since the germinal mutations in SUFU are responsible for up to 50% of all desmoplastic medulloblastomas in children under three years of age, genetic testing of SUFU should be encouraged in this population of patients.

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.

[Radiotherapy induced glioblastoma in a child previously treated for cerebellar medulloblastoma (case report and review of the literature)]. / Radioterapií indukovaný glioblastom u dítete léceného pro meduloblastom mozecku (kasuistika a prehled literatury).

Malignant gliomas represent the most frequent radiotherapy induced ("secondary") solid tumor. Their prognosis remains extremely poor despite of aggressive multimodal treatment. We present a case report of a 16 years old boy who developed cerebellar glioblastoma six years following the combined treatment for medulloblastoma. Clinical history, pathological and cytogenetic findings of the case are discussed along with treatment possibilities.

Longterm treatment results of childhood medulloblastoma by craniospinal irradiation in supine position.

Medulloblastoma, a primitive neuroectodermal tumor growing in cerebellum, is one of the most sensitive to radiation therapy childhood brain tumors. The radiotherapy is an essential method of treatment for these tumours, but the surgery is the primary treatment of choice in medulloblastoma. I this study between January 1997 and March 2005 were post-operative irradiated a total number of 33 pediatric patients aged under 15 years (median age 8.7 years) with medulloblastoma. All tumors were histologically proved and were localizated infratentorially in the posterior fossa. All of the patients were irradiated with a dose of 24-36 Gy to the whole craniospinal axis and boost with conformal therapy restricted to the tumor bed to the total dose of 50-54 Gy (30-36 Gy "high risk", 24-30 Gy "standard risk" group). Chemotherapy received 26 patients (78%). Patients with craniospinal irradiation were placed in supine position and fixed by a vacuum-form body immobilizer and head mask. Irradiation was performed using standard fractionation (5 fractions per week) with a single dose of 1.5-1.8 Gy for craniospinal axis by photon beam (6 MV) of the linear accelerator. The median overall survival for the whole group was 55.3 months. The median of disease-free survival was 20.6 months, 8 patients (24%) died. In our study the statistical difference in survival rate between standard and high-risk patients with medulloblastoma was not shown. No relationship was found between survival and age, sex or tumor size. Endocrine deficits occurred in 45% (8 patients of the group were hypothyroid, 6 patients needed growth hormone replacement therapy, 1 patient had early puberty). This results (results of overall and disease-free survival) and side-effects of technique of craniospinal axis irradiation in supine position are comparable with results of technique in prone position. Further evaluation of the effectiveness of our therapy is not feasible due to the small number of patients.

Concomitant radiotherapy and metronomic temozolomide in pediatric high-risk brain tumors.

Temozolomide, an oral alkylating agent has a significant activity in preclinical testing and in clinical trials in adults and children as well. Penetration across the blood brain barrier has been documented. In adult and pediatric phase I and II trials a five-day every 28 days schedule was first approved for clinical use. With respect to temozolomide proximal mechanism of resistance, and further to increase dose intensity, new schedules are proposed to use more prolonged drug exposure. Higher doses of metronomic temozolomide were piloted. Eight children with poor prognosis brain tumors were eligible. Treatment consisted from concomitant radiotherapy given 1x170 cGy, 5d/wk, for total dose 55/56 Gy, together with temozolomide 90 mg/m2/day for 42 days. No further dose escalation has been planned for this group of patients. Myelosuppression was the primary toxicity, occurring around day 21. Nonhematologic toxicities were infrequent and in no case dose limiting toxicity (DLT) occurred. The most common nonhematologic toxicity was vomiting, effectively managed with antiemetics. Six responses were documented. The best responses (CR) were seen in 2 patients with high-risk medulloblastomas, who have progressed early after neurosurgery. Furthermore, one more patient had CR and 3 patients PR at the end of temozolomide treatment. We have piloted novel dose schedule of temozolomide and evaluated clinical toxicities in a cohort of 8 children. This is the first study to report feasibility and tolerability of 90 mg/m2/day of temozolomide treatment in metronomic fashion. In addition, we have documented encouraging responses in children with medulloblastomas, progressing early after their initial surgery.