Summary Background International consensus recognises four medulloblastoma molecular subgroups: WNT (MBWNT ), SHH (MBSHH ), group 3 (MBGrp3 ), and group 4 (MBGrp4 ), each defined by their ...characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions. Methods In this retrospective cohort study, we assessed 428 primary medulloblastoma samples collected from UK Children's Cancer and Leukaemia Group (CCLG) treatment centres (UK), collaborating European institutions, and the UKCCSG-SIOP-PNET3 European clinical trial. An independent validation cohort (n=276) of archival tumour samples was also analysed. We analysed samples from patients with childhood medulloblastoma who were aged 0–16 years at diagnosis, and had central review of pathology and comprehensive clinical data. We did comprehensive molecular profiling, including DNA methylation microarray analysis, and did unsupervised class discovery of test and validation cohorts to identify consensus primary molecular subgroups and characterise their clinical and biological significance. We modelled survival of patients aged 3–16 years in patients (n=215) who had craniospinal irradiation and had been treated with a curative intent. Findings Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified. MBWNT remained unchanged and each remaining consensus subgroup was split in two. MBSHH was split into age-dependent subgroups corresponding to infant (<4·3 years; MBSHH-Infant ; n=65) and childhood patients (≥4·3 years; MBSHH-Child ; n=38). MBGrp3 and MBGrp4 were each split into high-risk (MBGrp3-HR n=65 and MBGrp4-HR n=85) and low-risk (MBGrp3-LR n=50 and MBGrp4-LR n=73) subgroups. These biological subgroups were validated in the independent cohort. We identified features of the seven subgroups that were predictive of outcome. Cross-validated subgroup-dependent survival models, incorporating these novel subgroups along with secondary clinicopathological and molecular features and established disease risk-factors, outperformed existing disease risk-stratification schemes. These subgroup-dependent models stratified patients into four clinical risk groups for 5-year progression-free survival: favourable risk (54 25% of 215 patients; 91% survival 95% CI 82–100); standard risk (50 23% patients; 81% survival 70–94); high-risk (82 38% patients; 42% survival 31–56); and very high-risk (29 13% patients; 28% survival 14–56). Interpretation The discovery of seven novel, clinically significant subgroups improves disease risk-stratification and could inform treatment decisions. These data provide a new foundation for future research and clinical investigations. Funding Cancer Research UK, The Tom Grahame Trust, Star for Harris, Action Medical Research, SPARKS, The JGW Patterson Foundation, The INSTINCT network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK).
Historical risk stratification criteria for medulloblastoma rely primarily on clinicopathological variables pertaining to age, presence of metastases, extent of resection, histological subtypes and ...in some instances individual genetic aberrations such as
MYC
and
MYCN
amplification. In 2010, an international panel of experts established consensus defining four main subgroups of medulloblastoma (WNT, SHH, Group 3 and Group 4) delineated by transcriptional profiling. This has led to the current generation of biomarker-driven clinical trials assigning WNT tumors to a favorable prognosis group in addition to clinicopathological criteria including
MYC
and
MYCN
gene amplifications. However, outcome prediction of non-WNT subgroups is a challenge due to inconsistent survival reports. In 2015, a consensus conference was convened in Heidelberg with the objective to further refine the risk stratification in the context of subgroups and agree on a definition of risk groups of non-infant, childhood medulloblastoma (ages 3–17). Published and unpublished data over the past 5 years were reviewed, and a consensus was reached regarding the level of evidence for currently available biomarkers. The following risk groups were defined based on current survival rates: low risk (>90 % survival), average (standard) risk (75–90 % survival), high risk (50–75 % survival) and very high risk (<50 % survival) disease. The WNT subgroup and non-metastatic Group 4 tumors with whole chromosome 11 loss or whole chromosome 17 gain were recognized as low-risk tumors that may qualify for reduced therapy. High-risk strata were defined as patients with metastatic SHH or Group 4 tumors, or
MYCN
-amplified SHH medulloblastomas. Very high-risk patients are Group 3 with metastases or SHH with
TP53
mutation. In addition, a number of consensus points were reached that should be standardized across future clinical trials. Although we anticipate new data will emerge from currently ongoing and recently completed clinical trials, this consensus can serve as an outline for prioritization of certain molecular subsets of tumors to define and validate risk groups as a basis for future clinical trials.
Neuroblastoma is a paediatric cancer that despite multimodal therapy still has a poor outcome for many patients with high risk tumours. Retinoic acid (RA) promotes differentiation of some ...neuroblastoma tumours and cell lines, and is successfully used clinically, supporting the view that differentiation therapy is a promising strategy for treatment of neuroblastoma. To improve treatment of a wider range of tumour types, development and testing of novel differentiation agents is essential. New pre-clinical models are therefore required to test therapies in a rapid cost effective way in order to identify the most useful agents.
As a proof of principle, differentiation upon ATRA treatment of two MYCN-amplified neuroblastoma cell lines, IMR32 and BE2C, was measured both in cell cultures and in tumours formed on the chick chorioallantoic membrane (CAM). Differentiation was assessed by 1) change in cell morphology, 2) reduction in cell proliferation using Ki67 staining and 3) changes in differentiation markers (STMN4 and ROBO2) and stem cell marker (KLF4). Results were compared to MLN8237, a classical Aurora Kinase A inhibitor. For the in vivo experiments, cells were implanted on the CAM at embryonic day 7 (E7), ATRA treatment was between E11 and E13 and tumours were analysed at E14.
Treatment of IMR32 and BE2C cells in vitro with 10 μM ATRA resulted in a change in cell morphology, a 65% decrease in cell proliferation, upregulation of STMN4 and ROBO2 and downregulation of KLF4. ATRA proved more effective than MLN8237 in these assays. In vivo, 100 μM ATRA repetitive treatment at E11, E12 and E13 promoted a change in expression of differentiation markers and reduced proliferation by 43% (p < 0.05). 40 μM ATRA treatment at E11 and E13 reduced proliferation by 37% (p < 0.05) and also changed cell morphology within the tumour.
Differentiation of neuroblastoma tumours formed on the chick CAM can be analysed by changes in cell morphology, proliferation and gene expression. The well-described effects of ATRA on neuroblastoma differentiation were recapitulated within 3 days in the chick embryo model, which therefore offers a rapid, cost effective model compliant with the 3Rs to select promising drugs for further preclinical analysis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To compare event-free survival (EFS), overall survival (OS), pattern of relapse, and hearing loss in children with standard-risk medulloblastoma treated by postoperative hyperfractionated or ...conventionally fractionated radiotherapy followed by maintenance chemotherapy.
In all, 340 children age 4 to 21 years from 122 European centers were postoperatively staged and randomly assigned to treatment with hyperfractionated radiotherapy (HFRT) or standard (conventional) fractionated radiotherapy (STRT) followed by a common chemotherapy regimen consisting of eight cycles of cisplatin, lomustine, and vincristine.
After a median follow-up of 4.8 years (range, 0.1 to 8.3 years), survival rates were not significantly different between the two treatment arms: 5-year EFS was 77% ± 4% in the STRT group and 78% ± 4% in the HFRT group; corresponding 5-year OS was 87% ± 3% and 85% ± 3%, respectively. A postoperative residual tumor of more than 1.5 cm(2) was the strongest negative prognostic factor. EFS of children with all reference assessments and no large residual tumor was 82% ± 2% at 5 years. Patients with a delay of more than 7 weeks to the start of RT had a worse prognosis. Severe hearing loss was not significantly different for the two treatment arms at follow-up.
In this large randomized European study, which enrolled patients with standard-risk medulloblastoma from more than 100 centers, excellent survival rates were achieved in patients without a large postoperative residual tumor and without RT treatment delays. EFS and OS for HFRT was not superior to STRT, which therefore remains standard of care in this disease.
Medulloblastoma is the most common malignant brain tumour of childhood and accounts for around 10% of all childhood cancer deaths. Despite recent improvements in survival rates, the delivery of ...individualised therapies based on disease-risk remains a major goal; intensified treatment for poor-risk disease, whilst reducing therapy for favourable-risk cases, with the overall aim of maximising survival whilst minimising late effects. Current clinical indices for the prediction of disease course are imprecise, however a series of molecular and histopathological biomarkers have been identified recently, which may allow a more accurate prediction of disease outcome (e.g., β-catenin status as a favourable-risk marker, MYC gene amplification and large-cell histology as high-risk markers). Pan-European clinical trials being planned for medulloblastoma by the SIOP Brain tumour group will assess the stratification of patients using molecular and histological biomarkers, alongside clinical indices, to select favourable, standard and high-risk treatment groups. This selection will underpin two concurrent trials; PNET 5, which will test whether treatment can be reduced for a favourable-risk disease sub-group, with the aim of maintaining survival rates while reducing late-effects, and PNET 6, which will aim to improve survival rates in the standard-risk group. The implementation of these trials presents important new logistical challenges within routine practice, involving (i) the development of quality-controlled sample collection and handling systems across multiple treatment centres, including the mandatory ascertainment of fresh-frozen tumour material, and (ii) the delivery of standardised central biomarker analysis and histopathological review, within the approximately 30-day post-surgical window, prior to the selection and commencement of adjuvant therapy. Feasibility studies to establish these systems are underway across SIOP Europe national groups. Their success will require a coordinated approach by the entire multidisciplinary team, including neurosurgeons, oncologists and neuropathologists, with the common aim of facilitating targeted delivery of individualised risk-adapted therapies for children with medulloblastoma.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Solid tumours are less oxygenated than normal tissues. This is called tumour hypoxia and leads to resistance to radiotherapy and chemotherapy. The molecular mechanisms underlying such resistance have ...been investigated in a range of tumour types, including the adult brain tumours glioblastoma, yet little is known for paediatric brain tumours. Medulloblastoma (MB) is the most common malignant brain tumour in children. We aimed to elucidate the impact of hypoxia on the sensitivity of MB cells to chemo- and radiotherapy.
We used two MB cell line (D283-MED and MEB-Med8A) and a widely used glioblastoma cell line (U87MG) for comparison. We applied a range of molecular and cellular techniques to measure cell survival, cell cycle progression, protein expression and DNA damage combined with a transcriptomic micro-array approach in D283-MED cells, for global gene expression analysis in acute and chronic hypoxic conditions.
In D283-MED and U87MG, chronic hypoxia (5 days), but not acute hypoxia (24 h) induced resistance to chemotherapy and X-ray irradiation. This acquired resistance upon chronic hypoxia was present but less pronounced in MEB-Med8A cells. Using transcriptomic analysis in D283-MED cells, we found a large transcriptional remodelling upon long term hypoxia, in particular the expression of a number of genes involved in detection and repair of double strand breaks (DSB) was altered. The levels of Nibrin (NBN) and MRE11, members of the MRN complex (MRE11/Rad50/NBN) responsible for DSB recognition, were significantly down-regulated. This was associated with a reduction of Ataxia Telangiectasia Mutated (ATM) activation by etoposide, indicating a profound dampening of the DNA damage signalling in hypoxic conditions. As a consequence, p53 activation by etoposide was reduced, and cell survival enhanced. Whilst U87MG shared the same dampened p53 activity, upon chemotherapeutic drug treatment in chronic hypoxic conditions, these cells used a different mechanism, independent of the DNA damage pathway.
Together our results demonstrate a new mechanism explaining hypoxia-induced resistance involving the alteration of the response to DSB in D283-MED cells, but also highlight the cell type to cell type diversity and the necessity to take into account the differing tumour genetic make-up when considering re-sensitisation therapeutic protocols.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Central nervous system germ cell tumours (CNS GCT) form a diverse group of tumour entities, including germinoma, yolk sac tumour, embryonal carcinoma, choriocarcinoma, teratoma, and mixed tumours. ...Incidence peaks in the second decade, predominantly in males. Incidence rates vary globally, higher in Asia, suggesting genetic factors are important. CNS GCTs split into pure germinomas and non-germinomatous GCTs (NGGCT), influencing prognosis/treatment. Serum and CSF markers (alpha-fetoprotein, human chorionic gonadotropin) aid diagnosis, potentially avoiding neurosurgical biopsy. Histological features are distinguished by immunohistochemical staining. Studies have identified specific microRNAs in serum/CSF at diagnosis as promising biomarkers. Mutated pathways have been identified, but targeted therapies have shown limited success to date. Diagnosis involves recognising symptoms like raised intracranial pressure, endocrinological, and ophthalmological disturbances. MRI imaging is crucial for diagnosis and guiding treatment decisions. Treatment strategies vary, as pure germinomas respond well to chemotherapy and radiotherapy, or craniospinal radiotherapy alone, with excellent outcomes; in contrast NGGCTs demand aggressive combined chemo-radiotherapy, yielding generally inferior outcomes. Teratomas are typically chemo-/radio-resistant, requiring surgical intervention. Relapses need re-staging and (re-)biopsy consideration. Relapsed germinomas, though rare, may be cured with standard-dose chemotherapy and re-irradiation, or high-dose chemotherapy with stem-cell-transplantation, with/without further radiation. The more commonly observed NGGCT relapses have poor prognosis, even with thiotepa-based high-dose chemotherapy and stem-cell-transplantation delivered with curative intent. In summary, CNS GCT management integrates clinical, radiological, and histological findings, along with serum and CSF markers, for tailored treatment. Ongoing research aims to incorporate microRNA markers and molecular pathology for improved diagnosis, prognostication, and therapeutic intervention.
•Central nervous system (CNS) germ cell tumours (GCT) are a diverse group of neoplasms.•CNS GCTs comprise pure germinoma, nongerminomatous GCT (NGGCT), and pure teratoma.•Serum & cerebrospinal fluid (CSF) markers AFP and HCG aid diagnosis/prognostication.•Typical radiology and markers above thresholds may avoid need for surgical biopsy.•Pure germinomas are sensitive to chemo- and/or radio-therapy with > 90% cure rates.•For NGGCTs, more aggressive combined chemo-radiotherapy is required for cure.•Teratomas typically require surgical intervention only.•Multidisciplinary team working is essential for patients with CNS GCTs.
Chemotherapeutic drug resistance and relapse remains a major challenge for paediatric (medulloblastoma) and adult (glioblastoma) brain tumour treatment. Medulloblastoma tumours and cell lines with ...mutations in the p53 signalling pathway have been shown to be specifically insensitive to DNA damaging agents. The aim of this study was to investigate the potential of triggering cell death in p53 mutated medulloblastoma cells by a direct activation of pro-death signalling downstream of p53 activation. Since non-coding microRNAs (miRNAs) have the ability to fine tune the expression of a variety of target genes, orchestrating multiple downstream effects, we hypothesised that triggering the expression of a p53 target miRNA could induce cell death in chemo-resistant cells. Treatment with etoposide, increased miR-34a levels in a p53-dependent fashion and the level of miR-34a transcription was correlated with the cell sensitivity to etoposide. miR-34a activity was validated by measuring the expression levels of one of its well described target: the NADH dependent sirtuin1 (SIRT1). Whilst drugs directly targeting SIRT1, were potent to trigger cell death at high concentrations only, introduction of synthetic miR-34a mimics was able to induce cell death in p53 mutated medulloblastoma and glioblastoma cell lines. Our results show that the need of a functional p53 signaling pathway can be bypassed by direct activation of miR-34a in brain tumour cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects ...commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.
Display omitted
•Combined P53 and MYC family defects emerge at medulloblastoma relapse•P53-MYC defects are a biomarker for rapidly progressive relapsed disease•Trp53 and MYCN interact to drive aggressive medulloblastoma development in mice•Targeting MYCN or P53 pathway reactivation reduces tumor growth and prolongs survival
Hill et al. find that coincident MYC amplifications and p53 pathway defects are common in relapsed medulloblastoma (MB) and correlate with poor postrelapse prognosis. The authors go on to explore this MYC-p53 interaction in a mouse MB model and show that these tumors are dependent on both aberrations.
PDF
