[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The outcome of patients with anaplastic gliomas varies considerably. Whether a molecular classification of anaplastic gliomas based on large-scale genomic or epigenomic analyses is superior to histopathology for reflecting distinct biological groups, predicting outcomes and guiding therapy decisions has yet to be determined. Epigenome-wide DNA methylation analysis, using a platform which also allows the detection of copy-number aberrations, was performed in a cohort of 228 patients with anaplastic gliomas (astrocytomas, oligoastrocytomas, and oligodendrogliomas), including 115 patients of the NOA-04 trial. We further compared these tumors with a group of 55 glioblastomas. Unsupervised clustering of DNA methylation patterns revealed two main groups correlated with IDH status: CpG island methylator phenotype (CIMP) positive (77.5 %) or negative (22.5 %). CIMP(pos) (IDH mutant) tumors showed a further separation based on copy-number status of chromosome arms 1p and 19q. CIMP(neg) (IDH wild type) tumors showed hallmark copy-number alterations of glioblastomas, and clustered together with CIMP(neg) glioblastomas without forming separate groups based on WHO grade. Notably, there was no molecular evidence for a distinct biological entity representing anaplastic oligoastrocytoma. Tumor classification based on CIMP and 1p/19q status was significantly associated with survival, allowing a better prediction of outcome than the current histopathological classification: patients with CIMP(pos) tumors with 1p/19q codeletion (CIMP-codel) had the best prognosis, followed by patients with CIMP(pos) tumors but intact 1p/19q status (CIMP-non-codel). Patients with CIMP(neg) anaplastic gliomas (GBM-like) had the worst prognosis. Collectively, our data suggest that anaplastic gliomas can be grouped by IDH and 1p/19q status into three molecular groups that show clear links to underlying biology and a significant association with clinical outcome in a prospective trial cohort.
[Show abstract][Hide abstract] ABSTRACT: Genomics has illuminated the extensive intertumoural heterogeneity of medulloblastoma and identified at least four distinct molecular subgroups of the disease. Group 3 and Group 4 subgroup medulloblastomas account for the majority of pediatric cases, yet, oncogenic drivers for these subtypes remain poorly understood. Exome and genome sequencing studies have confirmed a paucity of recurrent gene-level mutations in Group 3 and Group 4, suggesting that alternative oncogenic mechanisms must account for the large fraction of cases that cannot currently be explained by single-nucleotide variants or insertions/deletions alone.
[Show abstract][Hide abstract] ABSTRACT: Although childhood malignancies have become curable in about 75% of cases due to empirically developed multi-modal therapeutic concepts applied in nation-wide collaborative trials, for children with a relapse, cure remains the exception. In the framework of the ICGC project PedBrain many new potentially druggable genetic lesions have been identified. However, it will not be feasible to conduct traditional phase I trials for all these new drugs in these overall rare entities. To still have our young patients participating in the recent advances in molecular targeted drug treatment, we initiated a novel innovative way of introducing these drugs in a clinical setting based on an individualized molecular rationale, a concept called INFORM (INdividualized therapy For Relapsed Malignancies in childhood).
[Show abstract][Hide abstract] ABSTRACT: According to the current WHO classification of CNS tumors, childhood CNS primitive neuro-ectodermal tumors (CNS-PNETs; WHO °IV) are poorly differentiated embryonal tumors with early onset and aggressive clinical behavior. Histological diagnosis can be complicated by morphological heterogeneity and divergent differentiation. Recent studies suggest the existence of molecular subgroups of CNS-PNETs sharing biological characteristics with other childhood CNS tumors. Here, we aimed at a comprehensive molecular characterization of CNS-PNETs and compared our results to profiles of other brain tumor entities in order to define the biological nature of tumors diagnosed as CNS-PNETs.
[Show abstract][Hide abstract] ABSTRACT: The current World Health Organisation (WHO) classification of central nervous system tumors comprises over 100 entities. Most of these are defined by purely histological criteria, with varying and sometimes overlapping spectra. Histological diagnosis is often challenging, however, especially in cases with limited or non-representative biopsy material. Thus, molecular technologies that can complement standard pathology testing have the potential to greatly enhance diagnostic precision and improve clinical decision-making. DNA methylation profiling, acting as a 'fingerprint' of cellular origin and molecular alterations, is one such promising technology.
[Show abstract][Hide abstract] ABSTRACT: Massive parallel sequencing (MPS) technologies have paved the way into new areas of research including individualized medicine. However, sequencing of trace amounts of DNA or RNA still remains a major challenge, especially for degraded nucleic acids like circulating DNA. This together with high cost and time requirements impedes many important applications of MPS in medicine and fundamental science. We have established a fast, cheap and highly efficient protocol called 'Capture and Amplification by Tailing and Switching' (CATS) to directly generate ready-to-sequence libraries for MPS from nanogram and picogram quantities of both DNA and RNA. Furthermore, those DNA libraries are strand-specific, can be prepared within 2-3 h and do not require preliminary sample amplification steps. To exemplify the capacity of the technique, we have generated and sequenced DNA libraries from hundred-picogram amounts of circulating nucleic acids isolated from human blood plasma, one nanogram of mRNA-enriched total RNA from cultured cells and few nanograms of bisulfite-converted DNA. The approach for DNA library preparation from minimal and fragmented input described here will find broad application in diverse research areas such as translational medicine including therapy monitoring, prediction, prognosis and early detection of various human disorders and will permit high-throughput DNA sequencing from previously inaccessible material such as minute forensic and archeological samples.
[Show abstract][Hide abstract] ABSTRACT: Medulloblastoma is a malignant embryonal brain tumor with highly variable outcome. In order to study the biology of this tumor and to perform preclinical treatment studies, a lot of effort has been put into the generation of appropriate mouse models. The usage of these models, however, has become debatable with the advances in human medulloblastoma subgrouping. This study brings together multiple relevant mouse models and matches genetic alterations and gene expression data of 140 murine tumors with 423 human medulloblastomas in a global way. Using AGDEX analysis and k-means clustering, we show that the Blbp-cre::Ctnnb1(ex3) (Fl/+) Trp53 (Fl/Fl) mouse model fits well to human WNT medulloblastoma, and that, among various Myc- or Mycn-based mouse medulloblastomas, tumors in Glt1-tTA::TRE-MYCN/Luc mice proved to be most specific for human group 3 medulloblastoma. None of the analyzed models displayed a significant match to group 4 tumors. Intriguingly, mice with Ptch1 or Smo mutations selectively modeled SHH medulloblastomas of adulthood, although such mutations occur in all human age groups. We therefore suggest that the infantile or adult gene expression pattern of SHH MBs are not solely determined by specific mutations. This is supported by the observation that human medulloblastomas with PTCH1 mutations displayed more similarities to PTCH1 wild-type tumors of the same age group than to PTCH1-mutated tumors of the other age group. Together, we provide novel insights into previously unrecognized specificity of distinct models and suggest these findings as a solid basis to choose the appropriate model for preclinical studies on medulloblastoma.
[Show abstract][Hide abstract] ABSTRACT: Background Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance. Methods We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis. Results We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib. Conclusions Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell-receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.).
New England Journal of Medicine 05/2014; · 54.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epigenetic alterations, that is, disruption of DNA methylation and chromatin architecture, are now acknowledged as a universal feature of tumorigenesis. Medulloblastoma, a clinically challenging, malignant childhood brain tumour, is no exception. Despite much progress from recent genomics studies, with recurrent changes identified in each of the four distinct tumour subgroups (WNT-pathway-activated, SHH-pathway-activated, and the less-well-characterized Group 3 and Group 4), many cases still lack an obvious genetic driver. Here we present whole-genome bisulphite-sequencing data from thirty-four human and five murine tumours plus eight human and three murine normal controls, augmented with matched whole-genome, RNA and chromatin immunoprecipitation sequencing data. This comprehensive data set allowed us to decipher several features underlying the interplay between the genome, epigenome and transcriptome, and its effects on medulloblastoma pathophysiology. Most notable were highly prevalent regions of hypomethylation correlating with increased gene expression, extending tens of kilobases downstream of transcription start sites. Focal regions of low methylation linked to transcription-factor-binding sites shed light on differential transcriptional networks between subgroups, whereas increased methylation due to re-normalization of repressed chromatin in DNA methylation valleys was positively correlated with gene expression. Large, partially methylated domains affecting up to one-third of the genome showed increased mutation rates and gene silencing in a subgroup-specific fashion. Epigenetic alterations also affected novel medulloblastoma candidate genes (for example, LIN28B), resulting in alternative promoter usage and/or differential messenger RNA/microRNA expression. Analysis of mouse medulloblastoma and precursor-cell methylation demonstrated a somatic origin for many alterations. Our data provide insights into the epigenetic regulation of transcription and genome organization in medulloblastoma pathogenesis, which are probably also of importance in a wider developmental and disease context.
[Show abstract][Hide abstract] ABSTRACT: p53 loss of heterozygosity (p53LOH) is frequently observed in Li-Fraumeni syndrome (LFS) patients who carry a mutant (Mut) p53 germ-line mutation. Here, we focused on elucidating the link between p53LOH and tumor development in stem cells (SCs). Although adult mesenchymal stem cells (MSCs) robustly underwent p53LOH, p53LOH in induced embryonic pluripotent stem cells (iPSCs) was significantly attenuated. Only SCs that underwent p53LOH induced malignant tumors in mice. These results may explain why LFS patients develop normally, yet acquire tumors in adulthood. Surprisingly, an analysis of single-cell sub-clones of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that p53LOH is a bi-directional process, which may result in either the loss of wild-type (WT) or Mut p53 allele. Interestingly, most BM progenitors underwent Mutp53LOH. Our results suggest that the bi-directional p53LOH process may function as a cell-fate checkpoint. The loss of Mutp53 may be regarded as a DNA repair event leading to genome stability. Indeed, gene expression analysis of the p53LOH process revealed upregulation of a specific chromatin remodeler and a burst of DNA repair genes. However, in the case of loss of WTp53, cells are endowed with uncontrolled growth that promotes cancer.Cell Death and Differentiation (2014) 0, 000-000.advance online publication, 16 May 2014; doi:10.1038/cdd.2014.57.
Cell death and differentiation 05/2014; · 8.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer stem cells (CSCs) have been suggested as potential therapeutic targets for treating malignant tumors, but the in vivo supporting evidence is still missing. Using a GFP reporter driven by the promoter of the nuclear receptor tailless (Tlx), we demonstrate that Tlx(+) cells in primary brain tumors are mostly quiescent. Lineage tracing demonstrates that single Tlx(+) cells can self-renew and generate Tlx(-) tumor cells in primary tumors, suggesting that they are brain tumor stem cells (BTSCs). After introducing a BTSC-specific knock-out of the Tlx gene in primary mouse tumors, we observed a loss of self-renewal of BTSCs and prolongation of animal survival, accompanied by induction of essential signaling pathways mediating cell-cycle arrest, cell death, and neural differentiation. Our study demonstrates the feasibility of targeting glioblastomas and indicates the suitability of BTSCs as therapeutic targets, thereby supporting the CSC hypothesis.
[Show abstract][Hide abstract] ABSTRACT: This study aimed to prospectively evaluate clinical, histopathological and molecular variables for outcome prediction in medulloblastoma patients. Patients from the HIT2000 cooperative clinical trial were prospectively enrolled based on the availability of sufficient tumor material and complete clinical information. This revealed a cohort of 184 patients (median age 7.6 years), which was randomly split at a 2:1 ratio into a training (n = 127), and a test (n = 57) dataset in order to build and test a risk score for this population. Independent validation was performed in a non-overlapping cohort (n = 83). All samples were subjected to thorough histopathological investigation, CTNNB1 mutation analysis, quantitative PCR, MLPA and FISH analyses for cytogenetic variables, and methylome analysis. By univariable analysis, clinical factors (M-stage), histopathological variables (large cell component, endothelial proliferation, synaptophysin pattern), and molecular features (chromosome 6q status, MYC amplification, subgrouping) were found to be prognostic. Molecular consensus subgrouping (WNT, SHH, Group 3, Group 4) was validated as an independent feature to stratify patients into different risk groups. When comparing methods for the identification of WNT-driven medulloblastoma, this study identified CTNNB1 sequencing and methylation profiling to most reliably identify these patients. After removing patients with particularly favorable (CTNNB1 mutation, extensive nodularity) or unfavorable (MYC amplification) markers, a risk score for the remaining "intermediate molecular risk" population dependent on age, M-stage, pattern of synaptophysin expression, and MYCN copy-number status was identified, with speckled synaptophysin expression indicating worse outcome. Test and independent validation of the score confirmed significant discrimination of patients by risk profile. Methylation subgrouping and CTNNB1 mutation status represent robust tools for the risk stratification of medulloblastoma. A simple clinico-pathological risk score was identified, which was confirmed in a test set and by independent clinical validation.
[Show abstract][Hide abstract] ABSTRACT: Mutations in TP53, NOTCH1, and SF3B1 were analyzed in the CLL8 study evaluating first-line therapy with fludarabine and cyclophosphamide (FC) or FC with rituximab (FCR). TP53, NOTCH1, and SF3B1 were mutated in 11.5%, 10.0%, and 18.4% of patients, respectively. While NOTCH1(mut) and SF3B1(mut) virtually showed mutual exclusivity (0.6% concurrence), TP53(mut) was frequently found in NOTCH1(mut) (16.1%) and in SF3B1(mut) (14.0%) patients. There were few significant associations with clinical and laboratory characteristics but genetic markers had strong influence on response and survival. In multivariable analyses, an independent prognostic impact was found for FCR, thymidine kinase (TK) ≥10, unmutated IGHV, 11q deletion, 17p deletion, TP53(mut), and SF3B1(mut) on progression-free survival; and of FCR, age ≥65 years, ECOG performance status ≥0, β2-microglobulin ≥3.5, TK ≥10, unmutated IGHV, 17p deletion, and TP53(mut) on overall survival. Notably, predictive marker analysis identified an interaction of NOTCH1 mutational status and treatment, in that rituximab failed to improve response and survival in patients with NOTCH1(mut). In conclusion, TP53 and SF3B1 mutations appear among the strongest prognostic markers in CLL patients receiving current standard first-line therapy. NOTCH1(mut) was identified as a predictive marker for decreased benefit from the addition of rituximab to FC (FCR). This study is registered at ClinicalTrials.gov, identifier: NCT00281918.
[Show abstract][Hide abstract] ABSTRACT: Smoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant.
Cancer cell 03/2014; 25(3):393-405. · 25.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.