Karine Jacob

Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

Hotspot Mutations in H3F3A and IDH1 Define Distinct Epigenetic and Biological Subgroups of Glioblastoma

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

BRAF-KIAA1549 Fusion Predicts Better Clinical Outcome in Pediatric Low-Grade Astrocytoma

Purpose: Recent studies have revealed that the majority of pediatric low-grade astrocytomas (PLGA) harbor the BRAF-KIAA1549 (B-K) fusion gene resulting in constitutive activation of the RAS/MAPK pathway. However, the clinical significance of this genetic alteration is yet to be determined. We aimed to test the prognostic role of the B-K fusion in progression of incompletely resected PLGA. Experimental Design: We retrospectively identified 70 consecutive patients with incompletely resected “clinically relevant” PLGA. We added 76 tumors diagnosed at our institution between 1985 and 2010 as controls. We examined BRAF alterations by reverse transcriptase PCR, FISH, and single-nucleotide polymorphism array analysis and correlated that with progression-free survival (PFS). Results: Overall, 60% of tumors were B-K fusion positive. All patients with B-K fused PLGA are still alive. Five-year PFS was 61% ± 8% and 18% ± 8% for fusion positive and negative patients, respectively (P = 0.0004). B-K fusion resulted in similarly significant favorable PFS for patients who received chemotherapy. Multivariate analysis revealed that B-K fusion was the most significant favorable prognostic factor in incompletely resected PLGA and was independent of location, pathology, and age. In vitro, BRAF overexpression resulted in growth arrest associated with DNA damage (γH2AX expression). Five-year PFS was 68% ± 15% and 0% for patients with B-K fused and γH2AX-expressing PLGA versus negative tumors (P = 0.001). Conclusion: These data suggest that B-K fusion confers a less aggressive clinical phenotype on PLGA and may explain their tendency to growth arrest. Combined analysis of B-K fusion and γH2AX expression can determine prognosis and may be a powerful tool to tailor therapy for these patients. Clin Cancer Res; 17(14); 4790–8. ©2011 AACR.

Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours

Background:Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location.Methods and results:Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 – 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30 – 80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16 – 62.5%) and is rare in hemispheric JPA (1 of 7 – 14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation.Conclusions and interpretation:Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway.

Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing†‡

Protein coding genes constitute approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease‐related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., “whole exome”) have the potential to contribute our understanding of human diseases. We used a method for whole‐exome sequencing coupling Agilent whole‐exome capture to the Illumina DNA‐sequencing platform, and investigated two unrelated fetuses from nonconsanguineous families with Fowler Syndrome (FS), a stereotyped phenotype lethal disease. We report novel germline mutations in feline leukemia virus subgroup C cellular‐receptor‐family member 2, FLVCR2, which has recently been shown to cause FS. Using this technology, we identified three types of genetic abnormalities: point‐mutations, insertions‐deletions, and intronic splice‐site changes (first pathogenic report using this technology), in the fetuses who both were compound heterozygotes for the disease. Although revealing a high level of allelic heterogeneity and mutational spectrum in FS, this study further illustrates the successful application of whole‐exome sequencing to uncover genetic defects in rare Mendelian disorders. Of importance, we show that we can identify genes underlying rare, monogenic and recessive diseases using a limited number of patients (n=2), in the absence of shared genetic heritage and in the presence of allelic heterogeneity. Hum Mutat 31:1–6, 2010.

Genetic Aberrations Leading to MAPK Pathway Activation Mediate Oncogene-Induced Senescence in Sporadic Pilocytic Astrocytomas

Purpose: Oncogenic BRAF/Ras or NF1 loss can potentially trigger oncogene-induced senescence (OIS) through activation of the mitogen-activated protein kinase (MAPK) pathway. Somatic genetic abnormalities affecting this pathway occur in the majority of pilocytic astrocytomas (PA), the most prevalent brain neoplasm in children. We investigated whether OIS is induced in PA. Experimental Design: We tested expression of established senescence markers in three independent cohorts of sporadic PA. We also assessed for OIS in vitro, using forced expression of wild-type and V600E-mutant BRAF in two astrocytic cell lines: human telomerase reverse transcriptase (hTERT)-immortalized astrocytes and fetal astrocytes. Results: Our results indicate that PAs are senescent as evidenced by marked senescence-associated acidic β-galactosidase activity, low KI-67 index, and induction of p16INK4a but not p53 in the majority of 52 PA samples (46 of 52; 88.5%). Overexpression of a number of senescence-associated genes [CDKN2A (p16), CDKN1A (p21), CEBPB, GADD45A, and IGFBP7] was shown at the mRNA level in two independent PA tumor series. In vitro, sustained activation of wild-type or mutant BRAF induced OIS in both astrocytic cell lines. Loss of p16INK4a in immortalized astrocytes abrogated OIS, indicative of the role of this pathway in mediating this phenomenon in astrocytes. OIS is a mechanism of tumor suppression that restricts the progression of benign tumors. We show that it is triggered in PAs through p16INK4a pathway induction following aberrant MAPK activation. Conclusions: OIS may account for the slow growth pattern in PA, the lack of progression to higher-grade astrocytomas, and the high overall survival of affected patients. Clin Cancer Res; 17(14); 4650–60. ©2011 AACR.

Genome-wide profiling using single-nucleotide polymorphism arrays identifies novel chromosomal imbalances in pediatric glioblastomas

Available data on genetic events in pediatric grade IV astrocytomas (glioblastoma [pGBM]) are scarce. This has traditionally been a major impediment in understanding the pathogenesis of this tumor and in developing ways for more effective management. Our aim is to chart DNA copy number aberrations (CNAs) and get insight into genetic pathways involved in pGBM. Using the Illumina Infinium Human-1 bead-chip-array (100K single-nucleotide polymorphisms [SNPs]), we genotyped 18 pediatric and 6 adult GBMs. Results were compared to BAC-array profiles harvested on 16 of the same pGBM, to an independent data set of 9 pediatric high-grade astrocytomas (HGAs) analyzed on Affymetrix 250K-SNP arrays, and to existing data sets on HGAs. CNAs were additionally validated by real-time qPCR in a set of genes in pGBM. Our results identify with nonrandom clustering of CNAs in several novel, previously not reported, genomic regions, suggesting that alterations in tumor suppressors and genes involved in the regulation of RNA processing and the cell cycle are major events in the pathogenesis of pGBM. Most regions were distinct from CNAs in aGBMs and show an unexpectedly low frequency of genetic amplification and homozygous deletions and a high frequency of loss of heterozygosity for a high-grade I rapidly dividing tumor. This first, complete, high-resolution profiling of the tumor cell genome fills an important gap in studies on pGBM. It ultimately guides the mapping of oncogenic networks unique to pGBM, identification of the related therapeutic predictors and targets, and development of more effective therapies. It further shows that, despite commonalities in a few CNAs, pGBM and aGBMs are two different diseases.

Abstract 3687: An integrative genomics approach identifies distinct molecular and epigenetic subgroups of pediatric glioblastoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pediatric glioblastoma (GBM) belongs to the comparably small group of childhood malignancies for which cure is still an exception. Histologically indistinguishable from their adult counterparts, they carry a similar dismal prognosis. Whereas genetic and epigenetic properties have been extensively studied in adult tumors, little is known about the molecular characteristics of pediatric GBM, although some reports indicate that it is likely a different entity in terms of tumor biology and molecular genetics. Thus, this study aimed to elucidate disease-defining molecular lesions by determining genomic, transcriptomic and epigenetic alteration profiles. Using an integrative genomics approach combining multiple screening strategies, we investigated primary tumor samples from 55 childhood GBM for copy-number aberrations (CNA), transcriptomic and epigenetic changes, complemented by sequencing analysis of TP53, IDH1/2 and further candidate genes. Methylome analysis revealed the existence of five separate clusters of childhood GBM with distinct molecular and clinico-pathological features. Methylation patterns correlated with novel recurrent, subgroup-specific driver mutations unique to the pediatric population, and with clearly distinguishable transcriptomic profiles. Integration of methylation and gene expression data suggested that different tumor subgroups are derived from at least two distinct precursor-cell populations, one of them without any signs of neural lineage commitment. Furthermore, distinct clusters were highly associated with the presence of balanced (13%) or aneuploid (33%) genomic profiles or with cases displaying highly-rearranged genomes (11%), or various high-level focal amplifications (43%) of known and novel oncogenes. Similar to adults, CNA frequently targeted GBM core signaling pathways such as RTK/PI3K, p53 and RB signaling. TP53 loss-of-function mutations were present in 46% of pediatric GBM. IDH1 mutations were detected in only six patients (11%), but these tumors displayed concerted hypermethylation at a large number of loci, resembling a CpG island methylator phenotype (CIMP). Relevant findings are being validated by immunohistochemistry or FISH analysis in an independent, large-scale cohort representing 130 uniformly-treated pediatric GBM. This study, one of the largest cohorts of pediatric GBM investigated for molecular alterations to date, describes frequent genetic and epigenetic features of this devastating disease and further emphasizes and differences between adult and pediatric GBM. The identification of distinct molecular subgroups and commonly altered pathways will help to characterize molecular biomarkers for improved prognostic assessment and risk-adapted treatment stratification, and may facilitate the development of suitable in vitro and in vivo models for defining novel therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3687. doi:1538-7445.AM2012-3687

Non-random aneuploidy specifies subgroups of pilocytic astrocytoma and correlates with older age.

Pilocytic astrocytoma (PA) is the most common brain tumor in children but is rare in adults, and hence poorly studied in this age group. We investigated 222 PA and report increased aneuploidy in older patients. Aneuploid genomes were identified in 45% of adult compared with 17% of pediatric PA. Gains were non-random, favoring chromosomes 5, 7, 6 and 11 in order of frequency, and preferentially affecting non-cerebellar PA and tumors with BRAF V600E mutations and not with KIAA1549-BRAF fusions or FGFR1 mutations. Aneuploid PA differentially expressed genes involved in CNS development, the unfolded protein response, and regulators of genomic stability and the cell cycle (MDM2, PLK2),whose correlated programs were overexpressed specifically in aneuploid PA compared to other glial tumors. Thus, convergence of pathways affecting the cell cycle and genomic stability may favor aneuploidy in PA, possibly representing an additional molecular driver in older patients with this brain tumor.