Koichi Ichimura

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.

Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase gene UTX, pointing to histone H3 lysine methylation deregulation in multiple tumor types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene.

IDH1 mutations are present in the majority of common adult gliomas but rare in primary glioblastomas.

We screened exon 4 of the gene isocitrate dehydrogenase 1 (NADP+), soluble (IDH1) for mutations in 596 primary intracranial tumors of all major types. Codon 132 mutation was seen in 54% of astrocytomas and 65% of oligodendroglial tumors but in only 6% of glioblastomas (3% of primary and 50% of secondary glioblastomas). There were no mutations in any other type of tumor studied. While mutations in the tumor protein p53 gene (TP53) and total 1p/19q deletions were mutually exclusive, IDH1 mutations were strongly correlated with these genetic abnormalities. All four types of mutant IDH1 proteins showed decreased enzymatic activity. The data indicate that IDH1 mutation combined with either TP53 mutation or total 1p/19q loss is a frequent and early change in the majority of oligodendroglial tumors, diffuse astrocytomas, anaplastic astrocytomas, and secondary glioblastomas but not in primary glioblastomas.

PARK2 deletions occur frequently in sporadic colorectal cancer and accelerate adenoma development in Apc mutant mice

In 100 primary colorectal carcinomas, we demonstrate by array comparative genomic hybridization (aCGH) that 33% show DNA copy number (DCN) loss involving PARK2, the gene encoding PARKIN, the E3 ubiquitin ligase whose deficiency is responsible for a form of autosomal recessive juvenile parkinsonism. PARK2 is located on chromosome 6 (at 6q25–27), a chromosome with one of the lowest overall frequencies of DNA copy number alterations recorded in colorectal cancers. The PARK2 deletions are mostly focal (31% ∼0.5 Mb on average), heterozygous, and show maximum incidence in exons 3 and 4. As PARK2 lies within FRA6E, a large common fragile site, it has been argued that the observed DCN losses in PARK2 in cancer may represent merely the result of enforced replication of locally vulnerable DNA. However, we show that deficiency in expression of PARK2 is significantly associated with adenomatous polyposis coli (APC) deficiency in human colorectal cancer. Evidence of some PARK2 mutations and promoter hypermethylation is described. PARK2 overexpression inhibits cell proliferation in vitro. Moreover, interbreeding of Park2 heterozygous knockout mice with ApcMin mice resulted in a dramatic acceleration of intestinal adenoma development and increased polyp multiplicity. We conclude that PARK2 is a tumor suppressor gene whose haploinsufficiency cooperates with mutant APC in colorectal carcinogenesis.

Molecular pathogenesis of astrocytic tumours

Our current knowledge of the molecular pathogenesis of the diffuse adult astrocytic tumours is vast if compared to 20 syears ago, yet we are far from understanding the details of this process at the molecular level and using such an understanding to logically and specifically treat patients tumours. In other astrocytic tumours we have little or no knowledge of the molecular processes. This article will attempt to summarise the histological classification criteria and genetic data for all the astrocytic tumours. The current World Health Organisation classification lists six entities, some with subgroups. Common problems associated with the diagnosis of these tumours are outlined. While the molecular findings are not as yet used clinically, we are approaching a time when the histological investigation will have to be supplemented with molecular data to ensure the best choice of treatment for the patient and as an accurate indicator of prognosis.

Feedback circuit among INK4 tumor suppressors constrains human glioblastoma development.

Summary We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18INK4C and p16INK4A codeletion. Functional reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18INK4C in p16INK4A-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16INK4A in primary astrocytes induced a concomitant increase in p18INK4C. Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18INK4C in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation.

An efficient method for derivation and propagation of glioblastoma cell lines that conserves the molecular profile of their original tumours

A growing body of evidence suggests that glioma stem-like cells are more representative of their parent tumours when cultured under defined serum-free conditions with the mitogens epidermal growth factor (EGF) and fibroblast growth factor (FGF). However, culturing these cells as free-floating spheroids can result in difficulty in efficiently deriving and propagating cell lines. We have combined neurosphere and monolayer culture techniques to improve the efficiency with which cells can be derived from clinical tumour samples under defined serum-free conditions. We have applied our protocol to consecutive samples of glioblastoma to show that they can form experimental tumours that recapitulate many of the histological features of the parent tumour. We go on to show that the tumour initiating cells also retain the cytogenetic abnormalities of the parent tumour. Finally we examined the cell lines for expression of markers associated with neural stem cells. Our results confirm the expression of transcription factors associated with neural patterning and specification including Sox2, Olig2, Pax6 and Nkx2.2. We went on to establish that these factors were also expressed in the parent tumour indicating that their expression was not a function of our culture conditions. The Cambridge Protocol is an efficient method of deriving stem-like tumour initiating cells from glioblastoma. Improving the efficiency of derivation will facilitate the improvement of in vitro and in vivo model systems to study disease mechanisms, screen drugs and develop novel therapeutic approaches in the future.

A20 deletion is associated with copy number gain at the TNFA/B/C locus and occurs preferentially in translocation-negative MALT lymphoma of the ocular adnexa and salivary glands

The genetic basis of MALT lymphoma is largely unknown. Characteristic chromosomal translocations are frequently associated with gastric and pulmonary cases, but are rare at other sites. We compared the genetic profiles of 33 ocular adnexal and 25 pulmonary MALT lymphomas by 1 Mb array–comparative genomic hybridization (CGH) and revealed recurrent 6q23 losses and 6p21.2–6p22.1 gains exclusive to ocular cases. High‐resolution chromosome 6 tile‐path array–CGH identified NF‐κB inhibitor A20 as the target of 6q23.3 deletion and TNFA/B/C locus as a putative target of 6p21.2–22.1 gain. Interphase fluorescence in situ hybridization showed that A20 deletion occurred in MALT lymphoma of the ocular adnexa (8/42 = 19%), salivary gland (2/24 = 8%), thyroid (1/9 = 11%) and liver (1/2), but not in the lung (26), stomach (45) and skin (13). Homozygous deletion was observed in three cases. A20 deletion and TNFA/B/C gain were significantly associated (p < 0.001) and exclusively found in cases without characteristic translocation. In ocular cases, A20 deletion was associated with concurrent involvement of different adnexal tissues or extraocular sites at diagnosis (p = 0.007), a higher proportion of relapse (67% versus 37%) and a shorter relapse‐free survival (p = 0.033). A20 deletion and gain at TNFA/B/C locus may thus play an important role in the development of translocation‐negative MALT lymphoma. Copyright

Clinical significance of EGFR amplification and the aberrant EGFRvIII transcript in conventionally treated astrocytic gliomas

The aim of this study was to evaluate the clinical value of assessing epidermal growth factor receptor (EGFR) amplification and the common 5′ rearrangement of EGFR resulting in the EGFRvIII transcript in astrocytic gliomas. Data from 221 tumours were correlated with patient survival. The majority of previous studies evaluated amplification alone and provided contradictory results. Amplification was analysed by a densitometry of Southern blot analysis or quantitative polymerase chain reaction (PCR). EGFR transcripts were examined by reverse transcription PCR and subsequent sequencing. A ribonuclease (RNase) protection assay was carried out on a subgroup to confirm PCR results. Amplification of EGFR was found in 41% (65/160) of glioblastomas (GBs) and 10% (4/41) of anaplastic astrocytomas (AAs). The EGFRvIII rearrangement was identified in 54% (35/65) of GBs and 75% (3/4) of AAs with amplification, as well as in 8% (8/95) of GBs and 5% (2/37) of AAs without amplification (confirmed by RNase protection assay). There were no abnormalities of the EFGR or its transcript in grade II astrocytoma (AII). We found no significant association between EGFR amplification or rearrangement, and age or survival in the 160 GB patients. We noted a tendency towards decreased survival with any EGFR abnormality in the 41 patients with AAs. This was most marked in the five cases with the EGFRvIII transcript (p=0.069), but these were significantly older than those without (p=0.023). No abnormalities of EGFR were identified in AII patients. We conclude that neither EGFR amplification nor the presence of the EGFRvIII transcript predicts patient outcome in conventionally treated GBs. However, in AAs, although uncommon, EGFR aberrations appear to be associated with shorter survival.

High-resolution array-based comparative genomic hybridization of medulloblastomas and supratentorial primitive neuroectodermal tumors

Medulloblastomas and supratentorial primitive neuroectodermal tumors are aggressive childhood tumors. We report our findings using array comparative genomic hybridization (CGH) on a whole-genome BAC/PAC/cosmid array with a median clone separation of 0.97 Mb to study 34 medulloblastomas and 7 supratentorial primitive neuroectodermal tumors. Array CGH allowed identification and mapping of numerous novel, small regions of copy number change to genomic sequence in addition to the large regions already known from previous studies. Novel amplifications were identified, some encompassing oncogenes MYCL1, PDGFRA, KIT, and MYB not previously reported to show amplification in these tumors. In addition, one supratentorial primitive neuroectodermal tumor had lost both copies of the tumor-suppressor genes CDKN2A and CDKN2B. Ten medulloblastomas had findings suggestive of isochromosome 17q. In contrast to previous reports using conventional CGH, array CGH identified 3 distinct breakpoints in these cases: Ch 17: 17940393-19251679 (17p11.2, n = 6), Ch 17: 20111990-23308272 (17p11.2-17q11.2, n = 4), and Ch 17: 38425359-39091575 (17q21.31, n = 1). Significant differences were found in the patterns of copy number change between medulloblastomas and supratentorial primitive neuroectodermal tumors, providing further evidence that these tumors are genetically distinct despite their morphologic and behavioral similarities.