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Pilocytic astrocytomas (PAs), WHO malignancy grade I, are the most frequently occurring central nervous system tumour in 5- to 19-year-olds. Recent reports have highlighted the importance of MAPK pathway activation in PAs, particularly through a tandem duplication leading to an oncogenic BRAF fusion gene. Here, we report two alternative mechanisms resulting in MAPK activation in PAs. Firstly, in striking similarity to the common BRAF fusion, tandem duplication at 3p25 was observed, which produces an in-frame oncogenic fusion between SRGAP3 and RAF1. This fusion includes the Raf1 kinase domain, and shows elevated kinase activity when compared with wild-type Raf1. Secondly, a novel 3 bp insertion at codon 598 in BRAF mimics the hotspot V600E mutation to produce a transforming, constitutively active BRaf kinase. Although these two alterations are not common, they bring the number of cases with an identified 'hit' on the Ras/Raf-signalling pathway to 36 from our series of 44 (82%), confirming its central importance to the development of pilocytic astrocytomas.

Chromosome translocations in the common epithelial cancers are abundant, yet little is known about them. They have been thought to be almost all unbalanced and therefore dismissed as mostly mediating tumour suppressor loss. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1 Mb resolution on bacterial artificial chromosome (BAC) arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2 kb resolution using oligonucleotide arrays. Many more of the translocations were balanced at 1 Mb resolution than expected, either reciprocal (eight in total) or balanced for at least one participating chromosome (19 paired breakpoints). Second, many of the breakpoints were at genes that are plausible targets of oncogenic translocation, including balanced breaks at CTCF, EP300/p300 and FOXP4. Two gene fusions were demonstrated, TAX1BP1-AHCY and RIF1-PKD1L1. Our results support the idea that chromosome rearrangements may play an important role in common epithelial cancers such as breast cancer.

Astrocytic, oligodendroglial and mixed gliomas are the commonest gliomas in adults. They have distinct phenotypes and clinical courses, but as they exist as a continuous histological spectrum, differentiating them can be difficult. Co-deletions of total 1p and 19q are found in the majority of oligodendrogliomas and considered as a diagnostic marker and a prognostic indicator. The 1p status of astrocytomas has not yet been thoroughly examined. Using a chromosome 1 tile path array, we investigated 108 adult astrocytic tumours for copy number alterations. Total 1p deletions were rare (2%), however partial deletions involving 1p36 were frequently identified in anaplastic astrocytomas (22%) and glioblastomas (34%). Multivariate analysis showed that patients with total 1p deletions had significantly longer survival (P=0.005). In nine glioblastomas homozygous deletions at 1p36 were identified. No somatic mutations were found among the five genes located in the homozygously deleted region. However, the CpG island of TNFRSF9 was hypermethylated in 19% of astrocytic tumours and 87% of glioma cell lines. TNFRSF9 expression was upregulated after demethylation of glioma cell lines. Akt3 amplifications were found in four glioblastomas. Our results indicate that 1p deletions are common anaplastic astrocytomas and glioblastomas but are distinct from the 1p abnormalities in oligodendrogliomas.

Deletions of chromosome 6 are a common abnormality in diverse human malignancies including astrocytic tumours, suggesting the presence of tumour suppressor genes (TSG). In order to help identify candidate TSGs, we have constructed a chromosome 6 tile path microarray. The array contains 1,780 clones (778 P1-derived artificial chromosome and 1,002 bacterial artificial chromosome) that cover 98.3% of the published chromosome 6 sequences. A total of 104 adult astrocytic tumours (10 diffuse astrocytomas, 30 anaplastic astrocytomas (AA), 64 glioblastomas (GB)) were analysed using this array. Single copy number change was successfully detected and the result was in general concordant with a microsatellite analysis. The pattern of copy number change was complex with multiple interstitial deletions/gains. However, a predominance of telomeric 6q deletions was seen. Two small common and overlapping regions of deletion at 6q26 were identified. One was 1,002 kb in size and contained PACRG and QKI, while the second was 199 kb and harbours a single gene, ARID1B. The data show that the chromosome 6 tile path array is useful in mapping copy number changes with high resolution and accuracy. We confirmed the high frequency of chromosome 6 deletions in AA and GB, and identified two novel commonly deleted regions that may harbour TSGs., (Oncogene (2006) 25, 1261-1271. doi:10.1038/sj.onc.1209156; published online 3 October 2005.)

The Mixed Lineage Leukemia (MLL) gene is commonly involved in translocations in infantile leukemia and is amplified in some cases of adult myeloid leukemia. A homolog of MLL denoted MLL2, which represents the second human homolog of the Drosophila trithorax gene, was characterized by assembling ESTs, the KIAA0304 cDNA clone, RT - PCR fragments and a new clone isolated from a cDNA phage library and compared to the available genomic sequence. The MLL2 gene maps to 19q13.1, a region of frequent rearrangement or amplification in solid tumors. MLL2 consists of an 8.5 - 9 kb transcript and spans 20 kb of genomic DNA. The predicted MLL2 protein possesses all of the major domains defined in MLL and the two genes have a similar genomic structure. We find that MLL2 is amplified in two of 14 pancreatic carcinoma cell lines and one of five glioblastoma cell lines and is a likely critical gene in 19q13.1 amplifications. It is also a candidate for chromosomal rearrangements involving this chromosome locus. MLL2 is one additional mammalian trithorax-group gene with involvement in human cancer.

The naevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple developmental defects and cancer susceptibility, in particular to basal cell carcinomas (BCCs). Medulloblastomas, primitive neuroectodermal tumours (PNETs) arising in childhood, occur in about 3-5% of NBCCS patients and a subset of PNETs was previously found with allelic imbalance at 9q22-q23, the region containing the gene for NBCCS (PTCH). We have analysed tumour DNA samples from 37 unrelated patients with sporadic PNETs and five medulloblastoma cell lines for PTCH mutations using an exon-by-exon single strand conformation polymorphism assay. We found three missense mutations, which affect conserved residues in transmembrane domains of the gene product and in the extracellular loop implicated in binding sonic hedgehog, one 2 bp deletion and an exon skipping splice site mutation. Most mutations were associated with the absence of the wild-type allele and were found in tumours exhibiting loss of heterozygosity (LOH) at loci flanking PTCH. The finding of LOH at 9q22-q23 in most mutated tumours while present in only three out of 26 tumours, in which a mutation was not identified, implicates PTCH as the target gene in PNETs with LOH at 9q22-q23 and deficient PTCH in the development of a subset of these tumours. Since all observed mutations were absent in the germ-line, a sporadic medulloblastoma developing as the first symptom of NBCCS is likely to be a very uncommon event.

A series of 195 human gliomas were studied as to the status of their CDKN2A, CDK4 and RB1 genes. Among 120 glioblastomas, 40% had no wild-type CDKN2A gene, 12% amplified the CDK4 gene, and 14% had no wild-type RBI gene. With two exceptions, each tumour had only one of these abnormalities. Thus the majority of the glioblastomas (64%) had distinct genetic aberrations which would obviously disrupt the control of transition from G1 to the S-phase of the cell cycle. A further 30% had loss of one allele of the CDKN2A and/or RBI genes. Only seven (6%) glioblastomas had no abnormalities of these genes. Anaplastic astrocytomas showed similar changes to the glioblastomas but at lower frequencies-34% showing no aberrations of the genes analysed. The astrocytomas showed solely loss of one allele of the RBI gene in 28% of tumours, with retention of one wild-type copy. In the glioblastomas with no alterations of CDKN2A, CDK4 or RB1, several other genes (CCND1, CCND2, CCND3, CDK6, E2F, CDK7, MYC and MYCN) whose products take part in cell cycle regulation were examined. No abnormalities were detected. Thus some aberration of the CDKN2A, CDK4 and RB1 genes appears to be almost obligatory in glioblastomas.

The epidermal growth factor (EGF) receptor is a membrane bound tyrosine kinase whose activity is initiated by ligand binding. The malignant brain tumour glioblastoma frequently shows amplification and rearrangements of the EGF receptor gene that are associated with the synthesis of a constitutively activated tyrosine kinase, lacking amino acids 6-273 near the protein's N-terminus. When expressed in Chinese hamster ovary (CHO) cells, this mutant receptor (p140EGFR) displays ligand-independent tyrosine kinase activity, stimulates DNA synthesis, and promotes cell proliferation. Here, we investigate the subcellular location of p140EGFR in CHO cell transfectants as well as in human glioblastoma tumours. p140EGFR had an intracellular location that contrasted sharply with the plasma membrane location of the wild-type EGF receptor. Endoglycosidase H sensitivity analysis and the pattern of p140EGFR immunoreactivity suggested that the aberrant tyrosine kinase resided primarily in the endoplasmic reticulum. The half-life of p140EGFR in the endoplasmic reticulum was extended several-fold over that of the ligand-activated wild-type receptor. The altered subcellular location of p140EGFR in combination with its prolonged half-life suggest that this activated tyrosine kinase may escape the regulatory mechanisms utilized for the attenuation of wild-type receptor signaling. Therefore, the previously reported growth stimulatory property of the ligand-independent p140EGFR may be attributed to a sustained tyrosine kinase activity resulting from an altered subcellular location.

The most common type of alteration of the epidermal growth factor receptor gene (EGFR) in human glioblastomas results in the synthesis of an aberrant mRNA lacking 801 bases that encode amino acids 6-273 of the receptor's extracellular domain. To study the effects of this mutation on receptor function, we have developed chinese hamster ovary cell transfectants which express the mutant EGF receptor. Comparison of wild-type and mutant receptor properties in this cell host indicates that the truncated receptor does not bind EGF or TGF-alpha and, consequently, DNA synthesis is not stimulated in cultures of mutant transfectants by either grown factor. However, levels of DNA synthesis determined for mutant transfectants in serum-free media are several-fold higher than those determined for corresponding cultures of wild-type transfectants. Western blot analysis with anti-phosphotyrosine antibody indicates that the mutant receptor is constitutively phosphorylated in CHO cells, and the same analysis applied to lysates of glioblastoma biopsies reveals the altered receptor is readily detectable as a phosphotyrosine protein in tumors for which there is evidence of corresponding EGFR gene and transcript alterations. In total, these results indicate that the aberrant EGF receptor synthesized in glioblastomas, and which lacks a portion of the extracellular domain necessary for ligand binding, is an activated tyrosine kinase.