Molecular characterization of disseminated pilocytic astrocytomas.

AIM: Pilocytic astrocytomas represent the most common paediatric tumours of the central nervous system. Dissemination through the ventricular system occurs rarely in patients with pilocytic astrocytomas; however, it is more common in infants with diencephalic tumours, and is associated with a poor outcome. Despite histological similarities with classic pilocytic astrocytomas, it is still unclear whether disseminated pilocytic astrocytomas may have specific molecular features. METHODS: Seventeen disseminated pilocytic astrocytomas were investigated using the molecular inversion probe array and screened for the presence of gene fusions (KIAA1549-BRAF) and mutations (BRAF, RAS and FGFR1). RESULTS: Along with evidence of a constitutive MAPK activation in all cases, the molecular inversion probe array, fluorescence in situ hybridization analysis and mutational study revealed KIAA1549-BRAF fusions in 66% and BRAF(V600E) mutations in 5% of cases. No KRAS, HRAS, NRAS or FGFR1 mutations were found. CONCLUSIONS: disseminated pilocytic astrocytomas showed genetic features similar to classic pilocytic astrocytoma, including a similar incidence of KIAA1549-BRAF fusions, BRAF mutations and a stable genetic profile. Given common activation of the MAPK pathway, the use of specific inhibitors can be hypothesized for the treatment of disseminated pilocytic astrocytomas, along with standard chemo- and/or radiotherapy.

Pathology of gliomas and developments in molecular testing.

Advances in technology are allowing a molecular characterisation of human brain tumours that is providing a wealth of new information. In this short overview, a summary of the histopathology of the common gliomas is integrated with some molecular data. In some instances, the data are proving clinically relevant with conventional therapies. Some single histological entities are being found to contain a number of molecular subtypes, whereas in others, different histological entities are found to be molecularly similar. The introduction of targeted therapies will necessitate a complete reassessment of the way we characterise tumours, and particularly the adequacy of our pathology reports in a new clinical environment.

Genomic differences in benign and malignant follicular thyroid tumours using 1-Mb array-comparative genomic hybridisation.

Currently there is a lack of objective markers that can reliably differentiate benign and malignant follicular thyroid tumours. Such markers are needed to avoid the morbidity and cost of diagnosing these lesions by a thyroid lobectomy and then a second operation to remove the remaining half of thyroid if cancer is found. The aim of this research was to look for genomic markers that might solve this important problem. Ethical approval for the project was obtained. DNA was extracted from formalin-fixed paraffin-embedded specimens and copy number analysed using an in-house produced 1-megabase genomic array by comparative genomic hybridization (1Mb-aCGH). Acceptable quality data were obtained in 25/26 (96 %) of adenomas and 17/28 (61 %) of carcinomas. Among the carcinomas, 11 were minimally invasive (MI), 5 widely invasive (WI) and there was one metastasis. Recurrent copy number changes distinguishing benign and malignant included +1p34.2-36.33, +1q, +13q12.11-14.3, +14q22.1-32.33, +20q and -22. +20q became more sensitive (36.4 %) for MI carcinomas, whereas +13q12.11-14.3 and +14q22.1-32.33 became more sensitive (66.7 %) for identifying WI cancers from adenomas. Only in the context of aneuploidy (3 adenomas, 3 MI, 3 WI) there were some specific copy number changes that could differentiate all aneuploid adenomas from carcinomas. This research is the first using 1Mb-aCGH to study benign and malignant follicular thyroid tumours. Overall, the incidence of any copy number changes is low, but there are a number of changes associated with different tumour types. Further research with a larger sample and better quality DNA will clarify these findings.

Fluorescence-guided surgical sampling of glioblastoma identifies phenotypically distinct tumour-initiating cell populations in the tumour mass and margin.

BACKGROUND: Acquiring clinically annotated, spatially stratified tissue samples from human glioblastoma (GBM) is compromised by haemorrhage, brain shift and subjective identification of 'normal' brain. We tested the use of 5-aminolevulinic acid (5-ALA) fluorescence to objective tissue sampling and to derive tumour-initiating cells (TICs) from mass and margin. METHODS: The 5-ALA was administered to 30 GBM patients. Samples were taken from the non-fluorescent necrotic core, fluorescent tumour mass and non-fluorescent margin. We compared the efficiency of isolating TICs from these areas in 5-ALA versus control patients. HRMAS (1)H NMR was used to reveal metabolic alterations due to 5-ALA. We then characterised TICs for self-renewal in vitro and tumorigenicity in vivo. RESULTS: The derivation of TICs was not compromised by 5-ALA and the metabolic profile was similar between tumours from 5-ALA patients and controls. The TICs from the fluorescent mass were self-renewing in vitro and tumour-forming in vivo, whereas TICs from non-fluorescent margin did not self-renew in vitro but did form tumours in vivo. CONCLUSION: Our data show that 5-ALA does not compromise the derivation of TICs. It also reveals that the margin contains TICs, which are phenotypically different from those isolated from the corresponding mass.

CpG island hypermethylation of the neurofibromatosis type 2 (NF2) gene is rare in sporadic vestibular schwannomas.

AIMS: Loss of both wild-type copies of the neurofibromatosis type 2 (NF2) gene is found in both sporadic and neurofibromatosis type 2-associated vestibular schwannomas (VS). Previous studies have identified a subset of VS with no loss or mutation of NF2. We hypothesized that methylation of NF2 resulting in gene silencing may play a role in such tumours. METHODS: Forty sporadic VS were analysed by array comparative genomic hybridization using 1 Mb whole genome and chromosome 22 tile path arrays. The NF2 genes were sequenced and methylation of NF2 examined by pyrosequencing. RESULTS: Monosomy 22 was the only recurrent change found. Twelve tumours had NF2 mutations. Eight tumours had complete loss of wild-type NF2, four had one mutated and one wild-type allele, 11 had only one wild-type allele and 17 showed no abnormalities. Methylation analysis showed low-level methylation in four tumours at a limited number of CpGs. No high-level methylation was found. CONCLUSIONS: This study shows that a significant proportion of sporadic VS (>40%) have unmethylated wild-type NF2 genes. This indicates that other mechanisms, yet to be identified, are operative in the oncogenesis of these VSs.

Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549:BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma.

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.

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-kappaB 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.

Real-time quantitative polymerase chain reaction (qPCR) analysis with fluorescence resonance energy transfer (FRET) probes reveals differential expression of the four ERBB4 juxtamembrane region variants between medulloblastoma and pilocytic astrocytoma.

AIMS: We report a comparative study on the mRNA expression of ErbB receptor tyrosine kinases, and in particular ERBB4 transcript variants, in two common paediatric brain tumours: medulloblastoma (MB) and pilocytic astrocytoma (PA). METHODS: While the conventional real-time quantitative polymerase chain reaction was used to measure the expression of ERRBs and ErbB4-processing protease genes, the LightCycler fluorescence resonance energy transfer probes were specifically designed to investigate all of the known ERBB4 juxtamembrane (JM) and cytoplasmic transcript variants. RESULTS: The overall expression of ERBBs suggests that ErbB2/ErbB4 heterodimers and ErbB4 homodimers may be major functional units of the ErbBs in MB, while ErbB2/ErbB3 heterodimers may play a more prominent role in addition to ErbB4-containing dimers in PA. Different expression patterns of ERBB4 JM transcripts in MB, PA and normal brain were observed. The JM-d variant was only detected in MBs, while JM-c was present in MB and PA but was not identified in normal brain. The expression of cleavable ERBB4 transcript variants was elevated in PAs and MBs compared with normal brain, while mRNA levels of ErbB4-processing proteases were similar in both tumour types and normal brain. This suggests that proteolytic cleavage of ErbB4 may be more common in MB and PA, which leads to signalling events divergent from those in normal brain. CONCLUSION: Taken together, these results suggest that ErbB4 processing and function may be altered in brain tumours, such as MB and PA, via differential expression of JM transcript variants.

p53-independent mechanisms regulate the P2-MDM2 promoter in adult astrocytic tumours.

The MDM2 gene is amplified and/or overexpressed in about 10% of glioblastomas and constitutes one of a number of ways the p53 pathway is disrupted in these tumours. MDM2 encodes a nuclear phosphoprotein that regulates several cell proteins by binding and/or ubiquitinating them, with p53 being a well-established partner. MDM2 has two promoters, P1 and P2 that give rise to transcripts with distinct 5' untranslated regions. Transcription from P2 is believed to be controlled by p53 and a single-nucleotide polymorphism (SNP309, T>G) in P2 is reported to be associated with increased risk for, and early development of, malignancies. The use of P1 and P2 has not been investigated in gliomas. We used RT-PCR to study P1- and P2-MDM2 transcript expression in astrocytic tumours, xenografts and cell lines with known MDM2, TP53 and p14(ARF) gene status. Both promoters were used in all genetic backgrounds including the use of the P2 promoter in TP53 null cells, indicating a p53-independent induction of transcription. Transcripts from the P1 promoter formed a greater proportion of the total MDM2 transcripts in tumours with MDM2 amplification, despite these tumours having two wild-type TP53 alleles. Examination of SNP309 in glioblastoma patients showed a borderline association with survival but no apparent correlation with age at diagnosis nor with TP53 and p14(ARF) status of their tumours. Our findings also indicate that elevated MDM2 mRNA levels in tumours with MDM2 amplification are preferentially driven by the P1 promoter and that the P2 promoter is not only regulated by p53 but also by other transcription factor(s).

Array painting reveals a high frequency of balanced translocations in breast cancer cell lines that break in cancer-relevant genes.

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.

1p36 is a preferential target of chromosome 1 deletions in astrocytic tumours and homozygously deleted in a subset of glioblastomas.

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.

Mutations in Rb1 pathway-related genes are associated with poor prognosis in anaplastic astrocytomas.

Anaplastic astrocytoma (AA, WHO grade III) is, second to Glioblastoma, the most common and most malignant type of adult CNS tumour. Since survival for patients with AA varies markedly and there are no known useful prognostic or therapy response indicators, the primary purpose of this study was to examine whether knowledge of the known genetic abnormalities found in AA had any clinical value. The survival data on 37 carefully sampled AA was correlated with the results of a detailed analysis of the status of nine genes known to be involved in the development of astrocytic tumours. These included three genes coding for proteins in the p53 pathway (TP53, p14(ARF)and MDM2), four in the Rb1 pathway (CDKN2A, CDKN2B, RB1 and CDK4) and PTEN and EGFR. We found that loss of both wild-type copies of any of the three tumour suppressor genes CDKN2A, CDKN2B and RB1 or gene amplification of CDK4, disrupting the Rb1 pathway, were associated with shorter survival (P=0.009). This association was consistent in multivariate analysis, including adjustment for age (P=0.013). The findings suggest that analysis of the genes coding for Rb1 pathway components provides additional prognostic information in AA patients receiving conventional therapy.

A PDGFRA promoter polymorphism, which disrupts the binding of ZNF148, is associated with primitive neuroectodermal tumours and ependymomas.

BACKGROUND: Platelet derived growth factor receptor alpha (PDGFRalpha) expression is typical for a variety of brain tumours, while in normal adult brain PDGFRalpha expression is limited to a small number of neural progenitor cells. The molecular mechanisms responsible for the PDGFRalpha expression in tumours are not known, but in the absence of amplification, changes in transcriptional regulation might be an important factor in this process. METHODS AND RESULTS: We have investigated the link between single nucleotide polymorphisms (SNPs) within the PDGFRalpha gene promoter and the occurrence of brain tumours (medulloblastomas, supratentorial primitive neuroectodermal tumours (PNETs), ependymal tumours, astrocytomas, oligodendrogliomas, and mixed gliomas). These SNPs give rise to five different promoter haplotypes named H1 and H2alpha-delta. It is apparent from the haplotype frequency distribution that both PNET (10-fold) and ependymoma (6.5-fold) patient groups display a significant over-representation of the H2delta haplotype. The precise functional role in PDGFRalpha gene transcription for the H2delta haplotype is not known yet, but we can show that the H2delta haplotype specifically disrupts binding of the transcription factor ZNF148 as compared to the other promoter haplotypes. CONCLUSIONS: The specific over-representation of the H2delta haplotype in both patients with PNETs and ependymomas suggests a functional role for the ZNF148/PDGFRalpha pathway in the pathogenesis of these tumours.

Global amplification of mRNA by template-switching PCR: linearity and application to microarray analysis.

Conventional approaches to target labelling for expression microarray analysis typically require relatively large amounts of total RNA, a serious limitation when the sample available is small. Here we explore the cycle-dependent amplification characteristics of Template-Switching PCR and validate its use for microarray target labelling. TS-PCR identifies up to 80% of the differentially expressed genes identified by direct labelling using 30-fold less input RNA for the amplification, with the equivalent of 1000-fold less starting material being used for each hybridisation. Moreover, the sensitivity of microarray experiments is increased considerably, allowing the identification of differentially expressed transcripts below the level of detection using targets prepared by direct labelling. We have also validated the fidelity of amplification and show that the amplified material faithfully represents the starting mRNA population. This method outperforms conventional labelling strategies, not only in terms of sensitivity and the identification of differentially expressed genes, but it is also faster and less labour intensive than other amplification protocols.

Treatment of graft-versus-host disease after liver transplantation with basiliximab followed by bowel resection.

Graft-versus-host disease (GVHD) after orthotopic liver transplantation (OLT) is a serious complication with mortality rates over 80%. Two patients with established GVHD after OLT were treated with Basiliximab, a chimeric murine human monoclonal antibody which binds to the alpha-chain of interleukin-2 receptor (IL-2R). Two males, aged 45 and 56 years, presented after OLT with a clinical picture consistent with GVHD. Quantitative measurements of recipient peripheral blood donor lymphocyte chimerism were carried out by flow cytometric analysis, and showed peak chimerism levels of 5% and 8%, respectively. Treatment comprised 3 doses of 1 g methyl prednisolone followed by 2 doses of 20 mg of Basiliximab. In both, treatment resulted in complete disappearance of macro-chimerism in blood. There was resolution of skin rash by day 7; however, diarrhea persisted. White cell scan showed increased uptake in the terminal ileum and small-bowel resection was performed in both patients. One patient is alive and well 36 months after OLT. The other patient had resolution of GVHD, but died of recurrent hepatitis C 1 year after OLT. The combination of immunological and surgical treatment for GVHD following solid organ transplantation has not previously been described.

Tuberous sclerosis presenting in late adult life.

A 59 year old woman presented with a three year history of left sided weakness. Magnetic resonance imaging of the brain showed a large high signal lesion occupying most of the right temporal lobe with mass effect. A probable diagnosis of low grade glioma led to temporal lobectomy. Histology revealed dysplastic cortical morphology typical of tuberous sclerosis. There were no clinical signs or family history of the disease. Ultrasound showed multiple bilateral renal angiomyolipomas, confirming the diagnosis of tuberous sclerosis. Molecular genetic analysis of peripheral white blood cells identified a novel mis-sense mutation R1409W in exon 33 of the TSC2 gene.

Cloning and chromosomal localization of human WIG-1/PAG608 and demonstration of amplification with increased expression in primary squamous cell carcinoma of the lung.

In this report, we describe the cloning of the coding region of human WIG-1 cDNA. The human 8 and 6 kb WIG-1 transcripts are both upregulated following ionizing irradiation of the human colon cancer cell lines HCT116 and LoVo which have wild type TP53 but not in DLD1 cells that lack wild type TP53. Basal levels of both WIG-1 transcripts were detected in human adult brain, kidney, and testis, but not in fetal brain, heart, pancreas, adrenal gland, fetal liver, and small intestine. FISH analysis mapped the human WIG-1 gene to 3q26.3. Investigation of squamous cell carcinomas of the lung by Southern blot and semiquantitative RT-PCR analysis showed amplification in combination with increased expression of WIG-1 in 1/7 tumors and increased expression in a further two cases.