[Show abstract][Hide abstract] ABSTRACT: Background
Genetic and epigenetic profiling of glioblastomas has provided a comprehensive list of altered cancer genes of which only O(6)-methylguanine-methyltransferase (MGMT) methylation is used thus far as a predictive marker in a clinical setting. We investigated the prognostic significance of genetic and epigenetic alterations in glioblastoma patients.Methods
We screened 98 human glioblastoma samples for genetic and epigenetic alterations in 10 genes and chromosomal loci by PCR and multiplex ligation-dependent probe amplification (MLPA). We tested the association between these genetic and epigenetic alterations and glioblastoma patient survival. Subsequently, we developed a 2-gene survival predictor.ResultsMultivariate analyses revealed that mutations in isocitrate dehydrogenase 1 (IDH1), promoter methylation of MGMT, irradiation dosage, and Karnofsky Performance Status (KFS) were independent prognostic factors. A 2-gene predictor for glioblastoma survival was generated. Based on the genetic and epigenetic status of IDH1 and MGMT, glioblastoma patients were stratified into 3 clinically different genotypes: glioblastoma patients with IDH1mt/MGMTmet had the longest survival, followed by patients with IDH1mt/MGMTunmet or IDH1wt/MGMTmet, and patients with IDH1wt/MGMTunmet had the shortest survival. This 2-gene predictor was an independent prognostic factor and performed significantly better in predicting survival than either IDH1 mutations or MGMT methylation alone. The predictor was validated in 3 external datasets.DiscussionThe combination of IDH1 mutations and MGMT methylation outperforms either IDH1 mutations or MGMT methylation alone in predicting survival of glioblastoma patients. This information will help to increase our understanding of glioblastoma biology, and it may be helpful for baseline comparisons in future clinical trials.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma is the most common malignant primary brain tumor. Temozolomide (TMZ) is the standard chemotherapeutic agent for this disease. However, intrinsic and acquired TMZ-resistance represents a major obstacle for this therapy. In order to identify factors involved in TMZ-resistance, we engineered different TMZ-resistant glioblastoma cell lines. Gene expression analysis demonstrated that EFEMP1, an extracellular matrix protein, is associated with TMZ-resistant phenotype. Silencing of EFEMP1 in glioblastoma cells resulted in decreased cell survival following TMZ treatment, whereas overexpression caused TMZ-resistance. EFEMP1 acts via multiple signaling pathways, including γ-secretase-mediated activation of the Notch pathway. We show that inhibition of γ-secretase by RO4929097 causes at least partial sensitization of glioblastoma cells to temozolomide in vitro and in vivo. In addition, we show that EFEMP1 expression levels correlate with survival in TMZ-treated glioblastoma patients. Altogether our results suggest EFEMP1 as a potential therapeutic target to overcome TMZ-resistance in glioblastoma.
[Show abstract][Hide abstract] ABSTRACT: The histopathological diagnosis of diffuse gliomas often lacks the precision that is needed for tailored treatment of individual patients. Assessment of the molecular aberrations will probably allow more robust and prognostically relevant classification of these tumors. Markers that have gained a lot of interest in this respect are co-deletion of complete chromosome arms 1p and 19q, (hyper)methylation of the MGMT promoter and IDH1 mutations. The aim of this study was to assess the prognostic significance of complete 1p/19q co-deletion, MGMT promoter methylation and IDH1 mutations in patients suffering from diffuse gliomas. The presence of these molecular aberrations was investigated in a series of 561 diffuse astrocytic and oligodendroglial tumors (low grade n=110, anaplastic n=118 and glioblastoma n=333) and correlated with age at diagnosis and overall survival. Complete 1p/19q co-deletion, MGMT promoter methylation and/or IDH1 mutation generally signified a better prognosis for patients with a diffuse glioma including glioblastoma. However, in all 10 patients with a histopathological diagnosis of glioblastoma included in this study complete 1p/19q co-deletion was not associated with improved survival. Furthermore, in glioblastoma patients >50 years of age the favorable prognostic significance of IDH1 mutation and MGMT promoter methylation was absent. In conclusion, molecular diagnostics is a powerful tool to obtain prognostically relevant information for glioma patients. However, for individual patients the molecular information should be interpreted with caution and weighed in the context of parameters such as age and histopathological diagnosis.Modern Pathology advance online publication, 22 February 2013; doi:10.1038/modpathol.2012.166.
[Show abstract][Hide abstract] ABSTRACT: Up till now, typing and grading of diffuse gliomas is based on histopathological features. However, more objective tools are needed to improve reliable assessment of their biological behavior. We evaluated 331 diffuse gliomas for copy number changes involving 1p, 19q, CDKN2A, PTEN and EGFR(vIII) by Multiplex Ligation-dependent Probe Amplification (MLPA®, Amsterdam, The Netherlands). Specifically based on the co-occurrence of these aberrations we built a model for the timing of the different events and their exact nature (hemi- → homozygous loss; low-level gain → (high-copy) amplification) in the course of molecular progression. The mutation status of IDH1 and TP53 was also evaluated and shown to correlate with the level of molecular progression. The relevance of the proposed model was confirmed by analysis of 36 sets of gliomas and their 39 recurrence(s) whereas survival analysis for anaplastic gliomas confirmed the actual prognostic relevance of detecting molecular malignancy. Moreover, based on our results, molecular diagnostic analysis of 1p/19q can be further improved as different aberrations were identified, some of them being indicative for advanced molecular malignancy rather than for favorable tumor behavior. In conclusion, identification of molecular malignancy as proposed will aid in establishing a risk profile for individual patients and thereby in therapeutic decision making.
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor receptor (EGFR) is commonly affected in cancer, generally in the form of an increase in DNA copy number and/or as mutation variants [e.g., EGFR variant III (EGFRvIII), an in-frame deletion of exons 2-7]. While detection of EGFR aberrations can be expected to be relevant for glioma patients, such analysis has not yet been implemented in a routine setting, also because feasible and robust assays were lacking. We evaluated multiplex ligation-dependent probe amplification (MLPA) for detection of EGFR amplification and EGFRvIII in DNA of a spectrum of 216 diffuse gliomas. EGFRvIII detection was verified at the protein level by immunohistochemistry and at the RNA level using the conventionally used endpoint RT-PCR as well as a newly developed quantitative RT-PCR. Compared to these techniques, the DNA-based MLPA assay for EGFR/EGFRvIII analysis tested showed 100% sensitivity and specificity. We conclude that MLPA is a robust assay for detection of EGFR/EGFRvIII aberrations. While the exact diagnostic, prognostic and predictive value of such EGFR testing remains to be seen, MLPA has great potential as it can reliably and relatively easily be performed on routinely processed (formalin-fixed, paraffin-embedded) tumor tissue in combination with testing for other relevant glioma markers.
[Show abstract][Hide abstract] ABSTRACT: Most human gliomas are characterized by diffuse infiltrative growth in the brain parenchyma. Partly because of this characteristic growth pattern, gliomas are notorious for their poor response to current therapies. Many animal models for human gliomas, however, do not display this diffuse infiltrative growth pattern. Furthermore, there is a need for glioma models that represent adequate genocopies of different subsets of human gliomas (e.g., oligodendrogliomas). Here, we assessed the intracerebral growth patterns and copy number changes [using multiplex ligation-dependent probe amplification (MLPA)/comparative genomic hybridization (CGH)] of 15 human glioma lines in nude mice. Most xenografts present with compact growing lesions intracerebrally. Only the E98 and, to a lesser degree, E106 xenograft lines (propagated through subcutaneous growth) consistently produced intracerebral tumors, displaying diffuse infiltrative growth in the brain parenchyma. In contrast, four xenograft lines (E434, E468, E473 and E478), established by direct intracerebral inoculation of human glioma cells and serially propagated intracerebrally, consistently showed extensive diffuse infiltration throughout the brain. After several passages, the neoplastic cells still carry typical chromosomal aberrations [(-1p/-19q in oligodendroglioma, +7/-10 in glioblastoma multiforme (GBM)]. Especially these latter four models and the E98 line thus represent adequate geno- and phenocopies of human gliomas and form an attractive platform to investigate different therapeutic approaches in a preclinical setting.
[Show abstract][Hide abstract] ABSTRACT: Expression of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT), encoded by the O6-methylguanine (O6-mG) -DNA-methyltransferase (MGMT) DNA repair gene, results in resistance to alkylating agents, and hypermethylation of the MGMT promoter is associated with chemosensitivity as it prevents AGT expression. As the interpretation of the results of immunohistochemistry to evaluate AGT expression proved to be difficult, the aim of our present study is to establish a feasible, reliable, and robust method for MGMT promoter hypermethylation testing that can be easily implemented in a diagnostic setting and is applicable to routinely processed tissue. MGMT hypermethylation analysis using methylation-specific (MS-) multiplex ligation-dependent probe amplification (MLPA) was performed on 62 glioma samples of 55 individual tumors (including 12 cell lines) and compared to the more conventionally used, but improved, MS-polymerase chain reaction (PCR). In contrast to MS-PCR, MS-MLPA (i) is not based on bisulfite conversion of unmethylated cytosines (a somewhat troublesome step in MS-PCR), (ii) provided methylation status of all samples, (iii) proved to be semiquantitative, (iv) can be used to evaluate methylation status of multiple sequences (CpG dinucleotides) simultaneously, and (v) allows for a combined copy number detection and methylation specific analysis. The potential therapeutic value of MGMT hypermethylation evaluation using MS-MLPA was shown in a group of 20 glioblastoma patients receiving temozolomide chemotherapy. We conclude that MS-MLPA is a robust and reliable method that can be easily applied to differently processed tissues, including those fixed in formalin and embedded in paraffin. The semiquantitative aspect of MS-MLPA may prove to be of great value, especially in predicting response to alkylating agents, not only for gliomas as evaluated in this study but also for tumors in general.
Preview · Article · Nov 2007 · Laboratory Investigation
[Show abstract][Hide abstract] ABSTRACT: Aberrant RAS/RAF signaling has been reported to be important for many tumor types including gliomas. Activation of the RAS/RAF pathway can result from oncogenic mutations of RAS/RAF itself. However, such mutations have only occasionally been reported in gliomas. In order to further elucidate the role of RAS/RAF pathway activation in a histopathological and genetic spectrum of glioma subtypes (n = 93), we evaluated different types of aberrations in this pathway. Hotspot mutation analysis of BRAF, NRAS, KRAS, and HRAS revealed only two mutations, V600M in BRAF and G10E in NRAS, both occurring in pure oligodendroglial tumors. However, CGH analysis of 87 tumors revealed copy number gains including the above mentioned oncogenes in 38 of the neoplasms (44%) and including the upstream growth factors EGF, PDGF, IGF, FGF, TGF and/or their receptors in 46 tumors (53%). Phosphorylated MAPK (i.e. the activated compound downstream the RAS/RAF pathway) was detected by immunohistochemistry using tissue micro-arrays in the majority of gliomas. Interestingly, a significant correlation was found for nuclear MAPK-P staining and the number of these copy number gains (<or= 2 and >or= 3). These results indicate that RAS/RAF pathway activation in gliomas is achieved much more frequently by copy number gains including RAS/RAF and/or upstream growth factor (receptor) than by activating RAS/RAF mutations.
No preview · Article · Sep 2007 · Acta Neuropathologica
[Show abstract][Hide abstract] ABSTRACT: Genetic aberrations in tumors are predictive for chemosensitivity and survival. A test is needed that allows simultaneous detection of multiple changes and that is widely applicable in a routine diagnostic setting. Multiplex ligation-dependent probe amplification (MLPA) allows detection of DNA copy number changes of up to 45 loci in one relatively simple, semiquantitative polymerase chain reaction-based assay. To assess the applicability of MLPA, we performed MLPA analysis to detect relevant genetic markers in a spectrum of 88 gliomas. The vast majority of these tumors (n = 79) were previously characterized by comparative genomic hybridization. With MLPA kit P088 (78 cases), complete and partial loss of 1p and 19q were reliably identified, even in samples containing only 50% tumor DNA. Distinct 1p deletions exist with different clinically prognostic consequences, and in contrast to the commonly used diagnostic strategies (loss of heterozygosity or fluorescent in situ hybridization 1p36), P088 allows detection of such distinct 1p losses. Combining P088 with P105 will further increase the accurate prediction of clinical behavior because this kit identified markers (EGFR, PTEN, and CDKN2A) of high-grade malignancy in 41 cases analyzed. We conclude that MLPA is a reliable diagnostic tool for simultaneous identification of different region-specific genetic aberrations of tumors.
Preview · Article · Oct 2006 · Journal of Molecular Diagnostics
[Show abstract][Hide abstract] ABSTRACT: Central nervous system (CNS) hemangioblastomas are highly-vascularized tumors occurring in sporadic form or as a manifestation of von Hippel–Lindau disease (VHL). The VHL protein (pVHL) regulates various target genes, one of which is the CCND1 gene, encoding cyclin D1, a protein that plays a critical role in the control of the cell cycle. Overexpression of cyclin D1 is found in many cancers. The CCND1 gene contains a common G → A polymorphism (870G > A) that enhances alternative splicing of the gene. CCND1 genotype is associated with clinical outcome in a number of cancers although prognostic significance varies with tumor type. In VHL disease, CCND1 genotype has been suggested as a genetic modifier that influences susceptibility to hemangioblastomas.
In order to analyze whether CCND1 genotype plays a role in sporadic CNS hemangioblastomas, we investigated CCND1 genotype in tumor tissue of 17 sporadic and also in five VHL-related CNS hemangioblastomas. In addition, in these tumors the extent and localization of cyclin D1 expression was investigated by immunohistochemistry. We found no deviation in CCND1 genotype distribution and allele frequencies from expected values. Also, there was no correlation between age at onset and CCND1 genotype. The expression of cyclin D1 as detected by immunohistochemistry was highly variable within and between tumors, without a clear correlation with CCND1 genotype. We conclude that, whereas variable but sometimes high cyclin D1 expression is a feature of sporadic hemangioblastomas, CCND1 genotype is unlikely to be an important genetic modifier in the oncogenesis of these tumors.
Preview · Article · Sep 2005 · Journal of Neuro-Oncology
[Show abstract][Hide abstract] ABSTRACT: Von Hippel-Lindau (VHL) disease is an autosomal dominant tumor syndrome, in which hemangioblastomas (HBs), clear cell renal cell carcinomas (RCCs), and pheochromocytomas are the most frequently encountered tumors. The differential diagnosis of dedifferentiated tumors in general can be difficult, as standard histologic and immunohistochemical investigations do not always allow a definitive diagnosis. We used molecular genetic analysis to resolve the differential diagnosis of sarcomatoid RCC versus pheochromocytoma of a (peri)renal tumor in a VHL patient. Germline mutation analysis identified the C407T mutation, which has been related to a VHL phenotype in which pheochromocytomas are rare. Chromosomal imbalances detected in the tumor by CGH showed a pattern typical for RCCs and not for pheochromocytomas. CGH analysis of the multiple tumors of this VHL patient revealed a comparable karyotype in the metastatic tumors and the (peri)renal tumor. Concordantly, although the germline mutation was detected in all analyzed tumors, LOH 3p was only detected in the (peri)renal mass and most metastases. Overall, based on all genetic data, this tumor corroborated a diagnosis of metastatic sarcomatoid RCC. In line with these observations is the immunopositivity for the RCC-specific RC38 detected in the (peri)renal mass and the metastases that was not detected in pheochromocytomas. The RCC specific marker G250 was uninformative as it stains positive in all types of VHL tumors. This case report illustrates the promising role of genetic analysis in the differential diagnosis of histologically dedifferentiated tumors.
[Show abstract][Hide abstract] ABSTRACT: Molecular analysis on formalin-fixed paraffin-embedded tissue is of increasing importance in diagnostic histopathology and tumor research. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can be used for detection of copy number alterations of up to 45 different DNA sequences in one experiment. It can be performed on partially degraded DNA, which makes this technique very suitable for analysis of formalin-fixed lesions. We tested the reliability of MLPA by analyzing DNA isolated from formalin-fixed melanomas that were previously characterized by comparative genomic hybridization (CGH), and additionally the applicability of MLPA was tested by analyzing 29 routinely processed melanocytic lesions. MLPA appears to be a reliable and efficient method to evaluate DNA copy number changes as 86% of the loci tested revealed concordant CGH results. Discordance mainly involved alterations that were detected by MLPA and not by CGH probably due to a combination of lower resolution of CGH and occasionally false positive MLPA results. For application of MLPA in a diagnostic setting, different probes on a specific region of interest should be used to prevent false positive MLPA results. In a research setting as well as in a diagnostic setting, MLPA is a fast technique to screen large numbers of formalin-fixed lesions for DNA gains and losses.