Distinguishing and grading human gliomas by IR spectroscopy

As a molecular probe of tissue composition, IR spectroscopy can potentially serve as an adjunct to histopathology in detecting and diagnosing disease. This study demonstrates that cancerous brain tissue (astrocytoma, glioblastoma) is distinguishable from control tissue on the basis of the IR spectra of thin tissue sections. It is further shown that the IR spectra of astrocytoma and glioblastoma affected tissue can be discriminated from one another, thus providing insight into the malignancy grade of the tissue. Both the spectra and the methods employed for their classification reveal characteristic differences in tissue composition. In particular, the nature and relative amounts of brain lipids, including both the gangliosides and phospholipids, appear to be altered in cancerous compared to control tissue. Using a genetic classification approach, classification success rates of up to 89% accuracy were obtained, depending on the number of regions included in the model. The diagnostic potential and practical applications of IR spectroscopy in brain tumor diagnosis are discussed.     

Expression of the neurotrophin receptors Trk A and Trk B in adult human astrocytoma and glioblastoma

Neurotrophins and their receptors of the Trk family play a critical role in proliferation, differentiation and survival of the developing neurons. There are reports on their expression in neoplasms too, namely, the primitive neuroectodermal tumours of childhood, and in adult astrocytic gliomas. The involvement of Trk receptors in tumour pathogenesis, if any, is not known. With this end in view, the present study has examined 10 tumour biopsy samples (identified as astrocytoma, pilocytic astrocytoma and glioblastoma) and peritumoral brain tissue of adult patients, for the presence of Trk A and Trk B receptors, by immunohistochemistry. The nature of the tumour samples was also confirmed by their immunoreactivity (IR) to glial fibrillary acidic protein. In the peritumoral brain tissue, only neurons showed IR for Trk A and Trk B. On the contrary, in the tumour sections, the IR to both receptors was localized in the vast majority of glia and capillary endothelium. There was an obvious pattern of IR in these gliomas: high levels of IR were present in the low-grade (type I and II) astrocytoma; whereas in the advanced malignant forms (WHO grade IV giant cell glioblastoma and glio-blastoma multiforme) the IR was very weak. These findings suggest that Trk A and Trk B are involved in tumour pathogenesis, especially in the early stage, and may respond to signals that elicit glial proliferation, and thus contribute to progression towards malignancy.     

综述与专论: 脊髓星形细胞瘤研究进展

脊髓星形细胞瘤是一种罕见的中枢神经系统恶性肿瘤,在流行病学、肿瘤临床学表型、分子遗传标记、治疗及研究方面有着独特特征。虽然随着手术技术的进步以及分子病理的发展,脑胶质瘤的研究和治疗取得较大进展,但脊髓星形细胞瘤的研究和治疗却发展缓慢。其原因一方面在于临床样本较少,难以开展研究,另一方面因其分子遗传独特性,对脑胶质瘤一线化疗药替莫唑胺敏感性差。因而亟需理清脊髓星形细胞瘤的研究现状,为改善其临床疗效梳理潜在方向。基于此,本文综述脊髓星形细胞瘤的临床特征、病理分型、分子遗传特征和当前治疗方法等方面的研究进展,在描绘脊髓星形细胞瘤的临床治疗现状和研究进展的基础上,提出了未来研究和治疗潜在方向。     

In vitro study of astrocytic tumour metabolism by proton magnetic resonance spectroscopy

In vivo magnetic resonance spectroscopy (MRS) studies of glial brain tumours reported that higher grade of astrocytoma is associated with increased level of choline-containing compounds (Cho) and decreased levels of N-acetylaspartate (NAA) and creatine and phosphocreatine (Cr). In this work, we studied the metabolism of glioma tumours by in vitro proton magnetic resonance spectroscopy (1H-MRS). 1H-MR spectra were recorded in vitro from perchloric acid extracts of astrocytoma (WHO II) and glioblastoma multiforme (WHO IV) samples. We observed differences between astrocytoma and glioblastoma multiforme in the levels of Cho, alanine, lactate, NAA, and glutamate/glutamine. In astrocytoma samples, we found higher MR signal of NAA and lower signal of Cho and alanine. MR spectra of glioblastoma samples reported significantly higher levels of lactate and glutamate/glutamine. In contrast, levels of Cr were the same in both tumour types. We also determined NAA/Cr and Cho/Cr ratios in the tumour samples. The NAA/Cr ratio was higher in astrocytomas than in glioblastomas multiforme. Conversely, the Cho/Cr ratio was higher in glioblastoma multiforme. The results indicate that MRS is a promising method for distinguishing pathologies in human brain and for pre-surgical grading of brain tumours.     

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.     

Modulation of HJURP (Holliday Junction-Recognizing Protein) Levels Is Correlated with Glioblastoma Cells Survival

Background

Diffuse astrocytomas are the most common type of primary brain cancer in adults. They present a wide variation in differentiation and aggressiveness, being classified into three grades: low-grade diffuse astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma multiforme (grade IV), the most frequent and the major lethal type. Recent studies have highlighted the molecular heterogeneity of astrocytomas and demonstrated that large-scale analysis of gene expression could help in their classification and treatment. In this context, we previously demonstrated that HJURP, a novel protein involved in the repair of DNA double-strand breaks, is highly overexpressed in glioblastoma.

Methodology/Principal Findings

Here we show that HJURP is remarkably overexpressed in a cohort composed of 40 patients with different grade astrocytomas. We also observed that tumors presenting the higher expression levels of HJURP are associated with poor survival prognosis, indicating HJURP overexpression as an independent prognostic factor of death risk for astrocytoma patients. More importantly, we found that HJURP knockdown strongly affects the maintenance of glioblastoma cells in a selective manner. Glioblastoma cells showed remarkable cell cycle arrest and premature senescence that culminated in elevated levels of cell death, differently from non-tumoral cells that were minimally affected.

Conclusions

These data suggest that HJURP has an important role in the maintenance of extremely proliferative cells of high-grade gliomas and point to HJURP as a potential therapeutic target for the development of novel treatments for glioma patients.     

Proteomic analysis of low‐ to high‐grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non‐neoplastic brain tissue as control using 2‐DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up‐regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p<0.05), whereas four proteins were down‐regulated, among them raf kinase inhibitor protein (RKIP) (p<0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real‐time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.     

CD90 is identified as a candidate marker for cancer stem cells in primary high-grade gliomas using tissue microarrays

Although CD90 has been identified as a marker for various kinds of stem cells including liver cancer stem cells (CSCs) that are responsible for tumorigenesis, the potential role of CD90 as a marker for CSCs in gliomas has not been characterized. To address the issue, we investigated the expression of CD90 in tissue microarrays containing 15 glioblastoma multiformes (GBMs), 19 WHO grade III astrocytomas, 13 WHO grade II astrocytomas, 3 WHO grade I astrocytomas and 8 normal brain tissues. Immunohistochemical analysis showed that CD90 was expressed at a medium to high level in all tested high-grade gliomas (grade III and GBM) whereas it was barely detectable in low-grade gliomas (grade I and grade II) and normal brains. Double immunofluorescence staining for CD90 and CD133 in GBM tissues revealed that CD133(+) CSCs are a subpopulation of CD90(+) cells in GBMs in vivo. Flow cytometry analysis of the expression of CD90 and CD133 in GBM-derived stem-like neurospheres further confirmed the conclusion in vitro. The expression levels of both CD90 and CD133 were reduced along with the loss of stem cells after differentiation. Furthermore, the limiting dilution assay demonstrated that the sphere formation ability was comparable between the CD90(+)/CD133(+) and the CD90(+)/CD133(-) populations of GBM neurospheres, which is much higher than that of the CD90(-)/CD133(-) population. We also performed double staining for CD90 and a vascular endothelial cell marker CD31 in tissue microarrays which revealed that the CD90(+) cells were clustered around the tumor vasculatures in high-grade glioma tissues. These findings suggest that CD90 is not only a potential prognostic marker for high-grade gliomas but also a marker for CSCs within gliomas, and it resides within endothelial niche and may also play a critical role in the generation of tumor vasculatures via differentiation into endothelial cells.     

Infrared spectroscopy with multivariate analysis potentially facilitates the segregation of different types of prostate cell

The prostate gland is conventionally divided into zones or regions. This morphology is of clinical significance as prostate cancer (CaP) occurs mainly in the peripheral zone (PZ). We obtained tissue sets consisting of paraffin-embedded blocks of cancer-free transition zone (TZ) and PZ and adjacent CaP from patients (n = 6) who had undergone radical retropubic prostatectomy; a seventh tissue set of snap-frozen PZ and TZ was obtained from a CaP-free gland removed after radical cystoprostatectomy. Paraffin-embedded tissue slices were sectioned (10-mum thick) and mounted on suitable windows to facilitate infrared (IR) spectra acquisition before being dewaxed and air dried; cryosections were dessicated on BaF(2) windows. Spectra were collected employing synchrotron Fourier-transform infrared (FTIR) microspectroscopy in transmission mode or attenuated total reflection-FTIR (ATR) spectroscopy. Epithelial cell and stromal IR spectra were subjected to principal component analysis to determine whether wavenumber-absorbance relationships expressed as single points in "hyperspace" might on the basis of multivariate distance reveal biophysical differences between cells in situ in different tissue regions. After spectroscopic analysis, plotted clusters and their loadings curves highlighted marked variation in the spectral region containing DNA/RNA bands ( approximately 1490-1000 cm(-1)). By interrogating the intrinsic dimensionality of IR spectra in this small cohort sample, we found that TZ epithelial cells appeared to align more closely with those of CaP while exhibiting marked structural differences compared to PZ epithelium. IR spectra of PZ stroma also suggested that these cells are structurally more different to CaP than those located in the TZ. Because the PZ exhibits a higher occurrence of CaP, other factors (e.g., hormone exposure) may modulate the growth kinetics of initiated epithelial cells in this region. The results of this pilot study surprisingly indicate that TZ epithelial cells are more likely to exhibit what may be a susceptibility-to-adenocarcinoma spectral signature. Thus, IR spectroscopy on its own may not be sufficient to identify premalignant prostate epithelial cells most likely to progress to CaP.     

Down-regulation of 14-3-3 zeta sensitizes human glioblastoma cells to apoptosis induction

Strong 14-3-3 zeta protein expression plays an important role in tumorigenesis, including in the maintenance of cell growth, resistance increase, and the prevention of apoptosis. In this study, we focus on two targets: (1) the expression of 14-3-3 zeta in the different grades of human astrocytoma (II–IV), (2) suppression of 14-3-3 zeta protein expression in glioblastoma derived astrocytes by 14-3-3 zeta shRNA lentiviral particles. The tissues of human astrocytoma were provided from 30 patients (ten of each grade of astrocytoma). Control tissues were obtained from the peritumoral brain zone of those patients with glioblastoma. The protein and mRNA expression levels of each astrocytoma grade were assessed via western blotting and RT-PCR, respectively. Results indicated that 14-3-3 zeta was significantly expressed in glioblastoma multiforme (grade IV) and 14-3-3 zeta expression levels enhanced according to the increase of astrocytoma malignancy. In the cellular study for knock down of the 14-3-3 zeta protein, surgical biopsy of glioblastoma was used to isolate primary astrocyte. Astrocytes were transduced with 14-3-3 zeta shRNA or non-targeted shRNA lentiviral particles. Furthermore, reduction of the 14-3-3 zeta protein expression in the astrocytes evaluated through qRT-PCR and western blot after transduction of 14-3-3 zeta shRNA lentiviral particles. Moreover, apoptosis properties, including DNA fragmentation and ratio increase of Bax/Bcl-2 were observed in astrocytes following reduction of 14-3-3 zeta protein expression. Further observation indicated that the mitochondrial pathway through release of cytochorome c and caspase-3 activity was involved in the apoptosis induction. Hence, this study demonstrates a key role of the 14-3-3 zeta protein in tumorigenesis but also indicates that 14-3-3 zeta can be considered as a target for the astrocytoma treatment specially glioblastoma.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) production by glioblastoma cells. Despite the presence of inducing signals GM-CSF is not expressed in vivo.

One of the morphologic hallmarks of human gliomas are inflammatory infiltrates with accumulation of macrophages in the tumor site. The signals leading to the macrophage response are only at the beginning of being understood. Novel chemotactic factors that have recently been characterized as secretory products of glioblastoma cells may attract mononuclear cells from the blood. Within the tumor tissue blood-derived monocytes and macrophages of the brain tissue, the microglial cells, may increase in cell numbers due to tumor-derived growth factors. Both astrocytoma cell lines and cultured astrocytes have been shown recently to produce granulocyte-macrophage (GM)-CSF. We show that in vitro not only astrocytoma but also glioblastoma cell lines secrete GM-CSF when stimulated with TNF-alpha or IL-1. However, there is no evidence for GM-CSF production by glioblastoma cells in vivo: fresh tumor samples lack the mRNA for GM-CSF and the protein is not detectable in the tumor cyst fluids or the cerebrospinal fluids of glioblastoma patients. This contrasts IL-1 and IL-6 that are detectable in the tumor cyst fluids and IL-6 also in the cerebrospinal fluids of the patients. Unlike GM-CSF, transforming growth factor-beta 2 mRNA is expressed in ex vivo tested glioblastoma tissues. Absence of GM-CSF in vivo may be explained by the presence of tumor-derived inhibitory factors, such as transforming growth factor-beta 2 and PGE which suppress GM-CSF production by glioblastoma cells in vitro. The accumulation of macrophages at the tumor site may be due to local elaboration of chemoattractants and/or not yet defined growth factors rather than due to GM-CSF production.     

Gene Expression Analysis of PTEN Positive Glioblastoma Stem Cells Identifies DUB3 and Wee1 Modulation in a Cell Differentiation Model

The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway.     

Characterization of lipids from human brain tissues by multinuclear magnetic resonance spectroscopy

Multinuclear ((1)H, (13)C, and (31)P) magnetic resonance spectroscopy are applied to the biochemical characterization of the total lipid fraction of healthy and neoplastic human brain tissues. Lipid extracts from normal brains, glioblastomas, anaplastic oligodendrogliomas, oligodendrogliomas, and meningiomas are examined. Moreover, the unknown liquid content of a cyst adjacent to a meningioma is analyzed. Two biopsies from glioblastomas are directly studied by (1)H-NMR without any treatment (ex vivo NMR). The (1)H- and (13)C-NMR analysis allows full characterization of the lipid component of the cerebral tissues. In particular, the presence of cholesteryl esters and triglycerides in the extracts of high grade tumors is correlated to the vascular proliferation degree, which is different from normal brain tissue and low grade neoplasms. The (31)P spectra show that phosphatidylcholine is the prominent phospholipid and its relative amount, which is higher in gliomas, is correlated to the low grade of differentiation of tumor cells and an altered membrane turnover. The ex vivo (1)H-NMR data on the glioblastoma samples show the presence of mobile lipids that are correlated to cell necrotic phenomena. Our data allow a direct correlation between biochemical results obtained by NMR and the histopathological factors (vascular and cell proliferations, differentiation, and necrosis) that are prominent in determining brain tumor grading.     

Correlation between preoperative magnetic resonance spectroscopic data on high grade gliomas and morphology of Ki-67-positive tumor cell nuclei

OBJECTIVE: To investigate possible statistical correlations between metabolic data from preoperative proton magnetic resonance spectroscopy (1HMRS) and morphology of proliferating tumor cell nuclei in anaplastic gliomas and glioblastomas. STUDY DESIGN: Ki-67-positive tumor cell nuclei in paraffin sections of surgical specimens from 36 patients (7 anaplastic gliomas, World Health Organization grade 3; 29 glioblastomas, World Health Organization grade 4) were investigated by means of a digital image analysis system. Stringent inclusion criteria were formulated for all cases with respect to histologic quality and spectroscopic examination. As morphometric variables, nuclear area, shape variables (roundness factor, size-invariate Fourier amplitudes) and density of Ki-67-positive tumor cell nuclei per reference area were determined. RESULTS: Correlation analysis according to Spearman revealed a significant positive correlation between the total creatine (TCR) peak and nuclear area (P = .005). This correlation was also found within the glioblastoma group (P = .019). There was also a significant negative correlation of nuclear area with the ratio between choline and TCR in all cases (P = .014) and within the glioblastoma group (P = .046). No significant correlation of spectroscopic data was found with nuclear shape or density of Ki-67-positive tumor cell nuclei. CONCLUSION: The results demonstrate a correlation between spectroscopic data and morphology of proliferating tumor cell nuclei (nuclear size) in high grade gliomas. This study is part of a detailed investigation of the interrelationship between preoperative 1HMRS and quantitative histomorphology of gliomas.     

First step toward the “fingerprinting” of brain tumors based on synchrotron radiation X-ray fluorescence and multiple discriminant analysis

Synchrotron-radiation-based X-ray fluorescence was applied to the elemental microimaging of neoplastic tissues in cases of various types of brain tumors. The following cases were studied: glioblastoma multiforme, gemistocytic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, ganglioglioma, fibrillary astrocytoma, and atypical transitional meningioma. Apart from neoplastic tissue, the analysis included areas of tissue apparently without malignant infiltration. The masses per unit area of P, S, Cl, K, Ca, Fe, Cu, Zn, Br, and Rb were used to construct a diagnostic classifier for brain tumors using multiple discriminant analysis. It was found that S, Cl, Cu, Fe, K, Br, and Zn are the most significant elements in the general discrimination of tumor type. The highest similarity in elemental composition was between atypical transitional meningioma and fibrillary astrocytoma. The smallest differentiation was between glioblastoma multiforme and oligodendroglioma. The mean percentage of correct classifications, estimated according to the a posteriori probabilities procedure, was 99.9%, whereas the mean prediction ability of 87.6% was achieved for ten new cases excluded previously from the model construction. The results showed that multiple discriminant analysis based on elemental composition of tissue may be a potentially valuable method assisting differentiation and/or classification of brain tumors.     

Infrared spectroscopic discrimination between the loop and alpha-helices and determination of the loop diffusion kinetics by temperature-jump time-resolved infrared spectroscopy for cytochrome c

The infrared (IR) absorption of the amide I band for the loop structure may overlap with that of the alpha-helices, which can lead to the misassignment of the protein secondary structures. A resolution-enhanced Fourier transform infrared (FTIR) spectroscopic method and temperature-jump (T-jump) time-resolved IR absorbance difference spectra were used to identify one specific loop absorption from the helical IR absorption bands of horse heart cytochrome c in D2O at a pD around 7.0. This small loop consists of residues 70-85 with Met-80 binding to the heme Fe(III). The FTIR spectra in amide I' region indicate that the loop and the helical absorption bands overlap at 1653 cm(-1) at room temperature. Thermal titration of the amide I' intensity at 1653 cm(-1) reveals that a transition in loop structural change occurs at lower temperature (Tm=45 degrees C), well before the global unfolding of the secondary structure (Tm approximately 82 degrees C). This loop structural change is assigned as being triggered by the Met-80 deligation from the heme Fe(III). T-jump time-resolved IR absorbance difference spectra reveal that a T-jump from 25 degrees C to 35 degrees C breaks the Fe-S bond between the Met-80 and the iron reversibly, which leads to a loop (1653 cm(-1), overlap with the helical absorption) to random coil (1645 cm(-1)) transition. The observed unfolding rate constant interpreted as the intrachain diffusion rate for this 16 residue loop was approximately 3.6x10(6) s(-1).     

Investigation of stretching vibrations of glycosidic linkages in disaccharides and polysaccharides with use of IR spectra deconvolution

The results are presented for the deconvolution of IR spectra of disaccharides and polysaccharides with alpha and beta configurations of the 1 --> 4 glycosidic linkage (maltose, cellobiose, amylose, and cellulose), as well as of their corresponding monosaccharides (alpha- and beta-D-glucose) in the 1200-920 cm(-1) frequency range. It is established that a characteristic of di- and polysaccharides with 1 --> 4 glycosidic linkage is the appearance of new absorption bands in the 1175-1140 cm(-1) spectral range, as opposed to the IR spectra of monosaccharides. This can be a spectroscopic manifestation of the glycosidic linkage formation. In the 1000-970 cm(-1) frequency range, absorption bands, which are not observed in the monomer spectrum, are separated as a result of the deconvolution of the IR spectra of cellobiose and cellulose. The number of bands in this range remains unchanged for maltose and amylose, as compared to the monomer spectra. It is shown that the application of the method of deconvolution leads to a considerable enhancement in the resolution of the absorption bands in the IR spectra of mono-, di-, and polysaccharides.     

A different view on DNA amplifications indicates frequent, highly complex, and stable amplicons on 12q13-21 in glioma

To further understand the biological significance of amplifications for glioma development and recurrencies, we characterized amplicon frequency and size in low-grade glioma and amplicon stability in vivo in recurring glioblastoma. We developed a 12q13-21 amplicon-specific genomic microarray and a bioinformatics amplification prediction tool to analyze amplicon frequency, size, and maintenance in 40 glioma samples including 16 glioblastoma, 10 anaplastic astrocytoma, 7 astrocytoma WHO grade 2, and 7 pilocytic astrocytoma. Whereas previous studies reported two amplified subregions, we found a more complex situation with many amplified subregions. Analyzing 40 glioma, we found that all analyzed glioblastoma and the majority of pilocytic astrocytoma, grade 2 astrocytoma, and anaplastic astrocytoma showed at least one amplified subregion, indicating a much higher amplification frequency than previously suggested. Amplifications in low-grade glioma were smaller in size and displayed clearly different distribution patterns than amplifications in glioblastoma. One glioblastoma and its recurrencies revealed an amplified subregion of 5 Mb that was stable for 6 years. Expression analysis of the amplified region revealed 10 overexpressed genes (i.e., KUB3, CTDSP2, CDK4, OS-9, DCTN2, RAB3IP, FRS2, GAS41, MDM2, and RAP1B) that were consistently overexpressed in all cases that carried this amplification. Our data indicate that amplifications on 12q13-21 (a) are more frequent than previously thought and present in low-grade tumors and (b) are maintained as extended regions over long periods of time.