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Precursor T-cell acute lymphoblastic leukemia (T-ALL) in children represents a clinical challenge, because relapses are usually fatal. It is thus necessary to identify high-risk patients as early as possible to effectively individualize treatment. We aimed to define novel molecular risk markers in T-ALL and performed array-based comparative genomic hybridization (array-CGH) and expression analyses in 73 patients. We show that DNA copy-number changes are common in T-ALL and affect 70 of 73 (96%) patients. Notably, genomic imbalances predicted to down-regulate the TGF-beta or up-regulate the PI3K-AKT pathways are identified in 25 of 73 (34%) and 21 of 73 (29%) patients, suggesting that these pathways play key roles in T-ALL leukemogenesis. Furthermore, we identified a deletion at 6q15-16.1 in 9 of 73 (12%) of the patients, which predicts poor early treatment response. This deletion includes the CASP8AP2 gene, whose expression is shown to be down-regulated. The interaction of CASP8AP2 with CASP8 plays a crucial role in apoptotic regulation, suggesting a functional link between the clinical effect of the deletion and the molecular mode of action. The data presented here implicate the TGF-beta and PI3K-AKT pathways in T-ALL leukemogenesis and identify a subgroup of patients with CASP8AP2 deletions and poor early treatment response.

Summary: Support vector machine (SVMs) classification is a widely used and one of the most powerful classification techniques. However, a major limitation is that SVM cannot perform automatic gene selection. To overcome this restriction, a number of penalized feature selection methods have been proposed. In the R package 'penalizedSVM' implemented penalization functions L(1) norm and Smoothly Clipped Absolute Deviation (SCAD) provide automatic feature selection for SVM classification tasks., Availability: The R package 'penalizedSVM' is available from the Comprehensive R Archive Network (http://cran.r-project.org/) under GPL-2 or later.

The molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis of DNA copy number aberrations in pediatric low-grade astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPK pathway due to gene duplication or mutation of BRAF as a molecular mechanism of pathogenesis in low-grade astrocytomas and suggest inhibition of the MAPK pathway as a potential treatment.

Unlabelled: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is characterized by aggressive tumor behavior coupled with poor prognosis. Various etiologies have been linked to HCC development, most prominently chronic hepatitis B and C virus infections as well as chronic alcohol consumption. In approximately 10% of HCCs, the etiology remains cryptic; however, recent epidemiological data suggest that most of these cryptogenic HCCs develop due to nonalcoholic steatohepatitis. To identify etiology-dependent DNA copy number aberrations and genes relevant to hepatocarcinogenesis, we performed array-based comparative genomic hybridization of 63 HCCs of well-defined etiology and 4 HCC cell lines followed by gene expression profiling and functional analyses of candidate genes. For a 10-megabase chromosome region on 8q24, we observed etiology-dependent copy number gains and MYC overexpression in viral and alcohol-related HCCs, resulting in up-regulation of MYC target genes. Cryptogenic HCCs showed neither 8q24 gains, nor MYC overexpression, nor target gene activation, suggesting that tumors of this etiology develop by way of a distinct MYC-independent pathomechanism. Furthermore, we detected several etiology-independent small chromosome aberrations, including amplification of MDM4 on 1q32.1 and frequent gains of EEF1A2 on 20q13.33. Both genes were overexpressed in approximately half the HCCs examined, and gene silencing reduced cell viability as well as proliferation and increased apoptosis rates in HCC cell lines., Conclusion: Our findings suggest that MDM4 and EEF1A2 act as etiology-independent oncogenes in a significant percentage of HCCs.

Supratentorial primitive neuroectodermal tumors (stPNETs) and medulloblastomas have long been thought to arise from a common cell type in the subventricular germinal matrix. Because of the infrequent occurrence of stPNETs, little is known about their genetic background. Here, we performed a genome-wide screening for DNA copy-number aberrations in 10 supratentorial PNETs using array-based comparative genomic hybridization (array-CGH). Comparing our findings with data from a previous array-CGH study on 47 medulloblastomas, we identified differences in the frequency of copy-number losses at chromosome regions 1p12-22.1 and 9p, and gains at 19p, all of them more frequently occurring in stPNETs. In contrast to previous reports, we detected chromosome 17 aberrations by array-CGH in 2/10 stPNETs. To validate our findings obtained by array-CGH, we analyzed the loci of interest by fluorescence in situ hybridization in an independent set of 11 stPNETs and found deletions of 9p21 in 5/11 tumors of the second set, three of them being homozygous. All 9p21 deletions were associated with loss of CDKN2A protein expression. Altogether, CDKN2A deletions were detected in 7/21 stPNETs including four homozygous deletions, whereas such deletions were only found in 4/112 medulloblastomas, all of these being heterozygous (P < 0.001). Gains of 19p (14% vs. 0% in medulloblastomas, P = 0.02) were found to be significantly more frequent in stPNETs, whereas gains of 17q (14% vs. 45% in medulloblastomas, P = 0.02) were confirmed to be more frequent in medulloblastomas. These data further support the hypothesis of two different tumor entities of embryonal neuroepithelial tumors with characteristic genetic aberrations. (c) 2007 Wiley-Liss, Inc.

We have comparatively analyzed mechanisms associated with chromosomal and microsatellite instability in giant cell glioblastoma multiforme (gcGBM) and classic GBM. This included microsatellite instability (MSI), loss of expression of four major mismatch repair (MMR) proteins, aberrations of five chromosomes, EGFR copy number, and TP53 mutations. MSI was more frequent among gcGBM (30 vs. 7.8%, P = 0.054). TP53 mutations were more commonly observed in gcGBM (83.3%), whereas EGFR was amplified in just one gcGBM (8.3%). By tumor cell phenotype-specific cytogenetic analysis of gcGBM, increased chromosome copy numbers were identified in 72-84% of giant cells but in only 4-14% of nongiant cells; in classic GBM, intermediate frequencies were noted (11-49%). Chromosome 10 deletions were found in nongiant cells of all gcGBM cases but in only approximately 45% of the cell population in classic GBM. The present study shows a distinct pattern of cytogenetic alterations in nongiant and giant cell phenotypes in gcGBM and suggests that multinuclear giant cells evolve from nongiant tumor cells at an early tumor stage. Furthermore, the data point to differences in the profile of chromosomal and microsatellite instability in gcGBM and classic GBM that might underscore the distinct pathological features of both tumor subtypes.

Loss of heterozygosity (LOH) on chromosomal arms 1p and 19q is the most common genetic alteration in oligodendroglial tumors and associated with response to radio- and chemotherapy as well as favorable prognosis. Using microsatellite analysis, we previously identified the chromosomal regions 1p36.22-p36.31 and 19q13.3, as candidate tumor suppressor gene regions being commonly deleted in these tumors. To identify genes within these regions that are downregulated in oligodendroglial tumors with LOH 1p/19q, we performed cDNA microarray-based RNA expression profiling of 35 gliomas with known allelic status on 1p and 19q, including 7 oligodendrogliomas and 8 diffuse astrocytomas of World Health Organization (WHO) grade II, as well as 14 anaplastic oligodendrogliomas and 6 anaplastic oligoastrocytomas of WHO grade III. The microarrays used for expression profiling carried approximately 7,000 gene-specific cDNAs, with complete coverage of the genes located in 1p36.13-p36.31 and 19q13.2-q13.33. Microarray analysis identified 8 genes from these regions (MGC4399, SRM, ICMT, RPL18, FTL, ZIN, FLJ10781 and DBP), which all showed significantly lower expression in 1p/19q-deleted gliomas when compared to gliomas without 1p/19q losses. Quantitative real-time reverse transcription-PCR analyses were performed for the MGC4399, ICMT and RPL18 genes and confirmed the microarray findings. In addition, we found that the cytosolic phospholipase A2 (PLA2G4C) gene at 19q13.3 demonstrated significantly lower expression in anaplastic oligodendrogliomas (WHO grade III) when compared to well-differentiated oligodendrogliomas (WHO grade II). Taken together, our study provides a set of interesting novel candidate genes that may play important roles in the pathogenesis of oligodendroglial tumors., (Copyright 2006 Wiley-Liss, Inc.)

Purpose: Primary systemic therapy (PST) with gemcitabine (G), epirubicin (E), and docetaxel (Doc) has resulted in a pathologic complete response (pCR) in 26% of primary breast cancer patients. This study was aimed at the identification of a gene expression signature in diagnostic core biopsy tissue samples that predicts pCR., Patients and Methods: Core biopsy samples from patients with operable primary breast cancer, T2-4N0-2M0, enrolled onto two phase I and II trials evaluating GEDoc (n = 48) and GE sequentially followed by Doc (GEsDoc; n = 52) as PST were snap frozen and subjected to RNA expression profiling. A signature predicting pCR was discovered in the training set (GEsDoc) applying a support vector machine algorithm, and performance of this classifier was validated on the independent test set (GEDoc) by receiver operator characteristics analysis., Results: We identified a signature consisting of 512 genes, which was enriched in genes involved in transforming growth factor beta and RAS-mediated signaling pathways, that predicts pCR with a sensitivity of 78%, a specificity of 90%, and an overall accuracy of 88% (95% CI, 75% to 95%). Apart from our signature, only HER2 overexpression was an independent predictor of pCR in multivariate analysis., Conclusion: In conclusion, our gene expression signature allows prediction of pCR to PST containing G, E, and Doc with unprecedented high overall accuracy and robustness.

Purpose: Pathogenesis of ependymomas is still poorly understood and molecular markers for risk-adapted patient stratification are not available. Our aim was to screen for novel genomic imbalances and prognostic markers in ependymal tumors., Experimental Design: We analyzed 68 sporadic tumors by matrix-based comparative genomic hybridization using DNA microarrays containing >6,400 genomic DNA fragments. Novel recurrent genomic gains were validated by fluorescence in situ hybridization using a tissue microarray consisting of 170 intracranial ependymomas. Candidate genes were also tested for mRNA expression by quantitative real-time PCR, and protein expression was determined by immunohistochemistry on the tissue microarray., Results: Chromosomal gain of 1q correlated with pediatric patients (P = 0.004), intracranial ependymomas (P = 0.05), and tumors of grade III (P = 0.002). Gain of 1q21.1-32.1 was associated with tumor recurrence in intracranial ependymomas (P < 0.001). Furthermore, gain of 1q25 as determined by fluorescence in situ hybridization represented an independent prognostic marker for either recurrence-free survival (P < 0.001) or overall survival (P = 0.003). Recurrent gains at 5p15.33 covering hTERT were validated by immunohistochemistry, and elevated protein levels correlated with adverse prognosis (P = 0.01). In addition to frequent gains and high-level amplification of epidermal growth factor receptor (EGFR) at 7p11.2, immunohistochemistry revealed protein overexpression to be correlated with poor prognosis (P = 0.002). EGFR protein status subdivides intracranial grade II ependymomas into two different risk groups (P = 0.03) as shown by multivariate analysis., Conclusions: Thus, the states of 1q25 and EGFR represent independent prognostic markers for intracranial ependymomas to identify patient subgroups with different risk profiles in further clinical investigations. Moreover, EGFR might serve as therapeutic target for more specific chemotherapy applications.

Medulloblastoma is a highly malignant embryonal tumor of the cerebellum that accounts for 20%-25% of all intracranial pediatric tumors. The most frequent chromosomal rearrangement in medulloblastoma is isochromosome 17, or i(17q). Its frequency suggests that it serves an important role in tumor pathogenesis, possibly mediated by the disruption or permanent activation of a gene at the breakpoint. To address this question, we performed a detailed analysis of chromosome 17 DNA copy number from 18 medulloblastomas previously shown to carry an apparent i(17q). We identified two breakpoint regions, one well within band 17p11.2 (n = 16) and a second within the pericentromeric region (n = 2). To map the breakpoints more precisely, we constructed a tiling-path matrix-CGH array covering chromosomal band 17p11.2 to the centromere and utilized it to delineate two small breakpoint intervals mapping at Mb 19.0 and 21.7 in seven of the medulloblastomas and in nine hematological neoplasias with i(17q). The former interval contains two breakpoint clusters that each colocalize with a pair of head-to-head inverted DNA sequence repeats, and the latter maps close to a region of alpha-satellite repeats. No consensus coding sequence localizes in these regions. Together, these data strongly suggest that the effects of i(17q) in medulloblastoma are mediated by gene-dosage effects of genes on 17p or 17q rather than by the disruption or deregulation of a "breakpoint" gene. Furthermore, we identified artifacts introduced in DNA copy number data by cross-hybridization of low-copy repeat sequences and discuss the challenge these can pose in the interpretation of diagnostic microarrays.

Chromosomal band 11q13 is frequently amplified in oral squamous cell carcinoma (OSCC) and assumed to be critically involved in tumor initiation and progression by proto-oncogene activation. Though cyclin D1 (CCND1) is supposed to be the most relevant oncogene, several additional putative candidate genes are inside this chromosomal region, for which their actual role in tumorigenesis still needs to be elucidated. To characterize the 11q13 amplicon in detail, 40 OSCCs were analyzed by comparative genomic hybridization to DNA microarrays (matrix-CGH) containing BAC clones derived from chromosomal band 11q13. This high-resolution approach revealed a consistent amplicon about 1.7 Mb in size including the CCND1 oncogene. Seven BAC clones covering FGF3, EMS1, and SHANK2 were shown to be frequently coamplified inside the CCND1 amplicon. Subsequent analysis of tissue microarrays by FISH revealed amplification frequencies of 36.8% (88/239) for CCND1, 34.3% (60/175) for FGF3, 37.4% (68/182) for EMS1, and 36.3% (61/168) for SHANK2. Finally, quantitative mRNA expression analysis demonstrated consistent overexpression of CCND1 in all tumors and of EMS1 and SHANK2 in a subset of specimens with 11q13 amplification, but no expression of FGF3 in any of the cases. Our study underlines the critical role of CCND1 in OSCC development and additionally points to the functionally related genes EMS1 and SHANK2, both encoding for cytoskeleton-associated proteins, which are frequently coamplified with CCND1 and therefore could cooperatively contribute to OSCC pathogenesis.

Purpose: Invasive ductal carcinoma and invasive lobular carcinoma (ILC) represent the major histologic subtypes of invasive breast cancer. They differ with regard to presentation, metastatic spread, and epidemiologic features. To elucidate the genetic basis of these differences, we analyzed copy number imbalances that differentiate the histologic subtypes., Experimental Design: High-resolution genomic profiling of 40 invasive breast cancers using matrix-comparative genomic hybridization with an average resolution of 0.5 Mb was conducted on bacterial artificial chromosome microarrays. The data were subjected to classification and unsupervised hierarchical cluster analyses. Expression of candidate genes was analyzed in tumor samples., Results: The highest discriminating power was achieved when combining the aberration patterns of chromosome arms 1q and 16p, which were significantly more often gained in ILC. These regions were further narrowed down to subregions 1q24.2-25.1, 1q25.3-q31.3, and 16p11.2. Located within the candidate gains on 1q are two genes, FMO2 and PTGS2, known to be overexpressed in ILC relative to invasive ductal carcinoma. Assessment of four candidate genes on 16p11.2 by real-time quantitative PCR revealed significant overexpression of FUS and ITGAX in ILC with 16p copy number gain. Unsupervised hierarchical cluster analysis identified three molecular subgroups that are characterized by different aberration patterns, in particular concerning gain of MYC (8q24) and the identified candidate regions on 1q24.2-25.1, 1q25.3-q31.3, and 16p11.2. These genetic subgroups differed with regard to histology, tumor grading, frequency of alterations, and estrogen receptor expression., Conclusions: Molecular profiling using bacterial artificial chromosome arrays identified DNA copy number imbalances on 1q and 16p as significant classifiers of histologic and molecular subgroups.

The comparison of gene expression measurements obtained with different technical approaches is of substantial interest in order to clarify whether inter-platform differences may conceal biologically significant information. To address this concern, we analyzed gene expression in a set of head and neck squamous cell carcinoma patients, using both spotted oligonucleotide microarrays made from a large collection of 70-mer probes and commercial arrays produced by in situ synthesis of sets of multiple 25-mer oligonucleotides per gene. Expression measurements were compared for 4425 genes represented on both platforms, which revealed strong correlations between the corresponding data sets. Of note, a global tendency towards smaller absolute ratios was observed when using the 70-mer probes. Real-time quantitative reverse transcription PCR measurements were conducted to verify expression ratios for a subset of genes and achieved good agreement regarding both array platforms. In conclusion, similar profiles of relative gene expression were obtained using arrays of either single 70-mer or multiple short 25-mer oligonucleotide probes per gene. Although qualitative assessments of the expression of individual genes have to be made with caution, our results indicate that the comparison of gene expression profiles generated on these platforms will help to discover disease-related gene signatures in general.

The genetic hallmark of retinoblastoma is mutation or deletion of the RB1 gene, whereas other genetic alterations that are also required are largely unknown. To screen for genomic imbalances on a genomewide level, we studied a series of 17 primary retinoblastomas by matrix-based comparative genomic hybridization (matrix-CGH). The matrix-CGH chip contained 6,000 immobilized genomic DNA fragments covering the human genome, with an average resolution of about 500 kb. The most frequent imbalances detected were gains on chromosome arms 1q (12 of 17), 6p (10 of 17), 2p (5 of 17), and 19q (4 of 17) and loss on 16q (7 of 17). Candidate regions could be narrowed to small intervals by the identified minimally overlapping regions on 1q22, 1q32.1q32.2, 2p24.1, and 6p21.33-p21.31. Furthermore, two as-yet-unknown high-level amplifications were detected, each in a single patient, on chromosome bands 1p34.2 and 1p33. Thus, this study identified new chromosomal regions and therefore potential candidate genes that may play a role in retinoblastoma., ((c) 2005 Wiley-Liss, Inc.)

Background: Interpreting the results of high-throughput experiments, such as those obtained from DNA-microarrays, is an often time-consuming task due to the high number of data-points that need to be analyzed in parallel. It is usually a matter of extensive testing and unknown beforehand, which of the possible approaches for the functional analysis will be the most informative., Results: To address this problem, we have developed the Flexible Annotation and Correlation Tool (FACT). FACT allows for detection of important patterns in large data sets by simplifying the integration of heterogeneous data sources and the subsequent application of different algorithms for statistical evaluation or visualization of the annotated data. The system is constantly extended to include additional annotation data and comparison methods., Conclusion: FACT serves as a highly flexible framework for the explorative analysis of large genomic and proteomic result sets. The program can be used online; open source code and supplementary information are available at http://www.factweb.de.

Gene expression analysis using microarrays of synthetic long oligonucleotides is limited in that it requires substantial amounts of RNA. To obtain these quantities from minute amounts of starting material, protocols were developed that linearly amplify mRNA by cDNA synthesis and in vitro transcription. Since orientation of the product is antisense (aRNA), it is inapplicable for dye-labelling by reverse transcription and hybridization to sense-oriented oligonucleotide arrays. Here, we introduce a novel protocol in which aRNA labelling is achieved by a combination of two reverse and one forward transcription reactions followed by dye-incorporation using Klenow fragment, generating fluorescent antisense cDNA. We demonstrate high fidelity in arrays using up to 10(5)-fold amplification, starting from 2 ng total RNA. The generated data are highly reproducible and maintain relative gene expression levels between samples. These results demonstrate that our protocol describes an efficient and reliable technique to expand the applicability of oligonucleotide arrays to studies where RNA is the limited source material.