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Supplementary Figure 2 from Epigenetic Downregulation of Mitogen-Activated Protein Kinase Phosphatase MKP-2 Relieves Its Growth Suppressive Activity in Glioma Cells

Supplementary Figure 1 from Epigenetic Downregulation of Mitogen-Activated Protein Kinase Phosphatase MKP-2 Relieves Its Growth Suppressive Activity in Glioma Cells

Supplementary Figure Legends 1-3 from Epigenetic Downregulation of Mitogen-Activated Protein Kinase Phosphatase MKP-2 Relieves Its Growth Suppressive Activity in Glioma Cells

Supplementary Figure 3 from Epigenetic Downregulation of Mitogen-Activated Protein Kinase Phosphatase MKP-2 Relieves Its Growth Suppressive Activity in Glioma Cells

The German Cancer Consortium (‘Deutsches Konsortium für Translationale Krebsforschung’, DKTK) is a long-term cancer consortium, bringing together the German Cancer Research Center (DKFZ), Germany's largest life science research center, and the leading University Medical Center-based Comprehensive Cancer Centers (CCCs) at seven sites across Germany. DKTK was founded in 2012 following international peer review and has positioned itself since then as the leading network for translational cancer research in Germany. DKTK is long term funded by the German Ministry of Research and Education and the federal states of each DKTK partner site. DKTK acts at the interface between basic and clinical cancer research, one major focus being to generate suitable multisite cooperation structures and provide the basis for including higher numbers of patients and facilitate effective collaborative forward and reverse translational cancer research. The consortium addresses areas of high scientific and medical relevance and develops critical infrastructures, for example, for omics technologies, clinical and research big data exchange and analysis, imaging, and clinical grade drug manufacturing. Moreover, DKTK provides a very attractive environment for interdisciplinary and interinstitutional training and career development for clinician and medical scientists. CA extern

The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome-activating protein and investigate its potential pro-apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two-hybrid screen and further studied by deletion mutants, glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock-down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GISTs and histogenetically related cells including interstitial cells of Cajal (ICCs), "fibroblast-like cells" (FLCs) and ICC stem cells (ICC-SCs) was investigated by Northern blotting, reverse transcription-polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC-SCs stably transduced to re-express FAM96A was studied by xeno- and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC-SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC-SCs. Re-expression of FAM96A in GIST cells and transformed ICC-SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro-apoptotic tumor suppressor that is lost during GIST tumorigenesis.

Cutaneous lupus erythematosus (CLE) is a heterogeneous autoimmune disease. Different pathogenetic mechanisms, including the accumulation of apoptotic keratinocytes in CLE, have been reported. Therefore, we investigated whether CLE and other inflammatory skin diseases differ with regard to the epidermal expression of molecules that are crucial for the initiation and regulation of apoptosis. In this study, 241 skin biopsies from patients with CLE, psoriasis (PSO), lichen planus (LP) and healthy controls (HCs) were analysed immunohistochemically using the tissue microarray (TMA) technique. The TUNEL assay and anti-activated caspase-3 antibodies revealed a significant increase of apoptotic keratinocytes in CLE lesions compared with HCs. Furthermore, we detected a significant increase in the epidermal expression of CD95 in CLE specimens compared with PSO, LP and HCs. These data suggest that the accumulation of apoptotic keratinocytes in CLE might be due to the increased epidermal expression of CD95, resulting in increased activity of the extrinsic apoptotic pathway in the disease.

Gene copy number aberrations are involved in oral squamous cell carcinoma (OSCC) development. To delineate candidate genes inside critical chromosomal regions, array-CGH was applied to 40 OSCC specimens using a microarray covering the whole human genome with an average resolution of 1 Mb. Gene copy number gains were predominantly found at 1q23 (9 cases), 3q26 (11), 5p15 (13), 7p11 (7), 8q24 (17), 11q13 (15), 14q32 (8), 19p13 (8), 19q12 (7), 19q13 (8), and 20q13 (9), whereas gene copy number losses were detected at 3p21-3p12 (15), 8p32 (11), 10p12 (8), and 18q21-q23 (10). Subsequent mRNA expression analyses by quantitative real time polymerase chain reaction found high mRNA expression of candidate genes SOX2 in 3q26.33, FSLT3 in 19p13.3, and CCNE1 in 19q12. Tissue microarray (TMA) analyses in a representative OSCC collection found gene copy number gain for SOX2 in 52% (115/223) and for CCNE1 in 31% (72/233) of the tumors. Immunohistochemical analyses on TMA sections of the corresponding proteins detected high expression of SOX2 in 18.1% (49/271) and of CyclinE1 in 23.3% (64/275) of tumors analyzed. These findings indicate that SOX2 and CCNE1 might be activated via gene copy number gain and participate in oral carcinogenesis. The combination of array-CGH with TMA analyses allows rapid pinpointing of novel promising candidate genes, which might be used as therapeutic stratification markers or target molecules for therapeutic interference.

Purpose Medulloblastoma is the most common malignant brain tumor in children. Current treatment decisions are based on clinical variables. Novel tumor-derived biomarkers may improve the risk stratification of medulloblastoma patients. Patients and Methods A model for the molecular risk stratification was proposed from an array-based comparative genomic hybridization (array-CGH) screen (n = 80). Fluorescence in situ hybridization (FISH) analyses for chromosome arms 6q, 17p, and 17q and the MYC and MYCN loci were performed in an independent validation set (n = 260). Copy number aberrations were correlated with clinical, histologic, and survival data. Results Gain of 6q and 17q and genomic amplification of MYC or MYCN were each associated with poor outcome in the array-CGH study (n = 80). In contrast, all patients with 6q-deleted tumors survived. Given these findings, the following hierarchical molecular staging system was defined: (1) MYC/MYCN amplification, (2) 6q gain, (3) 17q gain, (4) 6q and 17q balanced, and (5) 6q deletion. The prognostic value of this staging system was investigated by FISH analysis (n = 260). The addition of molecular markers to clinical risk factors resulted in the identification of a large proportion of patients (72 of 260 patients; 30%) at high risk for relapse and death who would be considered standard risk by application of clinical variables alone. Conclusion Genomic aberrations in medulloblastoma are powerful independent markers of disease progression and survival. By adding genomic markers to established clinical and histologic variables, outcome prediction can be substantially improved. Because the analyses can be conducted on routine paraffin-embedded material, it will be especially feasible to use this novel molecular staging system in large multicenter clinical trials.

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.

Background: Gene copy number gain of chromosomal arm 5p is frequently found in oral squamous cell carcinoma (OSCC) suggesting the activation of proto-oncogenes. TERT is a candidate gene encoding for human telomerase reverse transcriptase (hTERT). The aim of the present study was to elucidate the relevance of TERT copy number gain and high hTERT expression in OSCC. Methods: Fluorescence in situ hybridization (FISH) for TERT and immunohistochemistry (IHC) for hTERT were performed to analyze TERT copy numbers and hTERT expression, respectively, on tissue microarray (TMA) sections including n = 247 OSCC and n = 105 pharyngeal and laryngeal squamous cell carcinomas (PSCC/LSCC). Results: Increased hTERT protein expression was more frequently found in OSCC (71.1%, 155/218) than in PSCC/LSCC (36.0%, 35/89) (P

Oral squamous cell carcinoma (OSCC) is a solid neoplasm exhibiting aggressive tumor phenotypes with unpredictable biological behavior. Recent studies suggested that high expression of the antiapoptotic protein survivin might be associated with adverse outcome in oral cancer patients. To investigate, whether increased copy numbers of the survivin-encoding gene BIRC5 results in elevated survivin levels and whether BIRC5 and survivin could serve as progression markers in the clinical course of OSCC, tumor tissue microarray analysis was performed applying fluorescence in situ hybridization and immunohistochemistry to 296 OSCC specimens. Gene copy number gain of BIRC5 was detected in 33.9% (150/227) of cases, which correlated significantly with high UICC stage and the presence of lymph node metastases (p = 0.003 and p = 0.001, respectively), but not with unfavorable patients' outcome (p > 0.05) in multivariate analysis. High survivin expression was found in 67.3% (169/251) of cases to predict increased 5- and 10-year overall survival of patients in a multivariate model including UICC stage and age as covariables (p = 0.035 and p = 0.026, respectively). Within a subgroup of patients, who received radiation therapy (n = 121), high survivin expression was found to be the only predictor of favorable 3-, 5- and 10-year overall survival in a multivariate cox regression analysis including UICC stage and age as covariables (p = 0.001, p = 0.004 and p = 0.006, respectively). In conclusion, high survivin expression might be useful to identify OSCC patients, who would benefit from radiotherapy.

To identify candidate genes relevant for prostate tumour prognosis and progression, we performed an exhaustive gene search in seven previously described genomic-profiling studies of 161 prostate tumours, and four expression profiling studies of 61 tumours. From the resulting list of candidate genes, six were selected for protein-expression analysis based on the availability of antibodies applicable to paraffinised tissue: fatty acid synthase (FASN), MYC, beta-adrenergic receptor kinase 1 (BARK1, GRK2) the catalytic subunits of protein phosphatases PP1alpha (PPP1CA) and PP2A (PPP2CB) and metastasis suppressor NM23-H1. These candidates were analysed by immunohistochemistry (IHC) on a tissue microarray containing 651 cores of primary prostate cancer samples and benign prostatic hyperplasias (BPH) from 175 patients. In univariate analysis, expression of PP1alpha (P=0.001) was found to strongly correlate with Gleason score. MYC immunostaining negatively correlated with both pT-stage and Gleason score (P0.001 each) in univariate as well as in multivariate analysis. Furthermore, a subgroup of patients with high Gleason scores was characterised by a complete loss of BARK1 protein (P=0.023). In conclusion, our study revealed novel molecular markers of potential diagnostic and therapeutic relevance for prostate carcinoma.

The treatment of advanced stage thymomas and thymic carcinomas is a multimodal therapy. New therapeutic targets are currently under investigation, including the epidermal growth factor receptor (EGFR) as well as KIT. A number of studies have shown protumorigenic potential of Cyclooxygenase-2 (COX-2) in a variety of human malignancies, but so far it is unknown whether COX-2 is expressed in primary malignancies of the thymus. Using tissue microarrays, the expression of COX-2, microsomal-PGES-1 and -PGES-2 (mPGES-1 and mPGES-2), as well as EGFR was evaluated in different subtypes of thymoma and thymic carcinomas. COX-2 was expressed in all subtypes as determined by immunohistochemistry. Some cases of type B2 and thymic carcinomas had COX-2 staining levels classified as mild to moderate. However, when measuring the optical color intensity, no significant differences could be detected. Concerning the expression levels, a weak correlation between the expression of COX-2, mPGES-1 and mPGES-2 as well as EGFR was found. Furthermore, additional cases of thymomas and thymic carcinomas were analyzed by COX-2 Western immunoblot analysis and were compared to normal thymi. The analysis showed that thymomas and thymic carcinomas had a significantly stronger COX-2 expression than that of the normal thymi (p < 0.04). In summary, COX-2 is expressed in all subtypes of thymomas and thymic carcinomas and thus represents, in addition to EGFR and KIT, a potential therapeutic target. Further studies are needed in order to determine whether a combined therapy using COX-2 inhibitors in addition to the evolving anti-EGFR antibody therapy may be considered as a treatment option.

Two metachronous glioblastomas with different cerebral locations in a 53-year-old long-term survival patient were analyzed by multiple genetic approaches. Using comparative genomic hybridization a different pattern of chromosomal aberrations was observed, with 19 imbalances in the first tumor and only 2 imbalances in the second. Sequence analysis revealed a distinct mutation profile in each tumor, with amino acid substitutions in the p53 and PTEN genes only in the first tumor, ie, p53 in codon 273 (CGT-->TGT, Arg-->Cys) and PTEN in codon 336 (TAC-->TTC, Tyr-->Phe). A splicing acceptor site PTEN mutation (IVS8-2A>G) was observed only in the second GBM. EGFR amplification, mutations of p16INK4a/CDKN2A or p14ARF were not observed. According to the results of p53 mutational analysis and EGFR amplification studies, the first tumor is classified as a type 1 GBM, whereas the alterations in the second one are different from those typically encountered in type 1 or type 2 tumors. In conclusion, our data strongly suggest that the metachronous tumors in this patient are exceptional in that they developed independently from each other. Whether the molecular features of the first glioblastoma are associated with the notably extended recurrence-free period of 5 years remains to be elucidated.

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.

Transcription factor nuclear factor kappa B (NF-kappaB) plays a central role in the pathogenesis of classical Hodgkin lymphoma (cHL). In anaplastic large-cell lymphomas (ALCLs), which share molecular lesions with cHL, the NF-kappaB system has not been equivalently investigated. Here we describe constitutive NF-kappaB p50 homodimer [(p50)2] activity in ALCL cells in the absence of constitutive activation of the IkappaB kinase (IKK) complex. Furthermore, (p50)2 contributes to the NF-kappaB activity in Hodgkin/Reed-Sternberg (HRS) cells. Bcl-3, which is an inducer of nuclear (p50)2 and is associated with (p50)2 in ALCL and HRS cell lines, is abundantly expressed in ALCL and HRS cells. Notably, a selective overexpression of Bcl-3 target genes is found in ALCL cells. By immunohistochemical screening of 288 lymphoma cases, a strong Bcl-3 expression in cHL and in peripheral T-cell non-Hodgkin lymphoma (T-NHL) including ALCL was found. In 3 of 6 HRS cell lines and 25% of primary ALCL, a copy number increase of the BCL3 gene locus was identified. Together, these data suggest that elevated Bcl-3 expression has an important function in cHL and peripheral T-NHL, in particular ALCL.

PurposeMedulloblastoma is the most common malignant brain tumor in children. Despite multimodal aggressive treatment, nearly half of the patients die as a result of this tumor. Identification of molecular markers for prognosis and development of novel pathogenesis-based therapies depends crucially on a better understanding of medulloblastoma pathomechanisms.Patients and MethodsWe performed genome-wide analysis of DNA copy number imbalances in 47 medulloblastomas using comparative genomic hybridization to large insert DNA microarrays (matrix-CGH). The expression of selected candidate genes identified by matrix-CGH was analyzed immunohistochemically on tissue microarrays representing medulloblastomas from 189 clinically well-documented patients. To identify novel prognostic markers, genomic findings and protein expression data were correlated to patient survival.ResultsMatrix-CGH analysis revealed frequent DNA copy number alterations of several novel candidate regions. Among these, gains at 17q23.2-qter (P < .01) and losses at 17p13.1 to 17p13.3 (P = .04) were significantly correlated to poor prognosis. Within 17q23.2-qter and 7q21.2, two of the most frequently gained chromosomal regions, confined amplicons were identified that contained the PPM1D and CDK6 genes, respectively. Immunohistochemistry revealed strong expression of PPM1D in 148 (88%) of 168 and CDK6 in 50 (30%) of 169 medulloblastomas. Overexpression of CDK6 correlated significantly with poor prognosis (P < .01) and represented an independent prognostic marker of overall survival on multivariate analysis (P = .02).ConclusionWe identified CDK6 as a novel molecular marker that can be determined by immunohistochemistry on routinely processed tissue specimens and may facilitate the prognostic assessment of medulloblastoma patients. Furthermore, increased protein-levels of PPM1D and CDK6 may link the TP53 and RB1 tumor suppressor pathways to medulloblastoma pathomechanisms.

Adenoid cystic carcinoma (ACC) of the salivary gland is a neoplasm characterized by slow but inevitable local progression and terminal hematogenous metastasis. To detect novel imbalanced chromosomal regions associated with tumorigenesis, we used chromosomal comparative genomic hybridization to screen 27 ACC. The most common aberration was copy number gain of 22q13 (nine cases) followed by gains of 16p (seven cases) and 17q (four cases) and copy number losses on 6q (six cases). To further delineate the prevalence of 22q13 copy number gains in ACC, fluorescence in situ hybridization was performed for five bacterial/phage artificial chromosome (BAC/PAC) probes from the 22q13 consensus region with 57 ACC on a tissue microarray. The overall prevalence of copy number gains on 22q13 was 30% of the tumors in the fluorescence in situ hybridization analysis, irrespective of histologic differentiation (cribriform/tubular vs. solid) or tumor event (primary vs. recurrent). We therefore assume that copy number gain of 22q13 is a novel frequent finding in ACC that may be involved in the initial pathogenesis of this neoplasm by proto-oncogene activation.

Gliomas are the most frequent primary brain tumors and comprise a group of morphologically, biologically and clinically heterogeneous neoplasms. The different glioma types are associated with distinct genetic aberrations, which may provide useful information for tumor classification as well as prediction of prognosis and response to therapy. To facilitate the molecular classification of gliomas, we established a genomic microarray that consists of bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones representing tumor suppressor genes, proto-oncogenes and chromosomal regions frequently gained or lost in gliomas. In addition, reference clones distributed evenly throughout the genome in approximately 15 Mbp intervals were spotted on the microarray. These customized microarrays were used for matrix-based comparative genomic hybridization (matrix CGH) analysis of 70 gliomas. Matrix CGH findings were validated by molecular genetic analyses of candidate genes, loss of heterozygosity studies and chromosomal CGH. Our results indicate that matrix CGH allows for the sensitive and specific detection of gene amplifications as well as low-level copy number gains and losses in clinical glioma samples. Furthermore, molecular classification based on matrix CGH data closely paralleled histological classification and was able to distinguish with few exceptions between diffuse astrocytomas and oligodendrogliomas, anaplastic astrocytomas and anaplastic oligodendrogliomas, anaplastic oligodendrogliomas and glioblastomas, as well as primary and secondary glioblastomas. Thus, matrix CGH is a powerful technique that allows for an automated genomic profiling of gliomas and represents a promising new tool for their molecular classification.

Medulloblastoma, a primitive neuroectodermal tumor of the cerebellum, is one of the most common central nervous system malignancies of childhood. Despite aggressive multimodal therapy, including surgery, irradiation, and chemotherapy, 5-year survival rates have only approached 50–60%. To identify potential candidate genes that predict for overall survival (OS), we performed a gene expression profiling analysis in 35 newly diagnosed medulloblastoma neoplasms. Subsequently, the nine most promising candidate genes were analyzed by immunohistochemistry and fluorescence in situ hybridization on tumor tissue microarrays representing a series of 180 tumors. We found 54 genes in which expression levels predicted for unfavorable survival in medulloblastoma. In line with the gene expression profiling analysis, a positive staining for STK15 (P = 0.0006), stathmin 1 (P = 0.001), and cyclin D1 (P = 0.03) was associated with an unfavorable OS, whereas cyclin B1, DAXX, Ki-67, MYC, NRAS, and p53 showed no statistical significant effect. In comparison to clinically defined parameters such as gender, age, metastatic stage, extent of tumor resection, application of chemotherapy, and tumor grade, positive staining for STK15 was identified as an independent prognostic factor for OS (P = 0.026). Moreover, additional gene copy numbers of MYC (P = 0.003) and STK15 (P = 0.05) predicted for poor survival. The combination of gene expression profiling with tissue microarray experiments allowed the identification of a series of candidate genes that predicts for survival in medulloblastoma. Of the results highlighted by the various data analysis procedures, genes associated with cell proliferation (cyclin D1), transcription (MYC), and especially mitosis (stathmin 1, STK15) appear particularly intriguing with respect to medulloblastoma pathomechanism.

The Bcl-2 family consists of about 20 homologues of important pro- and anti-apoptotic regulators of programmed cell death. The established mode of function of the individual members is to either preserve or disturb mitochondrial integrity, thereby inducing or preventing release of apoptogenic factors like Cytochrome c (Cyt c) from mitochondria. Recent findings also indicate further Bcl-2-controlled mitochondria-independent apoptosis pathways. Bcl-2 represents the founding member of the new and growing class of cell death inhibiting oncoproteins. In this review, we try to briefly summarize current models of Bcl-2 family function and to outline the work demonstrating the influence of deregulated Bcl-2 family member expression on tumorigenesis and cancer therapy. Since several Bcl-2 homologues, in addition to influencing apoptotic behaviour, also impinge on cell cycle progression, we discuss possible implications of this additional role for the expression of Bcl-2 family members in tumor cells.

Neuere morphologische und molekulare Befunde haben unser Verstandnis uber gastrointestinale Stromatumoren (GIST) betrachtlich erweitert. Gastrointestinale Stromatumoren werden gegenwartig uber ihre Uberexpression von CD117 (KIT), dem Rezeptor des Stammzellfaktors, definiert und so gegenuber glattmuskularen Tumoren abgegrenzt. Zytogenetisch sind GIST bereits in fruhen Stadien durch haufige komplette oder partielle Verluste der Chromosomen 14 und 22 und terminale Deletionen des Chromosomenarms 1p charakterisiert. Im Verlauf der Tumorprogression wird eine Akkumulation von zusatzlichen chromosomalen Anomalien beobachtet. Basierend auf der Erstbeschreibung von aktivierenden KIT-Mutationen in GIST haben sich zahlreiche Studien mit der Rolle von wildtypischem und mutiertem KIT in GIST beschaftigt. Inzwischen wurden in der uberwiegenden Mehrheit der GIST KIT-Mutationen identifiziert und selbst in KIT-Mutation-negativen GIST eine konstitutive Phosphorylierung der KIT-Rezeptor-Tyrosinkinase nachgewiesen. Diese Befunde lassen darauf schliesen, dass KIT eine zentrale Rolle bei der Pathogenese von GIST spielt. Imatinib (STI571/Glivec®) inhibiert selektiv die BCR/ABL-, die PDGFR- und die KIT-Rezeptor-Tyrosinkinasen. Erste therapeutische Anwendungen von Imatinib bei Patienten mit progredienten GIST haben in dieser chemo- und radiotherapieresistenten Tumorentitat beachtliche Ergebnisse erzielt. In dieser Ubersicht werden die morphologischen Befunde und molekularen Grundlagen von GIST dargestellt, die eine neue therapeutische Perspektive eroffnet haben.

Four Hodgkin's lymphoma cell lines (KM-H2, HDLM-2, L428, L1236) were analyzed for cytogenetic aberrations, applying multiplex fluorescence in situ hybridization, chromosome banding and comparative genomic hybridization. Each line was characterized by a highly heterogeneous pattern of karyotypic changes with a large spectrum of different translocated chromosomes (range 22–57). A recurrent finding in all cell lines was the presence of chromosomal rearrangements of the short arm of chromosome 2 involving the REL oncogene locus. Furthermore, multiple translocated copies of telomeric chromosomal segments were frequently detected. This resulted in a copy number increase of putative oncogenes, e.g., JAK2 (9p24) in 3 cell lines, FGFR3 (4p16) and CCND2 (12p13) in 2 cell lines as well as MYC (8q24) in 1 cell line. Our data confirm previous cytogenetic results from primary Hodgkin's tumors suggesting an important pathogenic role of REL and JAK2 in this disease. In addition, they provide evidence for a novel cytogenetic pathomechanism leading to increased copy numbers of putative oncogenes from terminal chromosomal regions, most probably in the course of chromosomal stabilization by telomeric capture. © 2002 Wiley-Liss, Inc.

Summary Mediastinal (thymic) B-cell lymphoma (MBL) is a locally highly aggressive tumour that was first definitively described in the early 1980s. The incidence of MBL is low, which made disease characterisation difficult initally. However, MBL has several peculiar clinical, morphological, immunological, and genetic features. Collectively, these characteristics distinguish it from other diffuse, large B-cell lymphomas. Consequently, MBL has become a defined subtype of diffuse large B-cell lymphoma with its own code (9679/3) in the International Classification of Diseases. New insights into the biological and clinical aspects of MBL have been gained from the study of large numbers of cases. Nevertheless, the histogenesis of the disease is not yet fully understood. We review the available data on MBL with special emphasis on its morphological, immuno-logical, and genetic properties. Also discussed are recent data on molecular genetics, biology, and treatment.

Using comparative genomic hybridization, DNA copy number changes were studied in 14 pleomorphic liposarcomas and compared to those detected in high-grade areas of 9 dedifferentiated liposarcomas. A total of 251 gains and 84 losses were detected. The most frequent gains involved subregions of chromosomal arms 12q and 20q (70% each), 5p (57%), 6q and 9q (52% each), 1q, 7p and 17p (48% each), 1p (43%), 6p and 17q (39% each), 20p and 22q (35% each) as well as 7q and 12p (30% each). The same subregions were also affected by 30 high level amplifications. The most frequent losses were found in subregions of chromosomal arms 13q (35%) as well as 11q and 12p (30% each). Overall, gains of chromosomal material were more frequent than losses (p < 0.001). There were significant differences in the frequency and distribution of recurrent chromosomal imbalances between pleomorphic liposarcomas and the dedifferentiated areas of dedifferentiated liposarcomas. Gains of chromosomal material detected predominantly in pleomorphic liposarcomas involved subbands 5p13–p15 (p < 0.010), 1p21 (p < 0.019), 1q21–q22 (p < 0.040) and 7q22 (p < 0.049). Conversely, high level amplifications within chromosomal subregion 12q13–q21 were only found in the dedifferentiated components of dedifferentiated liposarcomas (p < 0.001). Overall, both gains and the less pronounced losses of chromosomal material were more frequent in pleomorphic than in dedifferentiated liposarcomas (p < 0.001 and p < 0.025, respectively). These results show that pleomorphic liposarcomas display a considerable number of recurrent chromosomal imbalances that are essentially different from those present in high-grade areas of dedifferentiated liposarcomas. Therefore, genetic data are considered as a helpful diagnostic adjunct for the discrimination between these 2 types of liposarcoma. The overall higher frequency of chromosomal imbalances in pleomorphic as compared to dedifferentiated liposarcomas could account for the more aggressive biological behavior of pleomorphic relative to dedifferentiated liposarcoma types. © 2002 Wiley-Liss, Inc.

Recently, it was discovered that the receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL) is part of an important signal transduction pathway for tissue homoeostasis. Therefore, we were interested in investigating RANKL expression in the epidermis of skin lesions from patients with different subtypes of cutaneous lupus erythematosus (CLE) and psoriasis as well as normal healthy donors. Using the tissue microarray technique, skin biopsy specimens were evaluated by immunohistochemistry. RANKL showed a significantly increased expression in the epidermis of skin biopsy specimens from patients with psoriasis (median: 4, range: 0–5) compared to patients with CLE (median: 0, range: 0–4) (P

Two [Cr,O2]+ isomers have been selectively produced and studied by FT-ICR mass spectrometry. The Cr(O2)+ complex was formed by supersonically expanding a plasma produced by laser vaporization of chromium metal with the helium carrier gas, which was seeded with traces of oxygen, while the chromyl cation is formed in an expansion with N2O. The complex is stable against thermal collisions, but in a bimolecular reaction with water it is rapidly converted to the chromyl cation, with ligand exchange being only a minor side reaction. Isotopic studies suggest a side-on geometry for Cr(O2)+, in accordance with density functional (B3LYP) calculations. The present work indicates that an investigation of the selected isomers can indeed be carried out, if appropriate chemical methods for the ion generation are applied.

Hodgkin's disease (HD) is the most common haematological malignancy after chronic lymphocytic leukaemia, but very little is known about its pathogenesis or the genetic events that contribute to the malignant phenotype of the tumour cells. p53 is assumed to play an important role in the pathogenesis of HD, based on the observation that p53 protein is frequently accumulated in Hodgkin and Reed-Sternberg (H & RS) cells. We investigated single H & RS cells from five different HD patients for point mutations at the genomic level using multiplex polymerase chain reaction amplification and subsequent sequencing. No point mutations were detected in 50 single H & RS cells analysed. Hence, accumulation of p53 protein cannot be explained by mutations within the gene. A genome-wide screening for genomic imbalances using comparative genomic hybridization revealed gain on chromosome 12q14, i.e. the mapping position of the MDM2 gene in several HD cases. Therefore, we assessed the copy number of the MDM2 gene using fluorescence in situ hybridization. In four out of six HD cases analysed, the copy number of the MDM2 gene was found to be increased. As gene amplification is frequently associated with protein overexpression, the observed accumulation of p53 in the nuclei of H & RS cells could be as a result of elevated MDM2 protein levels resulting in stabilization of p53 protein.

Primary mediastinal B-cell lymphoma (MBL) is an aggressive Non-Hodgkin's Lymphoma, which has been recognized as a distinct disease entity. We performed a comprehensive molecular cytogenetic study analyzing 43 MBLs. By comparative genomic hybridization (CGH), the most common aberrations were gains of chromosome arms 9p and Xq, which were present in 56% and 40% of cases, respectively. Based on the limited resolution of CGH, this technique may underestimate the real incidence of aberrations. Therefore, we also did an interphase cytogenetic study with eight DNA probes mapping to chromosome regions frequently altered in B-cell lymphomas. With this approach, both 9p and Xq gains were found in more than 70% of cases (75% and 87%, respectively). The findings were compared with results obtained in 308 other B-cell lymphomas. Gains in 9p were identified in only six of the 308 cases, and only one of these lymphomas with 9p gains was not primarily extranodal in origin (P < 10-(20) for CGH data and P < 10-(11) for fluorescence in situ hybridization data). We also present a novel MBL cell line, MedB-1, which carries the genetic aberrations characteristic of this entity.

Comparative genomic hybridization (CGH) has contributed significantly to the current knowledge of genomic alterations in hematologic malignancies. Characteristic patterns of genomic imbalances not only have confirmed recent classification schemes in non-Hodgkin's lymphoma, but they provide a basis for the successful identification of genes with previously unrecognized pathogenic roles in the development of different lymphomas. Based on its technical limitations, there is little reason to apply CGH to chromosomes of metaphase cells in routine diagnostic settings. However, the new approach of CGH to DNA microarrays, a procedure termed matrix-CGH, overcomes most of the limitations and opens new approaches for diagnostics and identification of genetically defined leukemia and lymphoma subgroups. Current efforts to develop leukemia specific matrix-CGH DNA chips, which are designed to meet the clinical needs, are presented and discussed.

Leiomyosarcomas comprise a group of malignant soft-tissue tumors with smooth-muscle differentiation. In this study, 14 cases of leiomyosarcoma were screened for changes in relative chromosome copy number by comparative genomic hybridization. A high number of imbalances (mean, 16.3; range, 6–26) was detected, with chromosomal gains occurring about twice as much as losses. The most frequent gains were found in 5p15, 8q24, 15q25→q26, 17p, and Xp (43% to 50%), whereas the most frequent losses were found in 10q and 13q (50% and 78%, respectively). Twenty high-level amplifications affecting 15 different chromosomal subregions were detected in nine different tumors. In three leiomyosarcomas, sequences on chromosome arm 17p were found to be highly amplified, with a minimal overlapping region on subbands 17p12→p11. We further discovered that the Smith-Magenis syndrome critical region on 17p11.2 is included in the 17p amplicons of two leiomyosarcoma cases. Using probes flanking this genetically unstable region, a mean of 14 and 22 signals per nucleus, respectively, was detected in both leiomyosarcomas by fluorescence in situ hybridization. In conclusion, this analysis identifies a number of characteristic chromosomal imbalances in leiomyosarcomas and provides evidence for the localization of potential oncogenes and tumor suppressor genes active in leiomyosarcoma genomes.

Reactions of platinum−argon complexes Pt+Arm, m = 1−6, with methane (CH4) and methane-d4 (CD4) were investigated by means of FT-ICR mass spectrometry and DFT calculations. Ligand exchange reactions are observed for Pt+Arm, m = 2−6, in which up to four argon ligands are replaced by methane. In contrast the bare platinum ion and platinum solvated with one argon ligand lead to the formation of a platinum−carbene complex. Gibbs free enthalpies from ligand exchange reactions of Pt+CH4 with CD4 and H2O provide evidence for the inserted hydrido−methyl complex HPt+CH3 which is corroborated by high-level DFT calculations. No isotopic scrambling is observed for the reaction of Pt+CH4 with CD4 (and the reverse reaction). This is attributed to the inability of the platinum cation to form more than three covalent bonds.

Isochromosomes are monocentric or dicentric chromosomes with homologous arms that are attached in a reverse configuration as mirror images. With an incidence of 3‐4%, the i(17q) represents the most frequent isochromosome in human cancer. It is found in a variety of tumors, particularly in blast crisis of chronic myeloid leukemia (CML-BC), acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), and medulloblastoma (MB), and indicates a poor prognosis. To determine the breakpoints on the molecular genetic level, we analyzed 18 neoplasms (six CML, four AML, one NHL, and seven MB) with an i(17q) and two MB with a pure del(17p) applying fluorescence in situ hybridization (FISH) with yeast artificial chromosome (YAC) clones, P1-artificial chromosome (PAC) clones, and cosmids from a well-characterized contig covering more than 6 Mb of genomic DNA. We identified four different breakpoint cluster regions. One is located close to or within the centromere of chromosome 17 and a second in the Charcot-Marie-Tooth (CMT1A) region at 17(p11.2). A third breakpoint was found telomeric to the CMT1A region. The fourth, most common breakpoint was detected in MB, AML, and in CML-BC specimens and was bordered by two adjacent cosmid clones (clones D14149 and M0140) within the Smith-Magenis syndrome (SMS) region. These results indicate that the low copy number repeat gene clusters which are present in the CMT and SMS regions may be one of the factors for the increased instability that may trigger the formation of an i(17q). Genes Chromosomes Cancer 25:230‐240, 1999. r 1999 Wiley-Liss, Inc.

Trapped hydrated aluminum cluster ions are studied by FT-ICR mass spectrometry on a time scale of several seconds. Blackbody radiation, besides causing the fragmentation of the cluster by stepwise loss of individual water molecules, induces in hydrated aluminum clusters Al+(H2O)n an intracluster reaction yielding hydrated hydroxide and releasing molecular hydrogen. Local deviations of the individual rate constants for the water loss process from a linear size dependence are due to increased rigidity of certain sizes due to the formation of stabilizing hydrogen-bonded bridges. In comparison with previously studied hydrated ions, surface versus internal solvation is discussed. The preferential occurrence of the intracluster reaction in the size region of n = 11−24 is attributed to a concerted proton-transfer mechanism, in which a chain of at least two water molecules is needed to transfer a proton between two first solvation shell water molecules, leading to formation of an Al(OH)2+(H2O)n hydrated aluminum ...

Biopsies routinely performed for the histopathological diagnosis of oral epithelial lesions before treatment were screened for chromosomal imbalances by comparative genomic hybridization. Comparative genomic hybridization was performed on 12 oral premalignant lesions (OPLs; dysplasias and carcinomas in situ) and 14 oral squamous cell carcinomas (OSCCs). Eight biopsies displayed areas of different histopathological appearance, so that OPLs and OSCCs from the same patient were analyzed. To avoid contamination with nonneoplastic cells, defined cell populations were isolated by micromanipulation with a glass needle. Before comparative genomic hybridization analysis, universal DNA amplification was performed using the DOP-polymerase chain reaction protocol. In the 14 OSCCs examined, the average number of chromosomal imbalances was significantly higher than in the 12 OPLs (mean +/- SEM: 11.9 +/- 1.9 versus 3.2 +/- 1.2; P = 0.003). The DNA copy number changes identified in more than one OPL were gains on 8q (3 of 12) and 16p (2 of 12), as well as losses on 3p (5 of 12); 5q (4 of 12); 13q (3 of 12); and 4q, 8p, and 9p (2 of 12 each). In more than 30% of OSCCs, gains of chromosomal material were identified on 20q (8 of 14); 8q, 11q, 22q (7 of 14 each); 3q, 15q, and 17p (6 of 14 each); and 14q, 17q, and 20p (5 of 14 each), and losses were identified on 3p and 4q (9 of 14 each), 5q (7 of 14), 13q (6 of 14), and 2q and 9p (5 of 14 each). These results were validated by positive and negative control comparative genomic hybridization experiments and microsatellite analysis for the detection of allelic loss. The vast majority of genomic alterations found in OPLs were again identified in OSCCs from the same biopsy, supporting the hypothesis that multiple lesions in the same patient are clonally related. In summary, we show that comprehensive information on the genomic alterations in oral epithelial lesions can be obtained from small biopsies. Such data may identify prognostic indicators that could eventually assist in designing therapeutic strategies.

The reactions of anionic niobium and rhodium clusters Mn−, M=Nb, Rh, n=3–28, with C6H6 are investigated under single collision conditions in a Fourier-transform ion-cyclotron-resonance mass spectrometer and compared with the results of previous studies on corresponding cationic species. This reveals strong effects of the cluster charge state on hydrocarbon activation as a function of cluster size. Both differences and parallels are observed for reactions of anions and cations. Niobium clusters with a given number of atoms react quite differently than those with a single atom more or less. The fact that almost identical such effects are in the present work found for anion clusters, as for cations with the same number of atoms but two less electrons, suggests that the observed reactivity patterns are more a function of the cluster shape and geometry, than of the details of their electronic structure. The variety of interesting trends and effects observed is interpreted in terms of simple physical models.

Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometric studies of both cationic water clusters H + (H2O)n, n ) 1-70, and anionic water clusters X - (H2O)n, n ) 1-30, X ) OH, O, with acetone and acetaldehyde are reported. For the cations a sequence of ligand exchange and fragmentation reactions results in "solvated proton" complex ion final products. In the anionic OH - (H2O)n clusters, on the other hand, an OH - catalyzed aldol addition of the carbonyl compounds with a true covalent bond formation is observed. The anionic and cationic water clusters thus behave similar to basic and acidic aqueous solutions. I. Introduction The rate or outcome of a condensed phase chemical reaction can be strongly affected by the solvent. Many important chemical reactions, including numerous industrially relevant processes, take place in solutions. The most important solvent for the processes in earth's troposphere is water, and the chemistry on which life itself is based takes place in aqueous solutions. Many organic and inorganic compounds are ionized in the polar water solvent, and the course of the reactions often can be altered by changing the composition of the solution and the ions present. Particularly important are the concentrations of the H + and OH - ions, which are usually described by the pH value of the solution. Numerous "acid catalyzed" or "base catalyzed" reactions depend sensitively on their concentrations. 1-4 We have recently initiated studies of hydrated H + and OH - ions in the gas phase and shown that they can be used as simple model systems for investigating charge-transfer processes and solution chemistry. 5,6 We have constructed a supersonic expan- sion discharge source and interfaced it to our FT-ICR mass spectrometer, an arrangement that permits us to produce, store, and mass-select ions solvated with up to about 100 water molecules. We have also been able to show that such ion clusters, once prepared, gradually lose the solvent ligands one by one due to the absorption of the ambient temperature infrared background radiation. 7 This provides us with a gentle way of removing the water ligands one at a time, and observing the effect of the removal of the stabilizing solvent upon the system properties and stability. In the present paper we wish to address the question if and to what extent the solvated H + and OH - ions can "catalyze" chemical reactions. Specifically, we investigate reactions of simple carbonyl compounds, acetaldehyde and acetone, with water clusters. Several related previous studies of similar organic species are available in the literature. 8-10 The dissocia