Biomedical subjects

Peter Lichter

Publications and source records attributed to Peter Lichter.

At least 73 records · Page 4Linked to original sources

PubMed2004

Genomic DNA-chip hybridization in t(11;14)-positive mantle cell lymphomas shows a high frequency of aberrations and allows a refined characterization of consensus regions.

Tumor samples of 53 patients with t(11;14)-positive mantle cell lymphomas (MCLs) were analyzed by matrix-based comparative genomic hybridization (matrix-CGH) using a dedicated DNA array. In 49 cases, genomic aberrations were identified. In comparison to chromosomal CGH, a 50% higher number of aberrations was found and the high specificity of matrix-CGH was demonstrated by fluorescence in situ hybridization (FISH) analyses. The 11q gains and 13q34 deletions, which have not been described as frequent genomic aberrations in MCL, were identified by matrix-CGH in 15 and 26 cases, respectively. For several genomic aberrations, novel consensus regions were defined: 8p21 (size of the consensus region, 2.4 megabase pairs [Mbp]; candidate genes: TNFRSF10B, TNFRSF10C, TNFRSF10D); 10p13 (2.7 Mbp; BMI1); 11q13 (1.4 Mbp; RELA); 11q13 (5.2 Mbp; CCND1); 13q14 (0.4 Mbp; RFP2, BCMSUN) and 13q34 (6.9 Mbp). In univariate analyses correlating genomic aberrations and clinical course, 8p- and 13q14- deletions were associated with an inferior overall survival. These data provide a basis for further studies focusing on the identification of pathogenetically or clinically relevant genes in MCL.

Chromosome Aberrations↗

PubMed2004

Gene expression patterns in acute myeloid leukemia correlate with centrosome aberrations and numerical chromosome changes.

Centrosomes, which mediate accurate chromosome segregation during mitosis, undergo duplication precisely once per cell division at the G1/S boundary. Recently, we described centrosome aberrations as a possible cause of aneuploidy in acute myeloid leukemia (AML) and found a correlation of the percentage of cells carrying abnormal centrosomes to their cytogenetic risk profile. To elucidate the molecular events responsible for the development of centrosome aberrations in AML, tumor RNA of 29 AML samples was hybridized to cDNA microarrays. The microarrays comprised some 2800 different genes with relevance to hematopoiesis, tumorigenesis and mitosis and included a set of 359 centrosome-associated genes. We identified two gene expression signatures, which allowed an accurate classification according to the extent of centrosome aberrations and the ploidy status in 28 of 29 patients each. Specifically, 18 genes were present in both signatures, including genes that code for cell cycle regulatory proteins (cyclin A2, cyclin D3, cyclin H, CDK6, p18INK4c, p21Cip1, PAK1) and centrosome-associated proteins (pericentrin, alpha2-tubulin, NUMA1, TUBGCP2, PRKAR2A). In conclusion, the high expression of centrosome-associated genes matches the description of centrosome aberrations in several tumor types. Moreover, in AML the identification of G1/S-phase stimulatory genes suggests that one mechanism of aneuploidy induction might be the deregulation of centrosome replication at the G1/S boundary.

Aneuploidy↗

PubMed2004

Automated array-based genomic profiling in chronic lymphocytic leukemia: development of a clinical tool and discovery of recurrent genomic alterations.

B cell chronic lymphocytic leukemia (B-CLL) is characterized by a highly variable clinical course. Recurrent chromosomal imbalances provide significant prognostic markers. Risk-adapted therapy based on genomic alterations has become an option that is currently being tested in clinical trials. To supply a robust tool for such large scale studies, we developed a comprehensive DNA microarray dedicated to the automated analysis of recurrent genomic imbalances in B-CLL by array-based comparative genomic hybridization (matrix-CGH). Validation of this chip in a series of 106 B-CLL cases revealed a high specificity and sensitivity that fulfils the criteria for application in clinical oncology. This chip is immediately applicable within clinical B-CLL treatment trials that evaluate whether B-CLL cases with distinct chromosomal abnormalities should be treated with chemotherapy of different intensities and/or stem cell transplantation. Through the control set of DNA fragments equally distributed over the genome, recurrent genomic imbalances were discovered: trisomy of chromosome 19 and gain of the MYCN oncogene correlating with an elevation of MYCN mRNA expression.

Automation↗

PubMed2004

Neoadjuvant therapy with gemcitabine in breast cancer.

Primary systemic therapy (ie, preoperative or neoadjuvant) increases the possibility for breast-conserving surgery in patients with primary breast cancer. Patients with pathologic complete response to primary systemic therapy have improved survival compared with those with persistent tumors. Several phase II trials have evaluated gemcitabine-containing doublet or triplet regimens as primary systemic therapy for breast cancer, results of which have shown promising clinical and pathologic response rates with manageable toxicity. Results of a phase I/II study of gemcitabine (Gemzar)/epirubicin (Ellence)/docetaxel (Taxotere), or GEDoc, with prophylactic filgrastim (Neupogen), as primary systemic therapy in 77 evaluable patients with primary breast cancer are reported herein. Dose-limiting toxicities were grade 3 febrile neutropenia (n = 1) and grade 3 diarrhea (n = 2) at the fourth dose level of GEDoc tested (gemcitabine at 800 mg/m2 days 1 and 8, epirubicin at 90 mg/m2 day 1, and docetaxel at 75 mg/m2 day 1). As assessed by ultrasound, 92% of patients responded overall (22% complete response), and 79% of patients could undergo breast-conserving surgery. The pathologic complete response rate in resected breast tissue was 26%.

Adult↗

PubMed2003

Diffusion-limited compartmentalization of mammalian cell nuclei assessed by microinjected macromolecules.

In order to investigate the accessibility of the nucleoplasm for macromolecules with different physical properties, we microinjected FITC-conjugated dextrans of different sizes as well as anionic FITC-dextrans and FITC-poly-L-lysine into mammalian cell nuclei. Small dextrans displayed a homogeneous nuclear distribution. With increasing molecular mass (42 to 2500 kDa), FITC-dextrans were progressively excluded from chromatin regions, accumulating in and thereby outlining an apparently extended interchromatin space. Anionic FITC-dextrans (500 kDa) showed complete exclusion from labeled chromatin regions, while the positively charged FITC-poly-L-lysine was to some extent present within the chromatin regions. Moreover, the FITC-poly-L-lysine preferentially localized at the nuclear periphery. We also found a size-dependent exclusion of FITC-dextrans from nucleoli regions, while the FITC-poly-L-lysine accumulated in the nucleoli. Thus, the distinct and restricted nuclear accessibility for macromolecules is dependent on molecule size and electrical charge.

Animals↗

PubMed2003

VH mutation status and VDJ rearrangement structure in mantle cell lymphoma: correlation with genomic aberrations, clinical characteristics, and outcome.

Immunoglobulin variable heavy chain gene (VH) mutation status and VDJ rearrangement structure were analyzed in 141 patients with mantle cell lymphoma (MCL) and correlated with biologic and clinical characteristics; 29% of the MCLs displayed mutated VH using a 98% germline homology cutoff. Striking differences occurred in the VH mutation subgroups with respect to the use of specific V genes. Rearrangements involving V4-34 and V3-21 were almost exclusively unmutated, whereas rearrangements using V4-59 and V3-23 were typically mutated. Significant association occurred between mutated VH with shorter CDR3 lengths and the use of JH4b. V3-21 and V4-59 were involved in highly characteristic rearrangements, implying that antigen specificity might have been involved in MCL development. There was no evidence for isotype switch recombination or Bcl-6 expression in any MCL. ZAP70 expression was not different in VH-mutated or -unmutated MCL. Although the deletions 11q- and 17p- showed a balanced distribution, an overrepresentation was observed for trisomies +3q, +8q, and tetraploidy in the VH-unmutated subgroup and +12q in the VH-mutated subgroup. Clinically, mutated VH was associated with a higher rate of complete remission, but there was no correlation between VH mutation status and other clinical characteristics or overall survival.

Chromosome Aberrations↗

PubMed2003

Optimization of high-density cDNA-microarray protocols by 'design of experiments'.

Expression analysis using microarray technology implies a complex experimental procedure with a large number of parameters affecting the final result. We have demonstrated that optimization of such a complex protocol can be far better handled using design of experiments (DOE) than by working on a single parameter at a time. Based on the results of a screening design, we developed a spotting buffer composed of formamide, betaine and nitrocellulose. This buffer provides a 2-fold increase in signal-to-background ratio compared to 3x SSC. Comparison to seven other buffers tested on 10 different substrates revealed it had the highest sensitivity. DNA dissolved in this buffer can be spotted on epoxysilane-coated microscope slides at a density of up to 70 000 spots per slide. A second DOE approach characterized the RNA labeling process with regard to the concentration of fluorescent dyes, dNTPs and reverse transcriptase. Adjust ments of the concentrations of dNTPs, as well as reverse transcriptase, towards the optimum, produced an improvement in the performance of the labeling procedure by a factor of 3 (Cy3) and 10 (Cy5). These results demonstrate that the process of establishing a stable expression profiling protocol and its further optimization can be significantly shortened and improved by DOE.

Buffers↗

PubMed2003

HMGB1 inhibits cell death in yeast and mammalian cells and is abundantly expressed in human breast carcinoma.

Apoptosis is a fundamental biological process used to eliminate unwanted cells in a multicellular organism. An increasing number of regulatory proteins have been identified that either promote or inhibit apoptosis. For tumors to arise, apoptosis must be blocked in the transformed cells, for example by mutational overexpression of anti-apoptotic proteins, which represent attractive target proteins for molecular therapy strategies. In a functional yeast survival screen designed to select new anti-apoptotic mammalian genes, we have identified the chromosomal high-mobility group box-1 protein (HMGB1) as an inhibitor of yeast cell death induced by the pro-apoptotic Bcl-2 family member Bak. The C-terminal 33 amino acids of HMGB1 are dispensable for this inhibitory function. HMGB1 is also able to protect mammalian cells against different death stimuli including ultraviolet radiation, CD95-, TRAIL-, Casp-8-, and Bax-induced apoptosis. We found high HMGB1 protein levels in human primary breast carcinoma. Hmgb1 RNA levels are changing during different stages of mouse mammary gland development and are particularly low during lactation and involution. These data suggest that HMGB1 may participate in the regulation of mammary gland apoptosis and that its high expression level promotes tumor growth because of its anti-apoptotic properties.

Animals↗

PubMed2003

Profile of gene expression induced by the tumour promotor TPA in murine epithelial cells.

Malignant transformation of mouse skin by chemical carcinogens and tumour promoters, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process that leads to squamous cell carcinoma (SCC) formation. In an effort to identify tumour-associated genes, we studied the influence of short-term TPA-treatment on the gene expression profile of murine skin. A comprehensive microarray with some 5,000 murine gene specific cDNA fragments was established and hybridised with pooled RNA derived from control and TPA-treated dorsal skin samples. Of these genes, 54 were up- and 35 were down-regulated upon TPA application. Additionally, we performed suppression subtractive hybridisation (SSH) with respective RNA pools to generate and analyse a cDNA library enriched for TPA-inducible genes. Expression data of selected genes were confirmed by quantitative real-time PCR and Northern blot analysis. Comparison of microarray and SSH data revealed that 26% of up-regulated genes identified by expression profiling matched with those present in the SSH library. Besides numerous known genes, we identified a large set of unknown cDNAs that represent previously unrecognised TPA-regulated genes in murine skin with potential function in tumour promotion. Additionally, some TPA-induced genes, such as Sprr1A, Saa3, JunB, Il4ralpha, Gp38, RalGDS and Slpi exhibit high basal level in advanced stages of skin carcinogenesis, suggesting that at least a subgroup of the identified TPA-regulated genes may contribute to tumour progression and metastasis.

Animals↗

PubMed2003

Tissue microarray analysis reveals site-specific prevalence of oncogene amplifications in head and neck squamous cell carcinoma.

Fluorescence in situ hybridization was applied on a collection of 609 squamous cell carcinomas of the head and neck (HNSCCs),including 511 primary carcinomas of different clinical stage and anatomical localization and 98 recurrent carcinomas, second primary carcinomas, and regional metastases on a tissue microarray. The overall prevalence of amplifications of five oncogenes analyzed was 34.5% for CCND1, 12.7% for EGFR, 8.8% for MYC, 6.2% for ZNF217, and 3.6% for ERBB2. CCND1 amplifications were associated with the pharyngeal site in primary carcinomas (P < 0.001), whereas amplifications of ZNF217 were less frequent in pharyngeal carcinomas as compared with primary oral and laryngeal carcinomas (P = 0.02). The amplification pattern of these oncogenes suggests that different molecular pathways are involved in HNSCCs of different localizations.

Carcinoma, Squamous Cell↗

PubMed2003

Hidden gene amplifications in aggressive B-cell non-Hodgkin lymphomas detected by microarray-based comparative genomic hybridization.

DNA amplifications are important mechanisms for proto-oncogene activation. Comparative genomic hybridization (CGH) to metaphase chromosome preparations has revealed amplifications in 10-20% of B-cell lymphomas (B-NHL). We analysed a series of 16 aggressive non-Hodgkin lymphomas by the new approach termed Matrix-CGH (M-CGH) using genomic DNA microarrays as hybridization target. For M-CGH, a dedicated B-cell lymphoma chip was constructed containing 496 genomic targets covering oncogenes, tumor suppressor genes as well as chromosome regions frequently altered in B-NHL. In 10 of 16 samples a total of 15 DNA amplifications were identified. The amplicons included BCL2, REL, CCND1, CCND2, JAK2, FGF4 and MDM2. Four of the 15 amplifications remained undetected by chromosomal CGH. The respective amplicons mapped to bands 2p13, 9p13-p21 and 12q24 and, were confirmed by fluorescence in situ hybridization. Furthermore, for four genomically amplified genes real-time quantitative reverse transcription polymerase chain reaction revealed elevated mRNA expression levels. These data show the superior diagnostic sensitivity of the newly developed diagnostic tool. As only a small portion of the genome (approximately 1.5%) has been analysed by the present DNA array, it is likely that gene amplifications are much more common in aggressive lymphomas than previously assumed.

Burkitt Lymphoma↗

PubMed2003

Comparative sequence and expression analyses of four mammalian VPS4 genes.

The VPS4 gene is a member of the AAA-family; it codes for an ATPase which is involved in lysosomal/endosomal membrane trafficking. VPS4 genes are present in virtually all eukaryotes. Exhaustive data mining of all available genomic databases from completely or partially sequenced organisms revealed the existence of up to three paralogues, VPS4a, -b, and -c. Whereas in the genome of lower eukaryotes like yeast only one VPS4 representative is present, we found that mammals harbour two paralogues, VPS4a and VPS4b. Most interestingly, the Fugu fish contains a third VPS4 paralogue (VPS4c). Sequence comparison of the three VPS4 paralogues indicates that the Fugu VPS4c displays sequence features intermediate between VPS4a and VPS4b. Using complete mammalian VPS4a and VPS4b cDNA clones as probes, genomic clones of both VPS4 paralogues in human and mouse were identified and sequenced. The chromosomal loci of all four VPS4 genes were determined by independent methods. A BLAST search of the human genome database with the human VPS4A sequence yielded a double match, most likely due to a faulty assembly of sequence contigs in the human draft sequence. Fluorescent in situ hybridization and radiation hybrid analyses demonstrated that human and mouse VPS4A/a and VPS4B/b are located on syntenic chromosomal regions. Northern blot and semi-quantitative reverse transcription analyses showed that mouse VPS4a and VPS4b are differentially expressed in different organs, suggesting that the two paralogues have developed different functional properties since their divergence. To investigate the subcellular distribution of the murine VPS4 paralogues, we transiently expressed various fluorescent VPS4 fusion proteins in mouse 3T3 cells. All tested VPS4 fusion proteins were found in the cytosol. Expression of dominant-negative mutant VPS4 fusion proteins led to their concentration in the perinuclear region. Co-expression of VPS4a-GFP and VPS4b-dsRed fusion proteins revealed a partial co-localization that was most prominent with mutant VPS4a and VPS4b proteins. A physical interaction between the mouse paralogues was also supported by two-hybrid analyses.

3T3 Cells↗

PubMed2003

Hodgkin's lymphoma cell lines are characterized by frequent aberrations on chromosomes 2p and 9p including REL and JAK2.

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.

Chromosome Aberrations↗

PubMed2003

Genomic organization, splice products and mouse chromosomal localization of genes for transcription factor Nuclear Factor One.

Transcription factor Nuclear Factor One (NFI) proteins are derived from a small family of four vertebrate genes (NFIA, B, C and X), all of which produce a fair number of protein variants by alternative splicing. In order to ultimately locate RNA signal sequences around exon/intron borders for the production of regulated splice variants, we have determined the exon structure of the chicken NFIB gene as the last of the four vertebrate genes for which the gene structure was not yet elucidated. This made it possible to compile nine newly isolated and sequenced mouse NFI cDNA sequences together with all previously available ones and to deduce corresponding splicing patterns for the orthologous vertebrate genes of all four paralogous gene types. Results from the analysis of alternative splicing and of NFI gene mapping in the genome of human and mouse argue for a phylogenetic route in which the four vertebrate NFI genes result from a single duplication of a genomic segment containing two NFI intermediate genes rather than from two independent duplications of two separated single ancestor genes.

Alternative Splicing↗

PubMed2003

QuickLIMS: facilitating the data management for DNA-microarray fabrication.

SUMMARY: QuickLIMS is a Microsoft Access-based laboratory information and management system, capable of processing all information for microarray production. The program's operational flow is protocol-based, dynamically adapting to changes of the process. It interacts with the laboratory robot and with other database systems over the network, and it represents a complete solution for the management of the entire manufacturing process. AVAILABILITY AND SUPPLEMENTARY INFORMATION: http://www.dkfz-heidelberg.de/kompl_genome/Other/QuickLims/index.html

Computer Communication Networks↗

PubMed2003

Gains of 2p involving the REL locus correlate with nuclear c-Rel protein accumulation in neoplastic cells of classical Hodgkin lymphoma.

Structural aberrations of the short arm of chromosome 2, mostly resulting in gains of 2p13 approximately 16, have recently been described as being highly recurrent in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). As these gains consistently lead to increased copy numbers of the REL oncogene locus, we investigated the expression of the c-Rel protein in a series of 30 cHL cases with known genomic REL status as determined by comparative genomic hybridization and interphase cytogenetics. Expression of the c-Rel protein was investigated in 26 biopsies by immunohistochemistry. Distinct patterns were observed in HRS cells with no staining, cytoplasmic, and/or nuclear staining for c-Rel. All 13 samples with additional copies of the REL locus displayed nuclear staining for c-Rel, while 13 cHL samples lacking chromosome 2 (2p) gains displayed a significantly lower proportion or complete absence of HRS cells with nuclear c-Rel expression. Detailed analysis using combined immunophenotyping and interphase cytogenetics of individual HRS cells demonstrated that REL gains correlated with the presence of nuclear c-Rel staining. Additionally, in 2 cHL samples with translocation breakpoints in 2p13 approximately 16, nuclear staining of c-Rel was observed; in one of them the staining pattern was indicative of a truncated c-Rel protein. The correlation between structural aberrations involving the REL locus and nuclear c-Rel accumulation in HRS cells qualifies REL as a target gene of the frequent gains in 2p in cHL. The data suggest that REL aberrations are a genetic mechanism contributing to constitutive nuclear factor (NF)-kappa B/Rel activation in cHL.

Cell Line↗

PubMed2003

Gene expression patterns in ependymomas correlate with tumor location, grade, and patient age.

To elucidate the molecular events responsible for tumorigenesis and progression of ependymomas, we analyzed molecular alterations on the gene expression level in a series of newly diagnosed ependymal neoplasms (n = 39). To this aim, tumor RNA was hybridized to microarrays comprising 2600 different genes with relevance to mitosis, cell-cycle control, oncogenesis, or apoptosis. For CLU, IGF-2, and RAF-1, which are apparent candidate genes because they had been previously described to be involved in tumorigenesis of other human malignancies, we found a high expression on the mRNA as well as the protein level. We identified gene expression signatures for the differentiation of tumors with respect to location, grade, and patient age. Spinal ependymomas were characterized by high-expression levels of HOXB5, PLA2G, and CDKN2A and tumors in young patients (< or =16 years of age) by high-expression levels of LDHB and STAM. Notably, we were able to classify supratentorial grade II and III tumors with 100% accuracy, whereas this did not apply for infratentorial ependymomas. The similar gene expression patterns of grade II and III infratentorial malignancies suggest that grade III tumors may develop through a secondary multistep transformation process involving genes that are related to cell proliferation (LDHA, cyclin B, MAT2A) or tumor suppression (PTEN). In summary, our results provide new insight in the biochemical pathways particularly intriguing in the pathomechanism of ependymomas and suggest that this entity comprises molecularly distinct diseases.

Adolescent↗

PubMed2003

Independent molecular development of metachronous glioblastomas with extended intervening recurrence-free interval.

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.

Brain Neoplasms↗