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Ulrich Lehmann

Publications and source records attributed to Ulrich Lehmann.

At least 19 recordsLinked to original sources
PubMed2006

Quantitative high-resolution CpG island mapping with Pyrosequencing reveals disease-specific methylation patterns of the CDKN2B gene in myelodysplastic syndrome and myeloid leukemia.

BACKGROUND: Gene silencing through aberrant CpG island methylation is the most extensively analyzed epigenetic event in human tumorigenesis and has huge diagnostic and prognostic potential. Methylation patterns are often very heterogeneous, however, presenting a serious challenge for the development of methylation assays for diagnostic purposes. METHODS: We used Pyrosequencing technology to determine the methylation status of 68 CpG sites in the CpG island of the CDKN2B gene [cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)], frequently hypermethylated in myeloid malignancies, in a series of bone marrow samples from patients with myelodysplasia and myeloid leukemia (n = 82) and from 32 controls. A total of 7762 individual methylation sites were quantitatively evaluated. Precision and reproducibility of the quantification was evaluated with several overlapping primers. RESULTS: The use of optimized sequencing primers and the new Pyro Q-CpG software enabled precise and reproducible quantification with a single sequencing primer of up to 15 CpG sites distributed over approximately 100 bp. Extensive statistical analyses of the whole CpG island revealed for the first time disease-specific methylation patterns of the CDKN2B gene in myeloid malignancies and small regions of differential methylation with high discriminatory power that enabled differentiation of even low-grade myelodysplastic syndrome samples from the controls, a result that was confirmed in an independent group of 9 control and 36 patient samples. CONCLUSION: The precise quantitative methylation mapping of whole CpG islands is now possible with Pyrosequencing software in combination with optimized sequencing primers. This method reveals disease-specific methylation patterns and enables the development of specific diagnostic assays.

Adult↗
PubMed2006

LEF-1 is crucial for neutrophil granulocytopoiesis and its expression is severely reduced in congenital neutropenia.

We demonstrate here that lymphoid enhancer-binding factor 1 (LEF-1) mediates the proliferation, survival and differentiation of granulocyte progenitor cells. We initially documented the importance of this transcription factor in the bone marrow of individuals with severe congenital neutropenia (CN) with a 'differentiation block' at the promyelocytic stage of myelopoiesis. LEF-1 expression was greatly reduced or even absent in CN arrested promyelocytes, resulting in defective expression of the LEF-1 target genes CCND1, MYC and BIRC5, encoding cyclin D1 (ref. 2), c-Myc and survivin, respectively. In contrast, healthy individuals showed highest LEF-1 expression in promyelocytes. Reconstitution of LEF-1 in early hematopoietic progenitors of two individuals with CN corrected the defective myelopoiesis and resulted in the differentiation of these progenitors into mature granulocytes. Repression of endogenous LEF-1 by specific short hairpin RNA inhibited proliferation and induced apoptosis of CD34(+) progenitors from healthy individuals and of cells from two myeloid lines (HL-60 and K562). C/EBPalpha, a key transcription factor in granulopoiesis, was directly regulated by LEF-1. These observations indicate that LEF-1 is an instructive factor regulating neutrophilic granulopoiesis whose absence plays a critical role in the defective maturation program of myeloid progenitors in individuals with CN.

Antigens, CD↗
PubMed2006

Testing the importance of p27 degradation by the SCFskp2 pathway in murine models of lung and colon cancer.

Decreased expression of the CDK inhibitor p27kip1 in human tumors directly correlates with increased resistance to chemotherapies, increased rates of metastasis, and an overall increased rate of patient mortality. It is thought that decreased p27 expression in tumors is caused by increased proteasomal turnover, in particular activation of the pathway governed by the SCFskp2 E3 ubiquitin protein ligase. We have directly tested the importance of the SCFskp-mediated degradation of p27 in tumorigenesis by analyzing the tumor susceptibility of mice that express a form of p27 that cannot be ubiquitinated and degraded by this pathway (p27T187A). In mouse models of both lung and colon cancer down-regulation of p27 promotes tumorigenesis. However, we found that preventing p27 degradation by the SCFskp2 pathway had no impact on tumor incidence or overall survival in either tumor model. Our study unveiled a previously unrecognized role for the control of p27 mRNA abundance in the development of non-small cell lung cancers. In the colon cancer model, the frequency of intestinal adenomas was similarly unaffected by the p27T187A mutation, but, unexpectedly, we found that it inhibited progression of intestinal adenomas to carcinomas. These studies may guide the choice of clinical settings in which pharmacologic inhibitors of the Skp2 pathway might be of therapeutic value.

Animals↗
PubMed2006

Bone marrow infiltration pattern in B-cell chronic lymphocytic leukemia is related to immunoglobulin heavy-chain variable region mutation status and expression of 70-kd zeta-associated protein (ZAP-70).

B-cell chronic lymphocytic leukemia (B-CLL) consists of at least 2 subtypes with either somatically mutated or unmutated immunoglobulin heavy-chain variable region (IgVH) genes. A prognostic significance of the infiltration pattern in bone marrow trephine biopsy has been described before. The combined pattern analysis and detection of 70-kd zeta-associated protein (ZAP-70) expression in formalin-fixed, paraffin-embedded bone marrow trephines has not been investigated so far. To evaluate the relationship between ZAP-70 expression, mutation status, and the infiltration pattern in B-CLL, we analyzed bone marrow trephine biopsies from B-CLL patients (n = 35). The expression of ZAP-70 was related to the infiltration type: in all samples with diffuse infiltration pattern, the leukemic cells showed ZAP-70 staining, whereas leukemic cells in a nodular infiltration pattern were negative. By contrast, the mixed-pattern type showed a variable ZAP-70 expression. Besides definitely negative or positive ZAP-70 expression, a few samples showed a faint ZAP-70 staining, and the classification into the positive or negative group was difficult. In addition, the infiltration type was related to the mutation status in a subset of samples: mutation of the IgVH gene was restricted to the nondiffuse infiltration pattern and was not found in cases with diffuse infiltration of bone marrow. The expression of ZAP-70, detected by an immunohistochemical assay and also by real-time quantitative reverse transcriptase-polymerase chain reaction assigned 83% of the chronic lymphocytic leukemia cases to the suspected immunoglobulin mutation subtype. In 2 patients with ZAP-70 expression, a mutated IgVH status was found. These cases exhibited a mixed pattern of infiltration. We conclude that the pure nodular type of marrow infiltration in B-CLL is associated with IgH hypermutation and ZAP-70 negativity, whereas the predominantly diffuse type of infiltration reveals unmutated IgH genes with ZAP-70 overexpression. The mixed type of infiltration is displayed by mutated as well as unmutated cases with a varying pattern of ZAP-70 expression.

Adult↗
PubMed2006

Fibroblasts of recipient origin contribute to bronchiolitis obliterans in human lung transplants.

RATIONALE: The participation of circulating precursor cells in the development of experimental pulmonary fibrosing lesions in mice has been recently demonstrated. OBJECTIVES: This study analyzes whether circulating, bone marrow-derived, fibroblastic precursor cells contribute to the development of fibrosing lesions in human lungs, especially bronchiolitis obliterans. METHODS: The occurrence of in situ microchimerism in bronchiolitis obliterans lesions of human lung allografts (n = 12) as well as of autologous lung tissue from patients post-bone marrow transplantation (n = 2) was analyzed using laser-assisted microdissection after immunohistochemical labeling of leukocytes followed by short tandem repeat-polymerase chain reaction-based genotyping. Combined immunofluorescence and fluorescence in situ hybridization for sex chromosomes was performed for independent confirmation in cases with appropriate sex mismatch (n = 2). MEASUREMENTS AND MAIN RESULTS: The bronchiolitis obliterans lesions of all 12 lung transplant patients contained considerable numbers of recipient-derived fibroblasts (mean, 32%). The fibrosing pulmonary lesions of the two bone marrow-transplanted patients also displayed clear in situ microchimerism. The in situ detection methodology confirmed these results, although to a lower degree (6-16%). CONCLUSIONS: These data clearly demonstrate the involvement of circulating fibroblastic precursor cells in the development of human fibrosing lung lesions and provide evidence that these cells are most probably bone marrow derived. These results may open new venues regarding the prevention of fibrosis in lung transplants and potentially in other organs.

Adult↗
PubMed2006

High-resolution genomic profiling reveals association of chromosomal aberrations on 1q and 16p with histologic and genetic subgroups of invasive breast cancer.

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.

Biomarkers, Tumor↗
PubMed2006

Laser-assisted microdissection and isolation of DNA and RNA.

One of the major challenges in molecular analysis of breast cancer specimens is tissue heterogeneity. The admixture of contaminating bystander cells might distort the results of quantitative molecular analyses. Therefore, pure tumor cell populations have to be isolated in order to obtain reliable molecular data. In this chapter, we present protocols for the laser-assisted microdissection of breast cancer tissue sections (using a laser microdissection microscope from P.A.L.M., Bernried, Germany) and the subsequent isolation of genomic DNA or total RNA. The protocols presented in here have been used in our laboratory for the exact quantification of gene copy numbers in intraductal and invasive tumor cells and for the quantitative assessment of promoter hypermethylation during breast cancer progression. We have added some guidelines for the organization of the laser-microdissection and polymerase chain reaction laboratory, which prevent crosscontamination of samples and carry-over contamination because of polymerase chain reaction products.

Breast Neoplasms↗
PubMed2005

Distinct methylation patterns of benign and malignant liver tumors revealed by quantitative methylation profiling.

PURPOSE: A comparative quantitative methylation profiling of hepatocellular carcinoma and the most frequent benign liver tumor, hepatocellular adenoma, was set up for the identification of tumor-specific methylation patterns. EXPERIMENTAL DESIGN: The quantitative methylation levels of nine genes (RASSF1A, cyclinD2, p16INK4a, DAP-K, APC, RIZ-1, HIN-1, GSTpi1, SOCS-1) were analyzed in hepatocellular carcinoma and adjacent normal tissue (n = 41), hepatocellular adenoma and adjacent normal tissue (n = 26), focal nodular hyperplasia (n = 10), and unrelated normal liver tissue (n = 28). Accumulated methylation data were analyzed using various statistical algorithms, including hierarchical clustering, to detect tumor-specific methylation patterns. RESULTS: Cluster analysis revealed that hepatocellular adenoma displays a methylation profile much more similar to that found in normal liver tissue and focal nodular hyperplasia than to that found in hepatocellular carcinoma. Many characteristic differences were not detected when using mere qualitative methylation assays. The cyclinD2 gene was identified as a new and frequent target for aberrant hypermethylation in hepatocellular carcinoma (68%). In the control group of 28 liver specimens from healthy donors, a clear correlation between age of patient and frequency and level of aberrant methylation was seen, which could not be detected in the group of hepatocellular carcinoma specimens. CONCLUSIONS: Methylation profiling can clearly contribute to the unequivocal classification of suspicious lesions, but only if done in a quantitative manner applying cell type and gene-specific thresholds. In hepatocellular carcinoma, the altered methylation patterns accompanying malignant transformation override the age-dependent increase in gene methylation.

Adenoma, Liver Cell↗
PubMed2005

Up-regulation of DNA methyltransferases DNMT1, 3A, and 3B in myelodysplastic syndrome.

Analyzing bone marrow trephines from (myelodysplastic syndrome) MDS patients we show for the first time strong over-expression of the DNA methyltransferases 1, and 3A (and 3B to a much lesser extent) in the myelodysplastic syndrome FAB subtypes refractory anaemia (RA) and refractory anaemia with excess of blasts (RAEB). The increase in mRNA expression was much less pronounced in refractory anaemia with ringed sideroblasts (RARS). Classification according to the new WHO guidelines revealed distinct differences between RCMD and RARS. This elevated mRNA expression most probably contributes to the frequently found aberrant hypermethylation in MDS and might explain the promising clinical response of MDS patients treated with DNMT inhibitors.

Biopsy, Needle↗
PubMed2005

Absence of p21(CIP 1), p27(KIP 1) and p 57(KIP 2) methylation in MDS and AML.

Transcriptional silencing of tumour suppressor genes (TSG) due to hypermethylation is a common event in human tumours. The three members of the KIP/CIP family of cyclin dependent kinase inhibitors (CDKIs), p21(CIP 1), p27(KIP 1), and p 57(KIP 2), play key roles in cell cycle regulation, but little is known about their methylation in myeloid neoplasia. Therefore, we analysed 9 haematopoietic cell lines, 67 myelodysplastic syndrome (MDS) and 26 acute myeloid leukaemia (AML) cases as well as 11 controls. p 57(KIP 2) hypermethylation was found in 4/9 cell lines, but methylation of p21(CIP 1) and p27(KIP 1) was infrequent. All patient samples analysed were methylation-negative for these three genes.

Acute Disease↗
PubMed2005

Hypermethylation of the suppressor of cytokine signalling-1 (SOCS-1) in myelodysplastic syndrome.

Transcriptional silencing because of hypermethylation is now recognised to be a hallmark of human tumours. In contrast to acute myeloid leukaemia (AML), comparably little is known about aberrant methylation in myelodysplastic syndrome (MDS), a heterogeneous clonal stem cell disorder with a risk of transformation into secondary AML of up to 30%. Recent evidence demonstrates that suppressor of cytokine signalling SOCS-1, a negative regulator of cytokine pathways, may act as a tumour suppressor gene, and inactivation because of hypermethylation was shown in various malignancies. Employing a newly developed quantitative real-time polymerase chain reaction-based methylation assay we analysed, for the first time, SOCS-1 methylation in MDS and found disease-specific hypermethylation in 27 of 86 MDS patients (31%). Demethylation experiments provided direct evidence that aberrant methylation of SOCS-1 induces transcriptional silencing in myeloid cells. In addition, by analysing the expression of signal transducers and activators of transcription (STAT)-induced genes we provide for the first time evidence that the activity of the Janus kinase/STAT pathway is increased in primary patient samples showing SOCS-1 hypermethylation.

Acute Disease↗
PubMed2005

Quantitative mRNA expression analysis of co-stimulatory molecules in sequential biopsies from heart allografts.

Allograft-specific T-cell activation requires two signals, "signal one" via the T-cell receptor and "signal two" via costimulatory pathways. Animal models provide promising data that blockade of costimulatory signals can lead to T-cell anergy and tolerance. We analyzed 72 formalin-fixed and paraffin-embedded sequential heart allograft biopsies from 22 patients by quantitative real-time reverse transcriptase-polymerase chain reaction for the mRNA expression of the costimulatory molecules CD28, CD80, CD86, CD40, and CD154. mRNA expression levels were correlated to histological grade and time-point of rejection. mRNA expression of costimulatory molecules predominantly expressed by antigen-presenting cells (CD80, CD86, CD40), correlated with histological grade of cell-mediated acute rejection. Costimulatory molecules were up-regulated not only during rejection episodes early after transplantation, but also at late occurring rejection. The present results suggest a simultaneous and long-term therapeutical blockade of more than one costimulatory pathway for the prevention of repetitive T-cell mediated acute rejection episodes after heart transplantation.

Adult↗
PubMed2005

Down-regulation of the IGF-2/H19 locus during normal and malignant hematopoiesis is independent of the imprinting pattern.

H19 and IGF-2 are two growth regulatory genes located on chromosome 11p15 implicated in tumorigenesis. Both genes are imprinted and regulated reciprocally under many circumstances. In order to elucidate the contribution of H19 and IGF-2 to leukemogenesis, the mRNA expression level of both genes were quantitated in bone marrow biopsies and peripheral blood samples from normal (n=98), chronic myelomonocytic leukemia (CMML, n=43), chronic myelogenous leukemia (CML, n=40) and, acute myelogenous leukemia (AML, n=32) cases. A concomitant reduction of H19 and IGF-2 expression was observed in all leukemic samples compared to the healthy controls. This down-regulation was not accompanied by changes in methylation of the differentially methylated region (DMR). Whereas the H19 gene showed strict monoallelic expression in all informative normal (n=31) and leukemic (n=54) samples, the imprinting pattern of the IGF2 gene was found to be heterogeneous. No correlations between imprinting status (mono- versus biallelic expression), quantitative mRNA expression levels and course of disease were found for the IGF-2 gene. The data suggest a disturbed regulation of the IGF-2/H19 locus in myeloid leukemias which is not caused by loss of imprinting.

Alleles↗
PubMed2005

Combined laser-assisted microdissection and short tandem repeat analysis for detection of in situ microchimerism after solid organ transplantation.

Following the transplantation of a solid organ leukocytes of donor origin migrate out of the organ, contributing to a chimeric blood cell population ("peripheral microchimerism"). At the same time, leukocytes and pluripotent precursor cells of the recipient migrate into the organ, creating an "in situ microchimerism." A method is described for the identification of cells with the recipient's genotype in the transplanted organ by combining laser-assisted microdissection and short tandem repeat analysis. The microdissection allows the contamination-free isolation of morphologically and immunohistochemically characterized cells or groups of cells from histological tissue sections. The subsequent analysis of highly polymorphic short tandem repeats enables unequivocal genotyping in nearly all donor-recipient instances. Employing this new methodological approach, we could identify in individual transplanted organs differentiated parenchymal cells of recipient's origin, which most probably are derived from circulating precursor cells from the bone marrow.

Humans↗
PubMed2004

Cell cycle and no end.

Our knowledge about the molecular circuits regulating the duplication of the genetic material and the subsequent division of a cell into two daughter cells has exploded over the last decade. Aberrations in the regulation of the cell cycle belong to the hallmarks of malignant transformation, leading, in turn, to the development of tumours. After introducing the basics of eukaryotic cell-cycle regulation and describing the four phases of the cell cycle (namely, G1, S, G2 and M) in more detail, alterations of key components of the cell-cycle machinery in human malignancies and their functional consequences are presented. Principally, deregulation of the cell cycle can be caused by unrestricted activity of cell-cycle promoting factors (many oncogenes fall into this class) or by inactivation of inhibitory factors (many tumour suppressor genes belong to this class). Both types of deregulation have been described in human tumours and are discussed in detail. Perspectives concerning the translation of this knowledge into daily routine practice and future applications are discussed at the end. The molecular mechanisms of actual cell division (sister chromatid segregation and cytokinesis) are mentioned only briefly.

Animals↗
PubMed2004

Overexpression of delta-like (Dlk) in a subset of myelodysplastic syndrome bone marrow trephines.

The mRNA expression level of the gene delta-like (Dlk), coding for a signal transducer related to the Delta-Notch family, was measured using real-time PCR methodology in a large series of control biopsies (n = 61) and bone marrow trephines from patients with myelodysplastic syndrome (MDS) (n = 93) and related myeloid disorders (AML, n = 16 and chronic myeloproliferative disease (CMPD), n = 38). It turned out that dlk is strongly overexpressed in a subset of MDS (16%) cases, however with higher frequency in blast rich MDS cases. Therefore, the quantitative detection of dlk mRNA can support the morphological diagnosis of MDS in selected cases.

Bone Marrow Examination↗
PubMed2004

Chimerism of metanephric adenoma but not of carcinoma in kidney transplants.

Recipient-derived cells integrate into renal allografts inducing organ-specific microchimerism. Circulating pluripotent progenitor cells with high plasticity for differentiation were suggested as a potential source of allograft chimerism. Whether or not these cells also contribute to tumor formation in renal transplants is unknown. We analyzed six histologically different tumors in renal allografts for the presence of recipient-derived cells. To circumvent dependency on gender mismatch, a polymerase chain reaction assay for highly polymorphic short tandem repeat marker (DNA fingerprinting) in combination with laser microdissection was applied. Pure tumor cell populations were harvested by laser microdissection after immunohistochemical (CD45/CD68) marking of contaminating leukocytes. In cases of gender mismatch (n = 2), results were confirmed by sex chromosome in situ hybridization. Two metanephric adenomas demonstrated microchimerism comprising both donor- and recipient-derived tumor cells. Two clear cell carcinomas, one transitional cell carcinoma, and one renal cortical adenoma were all of donor origin without chimerism. We conclude that except for metanephric adenomas, tumors arising in renal transplants originate completely from graft cells. The mixed derivation of metanephric adenomas indicates an incorporation of recipient-derived progenitor cells. This finding suggests that adult stem cells can assume neoplastic phenotypes.

Adenocarcinoma, Clear Cell↗