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Biomedical subjects

Stephan Stilgenbauer

Publications and source records attributed to Stephan Stilgenbauer.

At least 37 records · Page 2Linked to original sources
PubMed2004

Microarray gene expression profiling of B-cell chronic lymphocytic leukemia subgroups defined by genomic aberrations and VH mutation status.

PURPOSE: Genomic aberrations and mutational status of the immunoglobulin variable heavy chain (VH) gene have been shown to be among the most important predictors for outcome in patients with B-cell chronic lymphocytic leukemia (B-CLL). In this study, we report on differential gene expression patterns that are characteristic for genetically defined B-CLL subtypes. MATERIALS AND METHODS: One hundred genetically well-characterized B-CLL samples, together with 11 healthy control samples, were analyzed using oligonucleotide arrays, which test for the expression of some 12,000 human genes. RESULTS: Aiming at microarray-based subclassification, class predictors were constructed using sets of differentially expressed genes, which yielded in zero or low misclassification rates. Furthermore, a significant number of the differentially expressed genes clustered in chromosomal regions affected by the respective genomic losses/gains. Deletions affecting chromosome bands 11q22-q23 and 17p13 led to a reduced expression of the corresponding genes, such as ATM and p53, while trisomy 12 resulted in the upregulation of genes mapping to chromosome arm 12q. Using an unsupervised analysis algorithm, expression profiling allowed partitioning into predominantly VH-mutated versus unmutated patient groups; however, association of the expression profile with the VH mutational status could only be detected in male patients. CONCLUSION: The finding that the most significantly differentially expressed genes are located in the corresponding aberrant chromosomal regions indicates that a gene dosage effect may exert a pathogenic role in B-CLL. The significant difference in the partitioning of male and female B-CLL samples suggests that the genomic signature for the VH mutational status might be sex-related.

Adult↗
PubMed2004

Graft-versus-leukemia activity may overcome therapeutic resistance of chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy-chain gene status: implications of minimal residual disease measurement with quantitative PCR.

The aim of this study was to investigate if graft-versus-leukemia (GVL) activity conferred by allogeneic stem cell transplantation (allo-SCT) is effective in chronic lymphocytic leukemia (CLL) with unmutated V(H) gene status. The kinetics of residual disease (MRD) were measured by quantitative allele-specific immunoglobulin heavy chain (IgH) polymerase chain reaction (PCR) in 9 patients after nonmyeloablative allo-SCT for unmutated CLL. Despite an only modest decrease in the early posttransplantation phase, MRD became undetectable in 7 of 9 patients (78%) from day +100 onwards subsequent to chronic graft-versus-host disease or donor lymphocyte infusions. With a median follow-up of 25 months (range, 14-37 months), these 7 patients remain in continuous clinical and molecular remission. In contrast, PCR negativity was achieved in only 6 of 26 control patients (23%) after autologous SCT for unmutated CLL and it was not durable. Taken together, this study shows for the first time that GVL-mediated immunotherapy might be effective in CLL with unmutated V(H).

Adult↗
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

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

Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients.

Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples.

Boron Compounds↗
PubMed2004

Molecular genetics and its clinical relevance.

Molecular genetic methods such as fluorescence in situ hybridization and DNA sequencing have greatly improved our understanding of pathogenic events and prognostic markers in chronic lymphocytic leukemia (CLL). There are genomic aberrations detected in over 80% of CLL cases, and genes potentially involved in the pathogenesis were identified with ATM in a subset of cases with 11q deletion and p53 in cases with 17p13 deletion. Genetic subgroups with distinct clinical features have been identified, such as 11q deletion, which is associated with marked lymphadenopathy and rapid disease progression, whereas 17p deletion predicts for treatment failure with alkylating agents, fludarabine, and short survival times. There is mutation status of the VH genes that allows the separation into patients with long (mutated VH) or short (unmutated VH) survival times. V-gene usage, VDJ structure, and gene expression differences in the two subgroups allow insights into differential pathogenic mechanisms and provide further prognostic information (V3-21 usage, ZAP-70 expression). The VH mutation status and genomic abnormalities have been shown to be of independent prognostic value in multivariate analysis, seem to allow outcome predication irrespective of the clinical stage, and may therefore allow a risk assessment for individual patients early in the course of their disease.

Chromosome Aberrations↗
PubMed2004

Chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is one of the most commonly diagnosed leukemias managed by practicing hematologists. For many years patients with CLL have been viewed as similar, with a long natural history and only marginally effective therapies that rarely yielded complete responses. Recently, several important observations related to the biologic significance of V(H) mutational status and associated ZAP-70 overexpression, disrupted p53 function, and chromosomal aberrations have led to the ability to identify patients at high risk for early disease progression and inferior survival. Concurrent with these investigations, several treatments including the nucleoside analogues, monoclonal antibodies rituximab and alemtuzumab have been introduced. Combination of these therapies in clinical trials has led to high complete and overall response rates when applied as initial therapy for symptomatic CLL. Thus, the complexity of initial risk stratification of CLL and treatment has increased significantly. Furthermore, when these initial therapies do not work, approach of the CLL patient with fludarabine-refractory disease can be quite challenging. This session will describe the natural history of a CLL patient with emphasis on important decision junctures at different time points in the disease. In Section I, Dr. Stephan Stilgenbauer focuses on the discussion that occurs with CLL patients at their initial evaluation. This includes a review of the diagnostic criteria for CLL and prognostic factors utilized to predict the natural history of the disease. The later discussion of risk stratification focuses on molecular and genomic aberrations that predict rapid progression, poor response to therapy, and inferior survival. Ongoing and future efforts examining early intervention strategies in high risk CLL are reviewed. In Section II, Drs. Ian Flinn and Jesus G. Berdeja focus on the discussion of CLL patients when symptomatic disease has developed. This includes an updated review of monotherapy trials with nucleoside analogs and recent trials that have combined these with monoclonal antibodies and/or alternative chemotherapy agents. Appropriate application of more aggressive therapies such as autologous and allogeneic immunotherapy and less aggressive treatments for appropriate CLL patient candidates are discussed. In Section III, Dr. John Byrd focuses on the discussion that occurs with CLL patients whose disease is refractory to fludarabine. The application of genetic risk stratification in choosing therapy for this subset of patients is reviewed. Available data with conventional combination based therapies and monoclonal antibodies are discussed. Finally, alternative promising investigational therapies including new antibodies, kinase inhibitors (CDK, PDK1/AKT, PKC) and alternative targeted therapies (DNA methyltransferase inhibitors, histone deacetylase inhibitors, etc.) are reviewed with an emphasis on the most promising agents for this patient population.

Antineoplastic Agents↗
PubMed2003

The prognostic impact of autologous stem cell transplantation in patients with chronic lymphocytic leukemia: a risk-matched analysis based on the VH gene mutational status.

To assess the therapeutic value of sequential high-dose therapy (SHDT) including autologous stem cell transplantation in chronic lymphocytic leukemia (CLL) we performed a risk-matched comparison between 66 patients who had undergone a uniform SHDT regimen and a database of 291 patients treated conventionally. Matching variables were age, Binet stage, IgVH (variable region of the immunoglobulin heavy chain) gene mutational status, and lymphocyte count. Forty-four pairs fully matched for all 4 variables were identified. Patient groups were well balanced for additional risk factors including adverse genomic abnormalities and CD38 expression. With an overall median follow-up time of 70 and 86 months, respectively, survival was significantly longer for the SHDT patients than for the conventionally treated patients when calculated from diagnosis (hazard ratio [HR] 0.39; P=.03 [log rank]) or from study entry (HR 0.32; P=.006). The benefit for the SHDT group remained significant when the analyses were restricted to those 58 patients who had an unmutated VH status. Cox regression analysis confirmed SHDT as independent favorable prognostic factor for survival from diagnosis (HR 0.38, P=.04) as well as from study entry (HR 0.38, P=.03). These data suggest a survival benefit for patients with poor-risk CLL receiving SHDT during the course of their disease.

Adult↗
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

Capillary electrophoretic analysis of genomic DNA methylation levels.

Changes in DNA methylation have been found in the large majority of tumors. This phenomenon includes both genome-wide hypomethylation and gene- specific hypermethylation. However, the clinical relevance of either mechanism has remained contentious. In order to determine DNA methylation levels from a large number of clinical samples, we have established a method for accurate high-throughput quantification of 5-methylcytosine in genomic DNA. Our protocol requires a small amount (<1 micro g) of DNA that is enzymatically hydrolyzed to single nucleotides. Single nucleotides are then derivatized with a fluorescent marker and separated by capillary electrophoresis. After calibration of the method, we have determined cytosine methylation levels from tumor samples of 81 patients that had been diagnosed with chronic lymphocytic leukemia (CLL). These patients showed a high variability in their methylation levels with a general trend towards hypomethylation. Because of its high accuracy and throughput our method will be useful in determining the role of genomic DNA methylation levels in tumorigenesis.

Cytosine↗
PubMed2003

Value of comparative genomic hybridization and fluorescence in situ hybridization for molecular diagnostics in multiple myeloma.

Chromosomal abnormalities, such as 13q deletions, are emerging as important prognostic factors in multiple myeloma. Fluorescence in situ hybridization (FISH) using specific DNA probes is the technique most widely used for the determination of genomic aberrations in this disease. The utility of comparative genomic hybridization (CGH) for molecular diagnostics in plasma cell malignancies has not been systematically analysed. We investigated tumour samples of patients with multiple myeloma (n = 43) or plasma cell leukaemia (n = 3) using CGH and FISH with five DNA probes localized to chromosome bands 1p22, 6q21, 11q22-q23, 13q14 and 17p13. By CGH, the most frequent genomic changes were gains on chromosomes 1q, 9q and 11q, as well as losses on chromosomes 13q, 6q, Xp and Xq. By FISH, trisomy 11q was identified at a similar frequency to the 13q deletion (42%). Compared with FISH data, the sensitivity of CGH was 80.7% and the specificity was 97.5%. Thirty-two aberrations found by FISH were not identified by CGH, mostly as a result of the proportion of cells carrying the respective aberrations, or because of the limited spatial resolution of CGH. Our data indicate that, for clinical molecular diagnostics in multiple myeloma, FISH with a disease-specific DNA probe set is superior to CGH analysis.

Adult↗
PubMed2002

Unmutated immunoglobulin variable heavy-chain gene status remains an adverse prognostic factor after autologous stem cell transplantation for chronic lymphocytic leukemia.

An unmutated germ line configuration of the immunoglobulin variable heavy-chain gene (VH) has emerged to be a crucial adverse prognostic factor in chronic lymphocytic leukemia (CLL) under conventional treatment. The purpose of the present study was to investigate whether the VH mutational status retains its prognostic value in CLL also in the setting of autologous stem cell transplantation (SCT). Therefore, we investigated the mutational status in 58 patients with CLL who underwent myeloablative radiochemotherapy with SCT. Rearranged VH genes were analyzed by multiplex polymerase chain reaction (PCR) and direct sequencing using FR1 family-specific primers and JH consensus primers. Twenty patients (34%) showed less than 98% homology compared with germ line VH sequences and were considered as mutated, whereas 38 patients (66%) had an unmutated VH status (median mutational rate of 0%; range, 0%-1.7%). An unmutated VH configuration was strongly correlated with the presence of short lymphocyte doubling time (P =.003) and high lymphocyte count (P =.005). Time to clinical relapse and time to recurrence of monoclonal B cells as assessed by consensus IgH CDR3 PCR was significantly shorter in the group with unmutated VH genes (2-year probability 19% versus 0%, P =.0008, and 34% versus 9%, P =.0006, respectively). These results show that in CLL, an unmutated VH gene status of the tumor clone remains an adverse prognostic factor after SCT. Nevertheless, the hitherto only 3 deaths and the median treatment-free interval of 49 months in the unmutated cohort suggest a beneficial effect of SCT for this high-risk population in comparison to conventional treatment.

Adult↗
PubMed2002

Genetic analysis of de novo CD5+ diffuse large B-cell lymphomas suggests an origin from a somatically mutated CD5+ progenitor B cell.

CD5(+) diffuse large B-cell lymphomas (DLBLs) have recently been described as a particular subgroup of DLBLs. Classical banding and interphase cytogenetic analyses targeting ATM, TP53, and P16(INK4a) genes and the D13S25 locus from 13 CD5(+) DLBLs were compared with 55 CD5(-) DLBLs. Additionally, analysis of somatic mutations of the immunoglobulin heavy chain variable region (IgVH) genes were performed in CD5(+) DLBLs. CD5(+) DLBLs were somatically mutated (7 of 8 cases) and were negative for t(11;14)(q13;q32) and t(14;18)(q32;q21), whereas t(3;14)(q27;q32) was found in only one tumor. Trisomy 3 and gains on chromosomes 16/16p and 18/18q were significantly overrepresented in CD5(+) DLBLs. No ATM deletions were detected. The prevalence of deletions at the D13S25 locus was significantly higher in CD5(+) DLBLs (4 of 12 [33%]) compared with CD5(-) DLBLs (4 of 42 [10%]), as were p16(INK4a) deletions (33% versus 8%). On the basis of these findings, CD5(+) DLBLs are likely to arise from the same progenitor cell as the mutated variant of CD5(+) lymphocytic lymphoma/B-cell chronic lymphocytic leukemia (B-CLL).

B-Lymphocytes↗
PubMed2002

V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), biologic risk factors such as immunoglobulin variable heavy chain gene (V(H)) mutation status, CD38 expression level, and genomic aberrations have recently been identified, but the relative prognostic impact of the individual parameters is unknown. In the current study, we analyzed V(H) mutation status by polymerase chain reaction and sequencing (n = 300), genomic aberrations by fluorescence in situ hybridization (+3q, 6q-, +8q, 11q-, +12q, 13q-, t(14q), 17p-) (n = 300), and CD38 expression by triple-color FACS (CD5, CD19, CD38) (n = 157) in a unicentric CLL cohort. The prognostic influence of V(H) mutation rate and CD38 expression level was tested by maximally selected log-rank statistics. A corrected P value (P(cor)) for a cutoff level allowing the best separation of 2 subgroups with different survival probabilities was identified at 97% V(H) homology (95% confidence interval [CI], 96%-98% homology, P(cor) <.001) and at 7% CD38 expression (95% CI, 20%-71% expression, P(cor) =.02). In univariate analyses, unmutated V(H) genes and high CD38 expression levels predicted for shorter survival times. The overall incidence of genomic aberrations was similar in the V(H) unmutated and V(H) mutated subgroups. High-risk genomic aberrations such as 17p- and 11q- occurred almost exclusively in the V(H) unmutated subgroup, whereas favorable aberrations such as 13q- and 13q- as single abnormalities were overrepresented in the V(H) mutated subgroup. In multivariate analysis, unmutated V(H), 17p deletion, 11q deletion, age, WBC, and LDH were identified as independent prognostic factors, indicating a complementary role of V(H) mutation status and genomic aberrations to predict outcome in CLL.

ADP-ribosyl Cyclase↗
PubMed2002

Evidence for distinct pathomechanisms in B-cell chronic lymphocytic leukemia and mantle cell lymphoma by quantitative expression analysis of cell cycle and apoptosis-associated genes.

The B-cell lymphoproliferative malignancies B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL) share characteristics, including overlapping chromosomal aberrations with deletions on chromosome bands 13q14, 11q23, 17p13, and 6q21 and gains on chromosome bands 3q26, 12q13, and 8q24. To elucidate the biochemical processes involved in the pathogenesis of B-CLL and MCL, we analyzed the expression level of a set of genes that play central roles in apoptotic or cell proliferation pathways and of candidate genes from frequently altered genomic regions, namely ATM, BAX, BCL2, CCND1, CCND3, CDK2, CDK4, CDKN1A, CDKN1B, E2F1, ETV5, MYC, RB1, SELL, TFDP2, TNFSF10, and TP53. Performing real-time quantitative reverse transcription polymerase chain reaction in a panel of patients with MCL and B-CLL and control samples, significant overexpression and underexpression was observed for most of these genes. Statistical analysis of the expression data revealed the combination of CCND1 and CDK4 as the best classifier concerning separation of both lymphoma types. Overexpression in these malignancies suggests ETV5 as a new candidate for a pathogenic factor in B-cell lymphomas. Characteristic deregulation of multiple genes analyzed in this study could be combined in a comprehensive picture of 2 distinctive pathomechanisms in B-CLL and MCL. In B-CLL, the expression parameters are in strong favor of protection of the malignant cells from apoptosis but did not provide evidence for promoting cell cycle. In contrast, in MCL the impairment of apoptosis induction seems to play a minor role, whereas most expression data indicate an enhancement of cell proliferation.

Adult↗
PubMed2002

Down-regulation of candidate tumor suppressor genes within chromosome band 13q14.3 is independent of the DNA methylation pattern in B-cell chronic lymphocytic leukemia.

Loss of genomic material from chromosomal band 13q14.3 is the most common genetic imbalance in B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, pointing to the involvement of this region in a tumor suppressor mechanism. From the minimally deleted region, 3 candidate genes have been isolated, RFP2, BCMS, and BCMSUN. DNA sequence analyses have failed to detect small mutations in any of these genes, suggesting a different pathomechanism, most likely haploinsufficiency. We, therefore, tested B-CLL patients for epigenetic aberrations by measuring expression of genes from 13q14.3 and methylation of their promotor region. RB1, CLLD7, KPNA3, CLLD6, and RFP2 were down-regulated in B-CLL patients as compared with B cells of healthy donors, with RFP2 showing the most pronounced loss of expression. To test whether this loss of gene expression is associated with methylation of CpG islands in the respective promotor regions, we performed methylation-sensitive quantitative polymerase chain reaction analyses and bisulfite sequencing on DNA from B-CLL patients. No difference in the methylation patterns could be detected in any CpG island of the minimally deleted region. Down-regulation of genes within chromosomal band 13q14.3 in B-CLL is in line with the concept of haploinsufficiency, but this tumor-specific phenomenon is not associated with DNA methylation.

Base Sequence↗