Fluorescence in situ hybridization using the subtelomeric 11q probe as a diagnostic tool for congenital thrombocytopenia.
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Chromosome Aberrations↗
Biomedical subjects
B Schlegelberger
Publications and source records attributed to B Schlegelberger.
Consensus guidelines for microarray gene expression analyses in leukemia from three European leukemia networks.
A plethora of studies have documented that gene expression profiling using DNA microarrays for various types of hematological malignancies provides novel information, which may have diagnostic and prognostic implications. However, to successfully use microarrays for this purpose, the quality and reproducibility of the whole procedure need to be guaranteed. Critical steps of the method are handling, processing and storage of the leukemic sample, purification of tumor cells (or lack thereof), RNA extraction methods, quality control of RNA, labeling techniques, hybridization, washing, scanning, spot filtering, normalization and initial interpretation, and finally the biostatistical analysis. These items have been extensively discussed and evaluated in different multi-center quality rounds within the three networks, that is, I-BFM-SG, the German Competence Network 'Acute and Chronic Leukemias' and the European LeukemiaNet. Based on the exchange of knowledge and experience between the three networks over the last few years, we have formulated guidelines for performing microarray experiments in leukemia. We confine ourselves to leukemias, but many of these requirements also apply to lymphomas or other clinical samples, including solid tumors.
Data Interpretation, Statistical↗
Loss of heterozygosity on chromosome 6q14-q24 is associated with poor outcome in children and adolescents with T-cell lymphoblastic lymphoma.
Deletions of chromosome 6q have been reported in several hematological malignancies, but data are not conclusive regarding their biological and prognostic impact. Therefore, we focused on pediatric patients diagnosed with T-cell lymphoblastic lymphoma (T-LBL) treated uniformly according to the NHL-BFM95 protocol. We used loss-of-heterozygosity (LOH) analysis of 25 microsatellite markers located on chromosome 6q14-q24. Fragment-length analysis was performed on ABI-PRISM3100 Genetic-Analyzer. Eligibility criterion was > or =3 informative markers. Between April 1995 and March 2003, 185 T-LBL patients were treated according to the NHL-BFM95 protocol. Five-year event-free (EFS) and disease-free survival (DFS) were 79+/-3 and 87+/-3% (median follow-up 4.7 [1.2-10.1] years). Sixty-one patients were evaluable for LOH analysis, including 18 out of 23 patients with relapse. EFS and DFS were 67+/-6 and 69+/-6% for these 61 patients. Testing of 853 markers in the 61 patients identified the presence of LOH in 19 patients (31%): 13 of the 18 relapse patients and five of the 41 in complete remission (odds ratio 18.7, 95% confidence interval 4.7-75.3). One LOH-positive patient died from treatment-related toxicity. We conclude that LOH on chromosome 6q14-q24 may have conferred a high risk of relapse on our group of children with T-LBL treated according to the NHL-BFM95 protocol.
Adolescent↗
Generation and characterization of a novel hematopoietic progenitor cell line with DC differentiation potential.
Studies of hematopoietic stem cell (HSC)-derived dendritic cells (DCs) are often limited by the rarity of HSC. To facilitate the study of DCs, we have generated a novel cell line (CR1) by retroviral Notch(IC) gene transfer into Sca1(+)ckit(+)lin- HSC. CR1 cells proliferated in vitro in the presence of recombinant interleukin-3. They maintained an immature progenitor cell phenotype and an intact karyotype. In the presence of granulocyte-macrophage colony-stimulating factor or Flt3L, CR1 cells differentiated into myeloid and plasmacytoid DCs, respectively. Functionally, CR1 cells were comparable to primary bone-marrow-derived DCs with respect to Toll-like-receptor-mediated maturation, cytokine release and capacity to induce effective antitumor immunity. CR1 cells thus provide an elegant new cellular tool to study DC development, function and preclinical DC-based immunotherapies.
Cell Differentiation↗
Unsupervised proteome analysis of human leukaemia cells identifies the Valosin-containing protein as a putative marker for glucocorticoid resistance.
The response to initial glucocorticoid therapy in childhood acute lymphoblastic leukaemia (ALL) reliably predicts the response to multiagent chemotherapy. Patients resistant to glucocorticoids (prednisone poor responders (PPR)) have a poorer event-free survival compared to glucocorticoid-sensitive patients (prednisone good responders (PGR)). A case-control study was performed to investigate differential protein expression in leukaemic blasts from PGR and PPR childhood ALL patients. Two-dimensional gel electrophoresis (2-DE) was used for an unsupervised screening and surface enhanced laser desorption/ionisation-time of flight mass spectrometry (SELDI-TOF MS) for the characterisation of protein spots. In difference maps of average gels for the proteomes of each responder group, differentially expressed proteins were identified after tryptic digestion and spotting onto H4-SELDI-TOF-MS chips. Proteins overexpressed in PPR were Catalase, RING finger protein 22 alpha, Valosin-containing protein (VCP) and a G-protein-coupled receptor. Proteins overexpressed in PGR were protein kinase C and malate dehydrogenase. Valosin-containing protein was chosen for validation and quantification by Western blot analysis in a second case-control group of ALL patients. In this second independent cohort, median VCP expression (P25-P75) was 0.15 (0.11-0.28) in PGR and 0.34 (0.14-0.99) in PPR patients (P = 0.04). We conclude that high VCP expression is associated with poor prednisone response in childhood ALL patients.
Adenosine Triphosphatases↗
Preserved reticulin network in a case of hepatocellular carcinoma.
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Carcinoma, Hepatocellular↗
Jacobsen syndrome and Beckwith-Wiedemann syndrome caused by a parental pericentric inversion inv(11)(p15q24).
Here we report on a male infant presenting the typical pattern of Jacobsen syndrome including trigonocephaly, thrombocytopenia, congenital heart defect, urethral stenosis, and partial agenesis of the corpus callosum. Conventional karyotyping, FISH, SKY and CGH analyses showed that the region distal to the MLL locus on 11q23 was lost and replaced by the distal region of 11p, leading to a partial trisomy of 11p and a partial monosomy of 11q. According to ISCN (1995) the karyotype can be described as 46,XY,add(11)(q2?3). ish 11ptel(D11S2071x3),11qtel(VIJyRM2072x1). Array-CGH analysis allowed us to narrow down the breakpoints to 11p15.1 and 11q24.1. Methylation analyses of genes located on 11p showed an increased level of the non-methylated paternal allele of the KCNQ1OT1 gene, confirming the concomitant presence of Beckwith-Wiedemann syndrome (BWS). The phenotype resulting from the 11q deletion seems to dominate the phenotype due to the distal 11p trisomy. Investigation of the parents revealed that this chromosomal rearrangement was caused by a paternal pericentric inversion inv(11)(p15q24). Since chromosomal aberrations like the one described here can easily be overlooked during routine chromosome analysis, combined FISH analysis using subtelomeric and possibly additional probes should be applied if there is any doubt about the integrity of telomeric regions.
Abnormalities, Multiple↗
Different breakage-prone regions on chromosome 1 detected in t(11;14)-positive mantle cell lymphoma cell lines and multiple myeloma cell lines are associated with different tumor progression-related mechanisms.
To better define secondary aberrations that occur in addition to translocation t(11;14)(q13;q32) in mantle cell lymphomas (MCL) and in multiple myelomas (MM), seven t(11;14)-positive MCL cell lines and four t(11;14)-positive MM cell lines were analysed by fluorescence R-banding and spectral karyotyping (SKY). Compared with published data obtained by G-banding, most chromosome aberrations were redefined or further specified. Furthermore, several additional chromosome aberrations were identified. Thus, these cytogenetically well defined t(11;14)-positive MCL and MM cell lines may be useful tools for the identification and characterization of genes that might be involved in the pathogenesis of MCL and MM, respectively. Since MCL and MM were found to have different alterations of chromosome 1, these were investigated in more detail by fluorescence in situ hybridization (FISH) and multicolor banding (MCB) analyses. The most frequently altered and deletion-prone loci in MCL cell lines were regions 1p31 and 1p21. In contrast, breakpoints in MM cell lines most often involved the heterochromatic regions 1p12-->p11, and the subcentromeric regions 1q12 and 1q21. These data are in accordance with previously published data of primary lymphomas. Our findings may indicate that different pathways of clonal evolution are involved in these morphologically distinct lymphomas harboring an identical primary chromosome aberration, t(11;14).
Cell Line, Tumor↗
Cytogenetic characteristics of a murine in vitro model for the human anaplastic large cell lymphoma (ALCL).
Anaplastic large cell lymphoma (ALCL) is an entity of non-Hodgkin lymphomas (NHL) that often occurs in young children and adolescents. In the majority of cases, ALCL are of T-cell origin and contain the t(2;5)(p23;q35) leading to an NPM-ALK fusion or variant ALK translocations. In addition, there is an ALK-negative subtype of ALCL. The anaplastic lymphoid cell line TS1G6 established by interleukin (IL)-9 transfection of T-helper cells represents a murine model of this subtype. Here, we describe the cytogenetic features of this cell line using spectral karyotyping (SKY) and single-color fluorescence in situ hybridization (FISH). We show that TS1G6 cells exhibit a hypotetraploid karyotype with complex structural alterations. Several unbalanced translocations involved the chromosomal region 14E5, and different translocation partners, i.e. X?A6, 3A3 and 8A1. FISH analysis using a BAC clone containing c-myc confirmed the presence of six copies, but also demonstrated that two loci were irregularly located, indicating that additional intrachromosomal rearrangements had occurred. Moreover, a duplication of the region XF2 approximately 3 was identified. Furthermore, six chromosomes 15 were found, representing a trisomy 15 in a tetraploid chromosome complement, indicating an altered gene dosage of the oncogene c-myc located in region 15D3.
Animals↗
[Azathioprine-associated myelodysplastic syndrome with cytogenetic aberrations].
HISTORY AND ADMISSION FINDINGS: A 49-years-old female with a history of multiple sclerosis and on medication with azathioprine over 5 years amounting to a cumulative dosage of 45 g presented with fatigue and sinus tachycardia. INVESTIGATIONS: Haematological analysis revealed a pancytopenia with a sustained normochromic anemia (hemoglobin 6,2 g/dl), a mild leukopenia (leucocytes 3500/ microl) and thrombocytopenia (platelets 22000/ microl) requiring platelet substitution. Bone marrow aspirate revealed a dysplasia of all three lineages with reduced thrombopoiesis and ineffective erythropoiesis. Cytogenetic analysis detected a complex aberrant clone including loss of the critically deleted regions in 5q31 and 7q31, as well as structural changes of 12p. The diagnosis was refractory cytopenia with multilineage dysplasia according to the WHO classification of myelodysplastic syndromes. TREATMENT AND COURSE: While allogeneic sibling transplantation was planned the patient developed spontaneous recurrent subdural haematoma and died due to persistent bleeding refractory to platelet substitution. CONCLUSION: Treatment with azathioprine may - depending on its cumulative dosage - lead to pancytopenia and subsequent development of myelodysplasia or secondary leukaemia, respectively. Complex genetic alterations involving chromosome 7 are characteristic. Long-lasting treatment with azathioprine needs critical assessment.
Anemia↗
Molecular cytogenetic characterization of the mantle cell lymphoma cell line GRANTA-519.
Combining fluorescence R-banding, fluorescence in situ hybridization and spectral karyotyping allowed us to precisely define chromosomal breakpoints, gains, losses and a newly detected amplification in the human mantle cell lymphoma (MCL) cell line GRANTA-519. GRANTA-519 is characterized by the t(11;14)(q13;q32) resulting in overexpression of cyclin D1, a key player in cell cycle control. Hitherto unresolved complex rearrangements involve 1p, 1q, 3cen, 9p, 11q, 12p, 12q, 16p, 17p, and 18cen. Moreover, a 4- to 6-fold gain of sequences on 18q leads to a low-level amplification of the BCL2 gene and to an overexpression of the BCL2 protein. These results provide the basis for the identification of not only candidate oncogenes responsible for MCL in gained regions, but also for the identification of putative tumor suppressor genes in commonly deleted regions like 1p22, which would eventually enable functional studies of these genes.
Cell Culture Techniques↗
Clinical, morphological, cytogenetic, and prognostic features of patients with myelodysplastic syndromes and del(5q) including band q31.
We analyzed data of 76 consecutive patients with myelodysplastic syndrome (MDS) and isolated del(5q) (n=66) or del(5q) plus one additional chromosomal abnormality (n=10) included in our MDS database over the last 26 years. The median age of our patient population was 66.8 years. The male to female ratio was 1:1.7. In all, 14 patients (18%) had advanced MDS with an increased medullary blast count. A total of 17 patients (22%) had significant dysplasia in the nonmegakaryocytic cell lines. Nearly half of the study population showed erythroid hypoplasia in the bone marrow. The projected median survival of patients with isolated del(5q) is 146 months for a median follow-up of 67 months. Patients with an increased medullary blast count and those with an additional chromosomal abnormality have a significantly shorter overall survival (24 and 45 months, respectively) than patients with isolated del(5q). We did not find survival differences for different cytogenetic breakpoints, nor did the amount of dysplasia have an impact on survival in our population. In total, 29 patients have died. Deaths occurred primarily due to transformation into acute leukemia, infection, or cardiac failure. Our data support the current definition of a separate entity of MDS with del(5q) that has been suggested by the World Health Organization.
Adult↗
The German competence network 'Acute and chronic leukemias'.
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Acute Disease↗
Molecular cytogenetic detection of chromosomal breakpoints in T-cell receptor gene loci.
Chromosomal aberrations with breakpoints in T-cell receptor (TCR) gene loci are recurrent in several T-cell malignancies. Although the importance of interphase cytogenetics has been extensively shown in B-cell lymphomas, hardly any molecular cytogenetic tools are available for recurrent changes in T-cell disorders. Thus, we have established fluorescence in situ hybridization (FISH)-based break-apart assays for the TCRA/D (14q11), TCRB (7q34) and TCRG (7p14) genes and the TCL cluster (14q32). The assays were validated in normal controls as well as in 43 T-cell malignancies with cytogenetically proven 14q11, 7q34-35 or 7p13-21 aberrations. Breakpoints in TCRA/D, TCRB and TCRG could be diagnosed by these assays in 32/33 T-cell neoplasms with chromosome 14q11, 3/6 with 7q34-35 and 1/7 with 7p13-21 alterations, respectively. Application of the new FISH assays to a series of 24 angioimmunoblastic and 12 cutaneous T-cell lymphomas confirmed the cytogenetic evidence of lack of breakpoints in the TCRA/D or TCRB locus. Simultaneous detection of TCRA/D or TCRB breaks was achieved in a multicolor approach, which was further combined with detection of the T-cell-specific CD3 antigen in a multicolor FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasm) assay. These new FISH and FICTION assays provide sensitive, rapid and accurate tools for the diagnosis and biological characterization of T-cell malignancies.
Adolescent↗
[Counselling, genetic testing and prevention in women with hereditary breast- and ovarian cancer. Interdisciplinary recommendations of the consortium "Hereditary Breast- and Ovarian Cancer" of the German Cancer AiD].
Women with familial predisposition for breast and ovarian cancer represent a small group of patients with very high risk for developing breast and/ or ovarian cancer before the age of 50 years. The individual breast cancer risk can be assessed by genetic counselling and can be specified by genetic diagnostics. As part of the gynaecological consultation, adequate preventive measures are offered. Psycho-oncological counselling may help in decision making. For hereditary carcinomas, counselling is still not considered as a routine medical care, even though basic and routine preventive measures are insufficient for this group of high risk patients. Within the last years, 12 specialized centres in Germany have developed a patient care concept for women with a familial risk for breast and ovarian cancer. Establishment of these centres for familial breast and ovarian cancer and use of evidence-based medical care was initiated in a nationwide interdisciplinary joint research project and supported by the German Cancer AiD. These measures were integrated in a quality assurance concept for structure, process and result optimization. Thus, all requirements for introducing these services into routine patient management have been fulfilled.
Breast Neoplasms↗
Cancer genetics service provision: a comparison of seven European centres.
OBJECTIVE: To conduct a survey in seven European cancer genetics centres to compare service provision, organisation and practices for familial breast and colon cancer consultations and testing. Information was obtained on aspects of services both nationally and locally. METHODS: A detailed survey questionnaire was adapted collaboratively to obtain the required information. Initial survey data were collected within each centre and interim results were discussed at two European Workshops. Where differences in practice existed, details were clarified to ensure accuracy and adequacy of information. Participating centres were Haifa (Israel), Hannover (Germany), Leiden (The Netherlands), Leuven (Belgium), Manchester (UK), Marseille (France) and Milan (Italy), representing countries with populations ranging from 6.5 to 80 million. RESULTS: The European countries diverged in regard to the number of cancer genetics centres nationally (from 8 in Belgium to 37 in France), and the average population served by each centre (from 0.59 million in Israel to 3.32 million in Italy). All centres offered free care at the point of access, but referral to specialist care varied according to national health care provision. At a centre level, staff roles varied due to differences in training and health care provision. The annual number of counsellees seen in each participating centre ranged from 200 to over 1,700. Access to breast surveillance or bowel screening varied between countries, again reflecting differences in medical care pathways. These countries converged in regard to the wide availability of professional bodies and published guidelines promoting aspects of service provision. Similarities between centres included provision of a multidisciplinary team, with access to psychological support, albeit with varying degrees of integration. All services were dominated (70-90%) by referrals from families with an increased risk of breast cancer despite wide variation in referral patterns. Collection of pedigree data and risk assessment strategies were broadly similar, and centres used comparable genetic testing protocols. Average consultation times ranged between 45 and 90 min. All centres had access to a laboratory offering DNA testing for breast and bowel cancer-predisposing genes, although testing rates varied, reflecting the stage of service development and the type of population. Israel offered the highest number of genetic tests for breast cancer susceptibility because of the existence of specific founder mutations, in part explaining why the cancer genetics service in Haifa differed most from the other six. CONCLUSION: Despite considerable differences in service organisation, there were broad similarities in the provision of cancer genetic services in the centres surveyed.
Breast Neoplasms↗
TEGDMA causes apoptosis in primary human gingival fibroblasts.
Previous in vivo studies have revealed that resins may generate a persistent inflammation of oral tissues and cell death as well. Apoptosis is an important regulated process that results in rapid cell death. This study tested the hypothesis that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) causes apoptosis. The effects of TEGDMA on proliferation and apoptosis in primary oral fibroblasts were analyzed by light microscopy and flow cytometry (FACS; Annexin V-assay). TEGDMA at 5 and 7.5 mM inhibited proliferation after 24 hrs. No increased frequency of apoptosis or necrosis was observed with 1 mM or 2.5 mM TEGDMA after 24 hrs. Apoptosis and Annexin V-positive cells were observed with 5 mM and 7.5 mM TEGDMA by light microscopy after 24 hrs. A dramatic increase in apoptotic cells was detected by FACS after 24 hrs with 7.5 mM TEGDMA. Thus, TEGDMA was cytotoxic and "apoptotic" in a dose- and time-dependent manner.
Analysis of Variance↗
Identification and characterization of a novel human brain-specific gene, homologous to S. scrofa tmp83.5, in the chromosome 10q24 critical region for temporal lobe epilepsy and spastic paraplegia.
We describe the structure, genomic organization, and some transcription features of a human brain-specific gene previously localized to the genomic region involved in temporal lobe epilepsy and spastic paraplegia on chromosome 10q24. The gene, which consists of six exons disseminated over 16 kb of genomic DNA, is highly homologous to the porcine tmp83.5 gene and encodes a putative transmembrane protein of 141 amino acids. Unlike its porcine homolog, from which two mRNAs with different 5'-sequences are transcribed, the human gene apparently encodes three mRNA species with 3'-untranslated regions of different sizes. Mutation analysis of its coding sequence in families affected with temporal lobe epilepsy or spastic paraplegia linked to 10q24 do not support the involvement of this gene in either diseases.
Amino Acid Sequence↗