Double hybridization signals on the chromatid revealed by telomeric probes.
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Chromatids↗
Biomedical subjects
A Bernheim
Publications and source records attributed to A Bernheim.
CD4+ CD56+ cutaneous neoplasms: a distinct hematological entity? Groupe Français d'Etude des Lymphomes Cutanés (GFELC).
We report seven cases of particular cutaneous tumors selected from the register of the French Study Group on Cutaneous Lymphomas. The patients (three men, four women) were aged 37-86 years. They initially presented with cutaneous nodules or papules. Three cases presented with regional lymph nodes. Stagings were negative, except for one patient with bone marrow involvement. Histological features were relevant with pleomorphic medium T-cell lymphoma, but these cells exhibited a distinguishing phenotype. They were positive for CD4, CD56, and also CD45, CD43, and HLA-DR. All other T-cell and B-cell markers were negative. The myelomonocytic markers (CD13, CD14, CD15, CD33, CD117, myeloperoxidase, and lysozyme) were negative excepted CD68, which was clearly positive in four cases and weakly in two cases. Others natural killer cell markers (CD16, CD57, TiA1, granzyme B), TdT, and CD34 were negative. Polymerase chain reaction studies did not detect any B or T clonal rearrangement. The cytogenetic studies, performed in five cases, showed a del(5q) in two cases. All patients were treated successfully by polychemotherapy, but relapsed quickly in the skin, between 4 and 28 months. Five patients developed bone marrow involvement, with leukemia in three cases, and they died in 5-27 months. One patient died at 17 months with skin progression. The seventh patient is alive at 33 months, with cutaneous progression. The origin of these cells is unclear. Despite expression of CD4 or CD56, we failed to demonstrate a T-cell, natural killer cell origin. However, CD4 and CD56 are not specific for T or natural killer lineages. Although these two markers are also known to be expressed by monocytic cells, classic myeloid antigens were negative. These seven cases, together with other rare similar cases already reported, seem to represent a distinct entity likely developed from hematological precursor cells.
Adult↗
Generation of whole-chromosome painting probes specific to each chicken macrochromosome.
The realization of physical and genetic maps of the chicken genome is dependent on progress in cytogenetic knowledge of its karyotype. To help achieve this goal, we constructed amplified representative DNA samples of the chicken chromosomes 1, 2, 3, 4, 5, 6, 7, 8, Z, W and of the terminal heterochromatin part of Zq by chromosome microdissection and DOP-PCR amplification. These chromosome DNA samples, which represent about 75% of the chicken genome, were used to generate whole chromosome painting probes for FISH. The direct application of these chromosome specific probes is dual FISH localization and characterization of panels of chicken interspecific somatic hybrids. We discuss some aspects of the chicken genome and its repeated sequences.
Animals↗
[Exploration of the genome and malignant proliferations: cytogenetics and cytogenomics].
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Cell Division↗
[Comparative genomic hybridization (CGH): application to the study of neuroendocrine tumors].
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Chromosome Aberrations↗
Double telomeric signals on single chromatids revealed by FISH and PRINS.
FISH probes for all human telomeres and specific telomeric probes that hybridize to unique sequences on individual chromosomes have been used to characterize the telomeric hybridization pattern of human peripheral blood lymphocytes and bone-marrow cells in interphase and metaphase chromosomes. We have identified the existence of double hybridization signals on chromatids both with the (TTAGGG)n telomere repeat arrays and on non chromosome-specific subtelomeric regions as well as on chromosome-specific sequences located several kilobases from the end of chromosomes. Preliminary results using cosmid or YAC probes that hybridize to regions rich in GC sequences also revealed double fluorescent spots on a single chromatid. Double spots were detected by PRINS on terminal and interstitial telomeric sequences in avian cells. The significance of this phenomenon is discussed based on some models of chromatid and DNA organization such as uninemy, looped chromatid organization and quartet DNA structures. The occurrence of double spots should be taken into consideration for the clinical cytogenetic diagnosis of duplications.
Base Sequence↗
Isolation of tumor-specific cytotoxic CD4+ and CD4+CD8dim+ T-cell clones infiltrating a cutaneous T-cell lymphoma.
We have isolated several T-cell clones from lymphocytes infiltrating a human major histocompatibility class (MHC) II negative cutaneous T-cell lymphoma (CTCL). We describe here two of these clones, TC5 and TC7, with, respectively, a CD4(+)CD8dim+ and CD4(+)CD8(-) phenotype. Both clones mediated a specific MHC class I-restricted cytotoxic activity toward the fresh autologous tumor cells, and autologous tumor cell lines previously established with interleukin-2 (IL-2) and IL-7 from the skin and from the blood. Analysis of the T-cell receptor (TCR) Vbeta gene expression showed that the tumor cells, which were shown to have a trisomy 7 by fluorescent in situ hybridization, expressed Vbeta7/Jbeta2.3, Vbeta13/Jbeta2.5, and Vbeta22/Jbeta2.5 rearrangements. Phenotypic analysis using specific anti-Vbeta monoclonal antibodies indicated that only Vbeta13 could be detected on the cell membrane of the tumor cells. Analysis of the TCR Vbeta gene expression of the clones showed that TC5 and TC7 expressed a unique TCR-Vbeta transcript, corresponding, respectively, to Vbeta5/Jbeta2.3 and Vbeta17/Jbeta2.7 gene segments. To determine whether these reactive T lymphocytes were present in vivo, we used specific primers corresponding to TC5- and TC7-Vbeta TCR transcripts. The results showed that both cytotoxic T-cell clones were present at the lesional skin site and amplified in vitro. TC7 was found in the patient peripheral blood invaded by tumoral cells, whereas TC5 was not, indicating that the repertoire of the reactional lymphocytes differs in the blood and at the tumor site. These results show for the first time the presence of reactive T lymphocytes with CD4 or double-positive phenotype infiltrating a CTCL. These findings raise the question of the role of these antitumoral effector T cells in the tumor growth.
Aged↗
Comparative genomic hybridization analysis of sporadic neuroendocrine tumors of the digestive system.
Little information is available on the molecular mechanisms underlying neuroendocrine tumorigenesis. To obtain an overview of the genomic imbalances characterizing these tumors, we studied 20 benign or malignant sporadic endocrine gastroenteropancreatic tumors by comparative genomic hybridization. Chromosomal imbalances were found in all tumors. Gains of chromosomal material were more frequent than losses. The most frequent gains were of chromosomes and chromosome arms 5 (55%), 14 (55%), 17q (55%), and 7 (50%). Losses were most frequent from 11q (30%) and 16p (30%). Gains of chromosome 5 did not occur in nonmetastatic tumors, whereas losses of 9p were observed exclusively in intestinal tumors. In addition, we found two high-level amplifications, of 17q11-21 and 19q13. A complementary FISH analysis revealed that the gain in 17q11-21 included amplification of the protooncogene HER2/neu. As in multiple endocrine neoplasia type-1-associated tumors, deletions of chromosome band 11q13 appear to be involved in the development of sporadic digestive tract neuroendocrine tumors, but our results suggest that other chromosomal regions are also involved.
Adolescent↗
Non-Hodgkin's lymphomas and myeloid disorders: deletions associated with t(2;5) and t(3;5) detected by FISH.
The nucleophosmin (NPM) gene is involved in two recurrent translocations in hematological malignancies: t(2;5) (p23;q35) in anaplastic large cell lymphoma (ALCL) and t(3;5)(q25.1;q34-35) in myelodysplasia and acute non-lymphocytic leukemia (ANLL). Using eight YACs encompassing the 5q34-q35 region, we could easily detect these two translocations. In both types of translocation, probable unexpected deletions were also discovered downstream of the breakpoint at 5q35.
Adolescent↗
Overrepresentation of 7q31 and 17q in renal cell carcinomas.
Xenografts from four metastatic renal cell carcinomas (RCCs) were established in immunodeficient mice. All tumors exhibited cytogenetic features specific for the papillary subtype, namely, partial or total polysomy of chromosomes 7 and 17 and integrity of 3p. Cytogenetic analysis of the initial and xenografted tumors indicated that although clonal characteristics were consistently maintained in xenografts derived from metastases, a minor clone had been selected for in the xenografts derived from the primary tumors. Reverse painting and comparative genomic hybridization (CGH) allowed us to localize minimal overrepresented genomic regions to 7q31, where the MET protooncogene is located, and to 17q. Other overrepresented regions were 8q in all xenografts and Xq22-qter in three of them. The gain of genetic material from these regions may be a key factor ensuring the papillary nature of RCCs and their survival in xenografts.
Aneuploidy↗
[Pre-leukemic granulocytic sarcoma of the uterus. Report of a case].
A case of pre-leukemic granulocytic sarcoma (GS) of the uterus was found in a 73-year old woman. The diagnosis was suggested by vaginal cytology and the green color of the gross lesions, then confirmed by naphthol AS-D chloro acetate esterase stain and immunohistochemistry on fixed tissue with the anti-lysozyme, anti-myeloperoxidase, CD 43 and CD15 antibodies. At the time of GS discovery, the patient presented no evident leukemia but myelogram contained 20% of blast cells. She developed acute myeloid leukemia 2 months later. Cytogenetic study of the bone marrow revealed chromosome 21 trisomy. GS is frequently mistaken for malignant lymphoma since it expresses some of the leukocytic antigens (leukocyte common antigen, CD 45 RO (UCHL1), MB2). Therefore, the use of a large panel of antibodies, including anti-myeloperoxidase, anti-lysozyme and CD15, is recommended. Precise diagnosis is essential because all GS must be treated as acute myeloid leukemias.
Acute Disease↗
Characterization of cosmids and telomeres in cytogenetic preparations by 3D confocal fluorescence.
OBJECTIVE: To analyze, with fluorescent probes, by three-dimensional (3D) emission patterns, fluorescence in situ hybridization (FISH) preparations (cosmids, telomeres) and to perform factor analysis of medical image sequences (FAMIS); to use FISH to track relevant DNA sequences in cell nuclei during interphase and in mitotic chromosomes; and to use cytogenetic techniques, resulting in flat preparations of whole cells that are assumed to preserve probe access to their targets. STUDY DESIGN: The study design entailed labeling targets by probes (sequences labeled by fluorescein isothiocyanate) in nuclei and/or chromosomes stained by propidium iodide. Visualization of targets was improved when 3D sequences of images obtained on a single photomultiplier detector of the confocal microscope by z stepping were investigated by FAMIS. RESULTS: Factors and factor images showed that targets could be detected and differentiated in focal planes inside nuclei or chromosomes. CONCLUSION: It is possible to localize cosmids in cell nuclei at interphase and telomeres in mitotic chromosomes by means of 3D sequences of images.
Cosmids↗
A chromosome 14q11/TCR alpha/delta specific yeast artificial chromosome improves the detection rate and characterization of chromosome abnormalities in T-lymphoproliferative disorders.
The rate of detection of chromosome abnormalities in T-cell proliferations is lower than that observed in B-cell malignancies. The former frequently involve the TCR alpha/delta locus at chromosome band 14q11. We have identified a YAC encompassing 70% of the TCR alpha/delta locus, which has been used as a fluorescence in situ hybridization probe to detect chromosome rearrangements involving 14q11, both at metaphase and within interphase nuclei, in patients with a variety of T-lymphoproliferative disorders. Its use allowed detection of previously unsuspected TCR alpha/delta rearrangements in 4/13 (30%) immature T-lineage acute leukemias, including two t(10;14) and 2 minor inversion 14s. It also clarified interpretation of complex chromosome 14 abnormalities in mature T-cell proliferations (T-prolymphocytic leukemia and ataxia telangiectasia). Use of this probe will aid the detection and characterization of abnormalities involving the TCR alpha/delta locus, particularly in cases with normal or complex karyotypes and in those proliferations for which mitoses are difficult to obtain.
Chromosome Aberrations↗
Emergence and scattering of multiple neurofibromatosis (NF1)-related sequences during hominoid evolution suggest a process of pericentromeric interchromosomal transposition.
Type 1 neurofibromatosis (NF1) gene encodes for a member of the GTPase activating protein family and is considered to be a tumor suppressor gene. Its very high rate of de novo mutation in humans led us to study a specific feature of this gene: the presence of numerous NF1-related sequences. According to our results, the human genome contains at least 11 NF1-related sequences, nine of which are scattered near centromeric sequences of seven different chromosomes. These NF1-related sequences, whose extent is quite varied according to loci, are unprocessed copies of the NF1 gene, and bear numerous mutations. A phylogenetic analysis of the six largest sequences indicates that they are all derived from a common ancestor, which would have appeared 22-33 million years ago, and was subsequently duplicated several times during hominoid evolution. The most recent duplication and interchromosomal transposition occurred in the last million years suggesting that the process could still be ongoing. Intriguing similarities between the evolution of alpha-satellite DNA and NF1-related sequences suggest the involvement of a common genetic mechanism for the generation and pericentric spreading of these NF1 partial copies.
Animals↗
Recurrent chromosomal abnormalities in hepatocellular carcinoma detected by comparative genomic hybridization.
Comparative genomic hybridization (CGH) was used to evaluate and map genomic aberrations in 50 hepatocellular carcinomas (HCCs) from patients chronically infected with hepatitis B virus (HBV). CGH clearly detected nonrandom genomic imbalances. Losses were most prevalent on chromosome regions 4q (70%), 8p (65%), 16q (54%), 17p (51%), 13q and 6q (37% each), and lp (30%). The most frequent gains occurred on 8q (60%), 1q (58%), and 6p and 17q (33% each). In a few cases, sequence amplifications were detected that were mapped to bands 11q12, 12p11, 14q12, and 19q13.1. This study represents the first analysis of primary liver cancers by CGH, and it confirms the presence of previously known chromosomal aberrations in HCC and highlights new quantitative abnormalities and sequence amplifications. These findings should lead to the characterization of new loci involved in liver cancer pathogenesis.
Carcinoma, Hepatocellular↗
Confocal fluorescence analysis of the depth and focus of cytogenetic preparations.
OBJECTIVE: To characterize differences in the depth of fluorescent probes, to observe estimated depth levels (focal planes) on fluorescent in situ hybridization preparations by factor analysis of medical image sequences and to use cytogenetic techniques resulting in flat preparations of whole cells that are assumed to preserve the probes' access to their targets in the human nuclear interphase. STUDY DESIGN: We used labeling of the targets by the probes (sequences labeled by fluoroscein isothiocyonate [FITC]) in the nuclei, stained by propidium iodide. The investigation was performed on this model by three-dimensional (3-D) sequences of images obtained on a single photomultiplier detector of a confocal microscope by selection of emission between 510 and 550 nm and by (z) displacement. The investigation was also performed by obtaining sequences of images from spherical fluorescent beads to verify (z) focusing, to visualize depth differences and to analyze estimated factor images. RESULTS: Estimated images showed depth differences in FITC-stained targets as well as in nuclei, stained with propidium iodide, in interphase and in fluorescent beads, that could not be visualized by conventional 3-D reconstruction. CONCLUSION: It is possible to study 3-D architecture of flattened preparations and penetration of fluorophores inside the beads and to evaluate depth differences.
Cell Nucleus↗
Integrated physical, genetic, and genic map covering 3 Mb around the human NGF gene (NGFB) at 1p13.
We have characterized 11 overlapping yeast artificial chromosomes (YACs) in the 1p13 region, 8 of them containing the human nerve growth factor (NGF) gene (HGMW-approved symbol NGFB). Sequence-tagged sites (STSs) corresponding to YAC extremities have been designed and used for chromosome assignment on a panel of monochromosomic somatic cell hybrids to check for YAC chimerism, in parallel with analyses by fluorescence in situ hybridization. Determination of end STS content and restriction mapping of the YACs led to the construction of a 3-Mb YAC contig. Four microsatellite markers from the Généthon collection and seven genes known to map to the 1p13 region have been ordered on the contig around the NGF gene. A new gene transcript from the Genexpress catalog has been localized on the contig. This work provides an integrated physical, genetic, and genic map of this chromosome 1 region. It constitutes a basis for determining the structure of the NGF gene and for further characterizing its genic environment.
Animals↗
High incidence of the t(2;5)(p23;q35) translocation in anaplastic large cell lymphoma and its lack of detection in Hodgkin's disease. Comparison of cytogenetic analysis, reverse transcriptase-polymerase chain reaction, and P-80 immunostaining.
Fifty-six cases of anaplastic large cell lymphoma (ALCL), 23 cases of Hodgkin's disease, and 16 cases of diffuse large cell lymphoma were investigated for the t(2;5)(p23;q35) translocation. The translocation was detected by using cytogenetic analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry with P80 antibody directed against the kinase domain of anaplastic lymphoma kinase (ALK) of the chimeric NPM/ALK protein. In all but three cases of ALCL, we found an agreement between cytogenetic analysis, RT-PCR, and P80 staining. However, in one case, the t(2;5) translocation was detected with cytogenetic analysis, but RT-PCR and P80 staining were found to be negative. Conversely, in another case the karyotype was normal, but the hybrid mRNA and P80 staining were found to be positive. In one case, malignant cells showed a translocation involving chromosomes 1q25 and 2p23 and were strongly positive for P80 staining. Such a result could be expected because P80 antibody detects the kinase domaine of the ALK protein encoded by chromosome 2p23. Overall 73.2% (41 of 56) of cases were found to be positive. However, the highest percentage (23 of 26 cases; 88.5%) of P80 positive cases was found in children compared with 60% (18 of 30 cases) in adult ALCL (P < .05). In Hodgkin's disease, Reed-Sternberg cells were found to be clearly negative by RT-PCR and with P80 antibody. The latter results suggest that Hodgkin's disease and t(2;5)-positive ALCL are distinct biological entities and that the demonstration of the t(2;5) translocation is of diagnostic importance in differentiating these two entities. The results of the present study indicate that immunohistochemistry with P80 antibody is a reliable method for detecting NPM/ALK chimeric protein.
Adolescent↗