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PubMed1993

Interphase cytogenetics of gastric and esophageal adenocarcinomas.

Numerical changes affecting chromosomes 1, 2, 3, 4, 6, 7, 8, 10, 11, 12, 16, 17, 18, and the X and Y chromosomes have been analyzed using chromosome-specific centromeric alpha-satellite repeat DNA probes in a panel of biopsies of six gastric and three esophageal adenocarcinoma and one epidermoid carcinoma of esophagus obtained at surgery. For each case, with each probe, the number of hybridization signals were determined in 200 nuclei. Hybridization of each probe to phytohemagglutinin-stimulated normal peripheral blood lymphocytes served as controls. Monosomy was defined by loss of one signal in 15% or more cells and trisomy or tetrasomy was defined by the presence of 3 or 4 signals in 7% or more cells, respectively. The Y chromosome was lost in 6 of 8 cases and monosomy 10 was seen in 5 of 10 cases. Trisomy for chromosomes 17, 8, 7, 12, 11, and 1 was seen in 4 of 10, 4 of 10, 4 of 10, 2 of 10, 2 of 8, and 2 of 10 cases, respectively, and tetrasomy for chromosome 7 was seen in 1 of 10 cases. These data show that the Y chromosome and chromosomes 10, 8, 7, 17, and 12 are most frequently involved in nondisjunctional changes in these tumors. They also document the feasibility and utility of interphase cytogenetics of gastric adenocarcinomas.

Adenocarcinoma↗
PubMed1994

Detection of monosomy 7 and trisomies 8 and 11 in myelodysplastic disorders by interphase fluorescent in situ hybridization. Comparison with acute non-lymphocytic leukemias.

Cells from 50 patients with myelodysplastic syndrome (MDS) and 20 patients with acute non-lymphoblastic leukemia (ANLL) were studied by fluorescent in situ hybridization (FISH) using alphoid biotinylated probes to detect numerical chromosome 7, 8 and 11 aberrations in interphase nuclei. FISH data were compared with cytogenetic results. Both methods were in agreement in 25/50 MDS and 20/20 ANLL cases. Trisomy 11 was found neither by cytogenetic study nor by FISH. In 11 MDS patients the percentage of abnormal cells was higher by FISH than by classical cytogenetic analysis. FISH revealed monosomy 7 which was undetectable by karyotypic study in 5-22% cells from 15 MDS patients. It also allowed the identification of two small markers and a ring chromosome in two MDS cases. FISH hence appears to be useful for the detection of minor abnormal clones and is a convenient complement to conventional cytogenetic analysis in the study of MDS.

Aged↗
PubMedDate not supplied

A quantitative study of the interphasic nucleolar organizer regions in two human cervical carcinoma cell lines.

We have studied the relationship between interphase nucleolar organizer regions (NORs) distribution and nucleolar size in cancer cells under prolonged culture. Six morphometric parameters (NUCLEAR SIZE, NOR NUMB, AREA SUM, MEAN AREA, SIZE RATIO and SIZE RATIO MEAN) of AgNORs in cancer cells were analyzed by an image analyzing system. The results show that the parameters NORN NUMB, AREA SUM and NUCLEAR SIZE demonstrate the most consistent changes in the two cervical carcinoma cell lines studied. These three parameters of AgNORs appear to be correlated with the degree of "dedifferentiation" that occurred in tumor cells under prolonged culture.

Cell Division↗
PubMed1993

Interphase cytogenetics on agar cultures: a novel approach to determine chromosomal aberrations in hematopoietic progenitor cells.

We describe a novel approach to determine the presence of chromosomal aberrations in progenitor cells by in situ hybridization (ISH) on agar cultures. Bone marrow cells of 3 patients suffering from acute myeloid leukemia (AML) were selected to develop the method. In all 3 cases, numerical aberrations for chromosome 1 and/or 8 were detected by karyotyping and ISH using chromosome-specific centromeric-associated DNA probes. These aberrations were used as markers in this study. After in vitro culture of the bone marrow samples in agar, the cells were pretreated to perform ISH. This approach retains the cytological architecture of the agar assay, allowing discrimination between chromosomal aberrations detected in the clonogenic and non-clonogenic cells in culture. With this new technique, the presence of the cytogenetic aberration in clonogenic cells can be shown at the interphase level.

Bone Marrow↗
PubMed1993

Trisomy 12 in B-cell chronic lymphocytic leukemia: interphase study by in situ hybridization in 75 patients.

Trisomy 12 is the most common cytogenetic abnormality in chronic lymphocytic leukaemia (CLL) and may be a prognostic indicator. In the present study, fluorescence in situ hybridization (FISH) is shown to be a method of choice for detection of trisomy 12 in interphase cells. Seventy-five cases of B-cell CLL were analysed with a chromosome 12 specific alpha satellite DNA probe and results compared with those from cytogenetic analysis. FISH showed the three hybridization spots characteristic of trisomy 12 in 32/75 patients (42.6%). Sixty-three patients were also studied by conventional cytogenetics: failure in 7 cases, normal karyotype in 28, trisomy 12 in 9 (14.3%) and in 19 cases abnormalities other than trisomy 12. In these same 63 patients, trisomy 12 was detected on 29 occasions by FISH (46%): in one case of failure by cytogenetic analysis, in 9 cases thought to have a normal karyotype, in 10 cases carrying abnormalities other than trisomy 12 and in all 9 cases showing trisomy 12 by conventional cytogenetic investigation. Correlation between trisomy 12 and the three stages of the Binet classification indicated an increasing proportion of trisomy 12 from stage A to stage C. It is concluded that fluorescence in situ hybridization is a powerful and sensitive technique for detection of trisomy 12 in CLL and although more cases will be required to confirm a correlation between the incidence of trisomy 12 and the stage of the disease, this link could be important from a prognostic point of view.

Adult↗
PubMed1993

Interphase death and reproductive death in X-irradiated MOLT-4 cells.

The killing effects of ionizing radiation on MOLT-4 cells were studied using colony formation assays, dye exclusion tests, and cell growth analysis. When MOLT-4 cells were exposed to X rays, the fraction of cells stained with erythrosin B increased markedly during the incubation after irradiation at 37 degrees C and reached a maximum within 24 h. In contrast, no such increase was observed for L5178Y cells or their derivative, radiosensitive mutant M10 cells, at a dose resulting in the same level of cell survival as measured by the colony formation assay. Detailed analysis of cell survival as measured by the dye exclusion test in comparison with that measured by the colony formation assay in MOLT-4 cells indicated that MOLT-4 cells are subject to interphase death as well as reproductive death. Analysis of subclones showed a wide variety of differences in the level of stained cells using the dye exclusion test. The results confirmed the population heterogeneity of MOLT-4 cells. Moreover, none of the subclones showed exactly the same cell survival when this was measured with the two assays. The present results strongly suggest that both modes of cell death may occur in single MOLT-4 cells. Possibly apoptosis is the mechanism accounting for both modes of death in these cells.

Adult↗
PubMed1993

Hairy cell leukemia: an interphase cytogenetic study.

Malignant cells from 24 cases of hairy cell leukemia were studied by in situ hybridization for evidence of selective aneuploidy using alphoid and satellite probes specific for 16 human chromosomes. Based on these data, hairy cell leukemia appears to be diploid for the chromosomes studied and is a malignancy which displays the phenomenon of pairing of the centromere and p arm of chromosome 15 during interphase.

Centromere↗
PubMed1993

Increase of calcium levels at interphase in NIH 3T3 cells.

The fluorescent calcium ion indicator dye Fluo-3 and DNA-binding dye Hoechst 33342 were employed to determine, in a quantitative microspectrofluorometric study, the intracellular calcium ion concentration ([Ca2+]i) and the DNA content of individual living NIH3T3 cells. The well-separated excitation and emission properties of these dyes allowed us to establish for each cell both the phase of the cell cycle using DNA content and [Ca2+]i. We found that the transition from G1, through S, to the G2 phase is accompanied by a two-fold increase in [Ca2+]i. The [Ca2+]i was inhomologous in each phase of the interphase (G1, S and G2) although [Ca2+]i in the S and G2 phases was never lower than certain threshold values in the G1 and S phases respectively. [Ca2+]i in G0 cells was lower than that in G1 cells. These changes in [Ca2+]i suggest that [Ca2+]i may be an important regulator of cell cycle progression.

3T3 Cells↗
PubMed1993

Interphase cytogenetics by fluorescent in situ hybridization (FISH) for characterization of monosomy-7-associated myeloid disorders.

By circumventing the need for metaphase preparations, fluorescent in situ hybridization (FISH) on interphase nuclei using chromosome-specific probes is a promising tool for the study of numerical chromosome aberrations not only in proliferating, but also in non-dividing cells. We analyzed 15 cases of monosomy-7-associated myeloid disorders with a biotinylated probe to the (peri)centromeric region of chromosome 7. Monosomy 7 was readily confirmed in all cases during active disease. In two patients only a minority of nuclei was monosomic, whereas cytogenetics had shown all metaphases to be missing one chromosome 7. FISH in one of them was able to identify a small marker chromosome as isolated pericentromeric region of chromosome 7. Minimal residual disease however could not be detected in three remission samples analyzed, as percentages of disomic nuclei were within the range of normal controls (96.8% 2.1%). In order to determine lineage involvement of the monosomic clone, a recent technique combining immunophenotyping and FISH (FICTION) was performed in one patient with AML after MPD. Monosomy 7 was found in virtually all myelomonocytic and erythroid cells (as discriminated by lineage-specific antibodies), in a part of CD34-positive precursor cells, but not in lymphocytes. We conclude that monosomy 7 in this patient is restricted to an early committed progenitor cell capable of erythroid and myelomonocytic differentiation.

Adolescent↗
PubMed1995

[Changes in methylation of tumor cells: a new in situ quantitative approach on interphase nuclei and chromosomes].

DNA methylation is known to be related to the regulation of gene expression. DNA methylation patterns are modified in tumors compared to normal tissue, and in some cases, these variations are linked to cancer progression. Molecular biology techniques are generally used to evaluate DNA methylation status. We describe here a simple and fast immunofluorescent method to quantitate in situ DNA methylation using an image analyser and a CCD camera. Quantification of relative methylation levels in interphase nuclei and metaphase chromosomes was performed on digital images of two types of cells with known methylation levels. The results from image cytometry corresponded to those obtained by molecular studies. Advantages of this approach are that all the DNA of a cell may be examined rather than a limited restriction sequence, and that it may be done on a cell by cell basis rather than on a heterogenous population. In addition, with this method, changes in methylation patterns during tumor progression could be followed and eventually used as a marker for prognosis.

5-Methylcytosine↗
PubMed1995

Interphase fluorescence in situ hybridization (FISH) as a powerful tool for the detection of aneuploidy in multiple myeloma.

Conventional cytogenetic (CC) studies performed in multiple myeloma (MM) are difficult because of the low proliferation rate of plasma cells (PC). The purpose of this study was to compare results obtained by CC and by FISH for the detection of numeric chromosomal changes in patients with MM. PC DNA content, CC and interphase FISH analysis were performed on 29 consecutive patients with MM. Fifteen patients (control group) had known Cytogenetic abnormalities identified by CC. The other 14 patients (study group) had a normal karyotype but an abnormal DNA content. Bone marrow material prepared for CC or cytospin slides were probed with classical satellite III or alpha satellite DNA sequences for chromosomes 3, 7, 8, 9, 11 and 15 (chromosomes 3, 7, 9, 11, 15 probes for hyperdiploid patients and the chromosome 8 probe for hypodiploid patients). In the control group, an unexplained discrepancy between CC and FISH occurred for only one chromosome in one patient. Also in this group, four patients had only one abnormal cell by CC and the numeric changes in these patients were always confirmed by FISH analysis. In the study group, FISH analysis showed an abnormal result in all but one patient. From these data, we conclude that FISH improves the detection of cytogenetic abnormalities in multiple myeloma. Using commercially available DNA probes for the most frequent numeric changes and slides for CC or cytospin slides, we demonstrated abnormal cytogenetics by FISH in 28/29 patients. In further studies, use of FISH could permit a more accurate description of numeric changes and their prognostic value in MM as well as an approach to clonal evolution. It would also be of interest in the study of monoclonal gammopathies of undetermined significance.

Adult↗
PubMed1996

Translocation t(2;5) is not a primary event in Hodgkin's disease. Simultaneous immunophenotyping and interphase cytogenetics.

A number of neoplastic disorders are characterized by recurrent chromosome aberrations. One of these is the translocation t(2;5), which is found in a considerable percentage of large-cell anaplastic lymphomas. This translocation results in the fusion of two genes, alk and npm. The recent discovery of alk/npm mRNA in 11 of 13 cases of Hodgkin's disease has caused a controversial discussion concerning the question of whether t(2;5) is also present in Hodgkin and Reed-Sternberg cells. We tackled this problem on the molecular cytogenetic level by combined CD30 immunophenotyping and interphase cytogenetics. Using a pair of DNA probes flanking both sides of the npm gene breakpoint at 5q35 we were able to prove, at least in 12 of 13 cases of Hodgkin's disease, that all CD30-positive Hodgkin and Reed-Sternberg cells lacked the translocation t(2;5). Fifteen to forty-five Hodgkin/Reed-Sternberg cells were analyzed per case (mean, 27). Our findings indicate that this translocation is not a primary event in the development of Hodgkin's disease.

Adult↗
PubMed1995

Transcription specific differences visualized by fluorescence in situ hybridization pattern on interphase nuclei of different cell types.

Application of a "formamide free" and thus "material preserving" in situ hybridization technique using the cDNA of the myf3 gene revealed the following results: Human rhabdomyosarcoma cells, characterized by a high expression of myf3 show intensive hybridization signals in their interphase. RNase treatment prior to hybridization considerably reduces the size of this signals. In comparison, isolated nuclei of human lymphocytes in which no need for the expression of this gene exists, show barely hybridization signals. Correspondingly, RNase treatment had no effect on hybridization pattern at all. In conclusion an increased transcription efficiency of a cell type specific gene is accompanied by a higher hybridization accessibility in the corresponding cell nuclei.

Cell Nucleus↗
PubMed1996

Interphase cytogenetic study of endometrial stromal sarcoma by chromosome in situ hybridization.

Endometrial stromal neoplasms include a heterogenous group of tumors with different clinical behavior and response to treatment. Cytogenetic study of such tumors has been scanty. The aim of this study was to analyze and compare the chromosome composition in low-grade and high-grade endometrial stromal sarcoma using the technique of chromosome in situ hybridization. Eight cases of low-grade stromal sarcoma and three cases of high-grade stromal sarcoma were studied. Biotinylated DNA probes specific for the regions of chromosomes X, 11, 12, and 17 were used on formalin-fixed paraffin embedded material from these tumors. The in situ hybridization signals were visualized by immunoperoxidase technique. Four of the eight low-grade stromal sarcomas retained normal disomy of the chromosomes studied. The other four low-grade sarcomas showed a gain of from one to three chromosomes, whereas all of the three high-grade sarcomas showed polysomies in all of the four chromosomes being studied. No loss of chromosomes was detected. One case of high-grade sarcoma contained coexisting areas of low-grade sarcoma. Although aneusomy was found in the high-grade portion, disomy was noted in the low-grade areas. In the stromal sarcomas studied, there was no definite correlation between the presence of chromosome polysomies and the clinical progress of the tumors. This is the first interphase cytogenetic study of uterine stromal sarcoma, and the results support the concept that complex numerical chromosome abnormalities evolve during anaplastic transformation of endometrial stromal sarcoma.

Adult↗
PubMed1996

Analysis of loss of inactive X chromosomes in interphase cells.

We have developed a method that allows, for the first time, a specific analysis of the inactive X chromosome (Xi) in interphase cells. By combining immunolabeling of acetylated histone H4 with specific antisera and FISH with an X-chromosome centromere-specific DNA probe, micronucleated whole Xis in human female cells may be identified by their lack of histone H4 acetylation. As one example of the potential applications of this methodology in genetic studies in humans, an artifact-free X-chromosome aneuploidy detection in lymphocytes of women of different ages has been performed. Our results indicate that not only the Xi but also the active X chromosome is preferentially lost during aging, indicating that the high frequency of sex-chromosome aneuploidy in human females cannot be explained solely by a lack of negative selection of Xi aneuploid cells. Further applications of the proposed methodology in genetic studies are discussed.

Adult↗
PubMed1997

Evaluation of MYC and chromosome 8 copy number in breast carcinoma by interphase cytogenetics.

We used fluorescence in situ hybridization (FISH) to determine MYC and chromosome 8 copy number on whole nuclear imprint preparations of 24 breast carcinomas, seven benign breast samples, and two phyllodes tumors. None of the benign tissues and neither of the phyllodes tumors demonstrated an increased copy number for MYC or chromosome 8, which was defined as greater than two signals in > 10% of nuclei. In contrast, 22 of 24 carcinomas demonstrated an increased MYC copy number. The modal numbers of MYC copies/nucleus were 0-2 in seven cases (29%), 3-5 in seven cases (29%), 6-9 in five cases (21%), and > 9 in five cases (21%). An increased chromosome 8 copy number was observed in 21 of 22 carcinomas with MYC gain, and the modal number of signals/nucleus was either identical to (n = 14; 64%) or less than (n = 8; 36%) the number of MYC copies. The number of MYC copies correlated with cellular DNA content, as determined by using flow cytometry. In peridiploid tumors (DNA index 0.9-1.2; n = 7), the MYC copy numbers/nucleus were 0-2 in five cases and 3-5 in two cases. In contrast, the modal MYC copy numbers/nucleus among the 11 hyperdiploid tumors (DNA index 1.3-1.9) were 0-2 in one case, 3-5 in four cases, 6-9 in five cases, and > 9 in one case. All three tetraploid/hypertetraploid carcinomas exhibited > 9 MYC copies/nucleus. We conclude that an increased MYC copy number, as detected by using interphase cytogenetics, is extremely frequent in human breast carcinomas. However, in most cases, MYC gene duplication is probably secondary to polysomy of chromosome 8 and/or genomic endoreduplication (i.e., DNA aneuploidy).

Aneuploidy↗
PubMed1997

Diagnosis of CMT1A duplications and HNPP deletions by interphase FISH: implications for testing in the cytogenetics laboratory.

Charcot-Marie-Tooth (CMT) disease type 1A is an inherited peripheral neuropathy characterized by slowly progressive distal muscle wasting and weakness, decreased nerve conduction velocities, and genetic linkage to 17p12. Most (> 98%) CMT1A cases are caused by a DNA duplication of a 1.5-Mb region in 17p12 containing the PMP22 gene. The reciprocal product of the CMT1A duplication is a 1.5-Mb deletion which causes hereditary neuropathy with liability to pressure palsies (HNPP). The most informative current diagnostic testing requires pulsed-field gel electrophoresis to detect DNA rearrangement-specific junction fragments. We investigated the use of interphase FISH for the detection of duplications and deletions for these disorders in the clinical molecular cytogenetics laboratory. Established cell lines or blood specimens from 23 individuals with known molecular diagnoses and 10 controls were obtained and scored using a two-color FISH assay. At least 70% of CMT1A cells displayed three signals consistent with duplications. Using this minimum expected percentile to make a CMT1A duplication diagnosis, all patients with CMT1A showed a range of 71-92% of cells displaying at least three signals. Of the HNPP cases, 88% of cells displayed only one hybridization signal, consistent with deletions. The PMP22 locus from normal control individuals displayed a duplication pattern in approximately 9% of cells, interpreted as replication of this locus. The percentage of cells showing replication was significantly lower than in those cells displaying true duplications. We conclude that FISH can be reliably used to diagnose CMT1A and HNPP in the clinical cytogenetics laboratory and to readily distinguish the DNA rearrangements associated with these disorders from individuals without duplication or deletion of the PMP22 locus.

Cell Line↗