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PubMed1994

[Interphasic in situ fluorescent hybridization (FISH) in 4 cases of myeloid neoplasias with chromosome 7 changes].

The use of FISH as a complement to the conventional cytogenetic studies is of great help in attaining a better characterisation of the chromosome anomalies present in haematological malignancies, such as chromosome 7 monosomy. A study was carried out in three cases of acute non-lymphoblastic leukaemia and a myelodysplastic syndrome with chromosome 7 involvement, as shown by conventional cytogenetic studies. The Cocktail probe for chromosome 7 (DZ1, DZ2) was used (Oncor) in performing in situ hybridation. A monosomic cell line for chromosome 7, undetected by conventional techniques, was disclosed with this procedure in two of the cases. In the remaining two patients the monosomy of chromosome 7 was confirmed, although at percentages different from those attained with the conventional methods.

Chromosomes, Human, Pair 7↗
PubMed1993

[The dependence of the quantity of silver granules detectable in interphase nuclei after the silver nitrate staining of the chromosomal nucleolus-organizer regions on the conditions for the hypotonic treatment of the cells and the effect on this index of cell treatment by a pharmacological agent causing dissociation of the nucleolar components].

We investigated effects of hypotonic treatment of cultured HL-60 cells and human fibroblasts, and of DRB on the quantity of AgNORs revealed following AgNO3 staining. The average number of grains increases after KCl hypotonic treatment. The lower the concentration of KCl in the solution, the higher the number of silver grains revealed in the nucleoli due to their dissociation into substructures. The use of adenosine analogue (DRB) before the hypotonic treatment of cells facilitates the following count of silver deposits. A maximum increase in the mean number of silver grains was observed after a 1.5 h incubation.

Cells, Cultured↗
PubMed1994

Interphase cytogenetic analysis of prostatic carcinomas by use of nonisotopic in situ hybridization.

To gain a better understanding of chromosomal aberrations in direct correlation with histology, we studied tumor material from 35 patients (36 regions) with primary prostate carcinoma by nonisotopic in situ hybridization. Nine biotinylated DNA probes were used on serial paraffin sections (centromer-specific probes for X, Y, 1, 7, 8, 10, 17, and 18, and a telomer-specific probe for 1p; ONCOR). Of the 324 hybridized sections, 94% were suitable for evaluation. In 34 of the 35 cases (35 of 36 regions) 1-8 chromosomal aberrations were detected. Chromosome X showed supernumerary centromer copies in 44% of cases. The probes for chromosomes 1, 1p, 10, and 18 demonstrated deletions in 25, 23, 40 and 58% of cases, respectively. Gains as well as deletions were present for Y, 7, 8, and 17 in 31, 25, 36, and 58% of cases, respectively. In 27% of cases discordant copy numbers of the centromer- and the telomer-specific probes for chromosome 1 were observed. No aberration which might be specific for prostate cancer could be established. The rate of aneusomy increased significantly with histological grade. Intratumoral heterogeneity of chromosomal aberrations was revealed in one case. Due to the higher sensitivity of nonisotopic in situ hybridization, aneusomic cases outnumbered cases with cytometrically determined DNA aneuploidy. In view of published results of metaphase preparations, the high frequency of aneusomy and some of the chromosomal aberrations detected by nonisotopic in situ hybridization were unexpected.

Aged↗
PubMedDate not supplied

Interphase nucleolar organizer regions in the evaluation of serosal cavity effusions.

Argyrophilic nucleolar organizer regions (AgNORs) were determined in 38 pleural and peritoneal effusions from patients with malignant solid tumors. The results were correlated with the cytologic diagnosis and flow cytometric DNA analysis to determine the diagnostic potential of this technique. Cytologically, 13 effusions were benign, 23 malignant and 2 indeterminate (5.2%). The mean AgNOR counts for benign, malignant and indeterminate specimens were 2.99, 3.94 and 3.50, respectively. Low AgNOR counts (< 4.0) were obtained in 12 (90.9%) of the 13 benign, 16 (60.0%) of the 23 malignant and 1 of the indeterminate effusions. High AgNOR counts (> 4.0) were noted in 1 (9.1%) of the benign, 7 (35.0%) of the malignant and 1 (5.0%) of the indeterminate specimens. There was no statistically significant difference between the AgNOR count in benign and malignant effusions (P = .48). All 10 benign effusions analyzed with flow cytometry were DNA diploid. Seven (35.0%) of the 20 cytologically malignant effusions were DNA aneuploid, and 13 (70.0%) were DNA diploid. Proliferative activity was low (< 7.0%) in 23 cases and high (> 7.0%) in 7 (6 malignant and 1 benign). There was no statistical correlation between the AgNOR count and DNA ploidy (P = .11) or proliferative activity (P = .23). We conclude that the AgNOR method lacks sensitivity as a diagnostic tool for serosal cavity effusions.

Adenocarcinoma↗
PubMed1994

Simultaneous interphase cytogenetic analysis and fluorescence immunophenotyping of dedifferentiated chondrosarcoma. Implications for histopathogenesis.

Cytogenetic analysis of four specimens (biopsy, definitive surgical, and two separately occurring lung metastases) of a dedifferentiated chondrosarcoma with a rhabdomyosarcomatous component revealed clonal karyotypic abnormalities in each. Anomalies seen in all specimens included a structurally aberrant chromosome 17 and extra copies of chromosomes 5, 7, 12, and 20. The derivation of the chromosomally abnormal cells was determined by a combined immunocytochemical/cytogenetic approach that allowed simultaneous assessment of cytogenetic aberrations and immunophenotypic features of individual cells. S-100 protein and desmin antibodies were used to evaluate the chondrosarcomatous and rhabdomyosarcomatous components, respectively. A chromosome 7-specific centromeric probe was used for determination of aneuploidy. In both specimens obtained from the primary lesion, S-100 protein and desmin-positive and -negative aneuploid cells were observed. These findings: 1) suggest that both the chondrocytic and rhabdomyoblastic cells arose from the same abnormal clone, 2) support the theory of a common primitive mesenchymal cell progenitor with the ability to differentiate or express features of more than one line of mesenchymal differentiation, and 3) indicate that the term dedifferentiated may be an inaccurate designation for this neoplasm.

Bone Neoplasms↗
PubMed1993

Ratios of radiation-produced chromosome aberrations as indicators of large-scale DNA geometry during interphase.

Chromosome aberrations produced by ionizing radiation are assumed to develop from DNA double-strand breaks (DSBs) which interact pairwise. Stable chromosome aberrations that exemplify inter- and intra-chromosomal exchanges are, respectively, translocations and pericentric inversions. By comparing the number of these for each chromosome one can infer results on the randomness of DSB induction or exchange formation and on large-scale chromosome geometry. We analyze frequencies of translocations and pericentric inversions in lymphocytes from 38 A-bomb survivors, using G-banding. A total of 636 translocations and 102 pericentric inversions were found. The 636/102 ratio of translocations to pericentric inversions is approximately 14 times smaller than predicted by a random model, in general agreement with earlier results and results on the ratio of dicentrics to centric rings for in vitro irradiation. Presumably the excess of intra-chromosomal exchanges is due to a spatial proximity effect, implying a localization of chromosomes within the cell nucleus during and shortly after irradiation. The distribution of the pericentric inversions among different chromosomes indicates this proximity effect is roughly the same for all chromosomes, regardless of DNA content; i.e., the ratio of pericentric inversions for two different chromosomes approximately equals the ratio given by a model which takes into account chromosome lengths and centromere locations but otherwise assumes randomness. Possible exceptions are chromosomes 7 and 12, which show some excess of pericentric inversions. The percentage of translocations involving each chromosome corresponds roughly to the percentage expected assuming randomness, except that for chromosome 1 there is a significant excess.

Chromosome Aberrations↗
PubMedDate not supplied

[Comparative study of Y-chromatin and fluorocenters in the interphase nuclei of human cells].

Y-chromatin of nerve cells and fibroblasts in culture has a similar morphofunctional organization. The average number of fluorocentres in the above-mentioned cells is different, i.e. the number of fluorocentres in neurons is less than that in fibroblasts. The fluorescence intensity of fluorocentres does not depend upon the body size. The data of a comparative study of Y-chromatin permit two classes of bodies to be distinguished depending on fluorescence density and their size.

Cells, Cultured↗
PubMed1996

Interphase cytogenetic analysis of serous ovarian tumors of low malignant potential: comparison with serous cystadenomas and invasive serous carcinomas.

The cytogenetic and molecular genetic changes in serous tumors of low malignant potential (LMP) of the ovary have not been well characterized so far. Therefore, we analyzed 20 serous tumors of LMP, 10 invasive serous ovarian carcinomas, and 7 benign serous cystadenomas by nonisotopic in situ hybridization (seven different centromere-specific probes) as well as by flow and image DNA cytometry and compared the data with results of p53 and Ki67 immunohistochemistry, MYC DNA PCR analysis and with the clinical follow-up. All but two tumors of LMP were DNA cytometrically diploid; 9 of 10 invasive carcinomas proved to be DNA nondiploid (p < 0.0001). Nonisotopic in situ hybridization revealed a mean number of 1.5 chromosomal aberrations in tumors of LMP, which differed statistically significantly from cystadenomas (mean, 0.4) and from invasive carcinomas (mean, 3.4) (rho < 0.01). The main changes in tumors of LMP were +6 (7 of 18 cases) and +7 (6 of 19) followed by -3 (5 of 20), -1 (4 of 17) and +X (3 of 20). In the group of invasive carcinomas, the number of cases with signal gains for chromosomes 6 (5 of 8), 7 (7 of 10) and X (4 of 10) and signal loss for chromosome 1 (4 of 9) was even larger. In addition, statistically significantly more cases showed gain of 8 (5 of 10) and loss of 17 (5 of 10) (p < 0.05). Proliferative activity (Ki67 index) was positively correlated with the number of chromosomal aberrations (p < 0.05). There was no association between changes in the centromere signal number of chromosomes 8 and 17 and MYC DNA amplification and immunohistochemical p53 accumulation, respectively. Clinical follow-up showed prognostic differences between tumors of LMP and invasive carcinomas as expected (rho < 0.001) but did not reveal differences within the group of tumors of LMP with regard to the number or type of the chromosomal abnormalities detected. In conclusion, the patterns of chromosomal gains and losses in serous tumors of LMP and invasive serous carcinomas of the ovary do not seem random and suggest a close relation between these neoplasms compatible with sequential stages in a multistep model of ovarian carcinogenesis.

Adult↗
PubMed1996

Enumeration of interphase chromosomes. Comparison of visual in situ hybridization and confocal fluorescence in situ hybridization.

OBJECTIVE: To compare two visual enumeration methods to determine whether the confocal approach yielded better counts of chromosome-specific hybridization sites. STUDY DESIGN: Brightfield microscopy was used to count in situ hybridization (ISH) sites in 4-microns tissue sections. Confocal microscopy was used to collect three-dimensional (3D) data sets from fluorescence in situ hybridization (FISH) preparations made with sections of various thicknesses. Analysis of the confocal images relied on custom-built interactive visualization software. RESULTS: The confocal method yielded higher average counts of hybridization sites per nucleus due to fewer truncated nuclei in thicker sections and to visual exclusion of the truncated nuclei that remained. Optimal section thickness was 8-12 microns. Limited penetration by FISH reagents restricted the use of thicker sections. CONCLUSION: Analysis of intact nuclei visualized in three dimensions was more sensitive in demonstrating high centromere number than was brightfield ISH analysis of 4-microns sections. Improvements in semiautomated interactive software may make the confocal approach practical for accurate evaluation of chromosome number in precise histologic contexts.

Carcinoma, Transitional Cell↗
PubMed1979

Ultrastructural and autoradiographic study of the effects of bleomycin on the interphase nucleus of cultured normal cells.

Primary cultures of hepatocytes and epithelial endometrial cells were treated with bleomycin (10 to 200 microgram/ml) for 30 to 300 min. Structural changes were studied with a staining method which contrasts ribonucleoproteins. The earliest visible alteration was the accumulation of perichromatin granules in association with the nucleolus. This disturbance was frequently accompanied by modifications in the nucleolar architecture. After larger treatments, the most striking changes were nucleolar segregation and the appearance of spherical clear bodies in the nucleolus. In the extranucleolar area, a remarkable diminution of ribonucleoprotein fibrils and clustering of interchromatin granules were observed. Functional disturbances in the synthesis and transporting of RNA to the cytoplasm were studied by high-resolution quantitative autoradiography after labeling with tritiated uridine. Bleomycin produces a strong inhibition of RNA synthesis in nucleolar and extranucleolar areas. Important decreases of [3H]uridine incorporation were observed as early as 30 min after the administration of drug. Alterations of processing and/or transporting of RNA to the cytoplasm were found after treatments with bleomycin (100 microgram/ml) for 30 to 300 min. It is suggested that the diminution of ribonucleoprotein fibrils is related to the inhibition of RNA synthesis while the accumulation of perichromatin granules is connected to alteration of the transporting and/or processing.

Animals↗
PubMed1997

Clinicopathologic and interphase cytogenetic analysis of papillary (chromophilic) renal cell carcinoma.

Trisomy 7 and 17 with deletion of Y is typical of papillary renal cell adenoma (PRCA), and additional alterations occur in the putative genetic progression toward papillary renal cell carcinoma (PRCC). Our study correlated aneuploidy with clinicopathologic features in PRCCs. We used fluorescence in situ hybridization to assess copy number for chromosomes 7, 8, 10, 12, 16, 17, and Y in 16 PRCCs and surrounding benign tubular parenchyma from 15 patients by use of alpha satellite (centromere) probes on deparaffinized tissue sections. We then compared the pattern of monosomy/nullisomy or trisomy/polysomy/hemidisomy to clinicopathologic parameters. Nine tumors (58% Group 1) showed the numeric aberrations typical of PRCAs and PRCCs, with gains of 7 and 17 and loss of Y. We also identified four trisomies of 12 and 16 and one of 8 in Group 1. The remaining seven cases (Group 2) were cytogenetically atypical. Two displayed borderline loss of chromosome 7, although trisomy 17 was present in both. Five had trisomy 7, but none exhibited chromosome 17 alterations, and two exhibited a gain of Y. Neoplasms in Group 2 were less often multicentric than were Group 1 tumors, and they contained foamy macrophage infiltrates less often. One chromophilic carcinoma with abundant clear cells and another with oncocytic features exhibited Group 2 chromosomal profiles. One patient (nuclear grade 4) died from disease, and 14 had no evidence of carcinoma at the last follow-up. We concluded that PRCCs represent a histologically and genotypically heterogeneous group of tumors. If PRCAs consistently exhibit +7, +17, and -Y, it is uncertain whether PRCCs always evolve directly from such lesions. The presence of genotypic heterogeneity might reflect histologic variants of PRCCs, which overlap with other types of RCC. PRCC is generally an indolent neoplasm, despite a high frequency of chromosomal aneuploidy.

Adult↗
PubMed1997

[Contribution of phenotyping cells in metaphase and interphase (MAC and MACISH techniques) to the study of hematologic neoplasms].

BACKGROUND: Banding techniques are essential in the chromosomal analysis for the cytogenetic diagnosis. Even that, conventional cytogenetic techniques destroy the cytoplasmic membrane and the lineage involvement of the karyotyped cells is unknown. In this work the usefulness of a method that keeps the cell intact and allows the sequential application of immunological, cytochemical, morphological and cytogenetic techniques in the same cell is shown. This technique is called MAC for morphology, antibodies and chromosomes. The combination of MAC and in situ hybridization techniques (MACISH method) allows the detection of a chromosome abnormality in all the cells even when no mitosis are present. PATIENTS AND METHODS: The MAC method was applied in 51 patients and the MACISH method in 9 patients in order to identify the cells which karyotype is analyzed. We have studied 47 patients with normal karyotype (37 chronic lymphocytic leukaemia [CLL] and 10 essential trombocythaemias (ET) and 4 patients with different diseases and abnormal karyotype. RESULTS: Among 37 patients with CLL and normal karyotype, in 9 cases only normal T-cells were in mitoses and in 28 cases the normal karyotype belonged to neoplastic B cells. Trisomy 12 has been confined exclusively to the leukaemic B cells with the MACISH technique in 3 of these CLL cases. In 10 patients with ET and normal karyotype the MAC method showed that in any case the mitosis analyzed belonged to the megakaryocyte lineage. In 4 patients with different chromosomal abnormalities the haematological cell lines involved in the neoplasia were known with the MAC method. CONCLUSION: In this work is shown the usefulness of the combination of the MAC and MACISH techniques with conventional cytogenetics in order to complete the chromosomic study of the haematological neoplasms is confirmed. These methods are specially usefull when different cell lineages are involved in the neoplasia, reactive proliferations are suspected, or to discard false aneuploidies.

Hematologic Neoplasms↗