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1.
Endocr Regul ; 45(3): 113-24, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21793623

ABSTRACT

OBJECTIVE: The objective of this study was the detection of circulating tumor cells (CTCs) in metastatic breast cancer patients. METHODS: Since only small numbers of circulating tumor cells (CTCs) are found in peripheral blood, at first we performed immunomagnetic separation as a concentration method suitable for selecting circulating tumor cells in peripheral blood. This was followed by analysis of isolated cells with the aid of laser scanning cytometry (LSC). Twenty eight patients with metastatic breast cancer were enrolled in the study and the control group consisted of 19 clinically healthy women. Six milliliters of peripheral blood was drawn for the analyses, but only in two patients the blood has been drawn twice. Blood samples were taken when no chemotherapy was administered, but hormonal therapy has been allowed. RESULTS: The positivity for CTCs was found in 20 (50.0 %) patients with metastatic breast cancer patients, while in 6 (31.6 %) healthy controls false positive circulating epithelial-like cells were detected. Because we did not use CD45 staining, we could not distinguish these circulating epithelial-like cells from CTCs. In a majority of metastatic breast cancer patients we found a mixed population of HER-2 gene expressing CTCs. We found that HER2+ CTCs in high numbers are CK19 + CTCs, while almost all HER2-CTCs are CK19- CTCs. CONCLUSION: The described method was found promising for estimating HER2 status on CTCs from peripheral blood in metastatic breast cancer patients.


Subject(s)
Breast Neoplasms/blood , Breast Neoplasms/pathology , Neoplastic Cells, Circulating/pathology , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/blood , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Carcinoma, Ductal, Breast/blood , Carcinoma, Ductal, Breast/enzymology , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/pathology , Carcinoma, Lobular/blood , Carcinoma, Lobular/enzymology , Carcinoma, Lobular/genetics , Carcinoma, Lobular/pathology , Female , Gene Expression Profiling/methods , Genes, erbB-2 , Humans , Keratin-19/genetics , Laser Scanning Cytometry , Middle Aged , Receptor, ErbB-2/biosynthesis , Receptor, ErbB-2/genetics , Statistics, Nonparametric
2.
Bratisl Lek Listy ; 111(1): 4-8, 2010.
Article in English | MEDLINE | ID: mdl-20429304

ABSTRACT

OBJECTIVES: Laser scanning cytometry (LSC) is a slide-based technique capable of measuring a number of biological parameters both in immobilised cell suspensions and in formalin-fixed paraffin-embedded tissue sections. BACKGROUND: High proliferation rate in surgically removed breast tumours is an unfavourable prognostic factor. In node negative cases it can help distinguish patients with higher risk for distant metastases from those with a lower risk. PATIENTS AND METHODS: In a prospective study we investigated 140 breast tumours, of which 113 were invasive ductal carcinomas, 11 were invasive lobular carcinomas, and 16 tumours were of other histological types. Cells for LSC investigations were prepared from fresh, surgically removed tumours by mechanical disintegration. After fixation the cells were stained with FITC-conjugated anti-cytokeratin (CK-FITC) to distinguish CK+ tumour cells from CK- stroma, and with propidium iodide to stain DNA. RESULTS: We identified three S-phase fraction (SPF) groups, with low (30 patients), moderate (54 patients), and high SPF (51 patients). Thirty-seven tumours were diploid, 83 were aneuploid, while 5 tumours had a bimodal distribution of DNA content. Chromatin texture values were increasing in the respective subclasses from the hypodiploid group to the tetraploid/hypertetraploid group. CONCLUSION: The measurement of DNA content and SPF of tumours by LSC completed by and correlated with other biological properties of the tumour cells may be a useful tool in assessing prognosis and clinical outcome of patients with breast cancer. (Tab. 5, Fig. 4, Ref. 18). Full Text (Free, PDF) www.bmj.sk.


Subject(s)
Breast Neoplasms/pathology , Chromatin/pathology , DNA, Neoplasm/analysis , Laser Scanning Cytometry , S Phase , Adult , Aged , Aged, 80 and over , Breast Neoplasms/genetics , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/pathology , Carcinoma, Lobular/genetics , Carcinoma, Lobular/pathology , Female , Humans , Lymphatic Metastasis , Middle Aged , Ploidies
3.
Bratisl Lek Listy ; 111(1): 13-9, 2010.
Article in English | MEDLINE | ID: mdl-20429306

ABSTRACT

BACKGROUND: The aim of our study was the potential detection of circulating tumour cells (CTCs) in early stage breast cancer patients. Our approach was cell microfiltration through polycarbonate membrane as a concentration method suitable for CTC selection in peripheral blood. The isolated cells on membrane were further analysed by laser scanning cytometry. METHODS: Sixteen patients were enrolled in the study, of which 13 had early stage breast carcinoma and 3 patients had metastatic breast carcinoma. The analyses were performed from 9 ml of peripheral blood, in one patient blood was drawn twice. Blood samples were taken after adjuvant chemotherapy but prior to adjuvant radiotherapy. The control group consisted of 12 clinically healthy subjects. CONCLUSIONS: In the control group 3 subjects out of 12 had 1 CTC, the mean CTC numbers being 0.25 +/- 0.45. In the early stage breast cancer patients 0-36 CTCs were detected (mean 13.9 +/- 12.9 CTCs. 10 patients out of 13 had more than 2 CTCs (62%). The detection and measurement of cells on membrane is a simple and reproducible method of detection of CTCs in peripheral blood. Sensitivity of the method is 88.5%. Detection of CTCs seems to be a promising method for the monitoring of adjuvant therapy in early stage breast cancer patients and for the identification of high risk patients in whom elevated numbers of CTCs are persisting following the termination of adjuvant therapy (Tab. 1, Fig. 4, Ref. 35). Full Text (Free, PDF) www.bmj.sk.


Subject(s)
Breast Neoplasms/pathology , Laser Scanning Cytometry , Neoplastic Cells, Circulating/pathology , Adult , Aged , Biomarkers, Tumor/analysis , Breast Neoplasms/diagnosis , Breast Neoplasms/therapy , Cell Line, Tumor , Female , Humans , Middle Aged
4.
Bratisl Lek Listy ; 109(1): 3-7, 2008.
Article in English | MEDLINE | ID: mdl-18447252

ABSTRACT

Cytometry is becoming a standard method of examination not only in biology but also in various fields of experimental and clinical medicine. While in flow cytometry suspensions of cells are measured, laser scanning cytometers enable both the measurement of cells in single-cell suspensions (after immobilising the cells on a conventional glass slide) and in frozen or paraffin-embedded tissue sections. We discuss the possible fields of utilisation and future perspectives of laser scanning cytometry in medicine with special reference to clinical pathology and cytology (Fig. 3, Ref. 49). Full Text (Free, PDF) www.bmj.sk.


Subject(s)
Laser Scanning Cytometry , Flow Cytometry , Humans , Immunophenotyping
5.
Neoplasma ; 41(6): 331-6, 1994.
Article in English | MEDLINE | ID: mdl-7870216

ABSTRACT

In a group of 391 patients with primary breast cancer the cytosolic concentrations of ER, PS2, Cath-D, TPS, TK and cAMP were determined. PS2 production was found to be significantly dependent not only on ER but also on cAMP. A similar dependence on ER and cAMP production was found also in Cath-D. When the production of these prognostic factors was correlated with the occurrence of metastases to axillary lymph nodes, three prognostically unfavorable groups of primary breast carcinomas were revealed. The first group was represented by tumors with negative PS2 values (< or = 2.5 ng/mg) and elevated TPS values (> or = 4.0 kU/mg). The second group comprised prognostically very unfavorable tumors with moderately to highly elevated PS2 values and positive Cath-D values (> or = 35 pmol/mg), or positive TPS values (> or = 8.0 kU/mg). The third group was constituted by tumors with elevated TK (> or = 5.0 U/mg) and ER values (> or = 40 fmol/mg). The possible role of PS2 in the metastasizing of primary breast carcinomas is discussed.


Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/chemistry , Proteins , Adult , Aged , Aged, 80 and over , Breast Neoplasms/enzymology , Breast Neoplasms/immunology , Breast Neoplasms, Male/chemistry , Cathepsin D/analysis , Cyclic AMP/analysis , Cytosol/chemistry , Estrogens , Female , Humans , Male , Middle Aged , Neoplasm Proteins/analysis , Peptides/analysis , Prognosis , Receptors, Estrogen/analysis , Thymidine Kinase/analysis , Trefoil Factor-1 , Tumor Suppressor Proteins
6.
Neoplasma ; 36(4): 401-10, 1989.
Article in English | MEDLINE | ID: mdl-2770927

ABSTRACT

Growth, flow cytometric, and karyological characteristics were analyzed with respect to differences between sensitive L1210 cells and eight sublines resistant to cisplatin (DDP), 1,2-diaminocyclohexane(DACH)-Pt(II) citrate, DACH-Pt(II) glucarate, cis-dichloro-bis-(isopropylamine)-trans-dihydroxyplatinum (IV) (CHIP, iproplatin), and methotrexate (MTX). No great differences were found in growth properties. The sensitive and a majority of resistant sublines displayed similar flow cytometric and karyological characteristics, major differences were found only in the sublines resistant to CHIP. The relationship between the mechanism(s) of resistance and different pharmacokinetics of the drugs is discussed with special reference to DNA content and chromosome structure.


Subject(s)
Cell Division/drug effects , Cisplatin/analogs & derivatives , Cisplatin/pharmacology , Leukemia L1210/pathology , Animals , Antineoplastic Agents/pharmacology , DNA, Neoplasm/analysis , Drug Resistance , Drug Screening Assays, Antitumor , Flow Cytometry , Karyotyping , Methotrexate/pharmacology , Mice , Organoplatinum Compounds/pharmacology , Structure-Activity Relationship
7.
Neoplasma ; 35(6): 643-50, 1988.
Article in English | MEDLINE | ID: mdl-2851743

ABSTRACT

Previously we demonstrated differences in the organization and methylation pattern of integrated Rous sarcoma proviral sequences in helper-dependent virogenic rat TWERC cells. In the present study we attempted to induce changes in the integrated viral genome of TWERC cells using 5-bromodeoxyuridine (BrdU). Four clones (A, B, C, and F) derived from the parental cell line were treated for 10 months with different concentrations of BrdU. Restriction enzyme analysis of the parental cell line and its clones showed that the cells contained in their genomic DNA two copies of deleted provirus. In TWERC cells, the PR-RSV provirus lost the whole env gene and part of the 3' end of the pol gene. The proviral sequences in DNA from the TWERC-derived clones were found to be hypermethylated. A slightly different situation was seen in clone C where demethylation of the provirus in the 3' region and in the 5' end of the genome was found. The level of mRNA expression both in the parental cells and in the clones correlated with the methylation pattern of the PR-RSV provirus. Clone C was less methylated and expressed more virus-specific RNA. The possible role of BrdU in these events is discussed.


Subject(s)
Avian Sarcoma Viruses/drug effects , Bromodeoxyuridine/pharmacology , Proviruses/drug effects , Animals , Avian Sarcoma Viruses/genetics , Blotting, Southern , Cell Line , DNA, Viral/genetics , DNA, Viral/isolation & purification , Genes, Viral , Plasmids , Proviruses/genetics , RNA, Viral/genetics , RNA, Viral/isolation & purification , Restriction Mapping
8.
Neoplasma ; 34(2): 235-8, 1987.
Article in English | MEDLINE | ID: mdl-3600888

ABSTRACT

Human peripheral blood lymphocytes were tested for induction of micronuclei, chromosome aberrations and sister chromatid exchanges (SCEs) frequency after exposure to two platinum complexes (cis-DDP Platidiam and oxo-Pt) and a comparison was made with the inhibitory effect of these drugs on mitotic activity. When the cis-DDP cell samples were compared with the untreated controls, there was a distinct increase in the frequency of micronuclei and chromosome aberrations, and the statistically significant increase in SCE frequency was accompanied by a significant decrease in mitotic activity. In oxo-Pt cell samples, using an identical concentration of the drug, only a slight increase in micronuclei and chromosome aberration frequency was observed. However, the increase in the SCE frequency was not significant and neither was the decrease of mitotic activity when compared with the controls.


Subject(s)
Chromosome Aberrations , Cisplatin/adverse effects , Cell Division/drug effects , Humans , In Vitro Techniques , Lymphocytes/drug effects , Lymphocytes/ultrastructure , Sister Chromatid Exchange
9.
Neoplasma ; 28(6): 675-84, 1981.
Article in English | MEDLINE | ID: mdl-7339496

ABSTRACT

A new cell line (B-25F) obtained from a benign polypoid fibrous lesion of the mucosa of oral cavity is described. The cells at first grew in suspension but after a month of cultivation they began to adhere and subsequently formed a monolayer typical for fibroblastoid cells. Population doubling time both in lower and higher passages was 60 h. Electron microscopy studies failed to detect any viral particles or mycoplasmas. A comparison of cell surface glycoproteins of fibroblasts (B-41FB), fibroma cells (B-25F), and fibrosarcoma cells (B-6FS) was made. These lines share common traits in their surface membranes although distinct differences among the individual lines could be detected. Karyological analysis showed that 64% of cells contained 46 chromosomes. This number veiled both diploid and pseudodiploid karyotypes. An aberrant chromosome, t(13;18?) was found. For isoenzyme comparison of B-41FB, B-25F, B-6FS, and HeLa cell lines the mobility of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PD) was employed. All these lines had LDH patterns of human cells, and a G-6-PD pattern of phenotype B except that of HeLa cells that had phenotype A.


Subject(s)
Fibroma/pathology , Mouth Neoplasms/pathology , Adult , Cell Line , Chromosomes , Female , Fibroma/analysis , Fibroma/genetics , Glycoproteins/analysis , Humans , Isoenzymes/analysis , Mouth Neoplasms/analysis , Mouth Neoplasms/genetics
10.
Neoplasma ; 27(5): 557-66, 1980.
Article in English | MEDLINE | ID: mdl-6262670

ABSTRACT

Six established human sarcoma cell lines (giant cell tumor of bone B-5GT, fibrosarcoma, B-6FS, cystosarcoma phylloides B-19CS, synovial sarcoma U-4SS and two osteogenic sarcomas U-20S and U-393OS) have been studied and compared to the normal B-41FB fibroblastic cells and the HeLa cells. For cytogenetic and isoenzyme characterization both conventional and G banding techniques as well as the mobility of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PD) were employed. All six sarcoma cell lines had karyotype and LDH patterns of human cells. A study of the chromosome counts distribution revealed a large variability from one sarcoma cell line to the other. There was no evidence of any cross-contamination between the lines or by HeLa cells. This conclusion is based on the detection of G-6-PD with B phenotype, the lack of HeLa marker chromosomes in all sarcoma lines and the presence of Y chromosome in U-4SS and U-393OS derived from male donors. In addition, each sarcoma cell line revealed a group of distinctive marker chromosomes which can serve to identify them and help to control cell line specificity during in vitro culturing.


Subject(s)
Isoenzymes/analysis , Karyotyping , Sarcoma/pathology , Cell Line , Chromosomes , Female , Glucose-6-Phosphatase/genetics , HeLa Cells/pathology , Humans , L-Lactate Dehydrogenase/analysis , Male , Phenotype , Sarcoma/enzymology
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