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B Schutte

Publications and source records attributed to B Schutte.

At least 37 records · Page 2Linked to original sources
PubMed1997

Trivariate flow cytometric analysis of paraffin-embedded lung cancer specimens: application of cytokeratin subtype specific antibodies to distinguish between differentiation pathways.

The aim of the present study was to investigate whether trivariate FCM analysis, for the simultaneous detection of two different CK subtypes in combination with DNA content, can be applied to paraffin embedded samples of different types of non-small cell lung cancer in order to evaluate the cell cycle of individual sublines. Single cell suspensions were prepared from 50 microm thick paraffin sections of 22 lung carcinomas by pepsin digestion and immunostained with CK-antibodies which were chosen to distinguish glandular differentiation (adenocarcinomas) and squamous differentiation. There was a good correlation between the immunocytochemical results of the different CK antibodies in tissue sections and in the corresponding single cell suspensions. Gating for CK-positivity revealed a higher S-phase fraction as compared to the ungated cell population. The tumor cells in adenocarcinoma cases were specifically recognized by CK7 antibodies, while well-differentiated squamous cell carcinomas were specifically stained for CK14 and/or CK17. In poorly differentiated squamous cell carcinomas simultaneous expression of CK7 and CK17 was detected in a subpopulation of the tumor cells, next to cells positive for CK7 or CK17 alone. The trivariate FCM analysis allowed the separate estimation of ploidy status and cell cycle parameters in the three different cell populations of these, apparently (phenotypically) heterogeneous, malignancies.

Adenocarcinoma↗
PubMed1996

A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture.

Early during the process of apoptosis, cells lose their phospholipid membrane asymmetry and expose phosphatidylserine (PS) at the cell surface while maintaining their plasma membrane integrity intact. This process can be monitored for suspended cell types by using annexin V-FITC, which is a Ca(2+)-dependent, phospholipid-binding protein with high affinity for PS, and flow cytometry. If adherent cell types are to be studied for this apoptosis-associated phenomenon, then a problem is encountered, in that specific membrane damage occurs during harvesting. In this paper, a flow cytometric-based method is described that allows the measurement of loss of phospholipid asymmetry during apoptosis of adherent cells in culture. The method relies on the phospholipid binding property of biotinylated annexin V. Furthermore, the use of this conjugate allows tricolor flow cytometric analysis of apoptosis. Employing the method to MR65 cells, which were initiated by olomoucine to enter apoptosis, it is shown that PS exposure occurs early after the onset of apoptosis and, at the prevalent time-resolution, that PS exposure is accompanied by loss of both cytokeratin and DNA. The annexin V+ cells appear as a characteristic sub-G1 peak in the DNA histogram.

Annexin A5↗
PubMed1996

Flow cytometric analysis of consecutive lymph node samples from patients with Hodgkin's disease: reproducible within one biopsy?

In Hodgkin's disease DNA aneuploidy is not a prognostic factor. However, the prognostic significance of DNA content in Hodgkin's disease may be missed by either intratumor DNA heterogeneity or DNA analysis of limited samples. For flow cytometry usually one section of 40-60 microns is used for the analysis. In breast cancer this proved to be insufficient. In Hodgkin's disease no data are available. Therefore, we examined if analysis of more sections does increase the yield of aneuploidy. Archival, formalin-fixed, parafin embedded tissues were used. From 13 patients four sections of 50 microns could be analysed for DNA content. In 12 of 13 patients the results were consistent in all four sections of one patient case; seven diploid, four aneuploid and one multiploid. In one case ploidy status changed: two sections were diploid and two were aneuploid. The DNA-index of the aneuploid samples ranged from 0.75 to 1.38 and varied from 0.02 to 0.14 within one case. The S-phase fraction remained constant within all evaluable cases (sd: 0.5-1.5%), except for one (sd: 4.7%). In conclusion, in Hodgkin's disease the ploidy status of the first section can be regarded to represent the whole tissue sample. Therefore, the absence of prognostic value of ploidy status is not explained by sampling errors in tissues analysed.

Biopsy↗
PubMed1996

Subcellular localization of proteasomes in apoptotic lung tumor cells and persistence as compared to intermediate filaments.

We have studied the subcellular localization and expression levels of proteasomes during apoptosis in a lung cancer cell line. Apoptosis was induced by exposing the cells to 200 microM olomoucine, a specific cyclin-dependent kinase inhibitor. The morphological changes characteristic for apoptotic cells were visible: the cells reduced in size, the chromatin condensed and the membranes became convoluted. As the process continued, the nuclei became fragmented, and the cells broke up into cytoplasmic vesicles and apoptotic bodies. Immunocytochemically, apoptotic cells were detected by the ability to bind annexin V at their surface. During the initial stages of apoptosis, proteasomes were present in the nucleus as well as in the cytoplasm. Upon increased chromatin condensation, nuclear proteasomes were found predominantly surrounding the chromatin, while the chromatin itself remained devoid of staining. That the proteasomes persisted relatively long in the apoptotic cells was shown by immunoblotting of non-denaturing gels, which indicated that both 20S and 26S proteasomes were present in apoptotic cells. In immunofluoresence microscopy the proteasome fluorescence intensity of apoptotic cells seemed higher than that of non-apoptotic cells. These differences in intensity were even more pronounced after Triton X-100 extraction. Flow cytometry revealed that the absolute levels of proteasome staining in cells were decreased after Triton X-100 extraction. However, no differences in staining levels were detected between apoptotic and non-apoptotic cells. A relative increase of proteasome concentration through cell shrinkage or a concentration in certain cell compartments may be the origin of the apparently increased signal that was seen in immunofluorescence microscopy. Furthermore, proteasomes were clearly detectable in the apoptotic bodies and cytoplasmic vesicles at the time immunocytochemical reactivity for cytokeratins and lamins had diminished to a large extent. Immunoblotting of denaturing polyacrylamide gels confirmed the results obtained by flow cytometry. The proteasome content was retained only partially in the cells after Triton X-100 extraction, while the intermediate filaments were not detectable anymore in the apoptotic cells.

Apoptosis↗
PubMed1995

Three parameter flow cytometric analysis for simultaneous detection of cytokeratin, proliferation associated antigens and DNA content.

The analysis of the cell cycle distributions by univariate flow cytometric DNA measurement has been widely applied in the clinic to determine kinetic parameters of human malignancies. A common problem with measurements of cell cycle phase distributions in tumor biopsy material is the presence of non-malignant diploid cells. Furthermore, such a static measurement might not be accurate enough to describe the dynamic process of cell proliferation. For this purpose alternative methods have been developed to include BrdUrd incorporation or the presence of intrinsic proliferation associated markers such as PCNA or Ki67-Ag into the analysis. However, the presence of nonmalignant diploid cells will influence also these bivariate analyses, especially in case of DNA-diploidy of the tumor cells. Here we present a three parameter flow cytometric assay based on the simultaneous detection of cytokeratin, DNA and a proliferation associated marker, such as BrdUrd, PCNA or Ki67-Ag. Based on the presence of cytokeratin, epithelial cells can be selected for a detailed cell cycle analysis. This method can be applied to frozen tissue, which makes this assay useful for multicentre clinical studies.

Analysis of Variance↗
PubMed1995

S-phase arrest of nutrient deprived lung cancer cells.

The human small cell lung cancer cell line NCI-H82 was used to study the effect of nutritional status on cell proliferative parameters. Incorporation of bromodeoxyuridine (BrdUrd) was used to characterize actively proliferating cells and to obtain information on cell cycle dynamics. During several days, in which the culture medium was not changed, a gradual decrease in overall cell growth, labeling index, and vitality was observed. Simultaneously, an increase in the number of S-phase cells that did not incorporate BrdUrd was noticed. From a more detailed kinetic study on d 6 of nutrient depletion, it appeared that, although the cells incorporated BrdUrd, they stopped cycling. When the same cells were regrown in fresh culture medium, a delay of 10 h in G1-phase entry and exit was measured. After this delay the cells resumed the cell cycle at normal phase transit rates. In addition, BrdUrd unlabeled S-phase cells were gradually lost from the culture. Bivariate flow cytometric DNA/proliferating cell nuclear antigen (PCNA) and DNA/Ki67-antigen analyses confirmed a delay in G1 phase entry and exit. In this paper we show that nutrient depletion can cause cell cycle arrest as indicated by the occurrence of BrdUrd unlabeled S-phase cells. This arrest could lead to overestimation of kinetic parameters such as S-phase transit time (Ts) and potential time (Tpot) as determined after in vivo labeling of tumors.

Bromodeoxyuridine↗
PubMed1995

Evaluation of proliferation parameters in in vivo bromodeoxyuridine labelled lung cancers.

In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcinoma, the validity of proliferating cell nuclear antigen (PCNA) and Ki67 antigen as proliferative indicators was evaluated in ethanol fixed, paraffin embedded tissue. The percentages of cells positive for these markers were compared to the in vivo bromodeoxyuridine (BrdU) labelling index. A good correlation was found between PCNA immunoreactivity and BrdU labelling index, while Ki67-antigen expression showed a significant relation with BrdU labelling index and with PCNA expression. All three parameters showed a trend towards similar values for the individual cases. Based on the fact that Ki67 antigen is expressed in all cycling cells, whereas replicon-associated PCNA and BrdU only reflect the S-phase fraction, the differences between Ki67-antigen scores on the one hand and BrdU and PCNA scores on the other were smaller than expected. In order to determine the degree of concordance between immunohistochemically and flow cytometrically detected proliferation variables, BrdU incorporation was measured using both methods in duplicate bronchial specimens. Discrepancies in labelling indices were observed predominantly in DNA diploid samples, with consistently lower values in the flow cytometrically analysed specimens. In tumour specimens with an aneuploid DNA content, flow cytometric determination of proliferative activity yielded results similar to those obtained by tissue section examination. We conclude that the scores for PCNA and Ki67 antigen, immunohistochemically detected in ethanol fixed, paraffin embedded tissue reflect functional proliferative activity.

Biopsy↗
PubMed1995

Alterations in cytoskeletal and nuclear matrix-associated proteins during apoptosis.

Evidence exists that apoptosis or programmed cell death plays an important role in tumor growth. Morphologically apoptosis is characterized by membrane blebbing and nuclear fragmentation in individual cells. In this study, we investigated changes in the nuclear organization in spontaneously occurring apoptotic cells in several cancer cell lines using several antibodies to nuclear matrix constituents. It appeared that nuclear matrix components remained detectable in cells undergoing spontaneous apoptosis. The same results were found when apoptosis was induced by cycloheximide in the non-small cell lung cancer cell line MR65. Using this induction method, the percentage of apoptotic cells in MR65 cells increased, allowing a more detailed and extensive examination of nuclear matrix alterations together with cytoskeletal changes. To study the expression of cytokeratins, type A- and B lamins, a nuclear matrix-associated 13 kDa U1RNP particle and the Ki67-antigen, immunocytochemistry in combination with confocal scanning laser microscopy was used. Apoptotic cells were identified based on nuclear morphology and the in situ nick translation assay. Whereas immunoreactivity against lamins and Ki67-Ag was rapidly lost during apoptosis, expression of the 13 kDa protein and, in early apoptotic stages, also cytokeratin expression, was observed to remain present. Dead cells lacked reactivity with all the antibodies tested. The persistence of nuclear matrix components is therefore a useful marker for the detection of apoptosis.

Antigens, Nuclear↗
PubMed1994

DNA content as prognostic factor in cervix carcinoma stage IB-III treated with radiotherapy.

A prognostic value of DNA content in cervix carcinoma for local recurrence following radiotherapy is suggested by both retro- and prospective studies. The purpose of this study was to confirm these data. Flow cytometry and computerized image cytometry were used to measure DNA content of tumor cells retrospectively in paraffin-embedded archive material from 98 patients with carcinoma of the uterine cervix FIGO stage IB-III. All patients were treated with combined megavoltage irradiation and brachytherapy. Minimum follow-up has been 5.1 years. Pelvic recurrence was seen in 10, 26, and 54% of patients with stages IB II, and III, respectively (P = 0.003). DNA content, expressed as DNA index (DI), was not correlated with the FIGO stage (P = 0.25), histopathologic grading (P = 0.26), or age (P = 0.63) at diagnosis in this series. Using univariate analysis pelvic disease-free survival (PDFS) was best predicted by the clinical stage (P = 0.004) and by an age greater than 55 years (P = 0.04) before treatment. PDFS tended to be better for patients with aneuploid tumors (DI > 1.2), but this difference was not statistically significant (P = 0.17). Using Cox's regression model for multivariate analysis, only stage was independently correlated to PDFS (P = 0.009). After stratification for stage, ploidy level was significantly correlated with PDFS in stage II patients (P = 0.04). Overall results suggest aneuploid tumors to be more radiosensitive than diploid tumors in this series.

Adult↗
PubMed1994

Ploidy and S-phase fractions (SPF) of primary breast cancers and their nodal metastases.

Ninety-one cases of primary breast cancers and their nodal metastases were examined with DNA flow cytometry. No differences were found between the stemline distributions in the primary tumors and nodal metastases. At both sites stemlines clustered around a DNA index of 1.0 (33-40% of cases) and 1.8. The mean S-phase fractions were 7.9 in primary tumors versus 5.6% in nodal metastases (p = 0.02); this difference was also observed if the analysis was restricted to cases with DNA aneuploidy at both sites (10.2 versus 7.6%, p = 0.04). Our results indicate that axillary nodal ploidy and proliferation reflect primary tumor characteristics rather than displaying changes associated with selection during the lymphatic metastatic process. Lymph nodes may have a suppressive effect on the proliferation of tumor cells.

Aneuploidy↗
PubMed1994

Proliferating cell nuclear antigen (PCNA): a new marker to study human colonic cell proliferation.

Immunohistochemistry of the S phase related proliferating cell nuclear antigen (PCNA) was studied as an alternative to ex-vivo bromodeoxyuridine (BrdU) immunohistochemistry for assessment of human colonic cell proliferation. From 16 subjects without colonic disease biopsy specimens were collected from five different sites along the colorectum and processed for BrdU and PCNA immunohistochemistry. The mean proliferation index of PCNA was significantly higher at 133% of the value obtained with BrdU. There was, however, a good correlation between the results from both techniques (r = 0.6275; p < 0.05). Decrease in proliferation index along the colorectum was seen with both staining methods but was clearer with PCNA immunohistochemistry (caecum/ascending colon v rectum: 12.0 v 7.2; p < 0.004). The total number of crypt cells also decreased from proximal to distal (134 to 128; p < 0.06) but at no site correlated significantly with the proliferation index. It is concluded that in clinical cell kinetic studies staining for PCNA may serve as an attractive alternative to the BrdU incorporation assay.

Adolescent↗
PubMed1994

DNA aneuploidy in Hodgkin's disease: a multiparameter flow cytometric analysis.

Paraffin-embedded lymph nodes from patients with Hodgkin's disease were examined for flow cytometric DNA content. In order to increase the sensitivity of the assay we tried to enrich for the neoplastic cells by bivariate analysis using a polyclonal anti-nucleolar antibody (AN-AB) and the forward scatter (FSC). DNA aneuploidy was found to be present in 67 of all 137 cases (49%), in 24 cases only demonstrable by dual-parameter analysis. The DNA index varied from 0.69 to 1.89 with a total of 22 hypo-diploid cases. The number of aneuploid nuclei exceeded the expected frequency of Reed-Sternberg (RS) and Hodgkin (H) cells in most of the analysed specimens. In conclusion, flow cytometry in Hodgkin's disease appears to give useful information regarding the ploidy status and evidence has been provided that the malignant cell population in Hodgkin's disease is not limited to the classical RS/H cells.

Aneuploidy↗
PubMed1994

Comparison of image (CAS 200) and flow cytometry determined DNA content of paraffin-embedded Hodgkin's disease tissue.

To assess the reliability of DNA estimation using image cytometry, deparaffinized lymph nodes from 70 patients with Hodgkin's disease were examined and the results obtained were compared with those from flow cytometry. Image analysis without discriminating between the various cell types, as found in Hodgkin's disease, revealed no separate aneuploid peak. Selecting on morphologically defined nuclear types DNA aneuploidy was detected in 20% of the cases (14/70). The aneuploid populations were limited to the population of nuclei defined as Reed-Sternberg (RS)-like or medium-sized lymphocytes. Benign lymph nodes DNA aneuploidy was not found in any of the controls. Comparison of DNA histograms obtained by image and flow cytometry showed aneuploid peaks using image cytometry in 4 of 30 diploid and 10 of 40 aneuploid flow histograms. In conclusion, image analysis using the CAS 200 system as compared to flow cytometry is more time-consuming and less sensitive to assess ploidy status, although it may provide extra information in some selected cases. Evidence is obtained that DNA aneuploidy in Hodgkin's disease is preferentially expressed by cells with the RS/H-like and medium-sized lymphocyte morphology.

Aneuploidy↗
PubMed1993

Cytokinetic analysis of lung cancer by in vivo bromodeoxyuridine labelling.

Cytokinetic parameters of various types of lung cancer were determined in bronchoscopy specimens after in vivo labelling with the thymidine analogue bromodeoxyuridine (BrdU). The S-phase fraction and BrdU labelling index were measured flow cytometrically, allowing calculation of the S-phase transit time and potential tumour doubling time. The methodology used was found to be feasible for obtaining cytokinetic data from 76% of the bronchial biopsy samples. Despite the difference in clinical behaviour and growth pattern between small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), no significant differences were observed between the mean values of the cytokinetic parameters of SCLC and NSCLC. The estimated cell loss factor was higher in NSCLC than in SCLC. It appears that the growth of a tumour, as clinically observed, is to a considerable extent influenced by cell loss. In accord with this assumption is the fact that we have observed non-BrdU labelled S-phase cells, both in tumour biopsies and in apparently normal tissue. The presence of these so-called unlabelled S-phase cells in relation to cell loss is discussed.

Adult↗
PubMed1993

Aneuploidy fraction but not DNA index is important for the prognosis of patients with stage I and II breast cancer--10-year results.

BACKGROUND: Individual assessment of the prognosis of patients with breast cancer is crucial for the selection of risk-adapted adjuvant therapy and in follow-up. Parameters from DNA flow-cytometry have been shown to provide significant prognostic information, but published results are in conflict and there are only a few investigations with long-term follow-up. The aim of this study is to clarify the impact of tumor DNA data on the clinical course of stage I and stage II breast cancer patients. PATIENTS AND METHODS: Several flow-cytometry DNA analyses were performed on tumor samples derived from 191 breast cancer patients entered in a controlled clinical trial after a median follow-up of more than 10 years. In addition to DNA index (DNI) and the percentage of cells in S phase (SPF), an index, designated aneuploidy fraction (AF), was determined. It ascertains the percentage of aneuploid cells out of all cells in the DNA flow-cytometry histogram, and its reproducibility has been tested by measurements of AF in two different samples of the same tumor. Univariate analyses and, in the 122 patients for whom complete information was available, a Cox model, were performed to investigate the individual prognostic impact of flow-cytometry parameters compared with established clinical factors. RESULTS: AF proved to be a very valuable prognostic indicator both in univariate and multivariate analyses, whereas DNI and SPF failed to provide independent prognostic information. The combination of AF and lymph node status clearly identifies different prognostic subgroups in operable breast cancer. CONCLUSIONS: Routine evaluation of patients with breast cancer should include tumor DNA flow-cytometry. Aneuploidy fraction is a valuable tool in assessing an individual patient's prognosis and thus can help in the choice of the appropriate adjuvant treatment strategy. Whether it, rather than DNI and SPF should be used, as we found, needs to be validated in a larger prospective investigation.

Aged↗
PubMed1993

A radiation hybrid map of 15 loci on the distal long arm of chromosome 4, the region containing the gene responsible for facioscapulohumeral muscular dystrophy (FSHD).

A physical map of 4q35 was constructed through radiation hybrid analysis of 134 clones generated from the cell line HHW416, a chromosome 4-only human-hamster somatic cell hybrid. This subtelomeric region contains the as-yet-unidentified gene responsible for facioscapulohumeral muscular dystrophy. The most likely order of 15 loci within 4q35 was determined. The loci ordered on this radiation hybrid map include both genes and polymorphic loci, as well as monomorphic loci which cannot be placed on a genetic linkage map. The physical distance spanning these loci was estimated to be approximately 4.5 Mb, by using a kilobase/centiray conversion factor derived from 4p16.3 marker analysis through the same set of radiation hybrids. The comparison of this physical map to establish genetic maps suggests that this region is smaller than initially estimated and that recombination rates are increased near the telomere.

Animals↗
PubMed1992

DNA ploidy and other results of DNA flow cytometry as prognostic factors in operable breast cancer: 10 year results of a randomised study.

We evaluated breast cancer specimens from 241 patients of a controlled clinical trial by means of DNA flow cytometry. We report the correlations between DNA index (DNI) and fraction of cells in S-phase (SPF) and other prognostic parameters. Both univariately and in a Cox model, the predictive power of these factors is evaluated after a follow-up of more than 10 years. There are strong correlations between DNI and SPF (P = 0.0001) and between flow cytometry parameters and clinical and histopathological factors such as axillary lymph node involvement, tumour size and histological grade. In univariate analysis DNI fails to provide prognostic information, whereas SPF turns out to be able to differentiate between patients at high and low risk for relapse and death (P = 0.002). In the multivariate Cox model, too, SPF is an important prognostic parameter with respect to patient survival (relative risk: +86%), only surpassed by nodal involvement. DNI, however, turns out to be an independent predictor of relapse free survival and distant recurrence free survival. By combination of DNI and SPF, patients can be divided into three prognostic subgroups. We conclude that data from DNA flow cytometry can be of great importance for the decision on the level of aggressiveness of adjuvant therapy for an individual patient and therefore may help to avoid overtreatment and toxicity.

Adult↗