Static Cytofluorometry Research Articles

Low-grade gastric adenomas/dysplasias: Phenotypic expression, DNA ploidy pattern, and LOH at microsatellites linked to the APC gene

The adenoma–adenocarcinoma sequence in the colon is generally accepted. Regarding the stomach, however, opinions about tumorigenesis are different. We analyzed the phenotypes, DNA ploidy patterns, and microsatellite regions linked to adenomatous polyposis coli (APC) gene on chromosome 5q of low-grade gastric adenomas/dysplasias, Vienna Category 3, to investigate whether these lesions have the potential to become adenocarcinomas. Phenotypes were determined by immunohistochemical staining with monoclonal antibodies MUC2, MUC5AC, MUC6, CD10, and Cdx2. DNA ploidy patterns were determined by static cytofluorometry. Microsatellite analyses were performed using Genescan on genetic analyzer ABI PRISM 310. None of the 15 cases showed coexisting high-grade adenomas/dysplasias or adenocarcinoma. Eighty percent of the cases had the complete intestinal phenotype, and the remainder showed slight MUC6 positivity for the intestinal phenotype. All cases were positive for Cdx2 and showed diploid DNA. In addition, 46.7% of the cases exhibited loss of heterozygosity (LOH) of chromosome 5q. Except for one case, 5q-LOH was detected at a single locus in cases with the complete intestinal phenotype. According to previous studies, most gastric adenocarcinomas have the gastric phenotype and no 5q-LOH. These results suggest that low-grade gastric adenomas/dysplasias, Vienna category 3, seldom progress to gastric adenocarcinomas of the differentiated type.

Simultaneous Paired Analysis of Numerical Chromosomal Aberrations and DNA Content in Osteosarcoma

Relatively little is known about the biologic relevance of numerical chromosomal changes in relation to DNA content in osteosarcoma. In this study, by using a series of human osteosarcoma cell lines, we standardized a method for the assessment, on the same nuclei specimen, of both specific chromosome copy numbers by fluorescence in situ hybridization (FISH) and the DNA content by static cytofluorometry or image cytometry. On the same cell lines, we also evaluated the DNA content by using flow cytometry and the chromosome number distribution by metaphase analysis. Comparison between these different methods showed that DNA ploidy level as determined by FISH or metaphase analysis is frequently lower than the ploidy pattern as defined by cytometric methods. By using comparative genomic hybridization, we were able to demonstrate that these discrepancies were due to the presence of several unbalanced chromosome aberrations, specifically gains and high-level amplifications, which affect the total DNA content with less effect on the total chromosome number. Thus, evaluation of DNA ploidy in osteosarcoma cells is needed for a correct interpretation of FISH or cytogenetic data concerning numerical chromosomal changes. Evaluation of tumor ploidy in a series of clinical samples demonstrated that in high-grade osteosarcoma, flow cytometry sometimes may give false results because of the presence of high proportions of contaminating, nonneoplastic cells that cannot be excluded from the flow cytometric assessment but that do not interfere with the evaluation of DNA ploidy by static cytofluorometry or image cytometry, in which only tumor cells are selected for the analysis. The possibility of using this method to evaluate, on the same nuclei sample, both specific chromosomal aberrations and DNA ploidy may allow a better determination of numerical chromosomal changes that may be relevant for the biologic behavior of osteosarcoma.

Amplification of growth factor receptor genes and DNA ploidy pattern in the progression of gastric cancer.

To study the background of oncogene amplification in gastric cancers, we examined the correlation between occurrence of oncogene amplification and DNA ploidy pattern. In 57 primary gastric cancers, amplifications of c-erbB, c-erbB-2, c-met and K-sam genes were investigated by Southern blot analysis, and the DNA ploidy pattern was determined by static cytofluorometry and by flow cytometry. Oncogene amplification was detected in 11 cancers, 10 of which were advanced gastric cancers and 1 was an early differentiated type. The amplification of c-erbB-2 and K-sam genes was found exclusively in differentiated- and undifferentiated-type cancers, respectively. Of the 11 cancers, 5 were DNA-diploid and 6 were DNA-aneuploid. All the 11 tumours with oncogene amplification contained polyploid cell populations (polyploidy), whereas none of the tumours without polyploidy showed oncogene amplification. In differentiated-type cancers the incidence of polyploidy was high in both early and advanced stages, while in undifferentiated-type cancers it was low in early stages but significantly higher in advanced stages. It was shown that amplification of growth factor receptor genes is closely related to the presence of polyploidy, irrespective of any different stemline DNA-ploidy mode. The time-course of oncogene amplification and kinds of genes amplified may differ between differentiated- and undifferentiated-type gastric cancers.

Studies on DNA ploidy of biopsy and surgical specimen for renal cell carcinoma

The object of this study is to evaluate the efficacy of selective renal tumor biopsy in patients with renal cell carcinoma. The accuracy of histological diagnosis and DNA content of biopsies in predicting those of surgical specimens was assessed. From 1985 to 1994, 41 patients with renal cell carcinoma underwent selective renal tumor biopsy before partial or radical nephrectomy. DNA content of renal cell carcinoma in 41 patients in biopsy specimens and surgical specimens were examined in paraffin blocks with static cytofluorometry (SCM) and flow cytometry (FCM). In 39 out of 41 cases, by SCM, as well as in 37 out of 41 cases by FCM, DNA content of biopsy could be analyzed. Findings of selective renal tumor biopsy for RCC coincided with those of surgical specimen in 27/39 (accuracy 69%, kappa 0.398) in cell types, 29/39 (accuracy 74%, kappa 0.432) in structural types, 26/39 (accuracy 67%, kappa 0.341) in gradings and 27/39 (accuracy 69%, kappa 0.532) in ploidy patterns. It was assumed that DNA histograms might help the histological diagnosis by biopsy.

Static cytofluorometry and fluorescence morphology of mitochondria and DNA in proliferating fibroblasts.

The shape, distribution, and content of mitochondria in individual cells were examined during the cell cycle phases (G(0)/G(1), S, G(2) mitosis) in living human fibroblasts by static cytofluorometry and fluorescence microscopy. The morphocytochemical evaluations were performed in cell cultures submitted to double supravital fluorochrome staining with Hoechst 33342 and DiOC(6) to label DNA and mitochondria, respectively. The staining modalities were based on the stability of mitochondrial labeling. The G(1) to early S phases were characterized by the presence of filamentous mitochondria, except during the early postmitotic period. During late S, G(2), and mitotic phases, mitochondrial mass reached its highest value and mitochondria became short and numerous. During the last stage of mitosis, mitochondria were distributed among daughter cells through a cytoplasmic bridge.

Tumor heterogeneity in DNA ploidy of renal cell carcinomas as revealed by static cytofluorometry and flow cytometry

DNA ploidy of 49 renal cell carcinomas of 46 patients were examined with static cytofluorometry (SCM) and flow cytometry (FCM). We used several paraffin blocks for each tumor (mean, 2.4), separated each block into several samples based on histological findings, and measured the DNA content of each sample. More samples could be analyzed with SCM than with FCM. With FCM, it was sometimes difficult to detect polyploid cells, or to determine whether diploid cells were tumor cells or stromal cells. DNA heterogeneity might thus be more accurately detected with SCM than with FCM. DNA aneuploidy was demonstrated for 59% of the tumors, and was significantly less common in grade 1 tumors than in higher-grade tumors. The incidence of polyploid cells in diploid tumors tended to increase with grade of tumor. Fifty-five percent of the tumors displayed DNA heterogeneity, the incidence of which tended to increase with grade of tumor. Ninety-four percent of the tumors were found to yield a diploid cell line. The findings of this study indicate that DNA content is associated with the histological grading. Diploid tumors with polyploid cells should be dealt with clinically in a separate fashion from diploid tumors without them. These findings suggest that diploid renal cell carcinomas with polyploid cells may be an intermediate stage between diploidy and aneuploidy.

Specific rearrangement of the microtubular network during the cell cycle phases and correlation with micronucleation and death induced by anticancer drugs in NIH/3T3 subcultures

The changes of the microtubular network induced by microtubule destabilizing (Vinblastine, VBL) and stabilizing (Taxol, TAX) agents were studied in NIH/3T3 fibroblastic cells in conventional culture conditions (NIHb) and in a subpopulation (NIHs), obtained after serum deprivation and expressing different morphofunctional features and higher cytokinetic activity. In this cell model, we analyzed VBL and TAX effects on cell cycle and microtubular network, in relation to cell death. In NIHb cells, VBL induced higher microtubule depolymerization, prevalence of tubulin paracrystals and micronucleation, while, in NIHs cells, lower depolymerization and appearance of tubulin spiral-like structures, with lower micronucleation, increase of apoptosis and disappearance of high polyploid cells. DNA static cytofluorometry of cells showing paracrystals or spirals permitted correlation of the appearance of these tubulin aggregation forms with the cell cycle phases. In NIHb cultures, the DNA content curves, in cells with paracrystals or spirals, showed a similar trend, with a higher frequency of the two anomalies in the G2/M phase. In NIHs cultures, paracrystals and spirals are found in G2/M cells, while G1 cells showed prevailingly paracrystals. TAX induced the appearance of microtubule bundles in the two cultures. The prevalence of circular bundles was found in NIHb cells, while a higher number of linear bundles was shown in NIHs cells. In NIHb cells, circular bundles were related to higher apoptosis and micronucleation. DNA cytofluorometry, in cells with linear or circular bundles, showed that the latter was present with high frequency in NIHb cells in all the cell cycle phases; in NIHs cells, they appeared, with lower frequency, prevailing in the S-G2/M phase. Furthermore, in NIHs cells, the appearance of linear bundles in G1 cells was related to a lower micronucleation. These finding showed that microtubule reorganization in different cell cycle phases could play a role in the progression of nuclear fragmentation/micronucleation relating to cell death.

S-phase fraction is a prognostic factor in stage I breast carcinoma.

The prognostic significance of cell proliferation, estimated as cytometric S-phase fraction (SPF), was investigated in node-negative breast cancer patients with small tumors (T1, NO). The 219 stage I patients originated from two series and were diagnosed either from 1978 to 1981 or from 1981 to 1985. The tumors were analyzed for estrogen receptors (ERs) by isoelectric focusing and for cellular DNA content by static cytofluorometry or flow cytometry. A high SPF correlated with the absence of ERs and abnormal DNA content, and was less often found in tumors smaller than 11 mm compared with those with a diameter between 11 and 20 mm. Among the variables age, tumor size, DNA ploidy, ER status, and SPF, only SPF showed a significant association with distant recurrence and breast cancer survival in systemically untreated patients. The relative recurrence rate for patients with an SPF of 10% or greater was three times that for patients with lower SPFs. Estimated 8-year breast cancer survival rates for the same groups were 72% and 91%, respectively. This study suggests that cytometric SPF has prognostic significance in stage I breast carcinoma.

Influence of S-phase fraction on metastatic pattern and post-recurrence survival in a randomized mammography screening trial.

Using static cytofluorometry, S-phase was determined on the primary tumors of 421 patients with breast carcinomas in stages I-III diagnosed 1981-85 during the second and third screening rounds of a randomized trial evaluating the effect of mammographic screening. Through December 1988, 82 patients had developed local and/or distant recurrence, 51 of whom had died of cancer during the same period. The distribution among sites of recurrence differed between patients with tumors detected by mammography screening and cancers diagnosed due to clinical symptoms. The mean S-phase fraction was highest in patients with liver or brain metastases and lowest in patients with metastases in subcutaneous and cutaneous tissue and lymph nodes only. In univariate analysis, survival after first recurrence was significantly associated with the site of primary recurrence, the disease-free interval, and node status and tumor size at diagnosis, as well as the S-phase level. The median survival period was 31.3. months for patients with a S-phase fraction below 6%, and 10.7 months in cases with S-phase exceeding 10%. Neither ploidy nor the estrogen receptor content had significant influence on post-recurrence survival. In Cox's multiple regression analysis, only metastatic site, disease-free interval, and S-phase fraction showed significantly independent prognostic value.

A Rapid System for Static Cytofluorometry Enabling the Simultaneous Determination of Nuclear Size and DNA Content

A measuring technique enabling the simultaneous determination of nuclear size and DNA content by single cell cytofluorometry is described. The system is based on a Leitz MPV cytophotometer, a Zeiss scanning stage and a microcomputer system. Cells to be measured are not positioned in the ordinary way, but are passed through the excitation light beam. In this way rapid measurements are achieved. Further, an estimate of the object diameter is calculated from the pulse shape obtained as the fluorescence is recorded continuously by using a circular measuring aperture, congruent with the excitation light field. Repeated measurements of nuclei stained with Hoechst 33258 showed a high reproducibility for DNA content with a coefficient of variation (CV) below 1% and an acceptable precision for nuclear area with a mean CV of 7%. Nuclear area estimated by the method was well correlated to area determined from photographs (r = 0.986). Disintegrated samples from eight patients with breast cancer were analyzed and compared to Feulgen-stained samples analyzed by scanning cytophotometry. Scatter diagrams illustrating DNA content and nuclear size showed essentially the same distribution with the two methods. We conclude that the system may be a rapid alternative when fluorescence together with object size are quantified in slide preparations.

Flow cytofluorometry of lysosomal acridine orange uptake by living cultured cells. Effect of trypsinization and starvation.

The uptake of the fluorescent, lysosomotropic weak base acridine orange (AO) by living cells in culture was studied by flow cytofluorometry. A mouse myeloma cell line (SP 2/0), growing in suspension, and an anchorage-dependent human malignant glioma cell line (U-251 MG), brought into suspension by trypsinization, were used. The consequences of trypsinization were also studied using static cytofluorometry. The lysosomal accumulation of AO by myeloma cells growing in suspension was found to be only moderately affected by starvation (i.e. incubation without medium change) for a period of up to five days. Trypsinization of the glioma cells after staining with AO caused pronounced release of the fluorescent dye while trypsinization before staining with AO did not significantly change the average lysosomal concentration of AO. We did, however, notice certain side effects of trypsinization in the form of both increased cellular green fluorescence and greater intercellular variability that reduce the validity of data obtained from cells detached by routine trypsinization. In conclusion, the condition of the lysosomal vacuome of living cultured cells growing in suspension may be studied by flow cytofluorometry after vital staining with the lysosomotropic weak base AO. Anchorage-dependent trypsinized cells, however, yield unsatisfactory results when examined in a flow cytofluorometer system and are better studied while still attached to their substratum, using static cytofluorometry.

Hypertetraploid cells in vocal cord epithelia.

DNA measurements yield information about the nature of cells and may provide diagnostic and prognostic information. Static cytofluorometry was performed on smears removed at microlaryngoscopy from 107 vocal cord lesions (96 patients). All stem cell lines were diploid except 3; 2 carcinomas and 1 severe dysplasia were polyploid. The mean proliferative activity (percentage of nuclei greater than diploid peak) was 2.1% for the group of epithelia with hyperplasia and mild dysplasia, 3.1% for those with moderate dysplasia, 4.0% for severe dysplasia, and 6.8% for carcinomas. Hypertetraploid cell nuclei (HT cells) were not found in epithelia with hyperplasia and mild dysplasia. Seven out of 15 patients with epithelia showing moderate dysplasia had HT cells; 5 of these patients developed a carcinoma. One of 8 without HT cells developed a severe dysplasia. Nine patients with severe dysplasia had HT cells; 4 had recurrences and 4 developed carcinoma within 4 years. In 14 patients without HT cells, 3 had recurrences and 1 developed a carcinoma 6 years later. HT cells were found in 15 patients with T1 & T2 carcinomas; residual carcinoma was present in 2 after radiotherapy and 4 had recurrences within 11 months. Fourteen patients with T1 & T2 carcinoma did not have any HT cells; one had residual carcinoma after radiotherapy and 3 had recurrences between 18 months and 4 years. DNA measurements and, especially, the demonstration of epithelia with HT cells prove to be of prognostic importance.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of Formalin Fixed, Paraffin Embedded Tumours for Estimation of Cellular Dna Content and S-Phase Fraction by Static Cytofluorometry

Sections with a thickness of 50 micron from 34 formalin fixed, and paraffin embedded tumours were deparaffinized and hydrated and the cells were disaggregated with pepsin. The cells were stained with Hoechst 33258 and a rapid method for static fluorometry was used. DNA ploidy and the fraction of cells in S-phase were estimated and compared with results obtained with freshly prepared cells. The coefficients of variation for the tumour stemlines for both the fresh and the embedded material were approximately 6 per cent. There was a good correlation between DNA ploidy determined from fresh and embedded material, respectively; there was also a close correlation between the fractions of S-phase cells estimated from fresh and embedded tumours. It was also possible to cut out small histologically defined lesions, such as carcinoma in situ of the breast from thick paraffin sections and obtain DNA histograms with high resolution.

A fast and reliable system for microcomputerized DNA cytofluorometry in tumour pathology.

A microcomputerized cytofluorometry system based on a Leitz MPV 3 cytophotometer, and intended for DNA measurements in tumour pathology is described. The system has been equipped with a reference channel for correction of excitation light instability. The importance of the adjustment of the epi-illumination for optimal performance of the reference channel is stressed, and a detailed description is provided. An apparatus variability well below 1% CV is obtained even during periods of marked instability of the arc-lamp. Software for conventional cytofluorometry with which about 200 cells can be measured in 20 min is presented. In addition a measuring technique where cells are not positioned, but are just passed through the excitation light spot is described. Preliminary results on cytocentrifuged specimens of human cancers indicate that this system is at least three times faster than conventional fluorometry. The increased speed of measurements considerably extends the possibilities to evaluate cell proliferation using static cytofluorometry. The measuring capacity of course is dependent upon the quality of the specimen. In well prepared cytocentrifuged specimens 1000 cells have been measured in 35 min.