Proliferation and apoptosis in solid tumors. Analysis by laser scanning cytometry

OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC). STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide. Proliferation was detected by Ki-67 antibody. Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides. RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0). Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0). Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination. The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5). Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%). CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation. However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells. Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices. Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data. Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.     

DNA ploidy analysis and cytologic examination of sorted cell populations from human breast tumors

Two different flow cytometric procedures were applied on cell samples from human breast tumors. One procedure involved DNA ploidy analysis on suspensions of isolated nuclei. The mean ploidy ratios of 27 benign breast lesions to chicken erythrocytes and rainbow trout erythrocytes were found to be 2.66 +/- 0.03 and 1.25 +/- 0.02, respectively. From the 45 stemlines found in a series of 43 carcinomas, 12 were diploid, 13 hyperdiploid and 20 near-tetraploid. No association was found between the lymph node status and the DNA ploidy level. The second procedure involved sorting fixed cells from DNA "windows" for the preparation of permanent cytologic specimens. The sorted cells appeared to be shrunken, but the morphologic quality was similar to that of imprint specimens from the same tumors, permitting discrimination between various types of normal cells and tumor cells. The combined use of both flow cytometric procedures may lead to greater insight into the relationship between the cytologic and cytogenetic heterogeneity of breast carcinomas.     

Flow cytometry DNA analysis on tumor cell subpopulation of human tumor specimens by exclusion of lymphohemopoietic cells

We describe a new flow cytometry procedure in which DNA analyses can be obtained selectively on pure, freshly obtained tumor cell subpopulations of human tumor specimens. This procedure is based on exclusion from analysis of the contaminating lymphohemopoietic cells mixed with tumor cells in tumor specimens. This exclusion is made possible by labeling all lymphohemopoietic cells with an antibody to HLe-1 (HLE), which is present on all lymphohemopoietic cells but on no other cells, and by gating against these labeled cells when analyzing for DNA. For the model system, a 1:1 mixture of normal human peripheral blood leukocytes and either of two human cancer cell lines, HEp-2 and MCF-7, normal leukocyte contamination can be reduced to 3.1% while retaining 94.7% of tumor cells for DNA analysis. Four examples of human tumor samples, two cases each of malignant effusions and lymph node metastases, were analyzed with this procedure. The results clearly indicate that this new method will improve ploidy analysis/aneuploidy detection and will make it possible to obtain more accurate cell-cycle analyses of tumor cells than have previously been possible. This new procedure will contribute to clinical and biological studies involving DNA of human tumors.     

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.     

A comparison between flow cytometric ploidy investigation and chromosome analysis of 32 human colorectal tumors

The correlation between flow cytometric ploidy investigation and classic chromosome analysis was studied in 32 human colorectal tumors. Flow cytometry was performed by nuclei isolation and DNA staining with ethidium bromide. Chromosome analysis was done after incubation with colcemid. In 12 cases, chromosome identification was possible by grouping according to the Denver system or by Q-banding. Generally, the measured DNA content corresponded well with the content expected from chromosome analysis, giving an average difference of 4%. In nine tumors, the measured DNA content was 4-18% higher than expected. Some of these discrepancies could be due to difficulties in identifying the corresponding cell populations in heterogeneous tumors. However, in general the number of cell populations and their quantitative representation by the two methods were statistically well correlated. The results indicate that flow cytometric ploidy investigation of colorectal tumors with the present technique is a reliable method, but also that a combination of both techniques may yield additional information about tumor cytogenetics.     

Use of leukocytes infiltrating bladder tumors as an internal reference for determining the DNA index by flow cytometry.

The use of leukocytes infiltrating bladder tumors as an internal standard for flow cytometric measurements of DNA and SG2M indices was investigated in comparison with the conventional use of chicken erythrocytes (chicken red blood cells; CRBC). The leukocytes were identified by indirect fluorescence, using a mouse anti-CD45 monoclonal antibody and a fluorescein isothiocyanate-conjugated goat anti-mouse Ig F(ab')2 fragment. DNA was stained with propidium iodide. The percentage of CD45-positive cells averaged 37% in 23 unimodal bladder tumors and 30% in 27 bimodal ones. The diploid peak of CD45-positive cells was always easily identified and permitted a more accurate calculation of the DNA index and the percentage of cells in SG2M phases than did the use of CRBC. Seven tumors that were classified as unimodal using CRBC as the standard did, in fact, contain a minor clone (with a DNA index of 1.80) that could be detected only when CD45-positive leukocytes were used as the internal standard.     

Comparison of DNA ploidy in routine fine needle aspiration biopsy samples and paraffin-embedded tissue samples

The nuclear DNA content was determined by flow cytometry (FCM) from unfixed fine needle aspiration (FNA) biopsy samples of 31 human tumors, and from the same tumors after their excision, fixation with formalin and embedding in paraffin. The ploidy of the histograms was the same in 29 (94%) of the 31 cases. The disagreement in two cases may be explained by clonal heterogeneity of the tumors. The DNA index of the aneuploid cases was identical in fresh and fixed samples. The coefficient of variation of the diploid peaks (P less than .001) and the mean percentage of S-phase cells (P = .06) were larger in the fixed samples. It is concluded that routine FNA biopsy is a practical and reliable method for collecting cells for FCM DNA ploidy determination.     

DNA ploidy in soft tissue sarcoma: comparison of flow and image cytometry with clinical follow-up in 93 patients

In soft tissue sarcoma, the prognostic importance of DNA ploidy status is limited. One possible explanation may be technical; small non-diploid stemlines will be diluted in relation to the presence of normal diploid cells and may not be detected by flow cytometry (FCM). We assessed DNA ploidy status in 93 tumors with both FCM and image cytometry (ICM). ICM may permit the exclusion of non-relevant cells. The ability of the two methods to detect non-diploid stemlines was compared, as were the prognostic consequences. The patients (54 males) had a median age of 69 years. Surgical procedures were performed on all patients. None of the patients had received preoperative radiotherapy or chemotherapy. FCM and ICM were performed with standard methods. The prognostic value was assessed with univariate and multivariate analysis. In 82 of the 93 tumors, a concordant ploidy status by FCM and ICM was found. In 5 FCM type 1-2 tumors (diploid), the identification of non-diploid stemlines by ICM did not influence the metastatic rates. Increasing tumor size, histotype other than liposarcoma, increasing malignancy grade, tumor necrosis, and ICM non-diploidy were univariate prognostic factors for metastasis. In a multivariate analysis, only tumor size larger than 9 cm was a prognostic factor. In about 10% of the tumors, a discrepancy between FCM and ICM ploidy status was found, but we could not find a consistent prognostic consequence of this. Neither FCM nor ICM ploidy status was an independent prognostic factor.     

Accuracy and reliability of flow cytometric DNA analysis using a simple, one-step ethidium bromide staining protocol

Sources of variation and error were investigated for a simple flow cytometric analysis of DNA content of detergent-isolated nuclei stained with ethidium bromide. Using the ploidy classes of mouse liver nuclei, deviations from linearity were assessed for three different instruments. In more extreme settings, the maximum deviations for a FACS instrument were up to 6 to 9%, but in general deviations were around 1% or lower for all instruments. As biological DNA standards, human peripheral lymphocytes and trout erythrocytes appeared to be suitable and easy to store frozen. The erythrocytes had dye-binding characteristics similar to those of human lymphocytes and a 20% lower fluorescence, thus being well suited as an internal standard, as was demonstrated in tumor ploidy analyses performed with varied tissue concentration. Staining homogeneity was improved when staining time was extended to 24 h, at which time male and female lymphocytes were completely separated with an average difference in DNA content of 1.9%. A small difference in fluorescence between mitogen-stimulated and unstimulated lymphocytes was reduced to less than 1% after 24 h of staining. In general, the manipulations of the conditions for the analysis resulted in maximum variations of around 1%, indicating the robustness and reliability of the technique.     

The clinical usefulness of determining ploidy patterns in human tumors as measured by slide-based Feulgen microspectrophotometry

Some aspects of the clinical value of the Feulgen microspectrophotometric assessment of DNA ploidy patterns in human tumors are reviewed. This method has been shown to be of predictive value for a number of tumor sites and may be independent of other prognostic indicators, such as the histopathologic grade. The association between ploidy and prognosis probably reflects the degree of chromosomal changes in the tumor cells; while it is probable that all malignant tumors are aneuploid, there is a tendency for the changes to be more extensive in more aggressive tumors. Thus, tumors with DNA modes that depart significantly from the diploid and tetraploid levels may have a worse prognosis than do tumors whose modes are at or close to these levels. This has clearly been shown for tumors of the breast, ovary, endometrium and several other sites. For some sites, including the cervix uteri and the large bowel, such a relationship is less clear, probably because tumors at these sites have frequently undergone extensive chromosomal changes that do not result in a significant deviation of the DNA mode from the euploid levels. The use of slide-based DNA analysis systems, in which the morphology of the cells being measured can be assessed, has advantages over flow cytometry that may be crucial in some situations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Image and flow cytometric analysis of DNA content in breast cancer. Relation to estrogen receptor content and lymph node involvement

A comparative flow-cytometric and image-cytometric study was performed on 166 human breast cancers. Parallel measurements of 67 cases showed a good correlation between the DNA indices measured with each of the techniques. However, minor ploidy abnormalities were detected with flow cytometry. Only with this technique about 70% of the tumors appeared to be aneuploid. DNA profiles obtained with image cytometry frequently contained DNA values above the modal G2 value of the tumor. At least part of these broadly scattered DNA profiles resulted from multiploid tumors, as was found with flow cytometry. The mean percentage of cells with DNA contents exceeding the modal G0G1 value in image-cytometric DNA profiles (non- G0G1 cells) appeared to be highest in aneuploid lymph-node-positive (N+) tumors and lowest in near-diploid lymph-node-negative (N-) tumors. Near-diploid tumors were more frequently (70%) estrogen receptor positive (ER+) than were aneuploid tumors (50%), whereas highly aneuploid tumors with low or negative ER contents tended to be N+. Tumors with a high percentage of non- G0G1 cells value were predominantly estrogen-receptor negative (ER-).     

Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells

We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obtained by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.     

DNA ploidy of bladder cancer using bladder biopsy supernate specimens

OBJECTIVE: To perform DNA image cytometry on 119 bladder biopsy supernate (BBS) specimens of transitional cell carcinoma (TCC) bladder to: (1) test the suitability of this cytologic specimen for use in DNA ploidy analysis, and (2) assess the value of DNA ploidy measured on this specimen as to the risk of tumor recurrence and survival. STUDY DESIGN: The histologic grade and cytologic grade were correlated, and the DNA ploidy produced was determined by image analysis of Feulgen-stained nuclei. Kaplan-Meier curves related age, sex, grade and DNA ploidy to recurrence of tumor and survival. Log rank analyses were used to ascertain the difference between the curves for each categorical variable. RESULTS: Urothelial cells derived from the BBS specimen were demonstrated to be representative of the tumor. The tumor recurrence rate was significantly higher (P = .0001) and the survival rate significantly lower (P = .0002) for patients with aneuploid tumors compared to those with diploid tumors. Patients with TCC 2 tumors had a significantly shorter time to recurrence (P = .003), although the relationship between ploidy and survival in this group was of marginal significance. CONCLUSION: The specimen was free of many of the problems associate with the other specimen types used for measuring DNA ploidy. The results show that the BBS specimen is diagnostically useful and suitable for DNA analysis, providing prognostically relevant information.     

Relationship of DNA ploidy to chemoresistance of tumors as measured by in vitro tests

To examine whether patients with aneuploid tumors might derive more benefit from chemotherapy than would patients with diploid tumors, predictive tests for determining resistance in human tumors were carried out and the test results compared with the DNA ploidy of the corresponding tumors. Multidrug-resistance in 15 kidney carcinomas grown as primary cultures was determined by immunofluorescence by Mab C219, which is specific for the plasma membrane glyco-protein P-170, and by the use of tritiated nucleotide incorporation after addition of doxorubicin. Aneuploid tumors had a higher tendency to be more sensitive than diploid tumors, but the correlation was not significant. This was confirmed by reanalyzing our earlier data on ovarian and lung cancers. In conclusion, DNA measurement using flow cytometry does not appear to be a suitable tool for prediction of resistance of human tumors to chemotherapy.     

Flow cytometric analysis of DNA content for ploidy determination in human solid tumors.

Flow cytometric studies of cellular DNA content were conducted in 26 patients with a variety of neoplasms. Cell dispersal was achieved with pepsin treatment, and a combination of ethidium bromide and mithramycin was used as DNA specific staining procedure. All measurements were conducted with a new sheath flow chamber in a PHYWE ICP 11 pulse cytophotometer. All but one patient with multiple myeloma had unimodal tumor cell DNA distributions. With human granulocytes as reference standard, 24 of 26 tumors were aneuploid; and of these, 23 showed varying degrees of hyperdiploidy. Except for one patient, ploidy abnormalities were stable on repeat examination.     

Gene dosage effects in human diploid and tetraploid fibroblasts

The effects of cell ploidy on the biochemical characteristics of cultured cells were compared using human diploid vs tetraploid fibroblasts isolated with a non-selective method. Their DNA replication was compared by thymidine incorporation, and DNA content by Feulgen staining and quantitative analysis. Their RNA and protein content, cell sizes and the specific activities of glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD) were assayed quantitatively. With the exception of RNA content, all other parameters demonstrated a 2-fold increase reflecting the increase in cell ploidy. These direct gene dosage effects on the genetic material and functional expression of the human genome were in contrast to previous observations in other species and validate the use of human intraspecific euploid hybrids for biochemical and genetic studies.     

The production and characterization of murine monoclonal antibodies to a DNA receptor on human leukocytes

Two murine mAb have been generated with a reactivity toward a 30,000 m.w. DNA binding protein found on the cell surface of human leukocytes; mAb 12A has an IgG1/k isotype, and mAb 24T has an IgG2b/k isotype. Both react with the DNA binding domain or adjacent region of the putative DNA receptor and inhibit the binding of [3H]DNA to PBMC at concentrations as low as 100 ng/ml. Stoichiometric studies indicate that both mAb react with monocytes and T cells with a kDa of 10(-7) M; about 0.5 x 10(6) molecules bind per cell at saturation. Flow cytometry indicated that 67% of lymphocytes and 98% of monocytes bore the DNA receptor. Dual labeling studies showed that 90% of B cells and 50% of T cells express the receptor; 50% of CD4+ T cells are receptor positive. Immunomatrices constructed with both mAb 12A and 24T allowed the receptor to be purified to a high degree of purity. A single protein of Mr 30,000 was readily observed after SDS-PAGE and silver staining of the gel; after electropheretic transfer of nitrocellulose this protein was shown to be a DNA binding molecule by use of a probe of biotin labeled DNA. These experiments provide further evidence to support the existence of a specific DNA receptor on human leukocytes; the availability of mAb to the receptor should be useful in its further characterization.     

DNA ploidy in colorectal cancer, heterogeneity within and between tumors and relation to survival

Flow cytometry was used to study the incidence of aneuploidy and to determine the significance of multiple sampling from colorectal tumors. DNA ploidy pattern has been proposed as a supplementary prognostic marker, but discrepancies in findings are major. DNA clonal heterogeneity, defined as two or more DNA aneuploid stemlines in the same tumor, is well established. However, most studies have been based on only one biopsy from each tumor. In our study multiple biopsies were taken from 163 patients (88 males and 75 females) electively operated for colorectal cancer. Tumor cells were harvested by fine needle aspiration from fresh frozen biopsies sampled at different sites of each tumor. DNA aneuploidy was detected in tumors from 145 patients (89%), and 18 patients (11%) had a solitary DNA diploid cell population. In a 79 month follow-up period 105 patients had died. Statistical analysis showed that distinction between diploidy and aneuploidy did not predict survival. However, grouping subpopulations into DNA diploid plus near diploid (DNA index (DI) 0. 97-1.15), DNA aneuploid with all aneuploid subpopulations in the interval 1.15-2.06, and DNA aneuploid with subpopulations with DI < 0.97 and/or DI > 2.06, showed a significant difference in survival in a Cox multivariate analysis including Dukes' stage P = 0.049 comparing the second group to the first and P = 0.01 comparing the third group to the first. In 21 (13%) patients only one subpopulation was found, 57 (35%) had two, 44 (27%) had three, and 41 (25%) had four or more different subpopulations. The association of DNA ploidy to survival is shown to be dependent on the number of biopsies analysed.     

Isolation and Characterization of a Near-Haploid Human Cell Line

Mammalian somatic cells are usually diploid. Occasional rare human tumors have been shown to have a hypodiploid karyotype. We have isolated a near-haploid subclone (P1-55) from a heterogeneous human leukemia cell line, KBM-7. These near-haploid cells have approximately half the human diploid DNA content and have a haploid karyotype except for a disomy of chromosome 8 (25, XY, +8, Ph(+)). This cell line maintains a majority of cells with a near-haploid karyotype for at least 12 weeks in culture. By serial subcloning, we have isolated near-haploid subclones that maintain ploidy for at least 8 months in culture. Near-haploid cells can also be efficiently isolated from mixed ploidy cultures by size selection. The availability of this human near-haploid cell line should facilitate the genetic analysis of cultured human cells.     

Flow cytometric analysis of DNA stemline heterogeneity in primary and metastatic breast cancer.

Flow cytometric DNA-ploidy analysis was used to investigate intratumor DNA stemline heterogeneity in primary breast carcinomas and lymph node metastases (LNM). The study was done in tumor specimens from 44 patients 35 of whom had LNM. In all, measurements were done in 214 different samples of primary tumors and 211 lymph nodes. Sixty-one percent (27/44) of the primary tumors were found to have multiple DNA aneuploid stemlines when the data of the separate samples per tumor (mean 4.9) were compared. Only five of 44 (11%) primary tumors were DNA diploid; two of these had DNA aneuploid metastases. Statistical analysis of these results indicated that, on average, four samples are needed for reliable determination of the DNA ploidy status of primary tumors by flow cytometry. In the majority of the cases (26/35), distinct tumor DNA stemlines found in LNM were also present in the primary tumor, which suggests that the generation of DNA ploidy diversity may have taken place prior to metastasis. Multiploidy was not related to tumor size but, particularly for LNM, was significantly correlated with age (r = 0.40, P = 0.02). The results of this study support the view that breast cancer is an extremely heterogeneous disease and that underestimation of this factor might account for the disagreement in literature about the prognostic value of DNA ploidy determinations.