DNA ploidy pattern and S-phase characteristics of metastatic melanoma.

Samples of 130 metastatic melanomas from 92 patients were analyzed by DNA flow cytometry. DNA aneuploidy was observed in 67% of the patients. DNA indices were evenly distributed from 0.6 to 2.6 Tumors originating from primary lesions in the lower extremities were more frequently DNA aneuploid than those of other sites. S-phase fraction (SPF) was evaluable from 73 tumors. DNA aneuploid tumors had a significantly higher SPF than diploid tumors, and females had a higher SPF than males. Furthermore, distant metastases had a higher SPF than metastases in regional lymph nodes and in transit metastases, probably indicating a higher growth potential in metastases spreading to distant sites.     

Optimizing flow cytometric DNA ploidy and S-phase fraction as independent prognostic markers for node-negative breast cancer specimens.

Developing a reliable and quantitative assessment of the potential virulence of a malignancy has been a long-standing goal in clinical cytometry. DNA histogram analysis provides valuable information on the cycling activity of a tumor population through S-phase estimates; it also identifies nondiploid populations, a possible indicator of genetic instability and subsequent predisposition to metastasis. Because of conflicting studies in the literature, the clinical relevance of both of these potential prognostic markers has been questioned for the management of breast cancer patients. The purposes of this study are to present a set of 10 adjustments derived from a single large study that optimizes the prognostic strength of both DNA ploidy and S-phase and to test the validity of this approach on two other large multicenter studies. Ten adjustments to both DNA ploidy and S-phase were developed from a single node-negative breast cancer database from Baylor College (n = 961 cases). Seven of the adjustments were used to reclassify histograms into low-risk and high-risk ploidy patterns based on aneuploid fraction and DNA index optimum thresholds resulting in prognostic P values changing from little (P < 0.02) or no significance to P < 0.000005. Other databases from Sweden (n = 210 cases) and France (n = 220 cases) demonstrated similar improvement of DNA ploidy prognostic significance, P < 0.02 to P < 0.0009 and P < 0.12 to P < 0.002, respectively. Three other adjustments were applied to diploid and aneuploid S-phases. These adjustments eliminated a spurious correlation between DNA ploidy and S-phase and enabled them to combine independently into a powerful prognostic model capable of stratifying patients into low, intermediate, and high-risk groups (P < 0.000005). When the Baylor prognostic model was applied to the Sweden and French databases, similar significant patient stratifications were observed (P < 0.0003 and P < 0.00001, respectively). The successful transference of the Baylor prognostic model to other studies suggests that the proposed adjustments may play an important role in standardizing this test and provide valuable prognostic information to those involved in the management of breast cancer patients.     

DNA ploidy pattern and S-phase characteristics of metastatic melanoma

Samples of 130 metastatic melanomas from 92 patients were analyzed by DNA flow cytometry. DNA aneuploidy was observed in 67% of the patients. DNA indices were evenly distributed from 0.6 to 2.6 Tumors originating from primary lesions in the lower extremities were more frequently DNA aneuploid than those of other sites. S-phase fraction (SPF) was evaluable from 73 tumors. DNA aneuploid tumors had a significantly higher SPF than diploid tumors, and females had a higher SPF than males. Furthermore, distant metastases had a higher SPF than metastases in regional lymph nodes and in transit metastases, probably indicating a higher growth potential in metastases spreading to distant sites.     

Malignant potential of dysplastic endocervical epithelium assessed by ploidy status,S-phase fraction and C-myc expression

The aim of this study was to determine if dysplastic endocervical cells (EC) posses a neoplastic potential as precursor lesions to adenocarcinoma in situ (AIS). The malignant potential was determined by assessing the ploidy status and proliferative activity by flow cytometry and c-myc expression by immunohistochemistry. The studied parameters were assessed separately in morphologically normal, dysplastic and malignant EC. The chi 2 test showed significant association of malignant EC with aneuploidy (p = 0.008) and high proliferative activity (p = 0.042). Since one third of the dysplastic EC are also aneuploid and show high mitotic activity, they probably have malignant potential as well. The dysplastic EC showed a significant association with c-myc oncogene expression (p = 0.028). Our results indicate the existence of pre-malignant glandular lesions, while the immunohistochemical detection of c-myc protooncogene could be helpful in detection of EC with malignant potential, even without any dysplastic morphological changes.     

MIB-1 and S-phase cell fraction predict survival in non-Hodgkin's lymphomas

The monoclonal antibody anti-Ki67 is used to detect proliferating cells, but its main limitation is the requirement of fresh-frozen material. On a series of patients with non-Hodgkin's lymphoma, we used a Ki67 equivalent monoclonal antibody, the recently proposed MIB-1, on formalin-fixed histopathological material using microwave antigen retrieval. MIB-1 expression was analysed in relation to other proliferation indices, such as autoradiographic ³H-thymidine labelling index (³HTL1) and flow cytometric S-phase cell fraction (FCM-S) and to pathological status. Moreover, the prognostic relevance of the cell kinetic indices was defined in uni- and multivariate analyses including histology and tumour stage. The relationship between MIB-1 index and the other proliferation indices was statistically significant even though the correlation coefficient was around 0.6. The MIB-1 index was also related to the REAL (Revised European American Lymphoma) classification, but not to the Ann Arbor stage classification. Univariate analysis showed that the MIB-1 index was a significant predictor of 6-year survival in the overall series and in distinctly analysed low-grade and high-grade lymphoma subgroups. With regard to S-phase indices, ³HTLI was a powerful prognosticator in patients with high-grade histologies and FCM-S in patients with low-grade histologies. Multivariate analyses revealed that MIB-1 indiex, ³HTLI and FCM-S retained their prognostic significance independent of histology. In conclusion, the MIB-1 antibody provides prognostic information in non-Hodgkin's lymphomas and has the main advantage that it can be used in formalin-fixed, paraffin-embedded specimens.     

Colorectal carcinoma. DNA ploidy pattern and prognosis with reference to tumor DNA heterogeneity.

Nuclear DNA content was measured in 72 colorectal carcinomas using single-cell microspectrophotometry on Feulgen-stained smears. Four samples were analyzed from each tumor. Patients were followed for 41-65 months (average, 53). DNA heterogeneity (both aneuploid and nonaneuploid patterns) was present in 44% of the cases. Sixty-eight percent of the tumors showed an aneuploid DNA pattern in at least one of the samples. Patients with nonaneuploid tumors tended to have a survival advantage over patients with homogeneously aneuploid tumors and demonstrated a significantly longer disease-free survival. The DNA ploidy pattern is of potential value in conjunction with histopathologic prognostic parameters in colorectal carcinoma. Since colorectal tumors exhibit pronounced DNA heterogeneity, multiple samples are required from each tumor to permit a proper evaluation of its DNA pattern. The DNA heterogeneity may represent tumor progression and can partly explain the conflicting results reported concerning DNA pattern and prognosis in colorectal carcinoma.     

DNA ploidy and survival in breast cancer patients

Flow cytometric DNA ploidy measurements using frozen or deparaffinized tumor specimens were performed on 565 primary breast cancers from patients treated in the period 1975-1984. Twenty-nine percent of the cases were diploid, 61% had a single aneuploid stemline, and 10% were multiploid. Aneuploid tumors more often had negative estrogen receptor values than diploid tumors, but no significant correlation was found between ploidy class and TNM stage. Patients with more than ten positive axillary lymph nodes had predominantly aneuploid tumors. Overall and distant relapse-free survival were higher for patients with diploid tumors and low-aneuploid tumors. Stratification of the patients according to degree of lymph node involvement, TNM stage, and menopausal stage showed that the prognostic effect of aneuploidy was apparent predominantly in patients with locally advanced disease. Postmenopausal node-positive patients with diploid tumors had a significantly better prognosis than those with aneuploid tumors, but this difference was not found for the comparable premenopausal group. Multivariate analysis with the Cox proportional hazards model indicated that ploidy is an additional, independent prognostic factor in postmenopausal patients.     

Intratumoral variations in DNA ploidy and s-phase fraction in human breast cancer.

To study intratumoral DNA ploidy heterogeneity and S-phase fraction (SPF) variability, we prospectively collected five different samples from 48 breast carcinomas and each sample was analysed separately by flow cytometry. Aneuploidy rate was 89.6% after analysis of four or five samples. DNA ploidy heterogeneity, i.e., different samples classified as either DNA euploid or DNA aneuploid in the same tumor was seen in 17%, and DNA index heterogeneity, i.e., tumor populations with different DNA indices (DIs) seen in different samples was 44%. A statistical model defining SPF heterogeneity is proposed. SPF heterogeneity as defined by us was 71%, and as expected the SPF heterogeneity rate increased significantly with increasing number of analysed samples. Four or more samples are needed to detect the most deviant (highest) SPF values. An unrecognized intratumor heterogeneity of DNA ploidy and SPF may partly explain the conflicting results reported in the literature on the above prognostic indicators.     

DNA Ploidy,Bromodeoxyuridine labelling index,S-phase fraction and AgNOR counts in brain tumours

DNA ploidy and the proliferative potential in 88 brain tumours were investigated using the bromodeoxyuridine labelling index (BrdUrd LI), S-phase fraction (SPF) and an argyrophilic nucleolar organizer regions (AgNOR) technique. The study included 65 highly malignant (AIII–AIV), and 23 low-grade (AI–AII) gliomas. One fragment of the tumour was fixed in Carnoy's solution for AgNOR test, while the other fragments were used for flow cytometric determination of the labelling index, SPF and DNA ploidy. For the BrdUrdLl, tumour samples from each patient were incubated in vitro for one hour at 37°C with BrdUrd using a high pressure oxygen method. After fixation and staining, the percentages of BrdUrd-labelied cells (BrdUrdLI) and unlabelled S-phase cells (SPF) were evaluated. The tumours showed variability in the BrdUrdLI values, SPF and AgNOR counts/cell nucleus. However, grade dependent differences in the proliferating rate were only found to exist on the basis of BrdUrdLI and AgNOR counts. The same percentage of DNA aneuploidy (56 %) was found in high-grade as well as in low-grade gliomas. A linear – regression analysis showed a significant correlation between the results of three applied methods: BrdUrdLl, SPF and AgNOR counts.     

S-phase cell fraction in the prognosis of Dukes' stage D colorectal carcinoma

Abstract. The S-phase cell fraction was prospectively defined as the [³H]-thymidine labeling index ([³H]dT LI) and flow cytometric S-phase (FCM-S) on 52 Dukes' D colorectal cancers. FCM-S estimates obtained by using different modeling systems were superimposible but they were weakly related to [³H]dT LI detected on the same tumour. Moreover, FCM-S values were higher in aneuploid than in diploid tumours, whereas [³H]dT LI values were independent of DNA-ploidy status. [³H]dT LI and FCM-S were also related differently to some clinical and pathological features such as tumour site and histology. [³H]dT LI and FCM-S were indicative of prognosis in terms of 2–year freedom from progression and overall survival. However, product-limit survival analysis showed an unexpectedly better freedom from progression and overall survival for patients with high S-phase tumours than for those with low S-phase tumours as defined by both cell kinetic variables.     

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.     

Prognostic biomarkers in renal cell carcinoma: relevance of DNA ploidy in predicting disease-related survival

OBJECTIVE: To investigate the prognostic value of DNA ploidy, Ki-67 index and p53 expression in relation to disease-related survival in a consecutive series of patients with renal cell carcinoma (RCC). MATERIAL AND METHODS: The study group consisted of 64 RCC patients treated by radical nephrectomy. Histological type, pathological staging and nuclear anaplasia were assessed according to the WHO classification, TNM system and Fuhrman grading criteria, respectively. Ploidy was determined by DNA flow cytometry using two sampling methods (frozen vs paraffin-embedded tissue). Ki-67 and p53 were evaluated by immunohistochemistry techniques using two cutoff points (10% vs mean value) for staining interpretation. Kaplan-Meier and Cox regression analyses were used for prognostic evaluation. RESULTS: Thirty-one tumors (48.4%) showed DNA diploidy and 33 (51.6%) were DNA aneuploid. Concordance between both ploidy measurement methods was found in 85.5% of cases (p=0.0455). The mean values for Ki-67 and p53 immunostaining were 3.65% (0-23.5%) and 5.90% (0-55.9%), respectively. DNA ploidy significantly correlated with staging, tumor size (pT), nuclear grading, and Ki-67 (mean value cutoff). Ki-67 (10% cutoff) correlated with staging and pT, while p53 (mean value cutoff) was associated with Ki-67 (mean value cutoff). There were significant differences between survival curves for pathological stage, pT, nuclear grade, ploidy, Ki-67 (both cutoffs), and p53 (10% cutoff). By univariate regression analysis, stage III and stage IV, pT3, aneuploidy, high Ki-67 (both cutoffs), and p53 overexpression (10% cutoff) showed significant correlations with worse disease-related survival. In addition, DNA aneuploidy significantly correlated with poor prognosis within stages I/II (p=0.0355) and stages III/IV (p=0.0138) of the disease. CONCLUSION: The results indicate that DNA ploidy has relevant prognostic value in RCC, adding useful information to the classic histopathological indicators of clinical outcome.     

Flow cytometric S-phase fraction measurement in breast carcinoma: Influence of software and histogram resolution

BACKGROUND: S-phase fraction (SPF) measurement by flow cytometry is a clinically useful prognostic factor in patients with breast carcinoma. Standardized SPF determination is essential. As part of a multicenter study, we evaluated the influence of the choice of software and histogram resolution (256, 512, or 1,024 channels) on SPF quantification. METHODS: One hundred thirty-three DNA histograms were analyzed in three laboratories with Modfit 5.2, Modfit LT, and Multicycle AV software. Strict rules for histogram interpretation and software management were applied. The following five options were compared: MF 5.2 1024, MF 5.2 256, MF LT 256, MC AV 256, and MC AV 512. RESULTS: In the DNA diploid and aneuploid groups, SPF distributions were not statistically different among the five options. Excellent quantitative correlations were obtained between pairs of options. When using tertiles as cutpoints for SPF classification, concordance rates ranged from 79.7% to 93.2% for DNA diploid samples and from 87.8% to 95.9% for DNA aneuploid samples, the best results being obtained with software working with a similar histogram resolution. CONCLUSIONS: Standardized use of commercially available software, including the choice of histogram resolution, provides comparable SPF results.     

Prevalence of gallstones and gallbladder disease in Canadian Micmac Indian women.

The prevalence of gallstones and gallbladder disease was studied between October 1973 and June 1976 in Canadian Micmac Indian women aged 15 to 50 years in an inland rural community near Shubenacadie, NS. Of 132 women at risk 98 underwent cholecystography, 6 had a history of cholecystectomy (verified from hospital records) and 3 had cholecystectomy because of cholecystitis during the 3 years of the study. Of the 17 abnormal cholecystograms 10 showed radiolucent gallstones, and repeated studies documented gallstones in 6 of the 7 radiographs on which the gallbladder was not visualized. The prevalence of gallstones was found to be 211/1000, and that of gallbladder disease, 240/1000. The peak prevalence was at 30 to 39 years of age. The women with gallbladder disease were significantly more obese and of greater parity than those without gallbladder disease even when age was controlled. The Micmac Indian women of Nova Scotia appear to be at a much higher risk for the development of cholesterol gallstones and gallbladder disease than Caucasian women in Framingham, Massachusetts.     

Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S-phase fraction, and surface phenotype using single laser instrumentation.

We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (greater than 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.     

DNA and S-phase distribution and incidence of metastasis in human primary lung carcinoma

The aim of the study was to investigate the relationship between DNA and S-phase distribution in primary non-small-cell lung carcinomas with the incidence of metastasis. Patients with non-small-cell lung carcinomas were divided into two groups depending on whether at time of surgery there were metastases or not, and these groups were correlated with the data obtained by flow cytometry or autoradiography. As expected from other studies, survival time was significantly longer for those patients without metastases at time of surgery (P = .0002) and the incidence of metastasis was significantly higher when the primary tumor was greater than or equal to 70 cm3 (P = .026). In this study, a total of 185 fresh specimens of lung carcinomas were investigated by flow cytometry. Patients with aneuploid tumors had a higher tendency to have metastases (P = .016). Patients with tumors with a higher proportion of S-phase cells measured by either flow cytometry or autoradiography demonstrated significant increase in the formation of metastases (P = .02 and P = .05). We feel that these results warrant further investigation with other primary tumors. A comparison of primary tumors that are known to rapidly metastasize vs. those that either slowly or rarely metastasize may prove to yield valuable insight into the important factors associated with metastatic potential.     

DNA content, kinetic complexity, and the ploidy question in Candida albicans.

Candida albicans is a dimorphic fungus that is pathogenic for humans. No sexual cycle has been reported for this fungus, and earlier reports have differed on whether typical strains of C. albicans are haploid or diploid. Previous estimates of the DNA content of C. albicans varied by one order of magnitude. We used three independent methods to measure the kinetic complexity of the single-copy DNA from a typical strain of C. albicans (strain H317) to determine the DNA content per haploid genote; we obtained values of 15 and 20 fg per cell by using S1 nuclease and hydroxyapatite assays, respectively. Optical assays for DNA reassociation kinetics, although not definitive in themselves, yielded values in this range. Chemical measurements of the DNA content of several typical strains, including strain H317, yielded values clustered about a mean of 37 fg per cell. We concluded that these strains are diploid.     

Computer-aided S-phase fraction determination in DNA static cytometry in breast cancer. A preliminary methodologic study on cytologic material.

This methodologic study was performed on a single-cell-cycle breast carcinoma to evaluate the feasibility of computer-aided S-phase fraction determination in DNA static cytometry. The investigation was performed on Feulgen-stained cytologic material in which the total optical density values of 1,000 consecutive, randomly selected nuclei were analyzed (MultiCycle software). A good correlation in the S-phase fraction value with flow cytometry was obtained when the G2/G1 ratio was fixed at 1.95, when the histogram data points were smoothed at least once and the coefficient of variation of the G2 peak was the same as that of G0-G1 or when a first-order S-phase polynomial model was used. The percentages of nuclei in G0-G1 and G2 were somewhat similar to those obtained with flow cytometry. The greatest discrepancy with flow cytometry was observed in the value of the coefficient of variation of the G0-G1 peak of the static cytometric data: it was at least twice as great. It always remained high despite the software options used. As for the influence of the sample size in the S-phase calculation, the software was also run on samples of 600 and 200 nuclei. When the G2/G1 ratio was fixed at 1.95, the data obtained from 600 nuclei did not differ from those obtained with 1,000 nuclei, whereas an analysis on 200 nuclei showed a substantial variation. The software also allowed calculation of the ratio of the G0-G1 peak of the neoplastic population against that of the diploid reference (DNA index), the value of which in flow cytometry was 1.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA content and survival in mammary carcinoma

In a retrospective study of archival fine needle aspiration biopsy material from 112 patients with primary mammary carcinoma, the DNA distribution patterns of the cancer cell populations were determined. A distinct correlation was found between the occurrence of certain types of DNA distribution histograms and the survival time of the patients. Thus, the data indicate that DNA determinations can give prognostic information, in the individual case, over and above that furnished by clinical staging and morphologic criteria.