DNA cytometry in pleomorphic adenomas with cytologic atypia

OBJECTIVE: To investigate the nuclear DNA content of pleomorphic adenomas with cytologic atypia. STUDY DESIGN: Analysis was performed on new, fuchsin-stained samples of 10 selected cases of pleomorphic adenoma with cytologic atypia. Morphometric analysis was done by a computer-assisted image cytometry system and consisted of the determination of DNA indices, Auer DNA histogram types, and 3c and 5c exceeding rates. RESULTS: Eight cases were diploid and two cases aneuploid according to the DNA index. The Auer histogram was type I in five cases and type III in the others. In the two aneuploid cases the 3c exceeding rate was > 10% and the 5c exceeding rate > 1%. CONCLUSION: Atypical cells in pleomorphic adenomas with cytologic atypia carry abnormal amounts of DNA. Image cytometry can make detecting very low numbers of aneuploid cells easier due to its higher resolution as compared to that of flow cytometry.     

Cell cycle flow cytometric analysis in the diagnosis and management of colorectal carcinoma

OBJECTIVE: To establish prognostic models and protocols for individualized management in colorectal carcinoma patients based on both clinical and DNA flow cytometric parameters. STUDY DESIGN: Prospective study of 88 colon carcinoma patients with a minimum follow-up of 12 months, operated on with the intent to cure and not treated with radiotherapy or chemotherapy. All the cases were subjected to a clinical evaluation: age, sex, tumor localization and size, histologic grade, tumor stage, disease-free interval, survival and flow cytometric study (ploidy, DNA index and S-phase fraction [SPF]). RESULTS: From the total of 88 neoplasms studied, 56 (63.6%) were from males and 32 (36.4%) from females; 30 (34%) were located in the right side of the colon, 7 (8%) in the transverse colon and 51 (58%) in the left side of the colon. Eleven (12.5%) were stage I, 52 (59.1%) stage II and 25 (28%) stage III. Forty-two (47.7%) were diploid and 46 (52.3%) aneuploid. The S-phase mean was 14.6% (12% for diploids and 16.9% for aneuploids). During the follow-up period, 26.1% of diploid tumors recurred, whereas aneuploid tumors recurred in 36.9% (P < .05). SPF from diploid and aneuploid tumors was analyzed separately. CONCLUSION: Regarding relapse-free interval, the behavior of diploid tumors with a high SPF was similar to that of aneuploid ones. Two kinetic profiles were established, favorable (diploid tumors with low S phase) and unfavorable (diploid with high S phase and all aneuploid tumors), that had significant prognostic value for progression and survival and that allowed identification of patients at high risk of recurrence. We formulated a prognostic index according to SPF and tumor stage that has discriminatory capacity for biologic behavior in colorectal tumors.     

Flow and image cytometry in thymic neoplasia: correlation with clinical outcome

OBJECTIVE: To compare nuclear DNA by flow (FCM) and image cytometry (ICM) in thymic neoplasms and to relate results to clinical outcome. STUDY DESIGN: DNA ploidy of 44 thymomas and 6 thymic carcinomas was studied by FCM and ICM of single nuclear suspensions from paraffin blocks. RESULTS: By FCM, 33 thymomas (75%) and one thymic carcinoma (17%) were diploid; 6 thymomas (14%) and 4 thymic carcinomas (67%) were aneuploid. By ICM, 36 thymomas (82%) were diploid; 7 thymomas (16%) and 6 thymic carcinomas (100%) were aneuploid. Mean follow-up in 44 cases was 46.2 months (range, 1-162). Ten patients with persistent/recurrent disease included four with thymic carcinoma, who died of the disease (two aneuploid by both techniques, two aneuploid by ICM with unsatisfactory/diploid FCM). Four had invasive thymoma and recurrence after 13-150 months (two diploid and two aneuploid by both methods), one had diploidy and noninvasive thymoma that recurred at 92 months, and one had an epithelial thymoma that recurred at 144 months (aneuploid by FCM, diploid by ICM). CONCLUSION: The results obtained in this preliminary, retrospective study show a high concordance between FCM and ICM; aneuploidy correlated with poor outcome by both methodologies. While these findings are encouraging, larger numbers of cases will be needed to define the role of FCM and ICM in predicting outcome in thymic tumors.     

Digital cell image analysis of Ewing's sarcoma.

We analyzed the lesional tissue in Ewing's sarcoma by means of a cell image processor computing 15 nuclear parameters in order to quantify the morphologic variability of the tumor cell nuclei. To this end we combined 32 cases (350-400 Feulgen-stained nuclei analyzed per case) in a data file, which was then subjected to principal component and canonical analyses. We found considerable heterogeneity within the cell nucleus population of Ewing's sarcomas. Indeed, after the arbitrary subdivision of the file into two cell classifiers (CC1 and CC2 cell types) on the basis of the first canonical function, the 32 Ewing's sarcomas showed a great deal of variability in the proportion of CC1 and CC2 cell nucleus types. The nuclei of the CC1 type had a more finely textured chromatin when compared to the CC2 type, the nuclei of which exhibited a more granular chromatin pattern. Additionally, these 32 Ewing's sarcomas were characterized by three distinct DNA histogram types. Eight tumors displayed a diploid nonproliferating DNA histogram pattern (type A), 11 a diploid proliferating (type B) and 13 an aneuploid (type C) DNA histogram profile. We found a highly significant relationship between these DNA histogram types and the CC1:CC2 cell type percentage ratio. The eight Ewing's sarcomas with a type A DNA histogram contained a significantly higher proportion of CC1 cell type nuclei as compared to the 13 tumors with a type C DNA histogram, which contained a great proportion of CC2 cell type nuclei.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Prognostic significance of DNA aneuploidy and p21 ras oncoprotein expression in colorectal cancer and their role in the determination of treatment modalities

The purpose of the present study was to investigate the prognostic significance of DNA ploidy, S-phase fraction and p21 ras oncoprotein expression in patients with colorectal cancer and to correlate these factors with the clinical behavior of the tumors and their response to therapy. Of 79 patients with colorectal cancer 57% (45/79) had early stage disease. Forty-one percent (32/79) had aneuploid tumors while 30% (24/79) of the tumors had a high (>10%) S-phase fraction. p21ras oncoprotein expression was detected in 38% (30/79) of tumors. Patients with aneuploid tumors had a worse prognosis than patients with diploid tumors (p=0.0002). Similarly, patients with high S-phase fraction tumors had a shorter survival than those with low S-phase fraction tumors (p=0.005). No such difference was found between p21 raspositive and p21 ras-negative tumor subgroups. In early stage colorectal cancer, aneuploidy was closely correlated with disease outcome (p=0.029). Early stage patients with diploid tumors who received radiotherapy and chemotherapy had a better prognosis than patients with aneuploid tumors. In conclusion, DNA ploidy is a significant and independent prognostic factor in colorectal cancer. Aneuploidy and genetic alteration of the p21 ras oncoprotein are important in determining the biological aggressiveness of colorectal cancer. Furthermore, DNA ploidy may identify those subgroups of patients with early stage disease who may benefit from more aggressive treatment.     

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.     

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.     

Comparison between flow cytometry and image cytometry in ploidy distribution assessments in gynecologic cancer.

The DNA content in 37 tumors from 34 women with gynecological cancer was measured by flow cytometry (FCM) and interactive image cytometry (ICM). Agreement was obtained in 81% of cases as regards ploidy levels, but seven tumors (19%) showed different ploidies. Of these, five were classified as diploid by FCM but either aneuploid (three cases) or polyploid (two cases) by ICM. Two other tumors were aneuploid by ICM but polyploid (one case) and unclassifiable (one case) by FCM. All tumors classified as aneuploid by FCM were also aneuploid by ICM, and all tumors classified diploid by ICM were also diploid by FCM. Of six patients whose tumors were classified as euploid (five diploid and one polyploid) by FCM but classified as aneuploid by ICM, five relapsed, and three of these have died of disease. On the basis of these findings, it is concluded that ICM must be performed in cases classified as diploid by FCM to ensure that small subpopulations of aneuploid tumor cells are not overlooked.     

Image cytometric DNA analysis in human breast cancer analysis may add prognostic information in diploid cases with low S-phase fraction by flow cytometry.

Measurements of DNA ploidy can be performed either with image cytometry (ICM) or flow cytometry (FCM); both methods provide independent prognostic information in primary breast cancer. The aim of the present investigation was to compare the two methods and to relate the findings to prognosis (median follow-up 42 months). Concordance in ploidy status (diploid, tetraploid, aneuploid) was obtained in 76% of the samples (168/222). When the fraction of S-phase cells (SPF) from FCM analysis was also taken into consideration, four different groups of samples were obtained (Flow I-IV), which were considered to correspond to the Auer classification (Auer I-IV) of DNA histograms obtained from image cytometry. Complete concordance between the two techniques now was 70% (155/222). Samples classified as Flow I (diploid or near-diploid with low SPF) and Auer I had a distant metastasis rate of 3/60 (5%), as compared to 62/154 (40%) for all other combinations of the Flow and Auer classifications taken together. Thus, the only findings of prognostic importance were that some samples were Flow I but not Auer I, or vice versa. These two groups represent 17 (7.7%) and 14 (6.3%), respectively, of the total number of samples, and had frequencies of distant metastasis similar to those of the other high-risk groups, namely, 7/17 and 5/14, respectively. In a multivariate analysis, flow cytometric S-phase value was a stronger prognostic factor than either the Flow and Auer classification. We conclude that when routine FCM DNA analysis is used, diploid or near-diploid samples with a low S-phase value should be reanalyzed with ICM.     

Cytophotomeric DNA-analysis of aspirated cells from prostatic carcinoma.

In the present study material obtained from prostatic lesions by transrectal aspiration biopsy was subjected to a comparative morphologic and cytophotometric DNA analysis. Based on the morphologic pattern, the clinical material was divided into benign lesions (prostatic hyperplasia), suspected prostatic malignancy and highly, moderately and poorly differentiated prostatic carcinoma. The cytochemical analysis, based on quantitative cytophotometric measurements of Feulgen stained nuclei, showed that cell nuclei from benign lesions (prostatic hyperplasia) exhibited the normal diploid amount of DNA. Contrary to this, cell populations from prostatic malignancies, were characterized by various degrees of heteroploidy, i.e. the existence of cells with nuclear DNA quantities increased above the normal diploid level. A general correlation between degree of heteroploidy (frequency of heteroploid cells) and degree of clinical malignancy seemed to exist; high grade malignant prostatic carcinoma (poorly differentiated) exhibited a pronounced degree of heteroploidy with more or less distinct aneuploid stemlines, whereas low-grade prostatic carcinomas (highly differentiated) were more similar to benign cell populations, in showing a large proportion of cells with a diploid DNA quantity suggesting the existence of diploid or near diploid stemlines. Cases morphologically classified as moderately differentiated prostatic carcinoma, which previously have been shown to exhibit individual variations in degree of clinical malignancy, also showed large individual variations in degree of hetyroploidy. Approximately half of these cases had cytochemical DNA characteristics similar to that of highly differentiated prostatic carcinoma in showing a modal DNA value in the diploid region, while the other half showed cytochemical characteristics similar to that of poorly differentiated prostatic carcinoma, i.e. aneuploid DNA distributions. However, no morphologiifferentiated prostatic carcinoma. In conclusion it can be stated that the present investigation suggests the possibility that quantitative cytochemical DNA analysis may be used in combination with, and offer additional information to, morphologic analysis in the malignancy grading of prostatic carcinoma. A future clinical follow-up, now in progress, will hopefully give a more definite answer to that idea.     

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.     

Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

OBJECTIVE: To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. STUDY DESIGN: Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. RESULTS: Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. CONCLUSION: There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.     

Flow cytometric analysis of DNA aneuploidy in primary and metastatic human solid tumors

DNA histograms were measured by flow cytometry for 656 human solid tumors (365 primary and 291 metastatic). The proportion of aneuploid cells in cell suspensions obtained by mechanical disaggregation was significantly higher than those obtained after enzymatic disaggregation (collagenase + DNAse) of the same tumor. A strong correlation was observed between the values of DNA-indices measured after staining with propidium iodide and with 4',-6-diamidino-2-phenylindole (r = 0.97). Aneuploid cells were observed in 430 tumors (66%); 30 of these had two aneuploid stemlines, and two had three aneuploid stemlines. The overall frequency of aneuploidy was 61% among primary and 71% among metastatic tumors. The median value of the DNA index was 1.67 for 224 primary aneuploid tumors and 1.68 for 206 metastatic aneuploid tumors. For most diseases, the largest proportion of aneuploid primary and metastatic tumors had DNA-indices in the hypertriploid region. No major differences in frequency and degree of aneuploidy was observed between primary and metastatic tumors. For carcinomas of the bladder and prostate, frequency of aneuploidy was higher among poorly differentiated, than among moderately and well-differentiated tumors. For carcinomas of the breast and for sarcomas, tumors with DNA-indices of greater than 2.0 were observed mostly in the poorly differentiated group. For patients with carcinomas of the bladder and prostate most tumors at earlier stages of disease were diploid; whereas most tumors at later stages of disease were aneuploid. For patients with carcinomas of the ovary, colon, and kidney, no relationship between stage of disease and aneuploidy was evident.     

Proliferative characteristics of primary and metastatic human solid tumors by DNA flow cytometry

The percentage of cells in S-phase (S-index) was calculated from DNA histograms of 453 primary and metastatic human solid tumors (predominantly bladder, breast, colorectal, renal, prostate, ovarian and lung carcinomas, melanomas, and sarcomas). S-indices varied widely among both primary and metastatic tumors (1-48%); there was no significant difference in S-indices between primary and metastatic tumors. The S-indices for aneuploid tumors were significantly higher than for diploid tumors. When data for all aneuploid tumors were analyzed collectively, there was no significant relationship between S-index and DNA ploidy index. However, for colorectal and ovarian carcinomas S-indices increased, and for lung carcinomas S-indices decreased with elevation in the degree of DNA-ploidy. Lung carcinomas had the highest S-indices. Comparison of flow cytometry (FCM) and cytology data indicated that for most diploid tumors S-indices reflect the proportion of S-phase cells among a mixed population of normal and tumor cells. For most aneuploid tumors, the proportion of tumor cells estimated cytologically was similar to the proportion of aneuploid cells estimated by FCM. For a small proportion of aneuploid tumors a comparison of cytology and FCM data indicated the presence of a predominant diploid tumor stemline and a minor stemline with aneuploid DNA content. There was a wide spread in the values of S-indices within tumor groups defined by degree of differentiation and stage of disease at surgery.     

The significance of DNA measurements in a histologically defined subset of infiltrating ductal carcinomas of the breast with long-term follow-up

The nuclear DNA content and other karyometric parameters were evaluated in a histologically homogeneous group of invasive ductal carcinomas of the breast from 13 patients who survived 25 years after radical mastectomy and from 13 controls matched for histologic tumor grade, lymph node status, tumor size and patient age. The nuclear DNA content and other morphometric features were evaluated by image analysis (using a modified TICAS system) on 12-microns-thick, Feulgen-stained sections. The DNA content of the tumors of both the long-term survivors and the controls varied from the diploid range to highly aneuploid (with a large proportion of the cells having a DNA content above 5N). Overall, the tumors of the controls exhibited a higher ploidy, a greater deviation from the diploid range and a greater variation of nuclear size than did the tumors of the long-term survivors. These results suggest that these measurements may be helpful in yielding prognostic information among sets of histologically identical breast tumors of similar pathologic stage.     

Comparison of quantitative histomorphometry and DNA ploidy in tissue sections of prostate carcinoma

OBJECTIVE: To examine the ability of quantitative histomorphometry to predict DNA ploidy of prostate carcinoma in biopsy tissue sections assigned after quantitation by nuclear digital image analysis. STUDY DESIGN: Thirty-five diploid, 35 tetraploid and 35 aneuploid prostatic carcinomas in biopsies, assessed by the CAS 200 image analyzer (Bacus Laboratories, Lombard, Illinois, U.S.A.), were reevaluated by the Bacus Laboratories Incorporated Slide Scanner, a microscope that quantifies histologic images. Thirty-one histomorphometric features from cancer cells were captured at 40 x magnification, averaged across tilesfor each case and incorporated into a multivariate discriminant model to determine which features predicted ploidy interpretation by nuclear image analysis using the CAS 200. RESULTS: On average, 60 and 15 minutes were required to perform nuclear image analysis and histomorphometry, respectively. The multivariate discriminant model identified configurable run length, difference variance, contrast, inverse difference moment, sum entropy and diagonal variance as histomorphometry features capable of distinguishing diploid from nondiploid tumors (P < .05). Cross-validation studies showed the model correctly classified 74.3% of the diploid and 57.1% of the nondiploid cases. CONCLUSION: Quantitative histomorphometry can predict the ploidy of prostate carcinoma in biopsy tissue sections. Quantitative histomorphometry has potential as a method of rapidly assessing DNA ploidy otherwise earmarked for nuclear image analysis, resulting in savings of time and expense.     

Combined DNA flow cytometry and sorting with k-ras2 mutation spectrum analysis and the prognosis of human sporadic colorectal cancer

BACKGROUND: Activation of the k-ras2 pathways and chromosomal instability leading to aneuploidy in human sporadic colorectal cancer (sCRC) is essential to the tumor cell ability to survive, grow, and metastatize. METHODS: The study included 135 patients with sCRC who were followed up for a median of 72 months. Multiple fresh-frozen fragments obtained from superficial and invasive areas of the tumors were mixed and used to detect the degree of DNA aneuploidy (DNA index [DI]) and S-phase fraction by two scatter signals and 4,6-diamidino-2-phenylindole-2-hydrocloride (DAPI) fluorescence flow cytometry (FCM). PCR amplification and k-ras2 mutation spectrum analysis were performed using enriched epithelial nuclei after sorting DNA aneuploid nuclei and DNA diploid nuclei from which tissue-infiltrating lymphocytes were absent. RESULTS: DNA aneuploidy was detected in 98 (73%) and k-ras2 mutations in 54 cases (40%). Univariate analyses of overall survival with both Dukes' A to D or B to C series of cases showed that DNA multiple aneuploidy, k-ras2 mutations, older age, and distal site, but not increased S-phase fraction, were predictive of worse outcome. Multivariate Cox models strongly indicated that k-ras2 mutations, but neither single nor multiple DNA aneuploidy, were an independent prognostic factor in both series of patients. In particular, with B and C Dukes' stage patients (n = 110), the relative risk (RR) of death was above 2.5 with k-ras2 mutations and above 3 with the G-->C/T transversions. CONCLUSION: Combined FCM and k-ras2 analysis may be used to predict long-term increased risk of death in sCRC patients.     

Ploidy and morphology in osteosarcoma

In a cytometric DNA study of high-grade osteosarcoma, the relationship between DNA content and morphology was analyzed. The investigation, based on microspectrophotometry of tissue sections and flow cytometry (FCM), included both primary lesions and recurrences. FCM analysis, applied to a consecutive series of 47 primary osteosarcomas, disclosed that 2 were diploid and 45 were nondiploid, 8 of which were tetraploid. Multiple aneuploid peaks were detected in 13 tumors. Among the nondiploid tumors, there was no clear relationship between the peak DNA value(s) and the histologic subtype (osteoblastic, chondroblastic, fibroblastic) or grade (III-IV). The proliferative activity, as reflected by the percentage of S-phase cells, could be determined in 38 of the 47 tumors analyzed by FCM. The percentage was higher for aneuploid than for tetraploid lesions; however, the distribution of S-phase cells was not related to the histologic subtype or the grade of the tumors. To assess the reliability of a single sample for FCM, the DNA content of biopsy and surgical specimens was compared in 20 tumors; there was complete agreement in all cases with respect to the classification of the lesion as diploid, tetraploid or aneuploid. Analysis by FCM or microspectrophotometry of 12 local recurrences and 16 metastases and the corresponding 19 primary tumors showed that an aneuploid characteristic of the primary lesion was retained during progression of the disease. In 12 tumors analyzed by microspectrophotometry in tissue sections, comparison of chondroblastic and osteoblastic/fibroblastic areas within the same lesion consistently disclosed hyperploidy in both areas.(ABSTRACT TRUNCATED AT 250 WORDS)