Diagnostic value of flow cytometric DNA determination combined with fine needle aspiration biopsy in thyroid tumors

The nuclear DNA content and the numbers of cells in the S and G2M phases of the cell cycle were determined by flow cytometry (FCM) in fine needle aspirates of 187 thyroid tumors to evaluate the diagnostic value of nuclear DNA content determination in combination with aspiration cytology. DNA aneuploidy was present in 4 of 5 follicular carcinomas, 2 of 3 anaplastic carcinomas, 5 of 15 excised follicular adenomas and 2 of 20 excised adenomatous goiters; all 7 papillary carcinomas and 4 lymphomas were diploid in the aspirate. Aneuploid carcinomas had easily distinguishable S and/or G2M phases, unlike the benign aneuploid tumors. None of the histologically benign tumors or the nonexcised tumors had greater than 6% S-phase cells, and only one benign tumor had greater than 9% G2M-phase cells. In contrast, all lymphomas had greater than 10% S-phase cells and four of seven papillary carcinomas had greater than 9% G2M-phase cells. The use of FCM determination in combination with fine needle aspiration biopsy cytology improved the diagnostic potential of the latter technique.     

The reliability of microspectrophotometric and flow cytometric nuclear DNA measurements in adenocarcinomas of the breast

The reliability of microspectrophotometric (MSP) and flow cytometric (FCM) nuclear DNA measurements has been studied in 50 human breast adenocarcinomas. The tumor material was obtained by means of fine-needle aspiration biopsy, and all samples except one were found to be highly representative. The results confirm earlier observations that a good correlation exists between modal value (MV) determined by MSP and DNA index (DI) determined by FCM. However, when tumors were classified into low and high malignant variants according to FCM/DI, FCM/S-phase percentages, and MSP histogram types, the concordance was less pronounced. This was found to be due mainly to the fact that in near-diploid tumors a discrepancy exists between MSP and FCM ploidy, as well as between MSP distribution pattern and the estimated percentages of cells in the S-phase region. Another source of discrepancy was observed in tumors with stemlines in the normal tetraploid region, including cells with highly scattered aneuploid DNA values. These tumors were judged by MSP as aneuploid/high malignant and by FCM as euploid/low malignant. In view of this discrepancy, we conclude that the simple determination of the stemline position by MSP/MV or FCM/DI is not sufficient for adequate cytochemical malignancy grading of breast carcinomas. We suggest that a combination of ploidy and percentage of cells scattered outside the modal peaks is a more sensitive method for optimal cytochemical malignancy grading in breast carcinomas.     

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.     

Two-color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte-common antigen

Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.     

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)     

Flow cytometric DNA measurements in human thyroid tumors

By means of flow cytometry (FCM), DNA distribution pattern and the fraction of cells in the various phases of the cell cycle were studied in 52 samples of normal thyroid tissues, follicular adenomas, follicular carcinomas, medullary carcinoma and fibrosarcomas. In the normal thyroid tissues and follicular adenomas DNA diploid cell populations only were found. Among 20 follicular carcinomas in 13 cases (65%) together with the DNA diploid cells, DNA aneuploid cell lines were also observed. S-phase fraction in follicular adenomas is higher than in the normal thyroid tissues and lower than those in thyroid carcinomas. The percentage of S-phase cells in DNA aneuploid populations is significantly higher (S = 19 +/- 9.3%) than in the diploid cell lines (S = 3.7 +/- 2.6%). DNA aneuploid cell populations were predominantly observed in carcinomas with a high degree of morphological anaplasia.     

Evaluation of tumor heterogeneity of prostate carcinoma by flow- and image DNA cytometry and histopathological grading.

BACKGROUND: Heterogeneity of prostate carcinoma is one of the reasons for pretreatment underestimation of tumor aggressiveness. We studied tumor heterogeneity and the probability of finding the highest tumor grade and DNA aneuploidy with relation to the number of biopsies. MATERIAL AND METHODS: Specimens simulating core biopsies from five randomly selected tumor areas from each of 16 B?cking's grade II and 23 grade III prostate carcinomas were analyzed for tumor grade and DNA ploidy by flow- and fluorescence image cytometry (FCM, FICM). Cell cycle composition was measured by FCM. RESULTS: By determination of ploidy and cell cycle composition, morphologically defined tumors can further be subdivided. Heterogeneity of tumor grade and DNA ploidy (FCM) was 54% and 50%. Coexistence of diploid tumor cells in aneuploid specimens represents another form of tumor heterogeneity. The proportion of diploid tumor cells decreased significantly with tumor grade and with increase in the fraction of proliferating cell of the aneuploid tumor part. The probability of estimating the highest tumor grade or aneuploidy increased from 40% for one biopsy to 95% for 5 biopsies studied. By combining the tumor grade with DNA ploidy, the probability of detecting a highly aggressive tumor increased from 40% to 70% and 90% for one and two biopsies, respectively. CONCLUSION: Specimens of the size of core biopsies can be used for evaluation of DNA ploidy and cell cycle composition. Underestimation of aggressiveness of prostate carcinoma due to tumor heterogeneity is minimized by simultaneous study of the tumor grade and DNA ploidy more than by increasing the number of biopsies. The biological significance of coexistent diploid tumor cell in aneuploid lesions remains to be evaluated.     

Flow cytometric DNA analysis of renal-cell carcinoma. A study of fine needle aspiration biopsies in comparison with multiple surgical samples

The flow cytometric (FCM) DNA analysis of fine needle aspiration (FNA) biopsy material was compared with the DNA analysis of multiple surgical biopsy material from 44 renal-cell carcinomas. Twenty tumors were heterogeneous with respect to their DNA content. Eleven of the 17 tumors that had both diploid and aneuploid tumor cell clones in the surgical specimens gave a diploid DNA content in the aspiration biopsies; the other 6 cases showed aneuploidy in the aspirate. The fine needle aspirates from 18 homogeneously diploid tumors and 9 tumors with an aneuploid DNA content in all eight surgical samples revealed DNA indices similar to those found in the surgical samples. These findings show that aneuploid clones of renal-cell carcinoma can remain undetected by the use of FNA biopsy as a method for obtaining samples for FCM DNA analysis.     

Flow cytometric study of DNA distribution in cytopunctures of benign and malignant breast lesions

One hundred seventy-eight cytopunctures of mammary lesions were obtained for cytologic diagnosis and flow cytometric (FCM) analysis of the nuclear DNA content. All lesions were excised and evaluated histologically; 106 were carcinomas and 72 were benign lesions. The benign lesions showed a diploid DNA content, with one exception. Among the 106 carcinomas, 35 (33%) were diploid, 14 (13%) were tetraploid and 57 (54%) were aneuploid. For 79 carcinomas, the relationship between ploidy and (1) "T" and "N" of TNM staging, (2) the histologic grading of Scarff, Bloom and Richardson, (3) axillary nodal involvement, (4) the presence of estrogen and progesterone receptors, (5) age and (6) menopausal status was investigated. The percentage of aneuploidy was significantly higher (P less than .05) in grade III tumors as compared to grade I tumors. There was no significant relationship between aneuploidy and the other factors. However, a trend was observed between the lack of steroid receptors and a high probability of the tumor being aneuploid. FCM DNA analysis was also carried out on breast carcinomas obtained at surgery in 40 patients for whom FCM DNA analysis had previously been performed on breast cytopuncture specimens. The FCM DNA analyses were found to be best performed on the samples obtained by cytopuncture, which may increase the yield of tumor cells.     

DNA image cytometry on machine-selected breast cancer cells and a comparison between flow cytometry and scanning cytophotometry

A DNA image cytometry method, implemented on the LEYTAS image processing system, has been applied to acriflavine-Feulgen-stained breast cancer cytology specimens. An essential feature of the LEYTAS image cytometry method (LCM) is the automated selection of single nuclei according to predetermined specifications. Visual interaction has been used to reject remaining artefacts like overlapping nuclei. DNA profiles obtained with LCM have been compared with DNA profiles obtained by scanning cytophotometry (SCM) or flow cytometry (FCM). The resolution of DNA profiles obtained with LCM is similar to that from SCM but lower than that from FCM. However, a high correlation is found for the DNA indices measured with LCM and FCM (r = 0.97). The LCM profiles of aneuploid tumours generally showed lower accessory diploid fractions than FCM profiles due to the automated rejection of leukocyte nuclei. Also, LCM profiles frequently showed the presence of minor subpopulations of highly aneuploid/polyploid tumour cells that could not be identified by FCM. Therefore, LCM appears to be supplementary to FCM for studying tumour cell stemline heterogeneity.     

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.     

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.     

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.     

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 ploidy pattern and amplification of ERBB and ERBB2 genes in human gastric carcinomas

The DNA ploidy pattern and amplification of ERBB and ERBB2 genes were examined in paraffinembedded tissue from gastric carcinomas using flow cytometry and a slot-blot hybridization technique. The incidence of aneuploidy in well differentiated adenocarcinomas (56%) was significantly higher (p<0.05) than that in poorly differentiated adenocarcinomas (21%). The DNA ploidy pattern was not remarkably different between the primary tumors and metastatic deposits in lymph nodes. Of the nine specimens having an aneuploid stem cell line in the primary tumor and/or in metastases, three showed ERBB2 gene amplification and one showed ERBB gene amplification. The incidence of epidermal growth factor (EGF) immunoreactivity in tumor cells showed no difference between diploid and aneuploid tumors. These findings indicate that aneuploidy is frequently associated with amplification of ERBB and ERBB2 genes.     

Bone metastases: prognostic applications of cytometric DNA analysis

OBJECTIVE: To examine DNA parameters as prognostic factors for developing metastases. STUDY DESIGN: Image cytometry was used to determine DNA content of 21 tumors and 28 metastases. DNA ploidy status, 2c deviation index (2cDI) and DNA malignancy grade (DNA-MG) (based on the variation of nuclear DNA content of tumor cells around the normal DNA [2c] peak) were examined for their prognostic value. RESULTS: Twenty of 21 tumors showed aneuploid content, and 1 tumor showed diploid DNA content. Twenty-one bone metastases showed aneuploid cells. In 6 cases both euploid and aneuploid cells were detected. In 1 metastasis only euploid cells were present. DNA-MG was increased in bone metastases (mean, 2.4) as compared to the corresponding primary tumor (mean, 2.2) in most of the cases. The mean value of the 2cDI was 30.07 in primary tumors and 42.5 in metastases. Twelve bone metastases had a higher 5cEE than did the primary tumor. CONCLUSION: Diploid and aneuploid cells were able to leave a tumor and establish metastases. DNA-MG and 2cDI were increased in metastases in comparison with the primary tumor, but even tumors with lower DNA-MG had metastatic potential.     

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.     

DNA quantification in colorectal carcinoma using flow and image analysis cytometry

Numerous studies using flow cytometry (FCM) have shown that DNA quantification and ploidy classification can provide information of prognostic significance for patients with colorectal carcinoma; recent advances in image analysis cytometry (image cytometry, ICM) provide a new, alternative technique for DNA quantification. This study investigated whether (1) patients with colorectal carcinomas that exhibit a diploid pattern of DNA distribution have improved five-year survival statistics as compared to their non-diploid counterparts and (2) ICM provides quantitative data comparable to that obtained by FCM. DNA quantification and ploidy classification of 27 cases of primary colorectal carcinoma was performed on archival paraffin-embedded tissue by both FCM and ICM; 70% (19) of the tumors were classified as nondiploid by ICM while 56% (15) were similarly classified by FCM. Diploid tumors were associated with Dukes' stage A while nondiploid tumors were associated with Dukes' stage D. The overall five-year survival rate was 75% for patients with ICM diploid tumors and 67% for patients with FCM diploid tumors. The five-year survival was only 53% for patients with nondiploid tumors identified by both techniques. This study confirmed that DNA quantification is an important prognostic indicator for patients with colorectal carcinoma. It also showed that ICM provides data comparable to that of FCM and may be more sensitive.     

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.     

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.