DNA ploidy level and S-phase fraction as prognostic factors in breast cancer

Imprint smears from sixty cases of breast cancer made after mastectomy were stained by the Feulgen method and the nuclear DNA content measured by a cytofluorometer equipped with an incident illumination system. After logarithmic transformation of the fluorescence intensity, the ploidy level and S-phase fraction (SPF) were calculated with a microcomputer and the correlation between the ploidy level or SPF and the clinicopathological prognostic factors studied. Patients with tumors of a larger diameter and more extensive lymph node involvement had higher levels of ploidy and SPF and the ploidy level in the metastatic lymph nodes was higher than that in the primary lesion. Moreover, a significant increase in SPF was observed in the metastatic lymph nodes and a high ploidy level found to be associated with tumors having a negative estrogen receptor. When the tumors were divided into a diploid group and an aneuploid group, the diploid group showed a significantly better prognosis than the aneuploid group, in 6-year survival. Similarly, the groups in which SPF was less than 20.0 per cent had significantly better prognoses than the group in which SPF was 20.1 per cent or more. These prognostic factors were evaluated with Cox's proportional hazard model and a significant correlation observed in lymph node status, ER status, ploidy level and S-phase fraction. It was thus concluded that ploidy level and SPF are important and independent prognostic factors for predicting the postoperative course of breast cancer patients.     

Relationship between DNA ploidy and survival in patients with primary breast cancer

The DNA ploidy of breast cancer tissue from paraffin blocks was measured by flow cytometry in 117 patients whose disease had been detected and treated with surgery between 1974 and 1976. Patients with aneuploid tumours had positive axillary nodes and distant metastases more often than those with diploid tumours. Aneuploid tumours were more common in postmenopausal than premenopausal women. The S-phase fraction (SPF) was significantly higher in aneuploid than in diploid tumours and positive axillary lymph nodes were found in 26 per cent of the patients who had a tumour with a SPF below the median (4.8 per cent) and in 48 per cent of those with tumours with SPF values above the median. At the primary clinical investigation 2 per cent of the patients with diploid tumours and 6 per cent of those with aneuploid tumours had distant metastases. During the follow-up, the proportion of patients with distant metastases increased to 42 and 72 per cent, respectively. With a follow-up of 11.5 years, the DNA aneuploidy of the tumour showed a significant association with decreased survival. Thirty-three per cent of patients with diploid and 65 per cent of patients with aneuploid tumours had died from breast cancer during the follow-up (P less than 0.001). All patients with hypertetraploid or multiploid tumours died from breast cancer. High SPF values were associated more closely with distant metastases or death during the follow-up than low SPF values. Our results suggest that DNA ploidy measured by flow cytometry from paraffin embedded tissue blocks of human breast cancer can be used to predict the aggressiveness of the tumour and the survival of the patients.     

乳腺癌原发灶及腋淋巴结FCM分析对预后的评价

作者采用流式细胞仪（ＦＣＭ）对５８例乳腺癌患者的原发灶及腋淋巴结共１１６份新鲜组织进行ＤＮＡ含量、倍体及Ｓ－期细胞百分率（ＳＰＦ）分析。经过５年随访,发现复发及转移（简称复发）２３例,其中２１例已死亡。结合各种因素进行分析,发现异倍体及高ＳＰＦ有较高的复发率,特别当病期较晚或腋淋巴结有转移时。作者还发现原发灶ＦＣＭ的分析结果比腋淋巴结的分析结果对预后有更大的价值。作者总结了二倍体肿瘤复发的可能原因,并提出了加强异倍体肿瘤术后治疗的建议。     

Prognostic indicators in invasive breast cancer

Tumor size and axillary lymph node involvement are the primary determinants of clinical course for most patients. Receptors for estrogen and progesterone are important additional prognostic factors for disease-free survival, overall survival, survival time after initial disease recurrence, and the likelihood of response to hormonal therapy. Histologic grading has merit as a prognostic factor, although poor reproducibility limits its broad application. Promising data have been emerging from the use of flow cytometry to analyze DNA content and proliferative rate. Patients with aneuploid tumors are more likely to have a shorter survival time than patients with diploid tumors. A high S-phase fraction also identifies a subset of patients at increased risk for early relapse. A combined index of ploidy and S-phase may be a more useful guide; together, diploidy and low S-phase identify a subgroup of node-negative patients at very low risk for disease recurrence. A number of oncogenes have been identified in breast cancer; amplification of the HER-2/neu gene or overexpression of the gene product may be an important prognostic indicator for node-positive patients.     

DNA ploidy, S-phase fraction and cytomorphometry in relation to survival of human penile cancer.

The prognostic significance of DNA ploidy, DNA index, S-phase fraction (SPF) and median nuclear size was studied in 11 patients with squamous cell carcinoma of the penis. These patients have been followed for a minimum of 7 months after diagnosis. Nuclear DNA content was determined by flow cytometry from paraffin-embedded tissue. Patients with DNA diploid cancer (n = 7, 64%) had a better survival rate than patients with aneuploid cancer, and a small SPF was associated with a favorable outcome. A statistically significant relation between median nuclear size and survival could be demonstrated. Small modal nuclear size associated with poorer prognosis. There was a significant difference in survival between metastatic and nonmetastatic groups of tumors during the follow-up period. This study suggests that flow cytometric determination of nuclear DNA ploidy from paraffin-embedded samples in penile cancer does add an additional prognostic determinant in addition to the clinical staging of tumors.     

DNA Flow Cytometry in Surgically Treated Lung Cancer: Prognostic Significance

Although DNA aneuploidy and high proliferative activity (S-phase fraction, SPF) of tumour cells, measured by flow cytometry, have proved to be indicators of poor prognosis in most solid tumours, there have been conflicting results in lung cancer studies. During a four-year period we studied the prognostic significance of DNA ploidy and SPF in 99 surgically treated lung cancer patients. Flow cytometric analysis was done from archival, formalin-fixed, paraffin-embedded tumour specimens. DNA index and SPF were determined, using MultiCycle software with sliced nuclear correction to compensate for debris. There were 61 DNA diploid and 38 DNA aneuploid tumours. The median SPF was 10.2%. Neither ploidy nor SPF was associated with previously known prognostic factors. Survival was poorer in patients with aneuploid tumours than in the other patients, but the difference was not statistically significant. DNA ploidy and SPF thus do not seem to be useful prognostic indicators in surgically treated lung cancer.     

DNA flow cytometry in surgically treated lung cancer--prognostic significance.

Although DNA aneuploidy and high proliferative activity (S-phase fraction, SPF) of tumour cells, measured by flow cytometry, have proved to be indicators of poor prognosis in most solid tumours, there have been conflicting results in lung cancer studies. During a four-year period we studied the prognostic significance of DNA ploidy and SPF in 99 surgically treated lung cancer patients. Flow cytometric analysis was done from archival, formalin-fixed, paraffin-embedded tumour specimens. DNA index and SPF were determined, using MultiCycle software with sliced nuclear correction to compensate for debris. There were 61 DNA diploid and 38 DNA aneuploid tumours. The median SPF was 10.2%. Neither ploidy nor SPF was associated with previously known prognostic factors. Survival was poorer in patients with aneuploid tumours than in the other patients, but the difference was not statistically significant. DNA ploidy and SPF thus do not seem to be useful prognostic indicators in surgically treated lung cancer.     

Proliferative activity as a marker for lymph node metastases on early gastric cancers--in vitro bromodeoxyuridine labeling for clinical use

We estimated the clinical significance of the DNA ploidy pattern and S-phase fraction (SPF) with in vivo administration of bromodeoxyuridine (BrdU) and flow cytometry on 61 early gastric cancers. Aneuploid tumors or tumors with high SPF had high incidence of lymph node metastases than diploid tumors or tumors with low SPF, respectively. Thirty cases of human gastric cancers were examined with both in vivo and in vitro labeling of BrdU. In early gastric cancers, DNA ploidy pattern was same in 11 cases out of 13 cases and SPF was correlated with in vivo and in vitro (p less than 0.01). However, in advanced gastric cancers, DNA ploidy pattern was same in only 3 cases out of 17 cases and SPF was not correlated with in vivo and in vitro. The reasons for no correlation with in vivo and in vitro was reduced to the existence of DNA ploidy heterogeneity which was observed 22.2% and 70.6% in early and advanced cancers, respectively. From the results, we considered that DNA ploidy pattern and SPF were useful marker for lymph node metastases and the preoperative in vitro BrdU labeling method was useful for the marker of lymph node metastasis in early gastric cancers.     

DNA ploidy pattern and tumour spread in gastric cancer

The DNA ploidy pattern of gastric cancer was studied in 58 patients to investigate the heterogeneity between primary tumour and metastases. In both primary tumours and lymph node metastases, diploid patterns accounted for 33 per cent, whereas all liver metastases were aneuploid. The percentage of polyploid cells was higher in the liver metastases than in primary tumours and lymph node metastases. When the heterogeneity of DNA ploidy pattern between primary tumour and metastasis was evaluated, diploid tumours had a significantly lower rate of lymph node metastasis heterogeneity than aneuploid tumours. When the DNA ploidy pattern and survival were evaluated, the patients who had a diploid pattern in both primary tumour and metastasis had a significantly higher survival rate than the patients who had an aneuploid pattern in the primary tumour and metastasis (57 per cent versus 26 per cent at 5 years). These data suggest that cell heterogeneity is a common phenomenon in gastric cancer, and this may be important in the evolution of the disease. Furthermore, the role of the DNA ploidy pattern as a prognostic factor is emphasized.     

Node negative breast cancer: the prognostic value of DNA ploidy for long-term survival.

The DNA content of breast tumours from 170 patients who presented between 1978 and 1980 was measured by flow cytometry. The relationship between tumour ploidy and disease outcome was assessed and its association with other prognostic factors evaluated. Compared with those with diploid tumours, patients with aneuploid tumours had significantly earlier relapse and shorter survival (P less than 0.0001). Tumour ploidy was strongly related to grade (P less than 0.001), but there was no significant association between DNA ploidy and c-erb-B-2 expression, lymph node status or tumour size. In lymph node negative and c-erb-B-2 negative patients, aneuploid tumours were associated with a poorer prognosis (P less than 0.001) than diploid tumours. Multivariate analysis showed that tumour ploidy gave independent information on disease free and overall survival. Tumour ploidy may be used as an independent prognostic variable in patients with breast cancer and it may be helpful in defining patients within the node negative or c-erb-B-2 negative groups likely to have a poor outcome who might benefit from adjuvant treatment.     

The importance of DNA flow cytometry in node-negative breast cancer

DNA flow cytometric analysis and conventional clinical factors were compared with disease outcome in 257 patients with node-negative infiltrating ductal carcinoma who had been treated between 1976 and 1983. Median follow-up was 80 months; none of the patients received adjuvant therapy. The relative prognostic importance of clinical variables, ploidy, and S-phase fraction was analyzed by Cox multivariate analysis. Ploidy was analyzable for 198 tumors and did not predict survival. Nuclear grade predicted disease-free survival for all patients. For 71 patients with diploid tumors, only high S-phase had a statistically significant association with relapse. For 127 patients with aneuploid tumors, tumor diameter predicted both disease-free survival and cancer death; histologic grade was also significant for predicting disease-free survival. In conclusion, flow cytometric determination of ploidy and S-phase fraction can provide valuable predictive information in node-negative breast cancer in addition to conventional variables.     

The relationship of estrogen receptor status to DNA ploidy in breast cancer

The relationship of estrogen receptor(ER) status to DNA ploidy was investigated in 121 patients with breast cancer who underwent surgery. Lymph node status was evaluated histologically and ER levels were determined by the dextran-coated charcoal method, with a level of 3 fmol/mg·protein being considered positive. Flow cytometric DNA content was analyzed using paraffin-embedded tissue blocks. Sixty-three per cent of the specimens were ER+, while 37 per cent were negative. Sixty-one patients (50.4 per cent) were diploid and 60 aneuploid. A statistically significant correlation between the ER positivity rate and diploid DNA was found. Higher ER levels were seen in the postmenopausal patients with diploid tumors than in those with aneuploid tumors and there was a significant tendency for ER levels to be higher in the diploid tumors. Nodal status was not correlated with ER positivity or ploidy pattern. The present results indicate that ER levels are correlated with DNA ploidy, and reflect the degree of functional differentiation.     

Flow cytometric analysis of DNA ploidy, cell cycle and cytomorphometry in sarcomatoid renal cell carcinoma.

In 5 patients with sarcomatoid renal cell carcinoma, the nuclear DNA content and size were determined by flow cytometry (FCM), and the prognostic value of DNA ploidy, the percentage of S-phase cells (SPF), and the ratio of modal nuclear size with clinicopathologic behavior was analyzed. Age and clinical stage have been shown to have a strong correlation with prognosis. Older patients with a high stage of cancer had poor outcome with a shorter survival time. In all 60% of the tumors were aneuploid. Tumor invasiveness was related to DNA ploidy. With increasing stage, the overall incidence of aneuploid rises. One alive patient had diploid DNA while 75% of the patients who died of sarcomatoid renal cell carcinomas had aneuploid DNA. Diploid sarcomatoid renal cell carcinomas show significantly lower SPF than aneuploid tumors. There was no significant association between the modal nuclear size and the invasiveness of tumors or survival time. This study suggests that FCM analysis of tumor DNA content and cell cycle could be regarded as an additional prognostic determinant of sarcomatoid renal cell carcinoma.     

DNA ploidy and the S-phase fraction and their prognostic significance in gastrointestinal tumors

The prognostic significance of nuclear DNA ploidy patterns and the S-phase fraction (labeling index; IL) were evaluated in 365 gastrointestinal tumors, and in 62 of them a combined analysis of DNA ploidy and the SPF was performed. For accurate evaluation, we used fresh frozen specimens, and we classified the ploidy pattern into 6 types; 1. diploid, 2. DS (diploid + high LI), 3. Notch, 4. Shoulder, 5. Tetraploid and 6. Aneuploid. Type 1 or type 2 tumors were classified as diploid, and the others were classified as aneuploid. In 103 cases of gastric cancer and 101 colon cancers due to the short observation period, but in 46 patients with operable primary liver cancer, a significant difference was observed. A high average of LI was detected in colon cancer (approximately 13%), but no relationship between LI and the ploidy pattern was found. This indicates that the LI may become an independent prognostic factor, and that the combined assay of DNA ploidy and the LI may offer a more precise evaluation of the malignant potential of gastrointestinal tumors.     

Sonographic blood flow measurements in malignant breast tumors

Background: The aim of this study was to find a possible relationship between the biological behavior of carcinomas of the breast and sonographically detectable blood flow after first studies showed a correlation between blood flow and prognostic factors. Method: 259 patients with ductal invasive breast cancer were examined using MEM (i.e., the Maximum Entropy Method), a new sonographic blood flow measurement technique capable of discerning considerably slower blood flow velocities than Doppler sonography. Due to the lack of objective methods for quantifying the blood flow, the findings were divided into three classes dependent upon the visual color information obtained. The blood flow was correlated with the size of the tumor, lymph node and receptor status, ploidy and S-phase fraction. Results: Most of the patients with small tumors, without lymph node metastases, with positive receptors, with a diploid genome, and with a low S-phase fraction belonged to the group with the lowest blood flow. Conclusion: The close relationship between the established prognostic factors and the sonographic blood flow measurements using MEM might be indicative of a new preoperative prognostic factor; this must, however, be confirmed by larger studies. Received: 16 August 1996/Accepted: 27 January 1997     

Prognostic value of MIB-1 index and DNA ploidy in resectable ampulla of Vater carcinoma

OBJECTIVE: To evaluate the prognostic value of the proliferative factors, MIB-1 index, DNA ploidy, and S-phase fraction, and further to determine the independent prognostic factors in ampulla of Vater carcinoma after pancreaticoduodenectomy. SUMMARY BACKGROUND DATA: Cell kinetics are important indicators of the biologic behavior of various human tumors, but only a few authors have reported the application of cell proliferative factors in ampulla of Vater carcinoma. METHODS: Patients undergoing pancreaticoduodenectomy for ampulla of Vater carcinoma were included. Proliferative factors, MIB-1 index, and DNA contents, measured by flow cytometry, were evaluated and compared with the conventional clinicopathologic factors. RESULTS: Ninety resectable ampulla of Vater carcinomas were included. By univariate analysis, MIB-1 index, DNA ploidy, S-phase fraction, stage, and lymph node status were significant prognostic factors. The 5-year survival rate was 40.7% for tumors with MIB-1 index < or =15% and 0% for those with MIB-1 index >15%. Diploid tumors had a significantly better prognosis than aneuploid. Outcomes of stage I and II tumors were more favorable than those of stage III and IV. After multivariate analysis, MIB-1 index, DNA ploidy, and stage remained as the independent prognostic factors. Among the three independent prognostic factors, MIB-1 index was the most powerful. CONCLUSIONS: Both MIB-1 index and DNA ploidy provide important prognostic value and potentially complement the conventional prognostic factors in resectable ampulla of Vater carcinoma. MIB-1 index is the most powerful independent prognostic factor.     

Molecular biological observations in some rare neoplasms: DNA ploidy and cell cycle data in liposarcoma and malignant fibrous histiocytoma

Introduction: The prognostic significance of tumor DNA ploidy and cell cycle analysis for long-term survival has been examined in 19 patients with liposarcoma or malignant fibrous histiocytoma. In many cases, different tumor areas of primary tumors and local recurrences have been analyzed to reveal intratumoral heterogeneity. Results: Among the primary tumors, there were eight aneuploid tumors, three of which showed diploid and aneuploid tumor regions. Correlations among DNA ploidy, grading, percentage of S-phase cells and infiltrative growth pattern of the tumors could be demonstrated. Poorly differentiated tumors (G3) showed aneuploidy in six of eight patients. Aneuploid tumors showed S-phase cells in 17.2% (range 3.2–38.1%), which was higher than the percentage of S-phase cells in diploid tumors (9.4%, range 2.1–27.4%). Aneuploid tumors showed a more infiltrative growth pattern (6 of 8 patients) than diploid tumors (6 of 11 patients). The median survival time of patients with diploid tumors was 86.5 months (8–144 months), compared with 40.9 months (11–54 months) for patients with aneuploid tumors. Conclusion: DNA ploidy and percentage of S-phase cells may be considered as prognostic factors. Received: 12 June 1998 Accepted: 14 September 1998     

Mammographic Density in Women on Postmenopausal Hormone Replacement Therapy

Background . Fine-needle aspiration (FNA) biopsy has been used increasingly in the diagnosis and biologic characterization of breast carcinomas in patients who receive preoperative chemotherapy. Because proliferative activity of breast carcinoma has been shown to be of prognostic significance, the authors compared immunocytochemical Ki-67 growth fraction and flow cytometric S-phase fraction (SPF), both evaluated on FNA samples. Methods . The proliferative activity of 134 FNA samples from primary breast carcinoma patients was studied using both immunocytochemistry with the monoclonal antibody Ki-67 and SPF determined by DNA flow cytometry. Results . Ki-67 and SPF were evaluable in 114 and 107 cases, respectively, and both were evaluable in 95 cases. Of the 134 FNA samples studied, 37% were diploid and 63% were aneuploid. The distribution of both Ki-67 and SPF was different in diploid and aneuploid tumors. The median Ki-67 value as well as the median SPF were significantly higher in aneuploid versus diploid tumors (p < 0.001). Median Ki-67 and SPF values were used to discriminate between low versus high proliferating tumors. The overall concordance between Ki-67 and SPF was 75% (p < 0.001). A good correlation was found between Ki-67 and SPF (correlation coefficient = 0.72; p < 0.001). Conclusions . The results of the current study suggest the Ki-67 growth fraction and SPF determined by FNA may be used as measurements of the proliferative activity of breast carcinoma. The authors recommend these determinations be used as preoperative procedures in patients with a cytologic diagnosis of breast carcinoma who are candidates for neoadjuvant chemotherapy and/or endocrine therapy.     

Value of DNA ploidy and S-phase fraction as prognostic factors in stage III cutaneous melanoma.

OBJECTIVE: To determine the prognostic value of flow cytometric analysis (S-phase fraction and DNA index) performed on lymph-node metastases of patients with stage III melanoma. DESIGN: A retrospective chart review with flow cytometric analysis of paraffin-embedded tissues. SETTING: A university teaching hospital. PATIENTS: Among 332 patients with cutaneous melanoma, 33 with stage III were identified. Distant metastases developed in 16 patients; 17 had no further recurrence. Charts were reviewed to obtain clinicopathologic parameters such as sex, age, location of the primary tumour, histologic features, presence or absence of ulceration, and Clark's and Breslow's levels. INTERVENTION: DNA ploidy and S-phase fraction were determined on the paraffin-embedded nodes. MAIN OUTCOME MEASURES: The groups with or without recurrence were compared in terms of disease-free survival (DFS) and overall survival (OS). These survival parameters were correlated with DNA ploidy and S-phase fraction. RESULTS: By univariate analysis, clinicopathologic factors did not predict OS. A higher Clark's level of invasion and more than 3 positive lymph nodes were associated with shorter DFS (p < 0.05). Tumour thickness and S-phase fraction did not correlate with either DFS or OS. Patients with diploid lymph-node metastases had an 87% 12-month survival compared with 41% for those with aneuploid tumours. CONCLUSIONS: DNA ploidy may be used as a prognostic index in patients with lymph-node metastases. This could be particularly useful in the context of sentinel lymph-node mapping by which more patients are being identified with single microscopic lymph-node involvement.     

Flow cytometric analysis of deoxyribonucleic acid ploidy and proliferation in choroid plexus tumors.

The deoxyribonucleic acid (DNA) content of 10 choroid plexus tumors, including 4 malignant tumors and 3 normal choroid plexus controls, was analyzed by flow cytometry to determine whether a ploidy or proliferation rate is a better predictor of tumor behavior than histological features. Nine of 10 neoplasms had both diploid and aneuploid modal populations. One neoplasm and all three control cases had only a diploid peak. Among the tumors, the DNA indices of the aneuploid peaks ranged from 1.1 to 2.2. The percentage of aneuploid cells ranged from 7 to 99, and no distinction was made between benign and malignant. Proliferation rates were estimated from the combined S-phase fractions (SPF). The mean SPF of the control group was 0.7% +/- 0.15% SD. The mean SPF of the benign tumors (1.1 +/- 0.82% SD) was significantly different from the malignant group (7.0 +/- 1.25% SD; range, 5.3 to 8.6%) P = 0.0095. Low SPF fractions always correlated with favorable outcome. Higher proliferation rates were generally associated with an aggressive course. Evaluation of proliferation rates may help predict the behavior of choroid plexus tumors, particularly when histological features are equivocal. Measurement of DNA ploidy does not appear to have a role in the evaluation of choroid plexus tumors.