Prognostic significance of flow cytometric DNA analysis in node-negative breast cancer patients

Flow cytometric DNA analysis using paraffin-embedded tumor blocks was done retrospectively on 155 node-negative breast cancers. The median duration of follow-up in patients still alive at the time of analysis was 10 years. Tumor aneuploidy was correlated significantly with increased tumor size (P = 0.003) and higher tumor grade (P less than 0.001). No significant correlation between tumor ploidy and patient age was found. Patients with diploid tumors had a significantly improved relapse-free and overall survival compared with patients with aneuploid tumors (P = 0.0001). In a Cox multivariate model with parameters including ploidy, histologic grade, tumor size, and patient age, ploidy (P = 0.02) and tumor size (P = 0.05) emerged as significant independent predictors of overall survival. Only ploidy was independently significant in the analysis of relapse-free survival. In conclusion, the current study indicates that flow cytometric measurement of DNA ploidy is a powerful prognostic indicator in node-negative breast cancer patients.     

Prognostic significance of flow cytometric DNA analysis in patients with malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) due to environmental exposure to asbestos and erionite is a relatively common cancer in Turkey. In this study, we investigated the value of flow cytometric (FCM) DNA analysis and other prognostic factors such as age and etiologic factor in the patients with MPM, treated with surgery+/-combination chemotherapy+/-radiotherapy. A total of 40 patients with a median age of 50 (range 30-68) were included in the study. Twenty-nine patients had asbestos exposure in etiology, while 11 had fibrous zeolite (erionite). Paraffin-embedded tumor specimens were studied by FCM for DNA analysis. Twelve patients (30%) had aneuploid tumors and 28 (70%) had diploid ones. Mean S-phase fraction (SPF; %) was 9.1+/-1.1 and proliferation index (PI, SPF+G2/M phase; %) was 11.3+/-0.9. While the median overall survival (OS) was 10+/-2 months (6-14; 95% CI), 1-year survival rate was 45.2%. Only PI was found to be statistically significant for OS in univariate analysis (P=0.013). PI was also found to be an independent prognostic factor for all patients (P=0.035). Aneuploidy was significantly higher in erionite group compared with asbestos group. Male predominance and poor survival were also prominent in erionite group, though not statistically significant. In conclusion, PI is an independent prognostic factor for patients with MPM and the biologic features of the disease may show differences with respect to different etiologies.     

DNA flow cytometric analysis and prognosis of axillary lymph node-negative breast carcinoma.

METHODS. The prognostic significance of flow cytometric analysis in patients with node-negative invasive breast carcinoma was evaluated in a retrospective series of 158 patients with a minimum follow-up study of 9 years. RESULTS. The ploidy status could be assessed in 147 specimens (93%), and the proliferative phase or S-phase fraction (SPF) could be assessed in 136 tumors (86%); 70 tumors (48%) were diploid, 49 tumors (33%) were aneuploid, and 28 tumors (19%) were tetraploid. Ploidy status and SPF were correlated significantly with tumor size, histologic grade, nuclear grade, and mitotic rate. By itself, ploidy was not a statistically significant prognostic factor, although all of the patients with multiploid and hypertetraploid tumors had recurrence of disease. The SPF was related significantly to recurrence of disease (P = 0.04). However, when multivariate analysis of various histopathologic variables was performed, SPF ceased to be a significant prognostic determinant, whereas peritumoral lymphovascular invasion was the most important variable. The combination of tumor size and flow cytometric parameters permitted stratification into three groups with different prognoses at the 9-year follow-up review (P less than 0.001). In the low-risk group (diploid tumors less than or equal to 2 cm in diameter with a low SPF or small tetraploid tumors), the recurrence rate was 12%. In the intermediate-risk group (diploid tumors greater than 2 cm in diameter with a low SPF or aneuploid tumors with a low SPF), the recurrence rate was 21%. In the high-risk group (diploid or aneuploid tumors with a high SPF or large tetraploid tumors), the recurrence rate was 49%. The high-risk group status remained a significant variable in the Cox proportional hazards multivariate analysis model. CONCLUSIONS. These results indicate that flow cytometry in breast carcinoma contributes useful but limited prognostic information and stress the importance of using multiple prognostic factors to improve prognostication and optimize patient management.     

Diagnostic and biological implications of flow cytometric DNA content analysis in lung cancer

Flow cytometric DNA content analysis, performed on clinical specimens from patients with lung cancer, was compared with clinical features, histological and/or cytological examination of the same specimen and, in some instances, to chromosome analysis and repeat DNA content analysis of short-term cultures. Tumors from 85% of non-small cell and 83% of small cell lung cancer patients had aneuploid DNA content; multiple aneuploid stem lines were present in 11% of patients. Comparisons with microscopic examination showed that aneuploid cells were detected in 122 of 167 clinical samples containing tumor cells and in 3 of 177 samples microscopically free of tumor. The high false-negative rate, shown to be due in large part to the presence of near-diploid tumor cells, makes single-parameter DNA analysis impractical for use in automated cytology. Short-term cultures of tumor cells, used to confirm that tumors had DNA content indistinguishable from diploid, demonstrated a single near-diploid peak on repeat DNA analysis with or without the addition of diploid lymphocytes and internal DNA standards. Chromosome banding studies showed clonal structural abnormalities with minimal numeric alterations. Survival of small cell lung cancer patients was similar for patients with near-diploid and aneuploid tumors. Cell cycle analysis could be performed in only a minority of samples, and there were no apparent differences in the proliferative fraction between lung cancer cell types. We conclude that flow cytometric DNA content analysis provides useful biological information and is a useful marker for following tumor cell cultures, but multiparameter analyses will be required for use in automated cytology and in cell kinetic studies.     

Prognostic value of flow cytometric DNA content analysis in granulosa cell tumor of the ovary

The nuclear DNA content and S-phase fraction of 23 ovarian granulosa cell tumors were measured from paraffin-embedded tissue with flow cytometry. Crude survival of the patients with a euploid tumor (17 diploid, one tetraploid) was more favorable than that of the patients with an aneuploid tumor (n = 5, P = 0.02). The percentage of S-phase cells was a good predictor of survival. If more than 6% S-phase cells were present in the DNA histogram, both crude survival (P = 0.0001) and survival corrected for intercurrent deaths (P = 0.0001) were clearly inferior as compared with tumors with less than 6% S-phase cells. The results indicate that DNA flow cytometric study may provide a rapid and valuable method to predict the biological behavior of granulosa cell tumors of the ovary.     

Prognostic and therapeutic significance of the flow cytometric nuclear DNA content in non-small cell lung cancer

To evaluate prognostic and therapeutic significance, tumor DNA content was determined by flow cytometry in 310 paraffin-embedded tissue samples obtained surgically from 130 patients with non-small cell lung cancer. Ninety-six (76.8%) patients had DNA aneuploid patterns that were statistically higher in adenocarcinoma than in squamous cell carcinoma. A better 5-year survival rate was observed in Group A (DNA diploidy, 69.6%) than in Group B (DNA aneuploidy and DNA peridiploidy, 33.2%; P less than 0.001). The survival curves of the patients in Group B continued to decrease during the next 2.5 years. Cox's model analysis showed that both the pathologic stage and the DNA content were the significant prognostic factors for survival. However, the DNA content was an independent prognostic factor in squamous cell carcinoma, but not in adenocarcinoma. These results indicate that DNA content analysis is useful for the evaluation of clinical behavior and prognosis, and that the clinical value of the DNA content must be differentiated between squamous cell carcinoma and adenocarcinoma.     

Prognostic significance of flow cytometric analysis of DNA content in colorectal cancer: A prospective study

We prospectively analyzed the tumor DNA content by flow cytometry in 100 patients who underwent a curative resection for colorectal cancer between August 1989 and May 1992 in order to evaluate the prognostic significance of DNA ploidy and the DNA index (DI). Patients with aneuploid tumors were found to have a significantly shorter disease-free survival than those with diploid tumors (P = 0.014). In addition, patients who had tumors with a DI greater than 1.6 had a significantly shorter disease-free survival than those who had tumors with a DI of less than 1.6 (P = 0.0001). After stratification by stage, this association was only seen in Dukes' stage C disease (P = 0.0065). Cox's regression analysis demonstrated that the DI (below or above 1.6) rather than DNA ploidy was an important independent predictor of disease-free survival. These results suggest that the DI rather than DNA ploidy provides us with important prognostic information in patients undergoing curative surgery for colorectal cancer. © 1993 Wiley-Liss, Inc.     

Renal cell carcinoma. A clinicopathologic and DNA flow cytometric analysis of 103 cases

Renal cell carcinoma is unpredictable in outcome, although the best predictor is tumor stage, followed by histologic grade. The authors retrospectively assessed the clinicopathologic features and DNA ploidy of 103 cases of renal cell carcinoma, the latter determined by flow cytometry of formalin-fixed, paraffin-embedded tissue. The study group comprised 63 men and 40 women (age, 28-80 years; mean, 57 years). Robson stage at diagnosis was Stage I in 52 patients, Stage II in 21, and Stage III in 30. Statistically significant variables in predicting outcome were Robson stage (P less than 0.0001), DNA ploidy (P = 0.0008), mitotic rate (MR, P less than 0.0001), worst nuclear grade (WNG, P = 0.00009), predominant nuclear grade (P = 0.019), and sex (P = 0.044). Tumor size, cell type, and architectural pattern were also assessed but did not prove to be significant. Statistically significant associations occurred between DNA ploidy and WNG (P less than 0.0001), stage (P = 0.0037), and MR (P = 0.015); between WNG and MR (P less than 0.0001) and stage (P = 0.0007); and between stage and MR (P = 0.002). Cox proportional hazards regression analysis of all significant variables showed Robson stage, tumor ploidy, and MR to be independent, significant predictors of outcome. If ploidy data had not been available, WNG would have been independently significant. The authors conclude that DNA ploidy analysis provides significant predictive information on renal cell carcinoma.     

Deoxyribonucleic acid (DNA) ploidy and proliferative characteristics of metastatic squamous cell carcinoma determined by flow cytometric analysis.

BACKGROUND: The deoxyribonucleic acid (DNA) index and the proliferative index (the fraction of cells in the S phase) can be independent prognostic indicators of the biologic aggressiveness of certain malignant neoplasms. OBJECTIVE: To determine whether the DNA index or proliferative index could predict metastases in cutaneous squamous cell carcinoma. METHODS: Nineteen different metastases from 15 patients with primary cutaneous squamous cell carcinoma (SCC) were reviewed, graded, and had DNA proliferative indexing performed by fluorescent activated cytometry. A control group of 13 patients with primary cutaneous SCC without metastases were studied in a similar manner. RESULTS: No significant difference between the metastatic and the nonmetastatic SCC groups for either DNA index or proliferative index (non-paired t-test) was observed. No correlative association with histologic grading with DNA index or proliferative index was observed for either group. CONCLUSION: We conclude that aneuploidy and S-phase fraction by fluorescent activated cytometry are not significant predictors of potential metastases in cutaneous squamous cell carcinoma.     

DNA ploidy of liver biopsies from patients with liver cirrhosis and hepatocellular carcinoma: a flow cytometric analysis.

Flow cytometric DNA analysis was used to assess cellular kinetics of needle liver biopsies from patients with liver cirrhosis and hepatocellular carcinoma (HCC). An abnormal DNA content was shown in 44.5% of liver cirrhosis cases and in 78.6% of tumor sites. The number of proliferating cells (S + G2M%) was significantly increased in cirrhotic liver (P < 0.05). Dysplasia was found in 66% of cirrhotic specimens. All negative dysplasia specimens showed a diploid pattern while 69% of positive dysplastic specimens were aneuploid (P < 0.001). In conclusion, cell proliferation, aneuploidy and liver cell dysplasia are important indicators in liver cirrhosis for the development of HCC.     

Prognostic potential of DNA flow cytometry measurements in node-negative breast cancer patients: preliminary analysis of an intergroup study (INT 0076).

An ancillary study (INT 0076) to the Intergroup clinical trial of node-negative breast cancer patients (INT 0011) was performed to retrospectively evaluate DNA flow cytometry measurements of ploidy (DNA content) and proliferative capacity (S-phase fraction) for their ability to predict time to recurrence. Of the 915 patients eligible for the clinical trial, 788 were registered for the ancillary flow cytometry study (INT 0076). Four hundred and three of these patients [estrogen receptor (ER)-positive, tumor size less than 3 cm] had been registered to the observation arm of the clinical trial and 385 (ER-negative and/or tumor size greater than or equal to 3 cm) had been randomly assigned to adjuvant chemotherapy (cyclophosphamide, methotrexate, fluorouracil, and prednisone for six cycles) or to observation. Paraffin blocks from 95% (748 of 788) of these patients were obtained, 712 of which had sufficient cancer tissue to be evaluable for the flow cytometric assay. DNA ploidy status (DNA diploid vs DNA aneuploid) was evaluable for 565 (79%) specimens, 64% of which were aneuploid. Proliferative capacity was estimated by the percentage of cells having an S-phase DNA content, using a trapezoidal modeling algorithm(s) as previously described. The median S-phase value for the entire group (both registered and randomly assigned patients) was 6.97%, which defined the cutoff for interpretation of high or low S-phase values. With a median follow-up time of 4.55 years, S-phase fraction, but not ploidy status, is a significant predictor for time to recurrence in both the randomly assigned and the untreated population (observed registered group and observed randomly assigned group).(ABSTRACT TRUNCATED AT 250 WORDS)     

乳腺不同类型病变与乳腺癌细胞DNA含量分析

目的：探讨乳腺不同类型病变与乳腺癌发生的关系。方法：应用流式细胞术（ＦＣＭ）测定细胞核ＤＮＡ指数（ＤＩ）、Ｓ期细胞比率（ＳＰＦ）和细胞增殖指数（ＰＩ）,对１２０例乳腺不同类型病变进行分析。结果：ＤＩ、ＳＰＦ及ＰＩ均随正常上皮→轻度增生→中度增生→重度增生→非浸润癌→浸润而信次增加,重芳增生是与乳腺癌最密切相关的癌前病变。结论：对癌前病变特别是重度增生进行ＦＣＭ分析,有利于发现早期乳腺癌。     

Effect of interferon-ß on the cell cycle of human glioma cell line U-251 MG: flow cytometric two-dimensional (BrdU/DNA) analysis

Summary This paper describes the determination of the effect of IFN-ß on U-251 MG cells using the bromodeoxyuridine (BrdU/DNA) analysis technique. The cell cycle perturbation of exponentially growing cells was estimated by a newly developed two-dimensional analysis of sequential BrdU/DNA distributions measured at 4-hr intervals after IFN-ß administration. The U-251 MG cell line was sensitive to IFN-ß, and cell proliferation was inhibited by 50.0% at 48 hr. Analysis of DNA histograms indicated that IFN-ß, accumulated the cells in the S-phase, from 16 to 48 hr after treatment. In the two-dimensional analysis, labeled cells treated with IFN-ß moved from the S-phase through the G₂M-phase and then entered the G₁-phase within 12 hr after the initial treatment, in a pattern similar to labeled cells untreated with IFN-ß. After 16 hr, labeled cells treated with IFN-ß, began to accumulate in the S-phase and remained there even after 48 hr. These results imply that IFN-ß may have an effect on the G₁-phase, thereby inducing S-phase accumulation of human glioma cell line U-251 MG.     

DNA ploidy and S-phase fraction in medullary carcinoma of the breast -a flow cytometric analysis using archival material

Summary In a population of 110 primary breast cancers with medullary features, registered in the Danish Breast Cancer Cooperative Group (DBCG) from 1977-82, we have determined ploidy and S-phase fraction (SF) by flow cytometry (FCM) on paraffin embedded tumour tissue. The distribution of DNA ploidy is not different from the distribution described for breast cancers in general. No difference is found between the subgroups of medullary and non-medullary cancer when using a new simplified histopathological definition of medullary carcinoma of the breast, recently proposed by us. When using the definition proposed by Ridolfiet al. in 1977, we find significantly more tumours with aneuploidy and high SF in the groups of typical medullary carcinoma (TMC) and atypical medullary carcinoma (AMC) than in the small group of non-medullary carcinoma (NMC), which seems a paradox, as patients with NMC have the worst prognosis. However, the number of patients in the NMC group is very small, and the percentage of aneuploid tumours is very low. In 84 protocolled patients we found no statistically prognostic importance of ploidy or SF, either in the whole group assessed or when stratifying for the histopathological subgroups. However, a prognostic influence of SF can be traced for the non-medullary cancers, according to the new definition, but not for the medullary cancers of the breast. The result emphasizes the impression of MC as being biologically different from other histological types of breast cancer.     

Evaluation of cell proliferation in breast carcinoma. Comparison of Ki-67 immunohistochemical study, DNA flow cytometric analysis, and mitotic count

Growth kinetics of 102 breast carcinomas were studied by immunohistochemical analysis with the monoclonal antibody Ki-67, which reacts with a nuclear antigen in proliferating cells. The results were correlated with ploidy and S-phase fraction (SPF) analyzed by DNA flow cytometric study and with mitotic count analyzed by light microscopic study. The proportion of Ki-67-positive cells (Ki-67 score) in breast carcinomas varied from 0.6% to 80% (median, 6.3%). Ki-67 scores were significantly higher in the DNA aneuploid than in the DNA diploid tumors. Ki-67 scores correlated significantly with tumor SPF in DNA aneuploid tumors. In DNA diploid tumors SPF showed no correlation with Ki-67 score. High Ki-67 scores were associated with high mitotic counts (P less than 0.0001) and histologic grade (P less than 0.0001). Nuclear pleomorphism, tubule formation, or lymph node status were not correlated with Ki-67 score. In conclusion, Ki-67 immunostaining correlates with other measures of cell proliferation (SPF, mitotic count) supporting its clinical significance.     

Expression of DNA methyl-transferase (DMT) and the cell cycle in human breast cancer cells

Estrogen receptor (ER)-negative breast cancer cells display extensive methylation of the ER gene CpG island and elevated DNA methyltransferase (DMT) expression compared to ER-positive cells. The present study demonstrates that DMT protein levels tightly correlate with S phase fraction in ER-positive cells, whereas ER-negative cells express DMT throughout the cell cycle. In addition, levels of p21CIP1, which disrupts DMT binding to PCNA, are inversely correlated with DMT levels. Therefore increased DMT expression in ER-negative cells is not simply due to elevated S-phase fraction, but rather to more complex changes that allow cells to escape normal cell cycle-dependent controls on DMT expression. Because ER-negative breast tumors often have activated growth factor pathways, the impact of these pathways on DMT expression was examined in ER-positive cells. Stable transfection with fibroblast growth factors (FGFs) 1 and 4 led to increased DMT expression that could not be accounted for by a shift in S phase fraction. Elevated DMT protein expression in FGF-transfectants was accompanied by a significant decrease in p21, again suggesting a reciprocal relationship between these two proteins. However, acquisition of an estrogen-independent phenotype, even in conjunction with elevated DMT levels, was not sufficient to promote ER gene silencing via methylation. These results indicate that multiple steps are required for de novo methylation of the ER CpG island.     

A flow cytometric analysis of DNA content in primary and metastatic lesions of esophageal squamous cell carcinoma.

BACKGROUND. DNA content of malignant tumors has been considered a significant prognostic factor, but intratumor variations in DNA content and differences in DNA content between primary and metastatic lesions have been found in various tumors. METHODS. DNA indexes of multiple samples obtained from different sites in each tumor were determined by flow cytometry (FCM) in 27 esophageal squamous cell carcinomas. RESULTS. Intratumor variation in DNA indexes in primary lesions was found in 10 (37%) of the 27 specimens. Of the rest, all DNA indexes were diploid in 5 and all identically aneuploid in 12 samples. A DNA index differing from that of the metastatic lesion was found in four (50%) of eight primary lesions; however, a component of the DNA index identical to that of metastatic lesion was found in seven (88%) of eight primary lesions. CONCLUSIONS. Recurrence after a "curative" operation was frequent in patients with a tumor that had an aneuploid DNA index, with or without a variation in DNA indexes. There was no recurrence in the five patients with tumors that had only a diploid DNA index. These results suggest that differences in DNA content between primary and metastatic lesions reflect intratumor variation in DNA content in the primary lesions. The presence of a tumor cell population with an aneuploid DNA content, even when the DNA content varied in the primary lesion, may indicate an aggressive clinical course in patients with esophageal squamous cell carcinoma.     

Image cytometric DNA analysis and proliferating cell nuclear antigen (PCNA) expression in transitional cell carcinoma of the bladder.

The proliferation rate as determined by Proliferating Cell Nuclear Antigen (PCNA) immunostaining and the DNA ploidy status as measured by static cytometry were studied in 70 transitional cell carcinomas of the bladder (TCCB) in relation to grade, stage, and recurrence. The follow-up period was 2 years. A significant difference was observed in PCNA expression among grades I, II, and III (P < 0.02), between superficial (pTa-pT1) and invasive (pT2-pT4) tumors (p < 0. 04), between recurring and non-recurring tumors (p < 0.001), and between tumors of the same grade with and without recurrence (p < 0. 05). A significant difference was also found in the ploidy pattern among grades I, II, and III (p = 0.002), and between superficial and invasive (p = 0.02) and recurring and non-recurring tumors (p < 0. 01). Finally, the recurrence status seems to be strongly influenced by the proliferation rate and ploidy of TCCB (p < 0.001). Our results suggest that the above-studied parameters may offer useful information on the biological behavior of TCCB.