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.     

Refining the prognostic significance of DNA ploidy status in colorectal cancer: a prospective flow cytometric study

A consecutive series of 123 colorectal cancers, prospectively followed for 3 years, was studied to determine if the prognostic significance of DNA ploidy related to: (a) thresholds used to define DNA aneuploidy, or (b) aneuploid sub-groups defined by DNA index (DI) and peak size. Aneuploidy was defined using 3 methods depending on the minimum proportion of nuclei considered to constitute an aneuploid peak; type 1, 5%; type 2, 10%; type 3, 10% if DI is 1.1-1.8, but 15% if DI is 1.9-2.1. DNA aneuploidy rates were type 1, 75%; type 2, 67%; type 3, 58%. The significance of clinical and pathological correlations varied with the use of different methods. All were associated with a significant DNA diploid survival advantage which was strongest for type 3 (p = 0.006). DI was unrelated to survival irrespective of the presence or absence of an associated S/G2; 33% with a DI of 1.1-1.8 and 35% with a DI of 1.9-2.1 survived. Prognosis was inversely proportional to aneuploid peak size, 48% with small peaks (less than 20%), 30% with intermediate peaks (greater than 20% less than 40%), but none with large peaks (greater than 40%) survived (p = 0.03). We conclude that: (a) thresholds used to define DNA aneuploidy affect the prognostic significance of DNA ploidy; (b) survival is independent of the DI of aneuploid peaks; and (c) measurement of aneuploid peak size refines the prognostic value of DNA ploidy.     

Cytogenetic and corresponding flow cytometric DNA analysis of renal cell neoplasms.

Results of flow cytometric DNA content and cytogenetic analyses of six neoplasms representing the spectrum of the morphologic subtypes of renal cell neoplasms are compared. Flow cytometric determinations of DNA diploidy and near diploidy in two renal cortical neoplasms correlated with modal chromosome numbers; however, in three of four neoplasms with a highly aneuploid DNA content, the modal chromosome numbers indicated diploidy or near-diploidy. Our data suggest that the short-term culture conditions used in cytogenetic analysis may favor growth of cells with diploid or near-diploid DNA content. This may explain the discrepancy frequently observed between results obtained using flow cytometry and karyotyping after short-term culture. We also observed (1) abnormalities in the short arm of chromosome 3 in all three nonpapillary neoplasms, (both clear and granular cell types); (2) aberrations in chromosomes 3 and 17 in the two papillary tumors; and (3) normal chromosome 3 in the presence of various random karyotypic anomalies including telomeric associations and a pseudo-diploid modal chromosome number in the only oncocytic neoplasm studied.     

Clinical flow cytometric DNA analysis of human solid tumors

Flow cytometry is widely used to analyze nuclear DNA content. Its clinical application in oncology is becoming increasingly important for detecting abnormalities in DNA content (DNA aneuploidy) of cells from solid tumors and for monitoring cell-growth kinetics in tumor cells (S-phase estimation), information which may be relevant for prognosis and evaluation of therapy. The risks of false DNA ploidy determination and inaccurate S-phase estimation are discussed. A methodological approach, criteria for histogram interpretation and criteria for histogram rejection are proposed in order to avoid these artifactual results.     

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.     

Flow cytometric DNA analysis: a prognostic tool in non-Hodgkin's lymphoma

Surgical biopsies from 234 untreated patients with non-Hodgkin's lymphoma (NHL), classified according to the Kiel nomenclature, were analysed with respect to proliferative activity (S-phase) and DNA content by flow cytofluorometric (FCF-DNA) analysis. The percentage of cells in S-phase was significantly higher in lymphomas of high compared to low grade NHL (p less than 0.001). Patients with lymphomas of high grade histology and low S-phase values (less than 5.6%) achieved complete remission (CR) more often (p less than 0.05) and survived significantly longer than those with high S-phase values (p less than 0.05). In the low grade NHL group the S-phase value did not correlate to response. S-phase correlated to survival for patients with the lymphocytic (CLL & IC) (p less than 0.05) and follicle center cell (FCC) derived (p less than 0.01) but not in blastic (LB, IB, Burkitt) NHL. DNA-aneuploidy was associated with poor response to therapy and shorter CR duration in low grade NHL (p less than 0.05 for both). However, the degree of DNA-ploidy (neardiploid or aneuploid) did not correlate to survival in any of the NHL groups analysed (high- or low grade, lymphocytic, FCC derived or blastic). The Cox regression analysis indicated that the S-phase value was a stronger predictor of survival than histopathology, stage or age, especially in low grade NHL. These results suggest that S-phase analysis should be included in the clinical evaluation of NHL patients as a prognostic indicator.     

脑膜瘤DNA流式细胞术分析的临床病理学意义

目的探讨脑膜瘤细胞DNA倍体和增殖活性与脑膜瘤组织病理分型及生物学特性的关系.方法采用流式细胞仪检测42例脑膜瘤细胞DNA倍体和S期比例(SPF)、增殖指数(PI),结合肿瘤病理学类型进行相关性分析.结果在不典型、恶性脑膜瘤细胞中,非整倍体比例、SPF、PI显著性增高(P＜0.01);在组织亚型之间,各参数无显著性差异(P＞0.05):SPF、PI在非整倍体细胞系和复发脑膜瘤细胞中显著性增高(P＜0.01).结论流式细胞术分析是研究脑膜瘤细胞动力学和生物学特性的重要方法.DNA非整倍体可作为判定肿瘤恶性程度的指标.SPF、PI作为细胞增殖的指标,对脑膜瘤恶性程度和预后的评估具有重要意义.     

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.     

Synovial sarcoma. A DNA flow cytometric study

The relationship between DNA content, clinicopathologic findings, and patient survival in synovial sarcoma was investigated. Patient age at diagnosis (P less than 0.001), tumor size (P less than 0.001), and ploidy status (P less than 0.003) correlated significantly with patient survival. A marginally significant correlation between mitotic count and patient survival was also observed (P = 0.04). Histologic subtypes (monophasic versus biphasic), mitotic count, and S-phase by flow cytometry had no significant influence on the clinical outcome of patients with synovial sarcoma in this study. The authors conclude that DNA ploidy analysis is a significant objective probe in the prognostication of patients with synovial sarcoma.     

Giant cell tumor of bone. A clinicopathologic and DNA flow cytometric analysis

Flow cytometric DNA analysis was performed on 60 cases of giant cell tumor of bone and the results were correlated with the clinicopathologic features. Tumors studied were from 31 men and 29 women whose ages ranged from 18 to 62 years (median, 29 years). The most common sites were the distal end of the femur and proximal end of the tibia, accounting for 75% of the lesions. Treatment consisted of resection in 29 patients (48%), curettage with bone chip packing in 15 patients (25%), or curettage with cement packing in 16 patients (27%). Ten patients (17%) had local relapse within 1 to 3 years, and two had lung metastases. Forty-two patients (70%) exhibited tumors with a diploid DNA content, 16 aneuploid (27%), and two tetraploid (3%). Six (37.5%) of the aneuploid patients had relapses: one of those had been treated by resection of the tumor and five by curettage. Of the remaining ten (62.5%) unrelapsed aneuploid patients, nine had been treated by resection of the tumor and one by curettage. Four of the 42 diploid patients (9.5%) had relapses; all had been treated by curettage of the tumor. The two tetraploid tumors were treated by resection and none relapsed. Histologic parameters did not correlate with relapse rate or DNA pattern. Although relapse was more common among aneuploid tumors, our study shows that this appears to be influenced by the treatment modality rather than the ploidy status. Based on this study the DNA analysis of giant cell tumor of bone has a limited utility for predicting the tumor's biologic behavior.     

Carcinoma of the anal canal and flow cytometric DNA analysis

Using flow cytometric DNA analysis of paraffin embedded tissue, DNA histograms were successfully obtained from the anal cancers of 117 patients. DNA diploid patterns were given by 82 cancers (70%) and DNA non-diploid patterns by 35 cancers (30%): 15 DNA aneuploid, 20 DNA tetraploid. Well differentiated squamous cell cancers were mainly DNA diploid, while a larger proportion of poorly differentiated and small cell cancers were DNA non-diploid. The large majority of stage A cancers were DNA diploid. A greater proportion of tumours that had invaded through the anal sphincter or had lymph node metastases or distant spread were DNA non-diploid. Prognosis was slightly poorer for patients with DNA non-diploid cancers when compared to patients with DNA diploid tumours (P = 0.08) and significantly poorer for individuals with DNA aneuploid anal cancers (P = 0.037). However, in a multivariate analysis model, the DNA ploidy pattern of an anal cancer was not of independent prognostic significance alongside tumour histology and tumour stage.     

Significance of flow cytometric DNA analysis from freshly aspirated samples

The procedure of aspiration biopsy cytology by fine needle aspiration (ABC) is as option in establishing definitive diagnoses for breast cancers. In this series, a needle size of 21G was considered most suitable for ABC as well as flow cytometric DNA analysis. Histograms from fresh samples aspirated by fine needle clearly delineated a sharp peak in G&sup0;G(1) phases and also a better CV was obtained than with paraffin-embedded preparations. In addition, fresh samples gave more reliable DI and suggested the value of measuring nuclear DNA contents. It is believed that the prognoses of breast cancers are closely associated with DNA ploidy patterns. In this sense, flow cytometric DNA analysis of fresh samples of ABC is regarded as important in clinical use.     

痰液细胞DNA含量分析对肺癌的诊断价值

目的　探讨痰液细胞DNA含量分析对肺癌的诊断价值。方法　用流式细胞仪对 44例患者 ( 2 9例肺癌、15例肺良性疾病患者 )痰液细胞DNA含量进行分析 ,并与痰细胞形态学检查结果作比较。对痰细胞学阴性而DNA含量分析阳性的对照患者组织切片行 p5 3、PCNA、Ki67免疫组化分析。 结果　以最后病理诊断为标准 ,肺癌患者异倍体检出的敏感性为 82 .8%,显著高于痰细胞学的敏感性 ( 2 7.6%) (P <0 .0 0 5 ) ;肺癌晚期异倍体阳性率高于早期肺癌 ( 87.5 %比 76.9%,P <0 .0 0 2 5 ) ,其中 1例肺癌患者提前临床 10月检出异倍体。部分炎性假瘤及结核球患者痰标本检出异倍体 ,其相应的组织切片PCNA、Ki67免疫组化染色亦阳性。结论　痰液细胞DNA含量分析是肺癌诊断有效的辅助方法 ,可能有利于肺癌的早期诊断。部分炎性假瘤及结核球存在细胞增殖异常     

Gastric lymphomas in Turkey. Analysis of prognostic factors with special emphasis on flow cytometric DNA content

BACKGROUND: In contrast to DNA ploidy, to the authors' knowledge the prognostic significance of S-phase fraction (SPF) in gastric lymphomas has not been determined. In the current study, the prognostic significance of various parameters including SPF and DNA aneuploidy were analyzed and some distinct epidemiologic and biologic features of gastric lymphomas in Turkey were found. METHODS: A series of 78 gastric lymphoma patients followed at Hacettepe University is reported. DNA flow cytometry was performed for 34 patients. The influence of various parameters on survival was investigated with the log rank test. The Cox proportional hazards model was fitted to identify independent prognostic factors. RESULTS: The median age of the patients was 50 years. There was no correlation between patient age and tumor grade. DNA content analysis revealed 4 of the 34 cases to be aneuploid with DNA index values < 1.0. The mean SPF was 33.5%. In the univariate analysis, surgical resection of the tumor, modified Ann Arbor stage, performance status, response to first-line chemotherapy, lactate dehydrogenase (LDH) level, and SPF were important prognostic factors for disease free survival (DFS). The same parameters, excluding LDH level, were important for determining overall survival (OS). In the multivariate analysis, surgical resection of the tumor, disease stage, performance status, and age were found to be important prognostic factors for OS. CONCLUSIONS: To the authors' knowledge the current study is the first to demonstrate the prognostic significance of SPF in gastric lymphomas. The distinguishing features of Turkish gastric lymphoma patients are 1) DNA indices of aneuploid cases that all are < 1.0, which is a unique feature; 2) a lower percentage of aneuploid cases; 3) a higher SPF; 4) a younger age distribution; and 5) lack of an age-grade correlation. The authors conclude that gastric lymphomas in Turkey have distinct biologic and epidemiologic characteristics.     

Correlation of DNA flow cytometric results and other prognostic factors in primary breast cancer

The percentage of cells in S-phase and DNA-ploidy have been measured in 300 primary mammary carcinomas by means of DNA-flow cytometry (FCM). The data were compared with the age and menopausal status of the patients as well as with the size, regional lymph-node involvement, histologic type, grade and concentration of estrogen (ER) and progesterone (PR) receptors of the tumors. A DNA-diploid distribution of the G0/1-peak was found in 37.6% of the cases. The mean percentage of S-phase fractions was 4.83. DNA-aneuploid tumors had significantly higher amounts of S-phase fractions (6.12%) than DNA-diploid tumors (2.66%). There was also a significant correlation between the DNA measurement data (DNA-ploidy and S-phase fractions) and histologic grade, as well as the content of ER and PR, but not between DNA-ploidy, S-phase fractions, tumor size (T) and evidence of axillary lymph-node metastases. DNA-FCM gives a biological characterization of the tumor in addition to the histopathologic examination. The method can be used as a routine procedure because of the reliability and reproducibility of the results as well as the short time needed for the measurements.     

A misleading flow cytometric analysis of DNA in an adenocarcinoma: a comparative flow cytometric and cytogenetic study

A case is presented in which flow cytometric and cytogenetic analysis was done on a biopsy from a highly anaplastic metastatic adenocarcinoma. Flow cytometric analysis of DNA content failed to show a significant population of aneuploid cells. However, histologic examination revealed a substantial number of tumor cells, and cytogenetic analysis produced chromosome counts ranging from 20 to 144.     

Renal oncocytoma: long-term follow-up and flow cytometric DNA analysis

We report a retrospective study on the clinicopathologic features and flow cytometric DNA analysis of ten renal oncocytomas compared with a control group of ten randomly selected renal cell carcinomas. Among the oncocytoma patients, no recurrences or metastases were noted over an average follow-up of 6.7 years (range = 6 months to 16 years). Reproducible, high-quality DNA histograms were obtained on the paraffin-embedded specimens by using our modified flow cytometric procedure. One aneuploid (10%) and two hyperdiploid tumors (20%) were found in the oncocytoma group. There was no correlation between these abnormal DNA histographic patterns and survival or tumor stages. On the contrary, a good correlation was found between tumor grades and DNA ploidy in the controls. We conclude that renal oncocytoma is a clinically benign tumor, yet it may exhibit varying degrees of flow cytometric DNA abnormalities, which have no predictive value on survival and probably reflect the characteristics of oncocytes rather than its malignant potential.     

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.     

DNA flow cytometric analysis of human nasopharyngeal carcinoma in nude mice

The tissue from a patient with nasopharyngeal carcinoma has been transferred to nude mice (BALB/c), and has successfully been growing through twenty passages. The tumors in the nude mice and primary human tumor have been examined for the cellular DNA content by FCM and also conventional pathological examination, chromosome analysis, and EBV test. The tumor take rate varied markedly in different passages with a mean value of approximately 70%, and showing a tendency to increase. The tumor doubling time within 6-12 weeks after transplantation of six tumors in 18 and 20 passages were 14.8 and 9.3 days respectively. However, the tumor volume at 12 weeks varied significantly, ranging from 438 to 1998 mm3. By FCM, it has been found that the values of DNA index were about the same in both primary tumor and the tumors in nude mice. The distribution of various phase cells in cell cycle was also about the same in both. In conclusion, the application of FCM to examine the cellular DNA content of the tumor in nude mice is a rapid and sensitive method, useful in the investigation on the stability of biological characteristics of human NPC in nude mice and in further studies on the effects by various therapeutic methods.