Flow cytometric DNA analysis of gastric smooth muscle tumors.

BACKGROUND. To better understand the malignant grade of gastric smooth muscle tumors, the DNA content of these tumors was studied. METHODS. In 43 patients with gastric smooth muscle tumors, the cellular DNA content was determined by flow cytometry and compared with the histologic classification and the prognosis. RESULTS. Flow cytometry indicated that all 21 leiomyomas and 9 of the 10 low-grade leiomyosarcomas were diploid; 10 of the 12 high-grade leiomyosarcomas were aneuploid. All patients with leiomyomas and 8 of the 11 patients with diploid leiomyosarcomas had neither local recurrence nor metastasis. By contrast, 8 of the 10 patients with aneuploid leiomyosarcomas died of their disease (mean survival, 41 months; range, 18-78 months). CONCLUSIONS. These results indicate that the DNA ploidy pattern shown by flow cytometry is related closely to the histologic classification and prognosis of gastric smooth muscle tumors.     

Flow cytometrically determined DNA content of breast carcinoma and benign lesions: correlations with histopathological parameters

The relative DNA content of cellular samples from 54 patients affected by breast carcinomas and 20 affected by benign breast lesions (including 11 fibroadenomas) was measured by flow cytometry. All normal tissue samples and 17/20 (85%) specimens from benign lesions exhibited a cytometrically diploid DNA distribution, 3/20 (15%) benign lesions an abnormal DNA content, and 35/54 (65%) carcinomas at least one aneuploid cell subpopulation. Furthermore, 9/54 (17%) tumors were characterized by the presence of more than one aneuploid cell subpopulation. The results also indicate that flow cytometry can be used to recognize lymph nodes infiltrated by aneuploid cells. Statistically significant correlations were evidenced between the occurrence of aneuploidy or the ploidy level measured as DNA index and the nodal infiltration status. The percentage of S cells can also be extracted from DNA content distribution histograms. Statistically significant differences (p less than 0.01) were also observed for the percentage of S cells between normal tissues (6.2 +/- 3.2 SD) and benign lesions (11.1 +/- 6.6 SD), normal tissues (6.2 +/- 3.2 SD) and aneuploid tumors (19.7 +/- 10.3 SD), benign lesions (11.1 +/- 6.6 SD) and aneuploid tumors (19.7 +/- 10.3 SD), and diploid (7.9 +/- 4.0 SD) and aneuploid tumors (19.7 +/- 10.3 SD).     

Flow cytometric analysis of nuclear DNA content in ovarian tumors. Association of ploidy with tumor type, histologic grade, and clinical stage.

The authors undertook a prospective, flow cytometric study of nuclear DNA ploidy in 140 fresh ovarian tumors. There were 43 benign tumors, 27 borderline tumors, and 70 malignant tumors. Results of DNA ploidy analysis were compared to age at diagnosis, menopausal status, tumor size, histologic type, grade, and International Federation of Gynecology and Obstetrics (FIGO) stage. Although the majority of benign tumors were diploid, 19% were aneuploid. Among the benign tumors, DNA ploidy was significantly associated with tumor type and tumor size. All borderline tumors were diploid. Of the 70 malignant tumors, 64% were aneuploid. In the malignant tumors, DNA aneuploidy had significant univariate associations with histologic type, grade, and FIGO stage. By multivariate analysis, DNA aneuploidy remained significantly associated with stage and grade, both known predictors of survival in ovarian cancer. These results indicate that DNA ploidy varies with the aggressive potential of an ovarian cancer and may, at the time of initial diagnosis, provide additional information about tumor prognosis.     

DNA aneuploidy and cell proliferation in breast tumors

The cellular DNA content of 30 benign and 180 malignant breast tumors was analyzed by means of flow cytometry (FCM). All benign tumors exhibited a normal DNA content (diploid), whereas 65% of the malignant tumors showed an abnormal DNA content (aneuploid). The ploidy distribution of malignant tumors was bimodal with an increasing frequency near diploid DNA index (DI), and a second group had a DI ranging from triploid to tetraploid. In estimating the degree of malignancy eight independent histomorphologic and cytologic criteria were introduced. A good correlation was observed between DNA content abnormalities and the grade of differentiation of breast carcinomas. The percentage of S-phase cells of DNA aneuploid cell lines was significantly higher than in the diploid ones. The highly differentiated breast carcinomas (Grade 1) indicated lower S-phase values as compared to the undifferentiated (Grade 3) ones. S-phase values estimated by FCM were about two times higher than the 3H-thymidine labeling index (LI) obtained by an in vitro procedure. The data estimated in this study showed that DNA determinations as an adjunct to conventional histopathologic assessment may provide objective clinically relevant information with respect to the degree of malignancy and prognosis of patients with breast carcinoma.     

Flow cytometric analysis of the nuclear DNA content of hepatoblastoma.

The nuclear DNA content of 15 hepatoblastoma cases was determined in paraffin-embedded tissues by flow cytometry. The DNA index (DI) was calculated, and the ploidy pattern of nuclear DNA was estimated. The correlation between the ploidy pattern and clinicopathologic findings was studied, and the prognostic significance of the ploidy pattern was investigated. An aneuploid pattern was seen in 50% of the lesions with histologic embryonal and anaplastic types. It was not seen in the fetal type. In the tumors with combined epithelial components, the fetal-type component had a diploid pattern in all five cases. The embryonal-type component was associated with aneuploidy in two of five cases. In aneuploid tumors, vascular invasion (tumor emboli in the vessels) was observed more frequently. The prognosis of the patients with an aneuploid tumor was significantly poorer. These results indicate that nuclear DNA ploidy pattern analysis might be useful in investigating the prognosis of hepatoblastoma.     

Variability in DNA measurements in multiple tumor samples of human colonic carcinoma

The DNA ploidy and cell-cycle distribution of three separate fresh tissue samples of 60 colorectal adenocarcinomas were analyzed by flow cytometry. DNA ploidy was concordant among the three samples in 38 cases (63.3%). In the remaining 22 cases (36.6%), the DNA histograms of two of the three multiple samples were similar; however, the ploidy of the third sample was discordant. No relationship was observed between Dukes' stage and histologic grade with concordance or discordance among samples. Thus, in about one third of the colonic carcinomas, a single sample showing either a diploid or diploid-cycling DNA histogram would not detect aneuploid DNA patterns. Comparison of scrapes and fine-needle aspirates of tumors as alternative sampling methods of tumors for DNA ploidy analysis indicated a strong association with the tumor ploidy (84% and 96%, respectively) only when the ploidy of the multiple samples was concordant. In about 25% of the cases, tumor scrapes and fine-needle aspirates did not correlate with the "most abnormal" ploidy observed in one of the three tissue samples. The data suggest that single or even double tissue samples may not show aneuploid DNA patterns in a substantial proportion of colorectal cancers.     

Flow DNA analysis of primary bone tumors. Relationship between cellular DNA content and histopathologic classification

The cellular DNA content of 15 benign and 34 malignant primary bone tumors was analyzed by means of flow cytophotometry. All benign tumors except one of questionable histologic type exhibited a normal DNA content (diploid), whereas 23 of 34 malignant tumors showed an abnormal DNA content (aneuploid). Closer analysis revealed that all supposedly highly malignant tumors, i.e., 16 osteosarcomas and 1 Ewing sarcoma were aneuploid, while 8 of 13 chondrosarcomas, 2 periosteal osteosarcomas, and 1 of 2 adamantinomas were diploid. Interestingly, these diploid malignant tumors represent tumor entities which are known to include variants of low-grade malignancy. Cell distribution analysis showed that the aneuploid tumors exhibited a higher proportion of S-phase and G2 + M cells than the diploid tumors, indicating differences in proliferative activity. However, no significant difference in this respect could be demonstrated between diploid benign and diploid malignant tumors. The current study clearly shows that flow DNA cytophotometry can be applied to most primary bone tumors despite a substantial content of hard tissue. The results also indicate that DNA determinations as an adjunct to conventional histopathologic assessment may provide objective clinically relevant information with respect to the degree of malignancy. Thus, regardless of histogenetic origin, it appears that benign bone tumors as well as malignant bone tumors of low-grade malignancy in general, are diploid, whereas highly malignant bone tumors in general are aneuploid.     

FLOW CYTOM ETRIC ANALYSIS OF CELLULAR DNA CONTENT IN EPITHELIAL OVARIAN TUMOR

The DNA content of tumor all was analyzed by flow cytometry on parafflnembedded specimens in 73 patients with epithelial ovarian tumor, and its clinical significance was evaluated. One of the 5 benign (20%), 2 of the 11 borderline (18.18%), and 30 of the 57 malignant (52. 63%) tumors were aneuplold. The occurrence rate of aneuploidy In malignant tumors was higher than In benign and borderline tumors ( P < 0. 05 ). Furthermore, aneuploidy was more frequently In the advanced stages (Ⅲ -Ⅳ ) (77. 7%) than in the early stages (Ⅰ - Ⅱ ) (9. 5%) (P<0. 005). The occurrence rate of DNA aneuploidy was higher in patients associated with ascites and the residual tumor≥.2 cm. Patients with aneuploid tumors had more of ten ascites (P<0. 005) and residual tumor size≥2cm (P< 0.005). There was no apparent correlation between the DNA ptoidy and the histologic grade, histologic type of the tumors. G0/G1 cell proportion of DNA diplold tumors in advanced carcinoma (64. 6%) was less than those of early stage carcinoma (     

Flow cytometric analysis of DNA and cell proliferation in ovarian tumors

DNA content in tumor cells from 50 patients with ovarian tumors was analysed by flow cytometry (FCM). Solid tissue samples were processed to obtain monodispersed cells. Staining for DNA analysis was achieved with ethidium bromide and mithramycin. Peripheral blood lymphocytes were used as reference diploid cell population. All benign ovarian tumors exhibited only diploid cells. DNA aneuploid cell lines were found 66.6% of serous carcinomas and in 80% of malignant granulosa cell tumors. The S-phase fraction of DNA diploid cells in benign ovarian tumors (S = 2.4 +/- 1.2%) was smaller than those of malignant tumors (S = 8.2 +/- 5.2%). DNA aneuploid cell populations in serous carcinomas display a higher S-phase fraction (S = 19.2 +/- 9.3%) than DNA diploid cells (S = 11.7 +/- 3.2%). No major differences were obtained between primary ovarian tumors and their metastases, as far as degree of aneuploidy and S-phase fraction are concerned. A high degree of correlation was established between the grade of differentiation of ovarian tumors and the DNA ploidy abnormalities.     

Relation between DNA ploidy and the clinical behavior of phyllodes tumors

We studied the DNA histograms obtained by flow cytometry from a series of six giant fibroadenomas and ten phyllodes tumors to determine if the analysis of DNA ploidy would help to predict clinical behavior. We were unable to document any relation between ploidy and histologic appearance, recurrence, metastasis, lesion size, or patient age. DNA aneuploid stem cell lines were seen in 75% of histologically benign phyllodes tumors, 50% of histologically malignant phyllodes tumors, and approximately 33% of giant fibroadenomas.     

Flow cytometric analysis of DNA content in human bladder tumors and irrigation fluids

Flow cytometry (FCM) was used to study the DNA distribution of 99 tumor biopsy specimens and 41 bladder irrigation samples from patients with transitional cell carcinoma of the bladder. For tumor biopsy and cystectomy specimens, the frequency of aneuploidy increased with advancing tumor stage and grade. All T0 tumors were diploid. Twenty-seven percent of T1, 71.4% of T2, and 75% of T3 and T4 tumors were aneuploid. All Grade I tumors were diploid. Thirty percent of Grade II and 76.9% of Grade III tumors were aneuploid. The frequency of aneuploidy of tumors in the early stages (Ta, T1) is similar to the incidence of subsequent progression by these tumors described in the literature. For irrigation fluids, the relationship between grade and stage and the frequency of aneuploidy was similar to the relationship seen with tumor specimens. All four patients with only carcinoma in situ had aneuploid cells in their irrigations. The comparison of FCM data of bladder biopsy and bladder irrigation from the same cystoscopic evaluation suggests adequate representation of tumor cells in the irrigation fluids for almost all cases. The authors conclude that DNA ploidy analysis by FCM appears useful in a clinically important group of patients with aneuploid superficial tumors of moderate or high grade. Bladder irrigation analysis appears useful in the follow-up of patients with a history of carcinoma in situ and those with aneuploid tumors.     

Prognostic significance of DNA ploidy in patients with stage II colorectal cancer

DNA ploidy status associated with colorectal cancer has been investigated widely in recent years. But the relationship between DNA analysis and prognosis has not been confirmed.[1(3] Some investigations showed that DNA aneuploid status was associated with lymph node involvement and poor differentiation, and thus may predict a poor clinical outcome in patients with colorectal cancer. These observations suggest that DNA ploidy abnormality may influence the development and progression of colo…     

Flow cytometric analysis of nuclear DNA content in esophageal cancer. Aneuploidy as an index for highly malignant potential

Flow cytometric analysis of nuclear DNA content was performed on excised malignant tissue of the esophagus. The DNA ploidy pattern was compared with a variety of histologic parameters and the subsequent clinical course to determine whether or not this pattern is associated with the mode of malignant potentiality. Of the 31 patients, eight had the diploidy DNA pattern and 23 the aneuploid DNA pattern. Tumors with the aneuploidy DNA pattern had a significantly higher frequency of lymph node metastasis than did those with the diploidy DNA pattern (P less than 0.01). Mitotic rates in the aneuploid tumors were significantly higher than was the case in diploid tumors (P less than 0.0005). The incidence of recurrence within 12 months after surgery was higher in patients with aneuploid tumors (83.3%) than in those with diploid ones (16.7%), with a statistical difference (P less than 0.05). Thus, the DNA aneuploidy based on flow cytometry closely correlates with the high frequency of nodal involvement and high mitotic rates, factors generally indicative of the aggressive behavior of the malignant tumors. DNA aneuploidy based on flow cytometric analysis is a pertinent index for determining the highly malignant potential in esophageal carcinoma.     

Ploidy assessment of benign and malignant ovarian tumors by flow cytometry. A clinicopathologic study

In a prospective study, 112 fresh ovarian tumor samples were collected from 83 consecutive patients. Cellular DNA content was measured by flow cytometry. All the benign (n = 24) and semimalignant (n = 6) tumors were diploid. Of 50 malignant tumors, 24 (48%) were diploid and 26 (52%) were aneuploid. Aneuploidy was more frequent in the advanced stages of the disease, in tumors of low degree of differentiation, and in older patients. The patients with aneuploid tumors had smaller primary tumors and more often ascites. The fraction of cells with S-phase DNA content was higher in the aneuploid tumors. No association was seen to the tumor type. Ploidy determination is objective and reproducible. Aneuploidy associates to most negative prognostic factors in ovarian carcinoma and may reflect the aggressiveness of the tumor. The ploidy status may be taken into consideration in the stratification of patients of comparable risk for treatment studies.     

Prognostic value of DNA content in colorectal carcinoma. A flow cytometric study with some methodologic aspects

The DNA content of 37 colorectal carcinomas was studied prospectively by flow cytometry. The DNA content from the same tumors was also studied in disintegrated paraffin sections. The methods gave similar information on the DNA content and the correlation was 0.94. In eight patients tumor imprints were prepared and the DNA content was analyzed with static cytometry. A correlation coefficient of 0.83 between flow cytometry on fresh tumor tissue and static cytometry was found. Sixty-two percent of the tumors were aneuploid and had significantly higher S-phase values than diploid tumors. Histologic grade was not related to ploidy level, whereas Dukes' C tumors often were aneuploid. When the clinical course of patients was analyzed by log rank test (mean follow-up, 30.4 months), the patients with diploid tumors showed a clear advantage in terms of survival; only two patients in this group developed progressive disease. It is concluded that the DNA content is an independent prognostic predictor of colorectal carcinoma and that DNA can be analyzed by several methods. Of these, the method using disintegrated paraffin sections appears most attractive.     

The prognostic significance of tumor ploidy and pathology in adenocarcinoma of the esophagogastric junction

Tumor DNA content has been advocated to be an important prognostic indicator in human malignancies. Paraffin-embedded specimens of 75 resected adenocarcinomas (AC) of the esophagogastric junction were studied by flow cytometric DNA analysis to determine whether tumor ploidy was a significant prognostic variable independent of stage and histologic grade of the tumor. Eighty-one percent of the tumors were aneuploid. More patients with aneuploid tumors had lymph node metastases than patients with diploid tumors (P = 0.007). Patients with aneuploid tumors had poorer 18-month disease-free and overall survival than patients with diploid tumors. Cox regression analysis demonstrated that the most important prognostic variables for predicting overall survival were lymph node status, depth of wall invasion, and tumor differentiation. Tumor ploidy was not an independent prognostic variable in predicting recurrent disease or death from AC of the esophagogastric junction. Tumor DNA content is valuable, however, as a marker for patients at increased risk of lymph node metastases, early recurrence, and poorer survival.     

Correlation of DNA ploidy and clinical outcome in Borrmann type 4 gastric carcinoma

The correlation between DNA ploidy pattern and clinical outcome was studied in 76 patients with Borrmann type 4 gastric carcinomas. Twenty-six tumors were diploid, and 50 tumors were aneuploid. There was no correlation among DNA ploidy and histologic type, lymph-node status, wall invasion, or clinical stage. The incidence of vascular invasion in the aneuploid tumors was significantly higher than that in the diploid tumors. Five year survival was achieved in 28% of the patients with diploid tumors and 8% of those with aneuploid tumors, respectively. Among the patients undergoing curative resection, 5 year survival rate was 54% in the patients with diploid tumors compared to 28% with aneuploid tumors. There was a significant survival advantage in patients with diploid tumors. These results indicate that DNA ploidy might be an important prognostic factor in Borrmann type 4 gastric carcinomas.     

Predicting outcome for patients with node negative breast cancer: a comparative study of the value of flow cytometry and cell image analysis for determination of DNA ploidy.

This study was aimed at determining whether tumour DNA content measured by cell image analysis could provide additional prognostic information when compared to that provided by flow cytometry. Sections cut from paraffin blocks of tumours from 101 patients with node negative breast cancer were analysed by both methods and the results related to other prognostic variables and to patient relapse and overall survival. DNA ploidy measured by flow cytometry classified 46 tumours as diploid and 55 as aneuploid, whereas by cell image analysis 30 were diploid and 71 aneuploid (P less than 0.002). There were 20 tumours with discrepancies between the two methods; 18 of these were tumours with only one peak in flow analysis, but determined to be aneuploid with image analysis. DNA content as measured by both methods was significant for predicting relapse and survival by log-rank test, as were tumour histological grade, c-erbB-2 expression and tumour size. Multivariate analysis showed DNA ploidy measured by flow cytometry to be the only variable of independent significance (P less than 0.02) for both relapse and overall survival. Compared with cell image analysis, flow cytometry demonstrated a significantly higher proportion of diploid tumours, which may be related to differences in the internal standards applied to each method. We suggest that cell image analysis techniques can provide more sensitive information on the DNA content of tumour cells by direct measurement of nuclear DNA density of both normal lymphocytes and tumour cells in the same section. However, although image analysis appears to be more sensitive than flow cytometry in detecting DNA aneuploidy, the image technique appears to lack the specificity of flow cytometry in correlation with clinical outcome.     

Flow cytometric DNA ploidy in salivary gland tumours

This study on 279 tumours of the salivary glands was conducted to analyse whether the assessment of DNA ploidy by flow cytometry may assist histopathology in discriminating benign from malignant types of tumours. The group of benign tumours included 164 pleomorphic adenomas, 51 Warthin's tumours, 7 basal cell adenomas, 2 lipomas as well as 5 other different tumours. All of the 229 benign tumours were diploid. The malignant tumours consisted of 18 adenoid cystic adenomas, 10 mucoepidermoid carcinomas, 5 acinic cell carcinomas, 5 carcinoma in pleomorphic adenoma as well as of 12 other malignancies belonging to 7 different tumour entities. Twelve of 50 malignant salivary gland tumours were aneuploid. There was no significant relationship between the DNA ploidy status and histopathological grading, lymph node metastasis and local recurrence development, respectively. In three cases which initially were taken for pleomorphic adenomas by routine histological examination, aneuploid cell populations exposed by DNA flow cytometric analysis gave rise to a closer inspection of the suspect lesions. Examination of consecutive slides actually revealed small assemblies of carcinoma cells that required a final diagnosis as non-invasive carcinoma in pleomorphic adenoma. The most obvious value of DNA flow cytometry in salivary gland tumours is thus its contribution to assist histopathology in identifying potentially malignant lesions.     

Prognostic implications of the ploidy pattern of the nuclear DNA in hepatocellular carcinomas

The nuclear DNA contents of paraffin-embedded specimens of 41 cases of a hepatocellular carcinoma have been measured by means of flow cytometry. Results have indicated that 25 cases (61%) were diploid and 16 cases (39%) were aneuploid. In the aneuploid cases, the serum AFP level was found to be higher and stage more advanced. We also found that patients with aneuploid tumors had a poorer prognosis than those with diploid tumors, this fact uncovered by means of a Cox's proportional hazard model. In conclusion, the ploidy pattern of the nuclear DNA may serve as a useful prognostic marker for a hepatocellular carcinoma.