DNA flow cytometry of canine mammary tumors: comparative aspects with human breast tumors

Flow cytometric analysis of DNA content was performed on 28 samples of canine mammary tumors. Nine of them were benign and 19 were malignant. All benign tumors and 11 malignant tumors (57.9 percent) were diploid (P<0.05). Form the aneuploid tumors, five (26.3 percent) were hyperdiploid, one (5.3 percent) hypodiploid, one (5.3 percent) near triploid and one (5.3 percent) multiploid. The analysis of the expression of the markers PR and CD31 revealed a significant difference between diploid and aneuploid tumors (P<0.05). The immunoreactivity of PR was higher in diploid tumors, while the immunoreactivity of CD31 was stronger in aneuploid tumors. No difference between the markers MIB-1, c-erbB2, p53 and Cyclin D1 was observed (P>0.05). Using the flow cytometry analysis and immunohistochemistry, it was found a close relationship between aneuploidy and malignant character of neoplasias, progesterone receptor (PR) negative immunostaining and higher microvases density. No correlation between DNA content and S phase or immunoreactivity for the markers MIB-1, p53, c-erbB2 and Cyclin D1 was observed

Análise por citometria de fluxo de DNA foi realizada em 28 amostras de tumores mamários de cadela. Nove eram benignos e 19 malignos, sendo todos os benignos e 11 malignos (57,9 por cento) diplóides (P<0,05). Dos tumores aneuploides, cinco (26,3 por cento) eram hiperdiploides, um (5,3 por cento) hipodiploide, um (5,3 por cento) triploide e um (5,3 por cento) multiploide. A análise dos marcadores de expressão PR e CD31 revelaram significativa diferença entre tumores diploides e aneuploides (P<0,05). A imunorreatividade do PR foi maior em tumores diplóides e a imunoreatividade do CD 31 maior em tumores aneuploides. Para os marcadores MIB-1, c-erbB-2, p53 e Ciclina D1 não foi observada diferença significativa (P>0,05). Pela citometria de fluxo e pela imunoistoquímica verificaram-se uma relação entre aneuploidia e características malignas das neoplasias, receptor de progesterona imunoreação negativa e alta densidade de microvascular. Não foi observada correlação entre conteúdo de DNA e a fase S ou imunorreatividade para os marcadores MIB-1, c-erbB-2, p53 e Ciclina D1

Relationship between Flow Cytometric Nuclear DNA Quantification and Clinicopathologic Parameters in Endometrial Cancer / 대한산부인과학회지

OBJECTIVE: The aim of this study was to investigate the relationship of the DNA ploidy, S-phase fraction to other clinicopathologic factors including age, stage, architecture grade, nuclear grade, lymph node involvement, myometrial invasion in patients with endometrial cancer. METHODS: A prospective analysis was performed on 66 endometrial cancer cases treated at our hospital from Jan. 2000 to Nov. 2004. Of these 66 cases, 41 cases were analyzed by flow cytometry. Fresh tissues for analysis were obtained by dilatation and curettage or surgery as hysterectomy. All specimens for histopathologic grading and stage were classified according to WHO criteria and FIGO stage. DNA ploidy groups were divided into two groups, diploidy and aneuploidy. Fraction more than 6% was classified as high percentage S-phase fraction (SPF). DNA ploidy and SPF were analyzed by flow cytometry in fresh surgical specimens from endometrial cancer. RESULTS: Of the 41 cases, 5cases were aneuploidy, and 16 cases were high percentage SPF. With regard to DNA ploidy and clinicopathologic prognostic factors, aneuploidy was significantly increased as stage, histological type, nuclear grade, architecture grade, and myometrial invasion increased. With regard to DNA ploidy and clinical prognostic factors, aneuploidy was not increased as age, lymph node involvement increased. With regard to SPF and clinicopathologic prognostic factors, high percentage SPF (>6%) was significantly increased as stage, histological type, and nuclear grade increased. With regard to SPF and clinicopathologic prognostic factors, high percentage SPF (>6%) was not increased as age, lymph node involvement, architecture grade, and myometrial invasion increased. CONCLUSION: The DNA ploidy by flow cytometry has shown to have a close relationship to stage, histological type, myometrial invasion, and nuclear and architecture grade. Also, the SPF has shown to have a close relationship to stage, histological type, and nuclear grade. Our results were consistent with the concept that aneuploidy or high percentage SPF could predict the poor prognosis of disease course. The flow cytometric DNA quantification in endometrial cancer may provide major information about tumor prognosis.

Prognostic Significance of Flow Cytometric Nuclear DNA Quantification in Ovarian Tumors / 대한산부인과학회잡지

OBJECTIVE: The aim of this study was to investigate the relationship of DNA ploidy, SPF to other surgicopathologic factors including stage, grade and CA-125 in ovarian cancer and to evaluate the association between CA-125, DNA ploidy, SPF and 2-year survival in ovarian cancer. METHODS: The study was prospective, which included 56 patients with ovarian tumors who were treated at the Department of Obstetrics and Gynecology, Yonsei University College of Medicine from Feb. 2000 to Jan. 2003. There were 7 benign tumors, 9 borderline tumors and 40 malignant tumors. All patients underwent surgical operation for fresh tissue. All specimens for histopathologic grading and stage were classified according to WHO criteria and FIGO stage. DNA ploidy groups were divided into two groups, diploidy and aneuploidy. DNA ploidy and S-phase fraction (SPF) were analyzed by flow cytometry in fresh surgicalspecimens from primary ovarian tumor. CA-125 was measured at diagnosis and after 6th courses of chemotherapy. RESULTS: Of the Benign tumors, 85.7% were diploidy, 14.3% were aneuploidy. Of the borderline tumors, 88.9% were diploidy, 11.1% were aneuploidy, 60.0% of malignant tumors were diploidy, 40.0% were aneuploidy. In relation between grade and DNA quantification, grade was not significantly associated with DNA ploidy (p=0.07), SPF (p=0.08). In the relationship of stage to DNA quantification, aneuploidy was associated with advanced stage (p=0.2), mean value of SPF was significantly high in advanced stage (stage III+IV) (p=0.04). In the DNA ploidy and SPF, mean value of SPF was 5.5 +/- 4.6% in diploidy and 13.6 +/- 12.8% in aneuploidy. The difference was significant (p=0.03). The serum CA-125 level after six courses was divided into two groups with a CA-12535 U/mL, aneuploidy and mean value of SPF were increased significantly in CA-125>35 U/mL (p=0.05, 0.04). In 2-year survival, CA-125>35 U/mL, aneuploidy and SPF>10% were poor prognostic parameters. CONCLUSION: CA-125 is an important prognostic factor in ovarian cancer. Our results were consistent with the concept that aneuploidy or high percentage of SPF could predict the poor prognosis of disease course. The flow cytometric DNA quantification in ovarian cancer may provide major information about tumor prognosis.

The Prognostic Value of DNA Flow Cytometry in Patients with Early Primary Oral Cavity and Oropharyngeal Squamous Cell Carcinoma / 대한이비인후과학회지

BACKGROUNDS AND OBJECTIVE: It has been known that tumor size, regional neck metastasis state and tumor thickness are the prognostic factors of oral cavity or oropharyngeal cancer. Additionally, DNA flow cytometry has also been reported to be one of the pronosic factors. We would like to evaluate the prognostic value of DNA flow cytometry in early oral cavity or oropharyngeal squamous cell carcinoma. MATERIALS AND METHODS: We analyzed the relation between tumor thickness or neck metastasis and survival rate in 59 patients with early oral cavity or oropharyngeal cancer. Among those patients, DNA flow cytometry was performed in 28 cases and the prognostic value of the parameters of DNA flow cytometry was analyzed. RESULTS: Overall, a 2-year survival rate of the patients was 90.1%. Neither tumor thickness nor neck metastasis state was related to the overall survival rate. Tumor thickness was not related to neck metastasis state, either. Of the parameters of the DNA flow cytometry, only the S phase fraction of aneuploidy was related to the overall survival rate (p=0.0288). Also the total proportion of aneuploidy was weakly related to the state of neck metastasis (p=0.0518). CONCLUSION: These results indicate that DNA flow cytometry can be used as a good complementary factor for predicting the overall survival or neck metastasis in patients with early primary oral cavity or oropharyngeal squamous cell carcinoma.

Prognostic Significance of DNA Content and S-Phase Fraction in Gastric Carcinomas

PURPOSE: DNA flow cytometry is a simple and easy method to assess the DNA content and the cell-cycle distribution of a tumor cell. The prognostic significance of the DNA content and the S-phase fraction in a gastric carcinoma has been controversial. The purpose of this study was to evaluate the prognostic significance of the nuclear DNA content and the S-phase fraction in patients with a gastric carcinoma. METHODS: Between May 1995 and March 1996, 94 patients who were underwent a gastric resection for a gastric carcinoma were evaluated with DNA flow cytometry. Of them, 88 patients underwent a gastric resection with curative intent. The relationship of variable clinicopathological factors and of recurrence pattern to survival and nuclear DNA content were assessed. RESULTS: The mean age was 55 years. 55 patients (58.5%) exbitied diploidy and 39 patients (41.5%) aneuploidy. There was no relationship between the clinicopathological factors and either the ploidy pattern or the S-phase fraction. Though the recurrence and its pattern were not different between the two ploidy group (p=0.860, 0.137), diploidy tended to recur locoregionally and aneuploidy hematogenously. CONCLUSION: The ploidy pattern was a significant prognostic factor in gastric carcinomas, but should be interpreted carefully.

Quantitative Study of Vascular Endothelial Growth Factor Expression in Malignant Bone Tumor / 中华骨科杂志

0. 05). Conclusion The expression of VEGF is highly related to the invasion, DNA ploidy and cellular proliferation activity of malignant bone tumor. It isn't related to the size of tumor. The expression of VEGF is probablya predictor for judgment of malignant degree of bone tumor.

Correlations between Flow Cytometric, Clinical, Pathologic Variables in 449 Breast Cancer Patients

BACKGROUND: Confusing data have been presented for the breast cancer cancer patients on correlations between DNA ploidy and the percentage of S-phase cells and other prognostic factors. The aim of this study was to compare DNA ploidy and the S-phase fraction with traditional prognostic factor to evaluate the prognostic significance of DNA ploidy and SPF. METHODS: We performed flow cytometry on archival paraffin blocks of primary breast cancers from 449 patients. The DNA ploidy and the SPF were examined along with tumor size, the status of lymph node metastasis, age, status of hormonal receptor, histologic grade, tumor type to evaluate clinical utility of DNA ploidy and SPF. RESULTS: The results of the DNA ploidy and SPF revealed 166 diploidy (37%), 283 (63%) aneuploidy tumors. The DNA ploidy status correlated significantly with the status of lymph node metastasis and tumor size. No significant correlations were found with age, histologic grade, or ER receptor status. The S-phase fraction correlated significantly with the ER receptot status. No significant correlations were found with age, tumor size, status of lymph node metastasis, histologic grade, tumor type. CONCLUSIONS: An increased incidence of aneuploidy was found in tumors from patients with positive axillary lymph nodes, larger tumor size and increased incidence of higher S-phase fraction was found in tumors from patients with negative ER receptor. To evaluate the clinical utility of these factors, it will be necessory to measure them on a larger number of patients, so that multivariate survival analyses can be performed.

Correlation between Proliferative Index by DNA Flow Cytometry and Histological Features in Stomach Cancer / 대한암학회지

PURPOSE: Stomach cancer is the most prevalent malignancy in Korea. The survival rate in advanced stage disease has stayed in less than 50%. One of the possible explanation for dismal outcome of stomach cancer is various biologic behavior of cancer cells of heterogeneous clones. Introduction of flow cytometric analysis has provided objective information of cancer cell kinetics, and it could help us in deciding the appropriate adjuvant therapy. The prospective study was undertaken to evaluate the clinical implication of DNA ploidy and each proliferative fraction by DNA flowcytometry. The other aim of the study was to evaluate which one is the most valuable index for proliferative activity of cancer cells. MATERIALS AND METHODS: One hundred and fifty-four patients who underwent gastric resection for primary stomach cancer were included in this study. Male to female ratio was 2.1: 1, and mean age was 58.2 years (range: 26-81). Resected cancer tissues were immediately transported to the flow cytometry laboratory, and analyses for DNA content and cell cycle distribution were carried out by FACScan. The results of flow cytometric analysis were studied in correlation with clinical and histologic parameters; depth of invasion, lymph node metastasis, distant metastasis, stage, Laurens classification, histologic types and grade. RESULTS: The frequency of aneuploid cancer was 40.3% (62 cases). The mean value of GO/Gl fraction was 75.9% and that of S-phase was 16.0%. Decrease of GO/Gl correlates with lymph node metastasis (p 0.015) and stage (p-0.046). Aneuploid cancer exhibited significant decrease of GO/Gl fraction. However, there was no significant conelation between decreased GO/Gl and depth of invasion, distant metastasis, Laurens classi- fication, differentiation of the cancer cells. Patients with metastasis to the lymph node or distant organs had increased S-phase fraction (p-0.032). High S-phase fraction also correlates with advanced stage (p-0.011) and ploidy of the oancer cells (p=0.001). When the ploidy of the tumor was analysed with clinical variables, aneuploid pattern was increased in cancer cells with intestinal type according to Laurens classificatian (p=0.042), Diploid cancer had significantly lower level of S-phase fraction than aneuploid cancer (p 0.001). CONCLUSION: Ploidy and growth fraction of the stomach cancer reflected the extent of disease in different aspects. However, there was no single parameter which reflected the extent of disease and degree of malignant potential. Furthermore, there is a possibility that S-phase & action alone is not an accurate parameter for the proliferative activity of stomach cancer cells. In conclusion, flow cytometric analyses is a valuable study providing us more precise information about biologic properties of cancer cells. However, further evaluation with longer follow-up period is imperative because the ultimate value as an prognostic factors can be estimated in respective of clinical outcomes.

The Significance of DNA Flow-cytometry in Breast Cancer / 대한암학회지

PURPOSE: To evaluate the relationship between nuclear DNA contents and prognostic factors and survival in breast cancer patients. MATERIALS AND METHODS: We determined nuclear DNA content from 91 paraffin-embedded malignant breast tumors and evaluated relationship between DNA nuclear content and well-known prognostic indicators of breast cancer and the survival of the patients by statistical analyses. RESULTS: Twenty nine (34.5%) of the 91 tumors examined were diploid, and the remainder (65.5%) contained one or more aneuploid clones. S-phase fraction (SPF) ranged from 1.4 to 68.3% (median 11.2%) and it was higher in aneuploidy tumors than in diploid tumors (p<0.05). Positive axillary lymph nodes were found in 72.7% of the patients who had a tumor with a high SPF (above the median 11.2%) and in 27.3% of those with tumor with low SPF (below median) (p<0.05). The overall survival rate was 96.1% in DNA diploid and 87.6% in DNA aneuploid tumors, showing that DNA ploidy had no prognostic significance in breast cancers. The actuarial survival rates were 96.4% and 86.3% for low and high SPF, respectively (p=0.28). The patients with high SPF showed high disease free survival rate compared to the patients with low SPF but the difference had no statistical significance. CONCLUSION: Our results indicate DNA aneuploid tumors were more prevalent in breast cancer patients with high SPF or lymph node metastasis and larger patient accumulation with longer follow-up period will be helpful to identifiy the relationship between flow- cytometrical analysis and prognosis.

The Prognostic significance of p53 Gene Mutation and DNA Ploidy and Their Correlation in Advanced Epithelial Ovariasn Canser / 대한산부인과학회잡지

Ovarian cancer has a higher mortality rate than any other gynecologic malignancy and the majority of the patients are in an advanced stage at the time of diagnosis. However, well-estagblished clinicopahtological prognostic factiors such as stage, hisgologic type, grade of differentiation, amonut of residual tumor, and age are insufficient for prediction survival, thus necessitating new, more objective, and reproducible biological prognostic varialbes. Mutation of p 53 tumor suppressor gene and DNA aneuploidy are known to be associated with development and progression of ovarian cancer, but their prognostic sinificance is not yet conclusive. The objectives of this study were to define the nature and the prevalence of p53 gene mutation and DNA aneuploidy, and to deteirmine thier prognostic implications and their correlation in advanced epithelial ovarian cancer. the study population implications and of p53 tumor suppressor gene and DNA anuuploidy, and to determine their prognostic implications and their correlation in advanced epithelial ovarian cancer. The study population comprised tiryty-two patients with stage II to IV epithelial ovarian cancer who were mangaged at Asan Medical Center between may 1993 and April 1996. Fresh frozen tumor samples of primary lesion were analysed by direct DNA sequencing technique using ploymerase chain reaction with primers for exon 5,6,7 and 8. Measurerements of the nuclear DNA content were performed on the same cytometry. Twelve (37.5%) point mutations were identified in all exons, 3,2,2and 5 cases in exon 5,6,7and 8 of p 53 gene respectively without any preferential pattern of nucleotide substitution. Twenty-Three (71.9%) cased of tumors were revealed to be aneuploid (DNA index > 1.05). there was no correlation between p53 gene mutation and DNA ploidy. The presence or absence of p53 gene mutation had no signficant correlation with FIGO stge, histologic grade, serum CA 125 level after second chemotherapy and residual tumor size after debulking surgery. Two-year survival rates of patients with and with out mutation showed no difference. On the other hand, patients with aneuploid tumors revealed to be significantly associated with more advanced stage (P=0.46) and higher level of serum CA 125 after second chemotherapy (P=0.18) and had shorter 2-year survival rate than shose with diploid tumor, although the statistical significance was marginal (P=0.057). In conclusion, p53 gene mutation and DNA ploidy show no correlation, and p53 gene mutation does not appear to be a marker prediction the biological behavior or the outcome of the disease. However, DNA ploidy was shown to be utilized as a prognostic fatoc for survival in advanced epithelial ovarian cnacer, althour further studies for longer period of time are required to confirm.

The Prognostic significance of p53 Gene Mutation and DNA Ploidy and Their Correlation in Advanced Epithelial Ovariasn Canser / 대한산부인과학회잡지

Ovarian cancer has a higher mortality rate than any other gynecologic malignancy and the majority of the patients are in an advanced stage at the time of diagnosis. However, well-estagblished clinicopahtological prognostic factiors such as stage, hisgologic type, grade of differentiation, amonut of residual tumor, and age are insufficient for prediction survival, thus necessitating new, more objective, and reproducible biological prognostic varialbes. Mutation of p 53 tumor suppressor gene and DNA aneuploidy are known to be associated with development and progression of ovarian cancer, but their prognostic sinificance is not yet conclusive. The objectives of this study were to define the nature and the prevalence of p53 gene mutation and DNA aneuploidy, and to deteirmine thier prognostic implications and their correlation in advanced epithelial ovarian cancer. the study population implications and of p53 tumor suppressor gene and DNA anuuploidy, and to determine their prognostic implications and their correlation in advanced epithelial ovarian cancer. The study population comprised tiryty-two patients with stage II to IV epithelial ovarian cancer who were mangaged at Asan Medical Center between may 1993 and April 1996. Fresh frozen tumor samples of primary lesion were analysed by direct DNA sequencing technique using ploymerase chain reaction with primers for exon 5,6,7 and 8. Measurerements of the nuclear DNA content were performed on the same cytometry. Twelve (37.5%) point mutations were identified in all exons, 3,2,2and 5 cases in exon 5,6,7and 8 of p 53 gene respectively without any preferential pattern of nucleotide substitution. Twenty-Three (71.9%) cased of tumors were revealed to be aneuploid (DNA index > 1.05). there was no correlation between p53 gene mutation and DNA ploidy. The presence or absence of p53 gene mutation had no signficant correlation with FIGO stge, histologic grade, serum CA 125 level after second chemotherapy and residual tumor size after debulking surgery. Two-year survival rates of patients with and with out mutation showed no difference. On the other hand, patients with aneuploid tumors revealed to be significantly associated with more advanced stage (P=0.46) and higher level of serum CA 125 after second chemotherapy (P=0.18) and had shorter 2-year survival rate than shose with diploid tumor, although the statistical significance was marginal (P=0.057). In conclusion, p53 gene mutation and DNA ploidy show no correlation, and p53 gene mutation does not appear to be a marker prediction the biological behavior or the outcome of the disease. However, DNA ploidy was shown to be utilized as a prognostic fatoc for survival in advanced epithelial ovarian cnacer, althour further studies for longer period of time are required to confirm.

Quantitation of spermatogenesis by DNA flow cytometry: Comparative study among six species of mammals.

Suspensions of testicular germ cells from six species of mammals were prepared and stained for the DNA content with a fluorochrome (ethidium bromide) adopting a common technique and subjected to DNA flow cytometry. While uniform staining of the germ cells of the mouse, hamster, rat and monkey could be obtained by treating with 0·5% pepsin for 60 min followed by staining with ethidium bromide for 30 min, that of the guinea pig and rabbit required for optimal staining pepsinization for 90 min and treatment with ethidium bromide for 60 min. The procedure adopted here provided a uniform recovery of over 80% of germ cells with each one of the species tested and the cell population distributed itself according to the DNA content (expressed as C values) into 5 major classes– spermatogonia (2C), cells in S-phase, primary spermatocytes (4C), round spermatids (1C), and elongating/elongated spermatids (HC). Comparison of the DNA distribution pattern of the germ cell populations between species revealed little variation in the relative quantities of cells with 2C (8–11%), S-phase (6–9%), and 4C (6–9%) amount of DNA. Though the spermatid cell populations exhibited variations (1C:31–46%, HCl:7– 20% and and HC2:11–25%) they represented the bulk of germ cells (70–80%). The overall conversion of 2C to 1C (1C:2C ratio) and meiotic transformation of 4C cells to 1C (1C:4C ratio) kinetics were relatively constant between the species studied. The present study clearly demonstrates that DNA flow cytometry can be adopted with ease and assurance to quantify germ cell transformation and as such spermatogenesis by analysing a large number of samples with consistency both within and across the species barrier. Any variation from the norms in germ cell proportions observed following treatment, for e.g. hormonal stimulation or deprivation can then be ascribed due to a specific effect of the hormone/drug on single/multiple steps in germ cell transformation.