Analysis and clinical significance of DNA ploidy, S-phase fraction and proliferating index in colorectal carcinoma / 中国医师进修杂志

ObjectiveTo study the changes and clinical significance of DNA ploidy,S-phase fraction(SPF),proliferating index(PI) in colorectal carcinoma.MethodsIn 40 cases of colorectal carcinoma (colorectal carcinoma group ),40 cases corresponding cancer-adjacent tissue (cancer-adjacent tissue group ),12 cases of precancerous lesions (precancerous lesions group) and 10 cases of normal colorectal mucosa coli (normal colorectal mucosa coli group),DNA ploidy,SPF and PI were detected with flow cytometry and compared.ResultsDNA diploid was 7 cases,DNA heteroploid was 33 cases in colorectal carcinoma group,35,5 cases in cancer-adjacent tissue group,10,2 cases in precancerous lesions group,10,0 case innormal colorectal mucosa coli group,there were significant differences among them(P＜ 0.01 ).SPF and PI in colorectal carcinoma group (35.36 ± 7.45,42.92 ± 6.81 ) were significantly higher than those in cancer-adjacent tissue group (20.82 ±5.51,31.34 ±4.88),precancerous lesions group (21.13 ± 5.07,31.70 ±5.59) and normal colorectal mucosa coli group ( 19.93 ± 3.73,32.01 ± 4.99),there were significant differences among them(P＜ 0.01 ).DNA ploidy was significantly correlated with Dukes staging (P=0.027) and the differentiation of colorectal carcinoma (P =0.030).ConclusionsDNA ploidy,SPF,PI may get some molecular biologycharacteristic and proliferation activity of colorectal carcinoma at molecule level,which may be helpful for treatment and evaluation of prognosis of colorectal carcinoma.

S-phase Fraction as an Independent Prognostic Factor in Invasive Breast Carcinoma -A Study of Long-term Follow-up / 한국유방암학회지

PURPOSE: To evaluate the significance of the S-phase fraction (SPF) and DNA ploidy, determined by DNA flow cytometry, as prognostic markers in invasive breast cancer. METHODS: Between October 1986 and June 1999, 143 breast carcinoma patients, treated by surgery, were analyzed. Flow cytometry was performed for the identification of the SPF and DNA ploidy, with immunohistochemistry performed on paraffin embedded material for the hormone receptor. Two SPF classes were defined on the basis of the median value (10) by using a log rank test (high SPF>10, low SPF<10). The correlation between SPF and the clinicopathological factors (tumor size, lymph node status, histological grade and steroid receptor status) and between the SPF and 5 yr disease-free survival (DFS) were investigated. RESULTS: DNA ploidy was not associated with tumor size, lymph node status, histological grade, overall survival and DFS. In a univariate analysis, high SPF values were associated with shorter 5 yr DFS in individual groups. In the node negative group, the 5 yr DFS of the low SPF group was higher than that of the high SPF group, but in the node positive group, the SPF values showed statistical significance with the 5 yr DFS. In a multivariate analysis, the SPF was independently associated with the 5 yr DFS in the node negative group. CONCLUSION: These results suggested the SPF is an independent prognostic factor in lymph node negative, estrogen receptor positive and progesterone receptor negative breast cancers.

The Prognostic Value of the S-phase Fraction and Ki-67 Expression in Early Breast Cancer

PURPOSE: The aim of this study was to compare the S-phase fraction (SPF) and Ki-67 with other biologic factors, and to assess the prognostic value of Ki-67 and SPF in 108 breast cancer patients. MEHOODS: The SPF and Ki-67 level were determined in formalin-fixed, paraffin-embedded tissue specimens from 108 patients with early breast cancer who underwent surgery between January 1997 and December 2000 at the Wonkwang University Hospital. The clinicopathological characteristics of the early breast cancer such as the tumor size, node status, histological grade, hormone receptor, various tumor markers and cancer recurrence were compared with the SPF and Ki-67 values. RESULTS: The median SPF was 9.03% (range 0~43%). The SPF correlated with CD31 (P=0.020) and DNA diploidy (P=0.000). Ki-67 correlated with the histological grade (P= 0.010) and p53 (P=0.035). No correlation was found between the SPF and Ki-67. Eight cases recurred during the follow-up period. Strong expression of Ki-67, p53, DNA aneuploidy and a young age were correlated with recurrence (P=0.001, P=0.029, P=0.021 and P=0.002, respectively). However, the SPF was not related to recurrence. CONCLUSION: In early breast cancer, Ki-67 expression is correlated with the histological grade and p53 expression. In addition, strong Ki-67 expression is associated with a recurrence. Further studies regarding the prognostic significance of the proliferation markers, such as Ki-67 and SPF, will be needed to confirm these results.

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 Value for the S-phase fraction in T1 T2, Node Negative Breast Cancer / 한국유방암학회지

PURPOSE: To evaluate the prognostic value of the S-phase fraction (SPF) and the correlation with other clinicopathologic factors in node negative breast cancer. METHODS: From 1995 to 1998, seventy one breast carcinoma tumors with T1-2N0M0 staging were prospectively sampled as fresh tumors for flow cytometric DNA analysis. We determined the nuclear DNA content, and the SPF was calculated from DNA histograms. We evaluated the relationship between the SPF and other clinicopathologic factors (age, tumor size, tumor grade and, steroid receptor status). The five year distant relapse free survival (DRFS) and overall survival (OS), according to the SPF, were determined. RESULTS: The SPF ranged from 0.1 to 50.9% (median: 13.4%). The SPF was dichotomized using the median value to divide patients into 38 patients (53.5%) having tumors with the low SPF and 33 patients (46.5%) having tumors with the high SPF. The patient's age and the tumor size were not significantly associated with the SPF. High SPF was associated with high tumor grade, but this did not reach statistical significance. There was a significant correlation between high SPF and estrogen receptor negativity; 34.4% of ER positive tumor had high SPF, whereas 58.3% of ER negative tumor had high SPF(p = 0.042). The mean follow up duration was 65.0 months (median: 62.3). Among 71 patients, there were 4 (5.6%) cases of local recurrence, 7 (9.9%) cases of systemic recurrence and 10 (14.1%) cases of disease related death. The patients with high SPF showed a poorer 5 years DRFS and OS than did the patients with low SPF (87.9% vs. 91.2%; 80.4% vs. 94.5%, respectively), but the difference had no statistical significance. CONCLUSION: The present data was insufficient to use SPF information for the selection of the type of adjuvant therapy, but SPF is a promising prognostic factor for node negative breast cancer. Further study with a sufficient number of patients is needed and this should lead to a better understanding of SPF in node negative breast cancer.

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.

Prognostic Value of Nuclear DNA Quantification and Cyclin A in Epithelial Ovarian Cancer / 대한산부인과학회잡지

OBJECTIVE: This study was carried out to investigate the relationship between DNA ploidy, S-phase fraction (SPF), expression of cyclin A and clinical prognostic factors including stage, grade, CA-125 and residual tumor size in epithelial ovarian cancer, and to evaluate the association between DNA ploidy, SPF, expression of cyclin A and 3-year survival. METHODS: Study group consisted of 31 cases of epithelial ovarian cancer, 10 of borderline ovarian tumor and 5 of benign ovarian tumor diagnosed at the department of Obstet. and Gynecol. in Yonsei University College of Medicine, Seoul, Korea from Feb. 2000 to Jan. 2003. All patients underwent staging-laparotomy and postoperative chemotherapy. The level of CA-125 was assessed after 6th postoperative chemotherapy with cut-off value of 35 U/mL. DNA ploidy and SPF were evaluated by flow-cytometry of fresh ovarian tissue obtained at the operative field. The expression of cyclin A was evaluated by immuno-histochemical stain. Expression of 5% was considered as positive. Statistical analysis was done by two-sample t-test, chi-square test, and Kaplan-Meier survival curve using SPSS ver 11.0 software. RESULTS: In 46 ovarian tumors aneuploidy, SPF and expression of cyclin A were significantly higher in epithelial ovarian cancer as compared with benign and borderline tumors (p=0.004, 0.001, 0.001, respectively). Number of aneuploidy, SPF and expression of cyclin A were significantly higher in patients with higher grade, more advanced stage, higher level of CA-125 (more than 35 U/mL) and more than 2 cm of residual tumor size (p=0.004, 0.009, 0.05, 0.002 in aneuploidy; p=0.06, 0.01, 0.04, 0.007 in SPF; p=0.03, 0.004, 0.06, 0.02 in cyclin A). Aneuploidy and expressions of more than 10% of SPF and cyclin A were also associated with poorer overall survival (p=0.02, 0.02, <0.0001, respectively). Significantly positive correlations were observed among these factors. CONCLUSION: Number of aneuploidy, percentage of SPF and expression of cyclin A were higher in more advanced stage, higher grade, higher CA-125 and more than 2 cm of residual tumor size and associated with poorer overall survival. Thus DNA flow-cytometry and estimation of expression of cyclin A may provide major information about prognosis of disease in epithelial ovarian cancer patients.

Possible factors influencing high serum Prostate-specific Antigen (PSA) in Indonesian patients with Benign Prostatic Hyperplasia (BPH).

Benign prostatic hyperplasia (BPH) cases in Indonesia frequently associated with high serum prostate specific antigen (PSA). To explore possible factors that could increase serum PSA level, we performed a retrospective, cross-sectional study on 805 consecutive patients in Sumber Waras and Dr. Cipto Mangunkusumo Hospitals from 1994 to 1997. Clinical manifestations were evaluated and prostate biopsies were performed if indicated. Complete histopathological data were only available in 82 BPH patients with no urinary retention from 1998-1999 and a thin section of paraffin blocks of BPH patients which still could be found from 1994-1999 was analyzed using flow cytometer to obtain the S-phase fraction as a parameter of proliferative activity, From 805 patients, 461 (57%) presented with urinary retention and need to be catheteized. Catheteization significantly increased PSA level if compared to noncatheterized patients (16.3 vs. 6,8 ng/mL, p= 0,000). Another data of 82 uncatheteized patients from 1998-1999 has revealed that 79 patients (96.3%) had chronic prostatitis and 19 (23.2%) showed the presence of prostatic-intraepithelial neoplasia (PIN) with an increase of PSA level (5.4 ng/mL). The S-phase fraction of BPH without PIN cases was significantly higher in cases with PSA > 4 ng/ml than patients with PSA ≤ 4 ng/ml (I3.1% vs. 8.9%, p=0,008). As conclusion, the high serum PSA level was mostly due to urethral catheteization and increased prostate volume. There was a tendency of increasing PSA in subclinical inflammation and PIN. Cases with high PSA also showed high proliferative activities which is suggestive of mitogenic activity.

Detection of Estrogen Receptor in Breast Cancer by Flow Cytometry / 中国医师杂志

Objective To detect estrogen receptor and DNA index in breast cancer by flow cytometry. Methods 32 cases of fresh specimens of breast cancer resected by operation were used in this study. Single cells suspension was prepared from those specimens, and estrogen receptor expression was analyzed by flow cytometry. At the same time, the status of ploid and S phase cell ratio were determined. Results Flow cytometry analysis could measure the expression level of estrogen receptor in the fresh breast cancer specimens, which was more sensitive and needed less volume of tumor tissue compared with immuohistochemical method. The information on the status of ploid and S phase cell ratio were simultaneously obtained. Conclusion The detection of estrogen receptor in breast cancers by flow cytometry was more helpful for evaluating prognosis and selecting treatment.

Study on Immunohistochemical Cyclin D1 Expression and Flow Cytometric DNA Analysis in Squamous Cell Carcinoma of the Larynx / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Cancer is a disease characterized by deregulation of cell cycle control. During the last decade, many alterations in the signaling pathways that ultimately lead to DNA replication and mitosis have been identified in various tumor types. DNA analysis by flow cytometry is considered to be of prognostic value in squamous cell carcinoma of the head and neck. However, a few and contradictory studies have been made on squamous cell carcinoma of the larynx. Expression of the cell cycle control gene cyclin D1 may, at least in some tumor types, provide a prognostic marker. Cyclin D1 is expressed during the G1 phase of the cell cycle and becomes associated with its catalytic partner CDK4 or CDK6. The authors evaluated the prognostic significance of cyclin D1, DNA ploidy and S-phase fraction (SPF) in the squamous cell carcinoma of the larynx to determine their relationship with the various clinicopathological parameters. MATERIALS AND METHODS: Paraffin embedded tissue specimens from 28 cases of squamous cell carcinoma of larynx were studied by the immunohistochemical method using cyclin Dl antibody and by flow cytometric DNA analysis. RESULT: The positive expression of cyclin D1 protein was 60.7% in squamous cell carcinoma of the larynx. In 28 cases of flow sytometric DNA analysis, 23 cases (82.1%) were diploidy and 5 cases (17.9%) were aneuploidy. The SPF ranged from 0.0% to 83.9% (mean 41.4, median 35.2). The mean SPF of DNA diploid cases was 34.2%, whereas that of DNA aneuploid cases was 74.7%. Expression of cyclin D1 protein was found in 52.2% of the diploid cases and in 100% of the aneuploid cases. This expression was statistically significant (p or = 35.2). The expression of cyclin D1 protein did not correlate with clinical features. CONCLUSION: The expression of cyclin D1 protein may be related with development of squamous cell carcinoma of the larynx, but not correlated with prognostic indicators. In cases of aneuploidy/higher SPF (> or = 35.2), The expression of cyclin Dl protein did not significantly correlate with lymph node metastasis, but showed a high expression rate of cyclin Dl protein. However, these correlations were not sufficient for the prognostic indicators in squamous cell carcinoma of the larynx.

EXPRESSION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR AND CELL CYCLE ANALYSIS IN THE HEAD AND NECK SQUAMOUS CELL CARCINOMAS

Growth factors and the receptors play an important role in the regulation of the growth and development of mammalian cells. In particular, epidermal growth factor is a polypeptide with potent mitogenic activity that stimulates proliferation of various normal and neoplastic cells through the interaction with its specific receptor(EGFR). EGFR has been described as a parameter of poor prognosis in many human neoplasms such as breast, bladder, and vulvar cancers. The objectives of this study are the evaluation of the expression of EGFR and cell cylce analysis in the head and neck squamous cell carcinomas(SCC), and the evaluation of the correlation between clinico-patholgic features and expression of EGFR and S-phase fraction. 37 head and neck squamous cell carcinoma specimens were evaluated for expression of EGFR by Western blot analysis and S-phase fraction by cell cycle analysis using the flow cytometry. The obtained results were as follows : 1. The expressions of EGFR were observed in 20 specimens(54%) among 37 head and neck SCC specimens. In case of oral SCC, 15 specimens(56%) out of 27 specimens were observed, and in case of nasopharyngeal SCC 5 specimens(50%) out of 10 specimens. 2. There was no correlation between clinical features(location, stage) of head and neck SCC and expression of EGFR (p>0.05). 3. There was a significant correlation between histo-pathological differentiation of head and neck SCC and expression of EGFR (p<0.02). 4. There was a significant correlation between expression of EGFR and S-phase fraction of cell cycle in the head and neck SCC (p<0.05). The above results suggest that expression of EGFR and S-phase fraction of cell cycle are adjunctive prognostic marker in the head and neck squamous cell carcinomas.

DNA Ploidy and S-phase Fraction in Breast Cancer

BACKGROUND: Histological differentiation, status of hormonal receptors, size of tumor, and status of axillary lymph nodes are known prognostic factors in breast cancer. Recently, the DNA ploidy and the S-phase fraction have been considered as relating to parameters affecting the prognosis of cancer patients. METHODS: The authors measured the DNA ploidy and the S-phase fraction and evaluated their significance as prognostic factors in breast cancer. Their correlation with tumor size, lymph-node involvement, pathologic differentiation, status of hormonal receptors, menopause, and survival duration were also analyzed. The DNA ploidy and the S-phase fraction were checked with an FAC scan (Becton Dikinson, U.S.A.) and a cell fit (Becton Dikinson, U.S.A.) using the fresh breast-cancer tissue from 104 patients. RESULTS: The ploidy analysis showed that 32% of the tumors were diploid and 68% were aneuploid. There was significance in the pathologic differentiation, but no significance in the other prognostic factors. The median S-phase fraction was 8.9%, and it was used as a cutoff point. Forty-nine percent (49%) of the tumors were greater than 8.9%, and 51% were less than it. There was significance in the status of hormonal receptors. There was no significance in the relation between these factors and the disease-free survival rate. CONCLUSIONS: Even though these results suggest that the DNA ploidy and the S-phase fraction determined by using flow cytometry are not clinically useful independent prognostic factors, it will be necessary to measure them in a large number of patients and then follow those patients so that a multivariate survival analyses can be performed to evaluate the clinical significance of these factors.

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.

Flow Cytometric Analysis of BRCA1 Protein in Sporadic Breast Cancer / 대한암학회지

PURPOSE: To study the subcellular localization with flow-cytometry and to evaluate their prognostic values. MATERIALS AND METHODS: The breast tissues were obtained from 28 patients with breast cancer and 6 patients with benign mass. The expression of BRCA1 protein was analyzed with the flow cytometry(Coulter Epics-XL, Coulter Corps, FL, USA) using the monoclonal antibody(BRCA1(Ab-1), Calbiochem, MA, USA) before and after nuclear and cytoplasmic permeabilization in association with DNA ploidy analysis. Several BRCA1 protein indices were derived including 95 percentile channel fluorescence(95% CF) and mean channel fluorescence(MCF) and percentage of BRCA 1 positive cell population arbitarily defined as those above 0.12 channel fluorescence. RESULTS: Cytoplasmic 95% CF were higher in breast cancer(n=28, 0.65+/-0.26) than in benign mass(n=6, 0.40+/-0.13, p=0.0211). Cytoplasmic BRCAl positive cell percentages were significantly higher in malignant tissues(24.0+/-10.3) than in benign mass(43.4+/-15.2, p=0.0059). Cytoplasmic BRCA1 positive cell percentages were significantly different according to the stages(stage I vs II, 32.6+/-9.8 vs 48.3+/-18.8, p=0.048, stage I vs stage III, 32.6+/-9.8 vs 47.0+/-10.9, p=0.010). The BRCA1 protein indices were not significantly correlated with histologic grades and DNA indices(aneuploidy, S phase and proliferation fractions). CONCLUSIONS: Flowcytometric assay offers an alternative approach to evaluating BRCA1 protein status of breast cancer tissue and detection of cytoplasmic BRCA1 protein by this method may help to understand the role of BRCA1 in breast cancer cell biology. The further study on cytoplasmic or nuclear BRCA1 protein in association with clinical therapeutic response or prognosis seems to be warranted.

Flow Cytometric DNA Content Analysis in Breast Cancer Comparison study of fresh and paraffin-embedded tissues

DNA content of 25 cases of breast carcinoma was analyzed by flow cytometry in both fresh and formalin-fixed, paraffin-embedded tissue. Aneuploidy in fresh tissue and paraffin-embedded tissues was 72% and 32%, respectively. There was a 52% agreement in analysis of DNA ploidy between fresh and paraffin-embedded tissues. Most of the discrepancies resulted from loss of aneuploid peaks on the histograms of paraffin-embedded tissue. Mean S-phase fraction was slightly higher in a paraffin-embedded tissue than that in the fresh tissue; 19.2 9.1% versus 16.1 8.8% and there was no significant correlation between the S-phase fractions. In statistical analysis, the histologic and nuclear grades were not correlated with ploidy or mean S-phase fraction. Therefore it is strongly recommended to use the fresh tissue in flow cytometric DNA content analysis of breast cancer.

Prognostic Significance of Flow Cytometric Nuclear DNA Content and S-phase Fraction in Non-small Cell Lung Cancer / 대한내과학회지

OBJECTIVES: In surgically treated non-small cell lung cancer, patients have a wide difference in prognosis even though they may be in the same stage. Therefore it is difficult to establish the prognosis for individual lung cancer patients. In this study, by using flow cytometric analysis of nuclear DNA content and S-phase fraction(SPF) of surgically treated non-small cell lung cancer patients, we proposed to establish other prognostic factors and their validity in comparison with the existing ones. METHODS: Paraffin-embedded tissue specimens from 81 surgically treated patients, diagnosed with non-small cell lung cancer ranging from stage I to stage IIIa, were analyzed by flow cytometrically determined nulear DNA content and S-phase fraction. Cellular DNA content stained with propidium iodide was analyzed by flow cytometry: histograms with a coefficient of variation exceeding 8% were not used. RESULTS: 1) DNA content analysis was carried out for 59 of 81 patients. Of the 59 patients who were investigated by flow cytometry, 45 (76.3%) of the tumors were DNA aneuploidy and 14 (23.7%) were DNA diploidy. The proportion of DNA aneuploidy tumors showed no significant difference between cell types or stage. 2) S-phase fraction was evaluated for 36 of 81 patients. Mean value of SPF was 19.2% (+/-12.62)%. The value of SPF had nothing to do with stage. 3) The proportion of the high SPF group (more than 10% of cell proliferation cycle) was 75% With advance staging, the proportion of the high SPF group increased. 4) Significant difference in the median survival time was observed between the low SPF group and the high SPF group (32 months in low SPF, 12 months in high SPF) (p<0.05). No significant difference in the median survival time was observed between the aneuploidy group and the diploidy group (19 months in aneuploidy, 34 months in diploidy). 5) Significant difference in the disease free median survival time was observed between the low SPF group and the high SPF group (5 months in low SPF, 19 months in high SPF) (p<0.05). No significant difference in the disease free median survival time was observed between the aneuploidy group and the diploidy group (12 months in aneuploidy, 34 months in diploidy). 6) Upon multivariate analysis, stage and high SPF (more than 10% of cell proliferation cycle) were significant prognostic factors in surgically treated non-small cell lung cancer patients. CONCLUSION: The TNM stage and high SPF were significant as prognostic factors in surgically treated non-small cell lung cancer patients. Therefore new treatment plan should be needed in the patients who have high SPF.

E-Cadherin Expression and DNA Ploidy Analysis in Invasive Squamous Cell Carcinoma of the Uterine Cervix Comparison with those of CIN

Epithelial cadherin (E-cadherin) is a Ca2+ -dependent cell-cell adhesion molecule that connects cells via homotypic interactions. Its function is critical in the induction and maintenance of cell polarity and differentiation, and its loss is associated with an invasive and poorly differentiated phenotype in a wide range of tumors. Formalin-fixed, paraffin-embedded tissue sections from 36 cases of cervical intraepithelial neoplasia (CIN) and 14 cervical squamous cell carcinomas were investigated for the expression of E-cadherin immunohistochemically. While E-cadherin expression was usually restricted on the cell membrane of basal and parabasal cells in normal cervix, the presence of cytoplasmic E-cadherin was found to be associated with its grade in CIN lesions. Also, marked cytoplasmic staining was commonly revealed in poorly differentiated ones than well-differentiated squamous cell carcinomas. More intense reactivity of cytoplasmic E-cadherin was frequently seen in the foci of invasion than adjacent carcinoma in situ, and in its periphery than the center of tumor islands. In addition, DNA ploidy and S-phase fraction of squamous cell carcinomas were analyzed and compared with those of CIN lesion. We found that invasive squamous cell carcinomas more frequently disclosed DNA aneuploidy than CIN lesions, and there was correlation between cytoplasmic E-cadherin expression and DNA aneuploidy. Also, cytoplasmic E-cadherin-reactive cervical neoplasms had a higher rate of cell proliferation than that of membranous E-cadherin-reactive cases. These data suggest that the increased cytoplasmic E-cadherin expression may represent one of the abnormalities underlying the loss of polarity and invasiveness of cancer cells, and the abnormal E-cadherin expression combined with/without DNA ploidy or S-phase fraction may serve as a prognostic indicator.

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.

Significance of S-phase Fraction and Nuclear Area in Classification and Prognosis of Non-Hodgkin Lymphoma / 대한혈액학회지

BACKGROUND: Non-Hodgkin lymphomas have morphologic and biologic variabilities and are classified by a variety of classification schemes. The authors studied the value of DNA content, S-phase fraction, and nuclear areas as a diagnostic discriminant in classification and predicting the clinical outcome of non-Hodgkin lymphoma. METHODS: Thirty cases of non-Hodgkin lymphoma and 13 cases of reactive lymph node specimens embedded in paraffin were used. DNA analysis was done by flow cytometric method using propidium iodide staining. Nuclear areas of lymphoma cells were measured by image analyzer. The results of DNA content, S-phase fraction, nuclear areas were statistically analyzed between groups of lymphoma classified according to the Rappaport classification and Working formulation. RESULTS: Aneuploidy patterns were identified in two cases (6.7%) of non-Hodgkin lymphoma (diffuse poorly differentiated lymphocytic and well differentiated lymphocytic). S-phase fraction and nuclear areas were high in lymphoblastic, diffuse poorly differentiated, diffuse lymphocytic undifferentiated, and diffuse mixed types (unfavorable prognostic group), and which had statistical significance against the low values of diffuse lymphocytic well differentiated type (favorable prognostic group). The S-phase fraction and nuclear area had significant correlation, so they could represent correlation between high proliferative activity and large nuclear area. High, intermediate, and low grades groups classified by Working formulation revealed statistically significant differences of S-phase fraction and nuclear areas between each group. CONCLUSION: The S-phase fraction and nuclear area measurement could be good diagnostic and prognostic factors in classification and clinical management of non-Hodgkin lymphoma.

Correlation of Proliferating Cell Nuclear Antigen (PCNA) Expression and S-phase Fraction, Survival Rate in Primary Non-Small Cell Lung Cancer / 결핵

BACKGROUND: To study the prognosis of patients with lung cancer, many investigators have reported the methods to detect cell proliferation in tissues including PCNA, thymidine autoradiography, flow cytometry and Ki-67. PCNA, also known as cyclin, is a cell related nuclear protein with 36KD intranuclear polypeptide that is maximally elevated in S phase of proliferating cells. In this study, PCNA was identified by paraffin-embedding tissue using immunohistochemistry which has an advantage of simplicity and maintenance of tissue architecture. The variation of PCNA expression is known to be related with proliferating fraction, histologic type, anatomic(TNM) stage, degree of cell differentiation, S-phase fraction and survival rate. We analyzed the correlation between PCNA expression and S-phase fraction, survival. METHODS: To investigate expression of PCNA in primary lung cancer, we used immunohistochemical stain to paraffin-embedded sections of 57 resected primary non-small cell lung cancer specimen and the results were analyzed according to the cell type, cell differentiation, TNM stage, S-phase fraction and survival. RESULTS: PCNA expression was dMded into five group according to degree of staging(-, +, ++, +++,++++). Squamous cell type showed high positivity than in adenocarcinoma. Nonsignificant difference related to TNM stage was noticed. Nonsignificant difference related to degree of cell differentiation was noticed. S-phase fraction was increased wit advance of PCNA positivity, but t could not reach the statistic significance. The 2 year survival rate and median survival time were -50% 13 months, +75% 41.3 months, ++73% 33.6 months, +++67% 29.0 months, ++++25% 9 months with statistic significance (P<0.05, Kaplan-Meier, generalized Wilcox). CONCLUSION: From this study. PCNA expression was high positive n squamous cell cancer. And, there was no relationship between PCNA positivity and TNM stage, cellular differentiation or S-phase fraction. But, the patients with high positive PCNA staining showed poor survival rate than the patients with lower positive PCNA. It was concluded that PCNA immunostaining is a simple and useful method for survival prediction in paraffin embedded tissue of non-small cell lung cancer.