Flow cytometric analysis of nuclear DNA content in gestational trophoblastic disease.

Flow cytometric analysis of nuclear DNA is a quick and accurate method of determining nuclear ploidy. This technique allows a clear distinction to be made between diploid and triploid hydatidiform moles. Additional ploidy aberrations, such as tetraploidy and aneuploidy, have also been reported in flow cytometric studies of hydatidiform moles. Recent investigations have suggested that aneuploidy in complete hydatidiform moles is associated with a high risk of persistent gestational trophoblastic disease. In addition to DNA ploidy analysis, cell cycle activity may be calculated from analysis of the DNA histogram. No significant correlation between clinical course and cell cycle kinetics has been demonstrated to date. By providing valuable DNA ploidy data, flow cytometric studies of hydatidiform moles may be helpful in the identification of patients at high risk of postmolar trophoblastic sequelae.     

Flow cytometric DNA ploidy analysis of ovarian granulosa cell tumors

The nuclear DNA content of 50 ovarian tumors initially diagnosed as granulosa cell tumors was measured by flow cytometry using paraffin-embedded archival material. The follow-up period of the patients ranged from 4 months to 19 years. Thirty-eight tumors were diploid or near-diploid, while 5 were aneuploid. DNA profiles of 7 tumors could not be evaluated. All 50 tumors were immunohistochemically tested for expression of intermediate filament proteins vimentin and cytokeratin and epithelial membrane antigen. The cells of all but 3 tumors expressed vimentin. These 3 vimentin-negative tumors were positive for cytokeratin and epithelial membrane antigen. They were highly aneuploid and though originally diagnosed as granulosa cell tumors, most likely represent undifferentiated carcinomas. Hence, only 2 typical granulosa cell tumors were aneuploid. In addition, frozen tissue samples from 9 of 10 granulosa cell tumors showed a DNA diploid content. Our results indicate that granulosa cell tumors tend to be diploid or have only minor ploidy abnormalities which is in line with their relatively benign character. An undifferentiated carcinoma should be considered in the differential diagnosis of tumors with a high DNA index.     

Flow cytometric analysis of DNA content in partial hydatidiform moles with persistent gestational trophoblastic tumor.

Hydatidiform moles may be classified as partial or complete based on genetic and pathologic criteria. Between January 1979 and January 1990, 17 (5.5%) of 310 patients followed for partial mole developed persistent gestational trophoblastic tumor. Tissues from 14 partial moles were available for flow cytometric analysis of DNA content. Eleven partial moles (85%) were triploid, two (15%) were diploid, and one DNA histogram was uninterpretable. All patients with triploid partial moles achieved complete remission with one course of single-agent chemotherapy. The two with diploid partial mole required multiple courses of chemotherapy to achieve gonadotropin remission. Although the DNA content of most partial moles with persistent gestational trophoblastic tumor was triploid, diploid partial moles with persistent tumor were less sensitive to single-agent chemotherapy.     

Flow cytometric DNA analysis of ovarian Brenner tumors and transitional cell carcinomas.

Flow cytometry has been previously used as a method of obtaining prognostic information about ovarian carcinomas using ploidy, DNA index, and S-phase fraction. DNA content has also been assessed in ovarian tumors of low malignant potential. Brenner tumor variants such as metaplastic, proliferating, and low malignant potential, recently designated as intermediate Brenner tumors, and malignant Brenner tumors are unusual tumors that present classification problems. Their histological appearance may not accurately reflect biological activity. We used flow cytometry to analyze paraffin-embedded tissue for DNA content and S-phase in 34 Brenner tumors, three ovarian transitional cell (urothelial) carcinomas (TCCs), and nine normal control ovaries. We correlated histological and clinical features with DNA analysis. Twenty-five Brenner tumors and three ovarian TCCs were acceptable for histogram analysis (coefficient of variation less than 7.0). Thirteen typical, three metaplastic (extensive mucinous or glandular metaplasia), and two proliferating (papillary formation with increased cellularity) Brenner tumors were diploid. One proliferating tumor was tetraploid. The single Brenner tumor of low malignant potential was diploid but had an increased S-phase. Four of five malignant Brenner tumors were aneuploid, and one was diploid. All the TCCs were aneuploid. S-phase was elevated in intermediate and malignant Brenner tumors and TCC. Limited numbers of cases available preclude prognostic prediction based on ploidy in malignant Brenner tumors or primary ovarian TCCs. DNA ploidy and S-phase reflect the intermediate status of metaplastic, proliferating, and low malignant potential Brenner tumors.     

Flow cytometric DNA analysis of gynecologic malignant tumors using paraffin-embedded tissue

We have studied flow cytometric DNA analysis of gynecologic malignant tumors using paraffin-embedded tissue for histopathology. A close correlation was observed between the DNA indices obtained from fresh unfixed tissue and from paraffin-embedded tissue (r = 0.993). The coefficient of variation (CV) for the diploid G0G1 peak of the paraffin-embedded tissue was one and half greater than that obtained from fresh tissue on the average. In the DNA analysis of 64 endometrial carcinomas, DNA aneuploidy was detected in 3% of nuclear grade 1, in 25% of nuclear grade 2 and in 63% of nuclear grade 3. That is, the more abnormal the nuclear findings, the higher the percentage of DNA aneuploidy. In the analysis of 48 ovarian tumors, DNA aneuploidy was detected in 79% of serous adenocarcinoma, in no of mucinous adenocarcinoma, in 17% of endometrioid carcinoma, in all of clear cell carcinoma and dysgerminoma, that is, its incidence was different among histological subtypes.     

Flow cytometric DNA analysis in gynecological oncology

The relevance of flow cytometric DNA analysis in neoplasia of the female genital tract is reviewed. The virtues and limitations of the technique are discussed. There is good evidence, mainly from retrospective studies, that DNA ploidy and/or the tumor S-phase fraction are valuable prognostic indicators in patients with carcinoma of the ovary and endometrium. Further prospective studies are needed, however, to establish the precise value of flow cytometric DNA analysis before it can be used safely for stratification of therapeutic regimes.     

Flow cytometric DNA analysis of uterine endometrial carcinoma

The DNA content in individual cells from 40 cases of histopathologically normal endometria, 15 of endometrial hyperplasia and 68 of endometrial adenocarcinoma was measured by flow cytometry. An aneuploid cell population was found in 50% of malignant endometria, but in the remaining endometrial carcinomas, the flow cytometrical findings showed no difference from those of benign tissue. Aneuploidy was more common (77.8%) in poorly differentiated tumors than in highly differentiated tumors (35.5%). Two and more aneuploid cell populations were found in 8 cases of 34. The DNA indices of aneuploid tumors were classified into 3 groups: hyperdiploidy, low hyperdiploidy (DNA index range: 1.04-1.2) and high hyperdiploidy (DNA index range: More than 1.2). The proportion of tumors with a high DNA index tended to increase as tumors became less differentiated. In normal endometria the fraction of cells with DNA content corresponding to the s-phase (s-fraction) was 8 +/- 3% on average in the proliferative phase. In well differentiated diploid tumors the s-fraction was 12 +/- 6%, but in moderately and poorly differentiated tumors it was higher (16.0% and 19.0%).     

Flow cytometric DNA analysis of early stage adenocarcinoma of the cervix.

OBJECTIVE: The aim of this study was to determine the utility of DNA flow cytometry as a prognostic indicator for risk of recurrence and overall survival in patients with early stage adenocarcinomas of the uterine cervix. METHODS: DNA flow cytometry was performed to determine ploidy, DNA index, and proliferative index in 66 women with stage IB and IIA pure mucinous adenocarcinomas of the cervix treated by primary surgical therapy with radical hysterectomy and pelvic lymphadenectomy. Fifty-seven of 66 (86.3%) tissue samples were analyzable. Three sections were obtained from paraffin-embedded tissue blocks containing primary tumor. Flow cytometric results, along with other known prognostic variables for risk for recurrent disease and survival, were analyzed using the Cox regression proportional hazards model and survival curves generated by the Kaplan-Meier method. RESULTS: Of 57 interpretable samples, DNA ploidy patterns were 18 (27%) diploid, 8 (12%) tetraploid, and 31 (47%) aneuploid. Thirteen of 66 patients (20%) experienced recurrence with a median time to recurrence of 1.6 years. No significant correlation was noted between DNA ploidy and risk of recurrence (P = 0.429). Multivariate analysis confirmed that positive metastatic lymph nodes were associated with risk of recurrence (P < 0.001). In node-negative patients, a high proliferative index (S% + G(2)M% > 20%), measured as a continuous variable, was the only significant factor for tumor recurrence (P = 0.002). CONCLUSION: DNA ploidy does not predict a patient's risk for tumor recurrence; however, a high proliferative index value warrants further investigation as a potential prognostic indicator for risk of recurrent disease in patients with adenocarcinoma of the uterine cervix.     

Flow cytometric DNA analysis of stage I endometrial carcinoma

Flow cytometric DNA analysis was performed on 203 paraffin-embedded archival specimens obtained from patients with surgical stage I endometrial carcinoma. Primary therapy for those patients (1979-1983) had been definitive extirpation with adjuvant therapy determined by histologic grade, histologic subtype, myometrial invasion, and peritoneal cytologic findings. Diploid DNA patterns were identified in 171 (84%) specimens and nondiploid characteristics were observed in the remaining 32 (25 DNA aneuploid, 7 DNA tetraploid). Although DNA nondiploid specimens accounted for only 16% of all stage I patients, they accounted for 50% of all relapses. Regardless of treatment or other pathologic features, progression-free 5-year Kaplan-Meier survival estimates were 92 and 63% for patients with DNA diploid and DNA non-diploid patterns, respectively (P less than 0.001). Overall 5-year progression-free survival for patients with grade 1 or 2 lesions was 90%; stratification by DNA diploid and DNA nondiploid patterns revealed progression-free survivals of 94 and 64%, respectively (P less than 0.001). Peritoneal cytologic study was positive in seven patients; none of the five with a DNA diploid pattern had a relapse and both with the DNA nondiploid pattern had relapses. These studies suggest that DNA ploidy status may be an objective prognostic determinant for patients with stage I endometrial carcinoma.     

Flow cytometric DNA analysis versus cytology in the evaluation of peritoneal fluids.

The use of flow cytometric DNA analysis as an adjunct to cytology in peritoneal fluid evaluation was studied. One hundred ninety-five fluids from 193 gynecologic patients were subjected to both DNA analysis and cytologic examination. It was found that 117/195 (60%) had invasive malignancies (50 ovarian, 48 endometrial, 17 cervical, and 2 miscellaneous); 34/117 (28%) patients with malignancies were positive by cytology, and 10/117 (8.5%) were positive (aneuploid) by DNA analysis. Of 34 cytologically positive cases, 7 (21%) were DNA positive, 25 (74%) were DNA negative, and in 2 (6%) insufficient cells were obtained. Only 3 fluids (3%) from malignancies were positive by flow cytometry and negative by cytology (1 stage I ovarian cancer, 1 stage I endometrial cancer, and 1 stage III ovarian cancer). No false-positive cytology and one probable false-positive flow result was obtained. If only those patients with histologically documented peritoneal involvement are considered, 29/43 (65%) had positive cytology and 8/43 (19%) had a positive flow result. We conclude that: (1) the high false-negative rate of flow cytometry (79%) versus cytology in this study may be related to a high percentage of diploid cancers, specimen preparation, or histogram interpretation, and (2) flow cytometry rarely adds to cytologic evaluation and is probably best reserved for use only in selected cases.     

Flow cytometric analysis of cellular DNA content in ovarian cancer

A total of 169 paraffin-embedded tissue sections from 42 patients with epithelial ovarian cancer were subjected to analysis of cellular DNA content by flow cytometry. Twenty-three cases were found to be homogenously diploid, whereas 19 cases were aneuploid. A "mosaic" type containing both aneuploid and diploid cell populations was found in 11 of 19 aneuploid cases. Clinical features showed significant correlation with tumor ploidy of FIGO stage and bulky disease. In evaluation of the prognostic value of tumor ploidy, "mosaic" tumors were frequently observed in women who died of disease (DOD), whereas women with diploid tumors survived longer than those with "mosaic" tumors. The present results suggest that determination of DNA heterogeneity may be a valuable parameter in the prognosis of epithelial ovarian cancer.     

Clinical and laboratory investigation of a virilized woman with placental-site trophoblastic tumor

The novel association of virilization and placental-site trophoblastic tumor, an uncommon form of gestational trophoblastic disease (formerly called "trophoblastic pseudotumor"), is described in a 32-year old woman. Multiple agent chemotherapy lowered serum concentrations of chorionic gonadotropin (hCG) (8.7 to 2.1 ng/mL), pregnancy-specific beta 1-glycoprotein (32 to 3.9 ng/mL), and testosterone (400 to 74 ng/dL). Subsequent hysterectomy revealed a 2-cm tumor nodule within the myometrium, and one week postoperatively serum concentrations of hCG (0.5 ng/mL), pregnancy-specific beta 1-glycoprotein (1.3 ng/mL), and testosterone (20 ng/dL) had all returned to normal. Percutaneous catheterization of the ovarian veins before chemotherapy demonstrated a 50-fold elevation of ovarian vein testosterone concentrations compared with normal women, whereas ovarian vein 17 beta-estradiol concentrations were only twofold higher than normal. Direct sampling of the uterine veins at the time of hysterectomy documented testosterone and estradiol production, presumably by the placental-site trophoblastic tumor. Ovarian vein testosterone concentrations at this time were only fourfold above normal, probably the result of falling serum concentrations of hCG. Wedge biopsy of the ovaries disclosed minimal histologic changes (thecal cell luteinization, focal thecosis) compatible with hCG stimulation. Failure of the placental-site trophoblastic tumor to produce large amounts of estrogen, in contrast to normal pregnancy and hydatidiform mole, resulted in marked androgen/estrogen imbalance, high circulating concentrations of free testosterone, and virilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric analysis of DNA content and RAS P21 oncoprotein expression in ovarian neoplasms.

Flow cytometric analysis of DNA content and ras p21 expression were studied in paraffin-embedded normal ovary (NO, n = 10), serous cystadenoma (SA, n = 11), serous tumors of low malignant potential (LMP, n = 13), and papillary serous cystadenocarcinoma (SCa, n = 7). Tissue for DNA analysis was processed via a modified Hedley technique; a separate aliquot of the same sample was stained with a monoclonal antibody directed to oncoprotein ras p21 (clone Y13259). NO (10/10) and SA (11/11) demonstrated diploid DNA stemlines; 4/12 LMP and 5/7 SCa were aneuploid. S-phase fraction varied with significant differences (p less than 0.001) for SA (mean = 4.4%), LMP (mean = 9.0%), and SCa (mean = 12.7%). When all cases were evaluated, p21 expression was relatively lower in NO and SA (mean = 4.2%) in contrast to LMP and SCa (mean = 13.0% and 8.1%). Diploid cases were examined separately, and lesions with high p21 expression were associated with a diagnosis of LMP/SCa (p less than 0.001), whereas diploid tissues with low p21 expression were associated with a nonmalignant diagnosis (NO/SA). This study suggests that aneuploidy or diploidy with high p21 expression (greater than 10%) may be associated with LMP or frankly malignant ovarian tumors.     

Flow cytometric analysis of DNA ploidy and S-phase fraction of Stage IIIB cervical carcinoma

OBJECTIVES: To evaluate the role of flow cytometry-measured DNA ploidy and S-phase fraction as survival prognostic indicators in women with FIGO Stage IIIB squamous cell carcinoma of cervix. METHODS: We retrospectively reviewed the medical and pathological records of women with Stage IIIB squamous cell cervical carcinoma treated between 1993 and 1996. Flow cytometric analysis of DNA ploidy and S-phase fraction was performed by the modified Hedley technique using paraffin-embedded tissue. Survival was calculated using the Kaplan-Meier life-table analysis. RESULTS: Of the 75 cases, 66 were analyzable. Diploid tumors were found in 73%. The mean S-phase fraction was 14% (SD = 5.4). The overall 5-year survival rate was 60%. The survival of patients with aneuploidy tumors was significantly worse than that of the diploid tumors (p = 0.001). The survival of the patients who had S-phase fraction > 12% was significantly worse than those who had S-phase fraction < or =12% (p = 0.04). CONCLUSIONS: In this homogeneous study population, we found that aneuploidy and S-phase fraction >12% correlated with poor survival. Identifying this poor prognostic group would be of benefit in considering additional treatment for a better outcome.     

Flow cytometric assessment of DNA ploidy is a useful prognostic factor for patients with granulosa cell ovarian tumors

Abstract. Holland DR, Le Riche J, Swenerton KD, Elit L, Spinelli J.J. Flow cytometric assessment of DNA ploidy is a useful prognostic factor for patients with granulosa cell ovarian tumors. Int J Gynecol Cancer 1991; 1 : 227–232.
A major obstacle to effective management for patients with ovarian granulosa cell tumors has been the lack of reproducible and useful prognostic factors. Thirty-nine patients with granulosa cell tumors were seen at our institution from 1974 to 1985. Median follow-up for all living patients is over 7 1/2 years. Tissue blocks obtained at primary diagnosis were available for 37 patients, of these 13 were DNA aneuploid and 24 were DNA diploid. The presence of residual disease was found to be the most important predictor of disease-specific and progression-free survival. DNA ploidy was found to be an independent prognostic factor of progression-free survival ( p = 0.023). Patients with residual-negative DNA diploid tumors have a particularly favorable outcome with a 10-year progression-free survival of 96% (95%CI 72–99%).     

Management of low-risk metastatic gestational trophoblastic tumors.

The clinical course of 48 patients with low-risk metastatic gestational trophoblastic tumors (GTTs) treated with primary single-agent chemotherapy was reviewed. All patients achieved sustained remission, although 25 (51%) required a second single-agent regimen, and 7 (14%) needed combination chemotherapy to achieve it. An average of 3.4 courses of chemotherapy were necessary to achieve remission, and 6 patients (12%) underwent resection of resistant tumor foci. Primary single-agent chemotherapy is a reasonable treatment option in patients with low-risk metastatic GTT.     

Flow cytometric DNA analysis and neoadjuvant chemotherapy of cervix cancer (stage IB2-IIIB)

The purpose of the present study is to determine the predictive role of the data of flowcytometric DNA-analysis--aneuploidity DNA-index, proliferative index in patients with squamous cervical cancer who was given chemotherapy as a initial treatment. The data of 12 patients in stage IB2-IIIB, who were divided into two groups according to the second-line treatment--surgery of definitive radiotherapy, were analysed. These data were correlated with the respond of the tumor to neoadjuvant chemotherapy (clinical and histopathological) as well as other factors such as stage, size of tumor lesion, grading disease free survival. The method of flowcytometric DNA-analysis was presented briefly. The results we have obtained, although in quiete limited number of patients, are interesting and justify prospective studies of the problem.