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ORIGINAL RESEARCH |
From the Departments of Oncology, Gynecology, and Pathology; University Clinic Hospital San Carlos, Madrid, Spain.
Address reprint requests to: Juan Jose Valverde, MD, PhD, Hospital Arrixaca, Servicio de Oncologia Medica, 30120 Murcia, Spain, E-mail: jjvalv{at}teleline.es
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Abstract |
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Methods: We retrospectively reviewed 77 cases of stages I–II ovarian cancer after comprehensive surgical staging. We recorded anthropometric data (age, menopausal status, weight loss, Karnofsky index) and pathologic variables (tumor size, bilaterality, capsular status, ascites, peritoneal cytology, histologic type, and grade). In 72 cases representative paraffin-embedded samples were available for DNA quantification and immunohistochemical evaluation of c-erbB-2 and p53 overexpression. Most women (87%) had received cisplatin-based adjuvant chemotherapy.
Results: The median follow-up was 90 months (range 50–148 months). The 6-year overall disease-free survival rate was 70% (95% confidence interval [CI] 60%, 81%), and overall global survival was 77% (95% CI 67%, 87%). Multivariable analysis using Cox stepwise regression identified DNA content (odds ratio [OR] 12.3; P < .001) and stage (OR 1.4, P = .09) as independent poor prognosis factors for relapse, and DNA content (OR 9.8, P < .001) as the main independent factor for survival. In stepwise discriminant analysis the combination of DNA content and stage provided a correct prediction of relapse in 78% of women.
Conclusion: Flow cytometric DNA quantification was the main independent prognostic factor of relapse and survival in these women with stages I–II epithelial ovarian cancer.
Epithelial ovarian carcinoma is the second most common gynecologic cancer and the leading cause of death from gynecologic malignancy.1 Survival is influenced primarily by extent of disease at diagnosis.2 International Federation of Gynecology and Obstetrics (FIGO) stages I and II3 account for 20–40% of all cases of epithelial ovarian cancer. Five-year survival rates range from 60% to 95% for stage I and from 35% to 70% for stage II.2 Survival differences might be explained in part by heterogeneity of women in the classic series because of inclusion of tumors of low malignancy potential and inclusion of stage III patients after incomplete surgical staging. Over the last decade, investigations have sought independent prognostic factors to define high-risk patients with early-stage disease who might benefit from adjuvant therapy.4
Classic diagnostic characteristics (FIGO substage, histologic grade and type, large volume ascites, dense adhesions, and capsule rupture) are far from sufficient for predicting prognoses of all ovarian cancer patients with early-stage disease. Histologic type and grade assessments are subjective and poorly reproducible5 and to date no consensus exists for relative importance of other prognostic factors.4 That has led to randomized trials with imbalances in stratification of prognostic variables and erroneous interpretations of treatment results. There is increased interest in identifying new prognostic factors with a more biologic rationale, so variables are more objective and quantifiably reproducible.
Our study was designed to analyze prognostic data of traditional clinicopathologic factors with a new set of biologic factors such as flow cytometry DNA quantification and immunohistochemical expression of oncogene c-erbB-2 and p53 in women with invasive epithelial ovarian cancer of stages I–II.
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Materials and Methods |
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Each woman had comprehensive staging laparotomy through a vertical midline incision long enough to allow access to the upper abdomen and diaphragm. The tumor capsule was examined for excrecences, dense adhesions, and rupture. Sample of ascitic fluid or peritoneal washings were collected for cytology examination. Bilateral salpingo-oophorectomy and total abdominal hysterectomy with complete removal of pelvic disease was done, as was infracolic omentectomy and appendectomy. Lymph node samples were collected from paraaortic, precaval fat pad, common, and external iliac nodes. Random biopsies were taken after meticulous peritoneal exploration (at least one sample each from pelvic walls, cul-de-sac of Douglas, urinary bladder, and paracolic and subdiaphragmatic spaces). Operative notes and pathology reports were reviewed to assure completeness of surgical staging.
Histologic Classification and Grading
All hematoxylin-eosin–stained histology slides of primary tumors were reviewed by two pathologists without knowledge of clinical data. Histology was typed according to World Health Organization (WHO) criteria for classification of common epithelial ovarian tumors. Grading was based on cytologic and architectural criteria as follows:
Grade 1 (low): papillary or glandular pattern, slight nuclear pleomorphism, and less than five mitoses per ten high-power microscope fields.
Grade 2 (moderate): papillary-glandular structures with some solid tumor areas, moderate nuclear pleomorphism, and five to 50 mitoses per ten high-power microscope fields.
Grade 3 (high): predominant solid tumor fields, severe nuclear pleomorphism, and more than 50 mitoses per ten high-power microscope fields.
DNA Flow Cytometry
Nuclear suspensions were prepared from paraffin-embedded tissue blocks of the primary tumors based on Hedley’s method.6 Paraffin blocks were checked for the presence of more than 50% tumor cells by examination of 5-µm hematoxylin-eosin slides; three 50-µm-thick sections were selected immediately adjacent ("sandwich technique"). In 21 cases, more than one representative sample of the same tumor was collected for DNA analysis. Sections were treated with xylene to remove paraffin then progressively rehydrated by repeated treatment with decreasing concentrations of alcohol in water (from 100% to 50% to distilled water alone).
The tissue was incubated for 20 minutes at 37C in 2 mL of 0.5% pepsin at pH 1.5. After disaggregation by mechanical homogenization, the slurry was filtered and centrifuged at 2000 rpm for 15 minutes to form the nuclear pellet. That solution was incubated with ribo-nuclease (180 U/mL in phosphate-buffered saline solution) at 37C for 20 minutes and stained with propidium iodide (100 µg/mL in phosphate buffered solution) in the dark for at least 4 hours at 4C before flow cytometry.
Nuclear DNA content was measured on an EPICS PROFILE II flow cytometer (Coulter Corporation, Hi-aleah, FL) with a sensor analytical rate of more than 10,000 nuclei per second. Cell cycle evaluation of DNA histograms was done with a Multicycle Computerized Software program (POWERPAK package). As an external control (diploid marker), a sample of nonneoplastic ovarian tissue was selected and analyzed.
The histograms were interpreted by one of the authors (JAG-A) without prior knowledge of clinical outcome. Tumors that showed a single G0-1 peak were classified as diploid, whereas DNA profiles with one or more additional peaks were considered aneuploid. The coefficient of variation (CV, standard deviation relative to mean value) was calculated on the G0-1 peak; the maximum acceptable value was 12%. The median CV in diploid tumors was 7.3% (range, 1.2–12%), and in aneuploid tumors 8.8% (range, 2.5–12%). The DNA index (DI) was calculated from the ratio of the mean channel number of the aneuploid peak and the reference diploid peak.
Immunohistochemistry
Expression of c-erbB-2 and p53 was detected by an indirect immunoperoxidase technique. The same blocks of paraffin selected for DNA analysis were sectioned at 5 µm and deparaffinized in three changes of xylene (10 minutes each) followed by 30 seconds’ incubation in absolute ethanol. To remove endogenous peroxidase activity, sections were treated with a 0.3% methanol/H2O2 solution (30 minutes at room temperature). The slides were hydrated in decreasing concentrations of alcohol and rinsed in 0.01 M citrate buffer (pH 0.6) before being heated in a microwave oven at 750 W for two cycles of 5 minutes each. Subsequently, the preparation was rinsed in phosphate buffered saline and allowed to cool for about 1 hour at room temperature before incubation for 24 hours with primary antibody. Monoclonal antibody CB-11 (BioGenex Laboratories, San Ramon, CA) recognizes the internal domain of the transmembrane c-erbB-2 oncoprotein. Monoclonal antibody DO-7 (Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) binds specifically to aminoacids 1–45 in the N-terminal region of the p53 nuclear gene protein product.
The sections were incubated with secondary rabbit antimouse antibody DAKO P-161 (Dako Corporation, Carpenteria, CA) at a dilution of 1:20 (in a 2:1 solution of phosphate buffered saline: human serum) for 45 minutes at room temperature, then washed again in phosphate buffered saline. The peroxidase reaction was viewed with the chromogen substrate diaminobenzidine tetrahydrochloride (3 mg in 5 mL Tris 0.2 M), with 0.03% hydrogen peroxidase added at the instant of reaction. The sections were then counterstained with hematoxylin for 30 seconds; after dehydration, cover slips were applied. To evaluate the specificity of the immunoreaction, a sample of human breast cancer (c-erbB-2) and colon cancer (p53) with known oncogene expression were used as positive controls. The staining in non-neoplasic areas in the same section served as the negative control.
Staining was evaluated microscopically by two independent observers at a multiheaded microscope without knowledge of clinical data. Three categories were defined: negative, 0%; positive-weak, up to 25% (inclusive); positive-strong, above 25%. Cytoplasmic staining was considered nonspecific.
Adjuvant Treatment and Follow-up
During the study, adjuvant treatment with cisplatin-based chemotherapy was offered to all women in the first 6 weeks after surgery. Fully informed written consent for chemotherapy administration was obtained. A second-look laparotomy was proposed to assess response to therapy no more than 6 months after adjuvant treatment, which included detailed inspection and surgical exploration of abdomen and pelvis, peritoneal washings, multiple peritoneal biopsies, and pelvic and para-aortic lymph node sampling.
Patients were monitored every 3 months for the first 3 years, and then every 6 months with complete clinical history and physical, rectovaginal, and pelvic examinations conducted routinely. A computed tomography scan of the abdomen and pelvis was done every 6 months for the first 3 years, and then annually. Recurrence was always confirmed by cytology or biopsy. Salvage therapy included surgery if disease was resectable, chemotherapy, or external radiotherapy.
Statistical Analysis
BMDP Statistical Software Inc. programs (1988 version, Los Angeles, CA) were used for statistical analysis. Life tables to examine relapse-free survival and survival rates were estimated by Kaplan-Meier method. Survival curves were compared with generalized Savage (Mantle-Cox)7 and generalized Wilcoxon (Breslow)8 methods. The independent effects of prognostic factors on survival function were determined by Cox stepwise regression analysis and stepwise discriminant analysis. Only factors found significant on univariable analysis were considered for inclusion in the multivariable models. Correlations between qualitative variables were examined by 
2 contingency tables with adjusted standardized deviates; Fisher exact test and Yates correction were used for two-by-two tables.
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summarizes the clinicopathologic characteristics of the study group. Capsule rupture was intraoperative in all registered cases.
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To evaluate tumor heterogeneity in relation to DNA content, we selected different representative samples of the same tumor in 21 subjects. Coexistence of DNA diploid and aneuploid cellular subpopulations were present in four cases (19%) that had been considered aneuploid. It was inadequate to determine S-phase fraction in most samples because of technical problems with paraffin-embedded tissue (high CV, debris, or aggregates). Tumor DNA content was statistically related to histologic grade: 15 of 20 low-grade tumors (75%) had diploid DNA content; ten of 12 high-grade tumors (83%) were aneuploid, P = .004 (Table 2). Moreover, DNA content information was more specifically related with relapse and death than histologic grade (Table 2).
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We did not find differences in flow cytometry DNA content or immunohistochemical staining for age of storage of the paraffin samples (42 cases from 1984–1987 versus 30 cases from 1988–1990).
Adjuvant Treatment and Second-Look Laparotomy
Sixty-three women received adjuvant treatment with four cycles of cisplatin-based chemotherapy. Fifty-four received cisplatin 80 mg/m2, doxorubicin 50 mg/m2, cyclophosphamide 500 mg/m2, every 21 days; nine received cisplatin 100 mg/m2, cyclophosphamide 600 mg/m2, every 21 days or carboplatin 300 mg/m2, cyclophosphamide 600 mg/m2, every 28 days. Two elderly women were treated with melphalan (0.2 mg/kg per day for 5 days, every 28 days, for four cycles). There were no deaths caused by toxicity. The remaining 12 women did not receive any postsurgical treatment because their prognoses were good in most cases (stage IA in nine cases).
Fifty-four women (70%) had second-look laparotomies, four (7.5%) had persistent or recurrent tumors. Locations of disease were pelvis (three cases), para-aorta (one case), and peritoneal washing cytology (one case). After salvage therapy, two women remain alive and disease-free. After a negative second-look laparotomy, nine of 50 had disease recurrence and eight had died by the conclusion of the study. There were no survival differences in the group of 20 who did not have second-look laparotomies.
Analysis of Relapse and Survival
The median follow-up of the study was 90 months (range 50–148 months). The overall 6-year survival rate and disease-free survival rate were 77% (95% confidence interval [CI] 67%, 87%) and 70% (95% CI 60%, 81%), respectively.
Univariable analysis was done to identify factors associated with relapse and survival. All clinicopathologic characteristics, DNA content, and immunohistochemical expression of c-erbB-2 and p53 were included (immunostaining analysis was negative versus positive and negative-positive weak versus positive strong). The factors that predicted relapse were presence of adhesions, histologic grade, FIGO stage, and DNA content (Table 3). Except for adhesions, the same factors predicted survival (Table 3). For aneuploid subjects, there were no statistically significant differences in relapse or survival with respect to DNA indices (cutoff of 1.4). Considering DNA content in stage I disease, it was possible to identify women with unfavorable prognoses (Figure 1).
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). The most determinant variables for relapse were DNA content (OR 12.3, 95% CI CI 2.7, 57) and FIGO stage (odds ratio 1.4, 95% CI 1.0, 2.2). DNA content remained the main independent prognostic factor of survival (OR 9.8, 95% CI 1, 91). To evaluate prognostic prediction of clinical outcomes, we did a stepwise discriminant analysis. DNA content and FIGO stage were the most discriminant parameters and their combination correctly predicted relapse in 78% of subjects. For overall survival, prognostic information of DNA content showed a correct prediction of outcome in 73% of cases.
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Discussion |
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Histologic grade is the most consistent classic prognostic factor despite a lack of consensus on criteria for tumor grade assignment.9–14 The existence of inter- and intraobserver diagnostic variability, not only in determination of histopathologic grading but also in diagnosis of histologic type has been shown.5 Recently, a new grading system based on architectural pattern, nuclear pleomorphism, and mitotic activity has been proposed, although some problems still remain in the reproducibility of classification and grading of clear cell carcinoma.14
Controversy abounds with respect to the prognostic role of other clinicopathologic factors. The FIGO staging system is based on the extent of spread noted at laparotomy and, although its clinical and prognostic value are well described, a lack of significant prognostic information of substage division IB-IC and IIB-IIC has been noted. Combining those divisions into respective single substages has gained support.2 Other variables with independent prognostic significance in some studies have been age,15 performance status,14 ascites,11,16 dense adhesions,16 and clear cell carcinoma type.9 Although in multivariable analysis the intraoperative capsule rupture has not been a significant factor,17–19 spontaneous preoperative rupture has had independent prognostic value.18,19
Accurate and comprehensive surgical staging is necessary before a patient can be truly classified as stage I–II. A retrospective multicenter study questioned the effect on survival of extensive surgery for pathologic staging20 and another study identified peritoneal washings as negative factors for survival.17 A prospective study that included 194 women with stage I epithelial ovarian cancer without adjuvant treatment administration identified histologic grade, surface tumor, and ascites as independent prognostic factors for relapse, while questioning the value of lymphadenectomy.11
Conflicting reports on immunohistochemical expression and prognostic relevance of biologic factors can be explained by differences in methods and subjective assessment of immunostaining. Overexpression of oncogene c-erbB-2 can be detected in ovarian cancer by immunohistochemistry from 9% to 32%, but correlation with tumor stage and histology grade has not been found.21 Although prognostic value in multivariate analysis has been postulated by some authors,22 that has not been confirmed by others.23
The significance of p53 mutations and expression of the protein products in women with ovarian carcinoma remains uncertain. Missense mutations can result in stable mutant proteins detectable by immunohistochemistry, but other mutations can be silent or not result in a stable protein product that is detectable by that method.24 Immunostaining of p53 has been observed from 30% to 60% in correlation with tumors of advanced stage and grade, but an independent prognostic significance has not been described consistently.25–27
Most studies of DNA cytometry for prognostic purposes have been limited to stage III and IV disease and few correlated DNA ploidy with more conventional prognostic factors or immunohistochemical expression of p53 or c-erbB-2. A strict definition of early disease requires an accurate surgical staging that has not been applied in other studies (Table 4). In the study of Vergote et al30 peritoneal washings, lymphadenectomy, or diaphragm scraping were not routine. Strict definition of surgical staging was omitted in other publications.28,29,31
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). Discrepancies regarding the relative importance of prognostic factors in different publications could be explained by lack of consistency in assessing histologic grades, technical or methodologic differences in analyzing DNA content, and variations in adjuvant treatment regimens. The frequency of aneuploidy in our study (29 of 68, 43%) was in concordance with previous reports (Table 4). Other studies found association of DNA content and FIGO stage, and the incidence of aneuploidy in advanced stages has been 70–80%.32 Vergote et al30 identified histologic grade as the most powerful prognostic indicator of disease-free survival, followed by DNA content and FIGO substage. Schueler et al31 reported DNA index the primary prognostic indicator for disease-free survival in a group with well-differentiated stage I disease. In both series, less than half of the subjects received cisplatin-based adjuvant treatment. An important issue related to DNA content heterogeneity has not been determined in previous prognostic studies.28–31 Different definitions of heterogeneity have been applied33 and their effect on predicting progression or survival has not been established, particularly in early-stage disease. After a meticulous review of our archived material we found coexistence of diploid/aneuploid populations in four of 21 cases (19%) with more than one representative sample. Because of extent of variations, caution must be exercised when interpreting prognosis from a single sample that showed diploid results (an undetected aneuploid population might be present).
Recommendations for the best quality and reproducibility of histograms for future studies include fresh as opposed to paraffin-embedded tumor tissue, evaluation of at least two representative biopsies because of the heterogeneity of DNA content, and supplementing flow cytometry measurements with image cytometry and morphometry analysis.32 Guidelines for implementation of clinical flow cytometry have been published and should they be followed, it would be possible to compare results from different institutions.34
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Received July 11, 2000. Received in revised form October 27, 2000. Accepted November 22, 2000.
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References |
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34. Shankey TV, Rabinovitch PS, Bagwell B, Bauer KD, Duque RE, Hedley DW, et al. Guidelines for implementation of clinical DNA cytometry. Cytometry 1995;14:472–7.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Analyses
