Large-scale genomic instability predicts long-term outcome for women with invasive stage I ovarian cancer

G. B. Kristensen1,+, W. Kildal2, V. M. Abeler2, J. Kaern1, I. Vergote1,3, C. G. Tropé1 and H. E. Danielsen2,4

1 Department of Gynecologic Oncology and 2 Department of Pathology, The Norwegian Radium Hospital, Oslo, Norway; 3 Department of Gynecologic Oncology, University Hospitals Leuven, Leuven, Belgium; 4 Division of Genomic Medicine, University of Sheffield, Sheffield, UK

Received 6 November 2002; revised 8 May 2003; accepted 3 June 2003


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background:

The objective was to evaluate the value of DNA ploidy using high-resolution image cytometry in predicting long-term survival of patients with early ovarian cancer.

Patients and methods:

A retrospective analysis of 284 cases with FIGO stage I ovarian carcinoma treated during the period 1982–1989 was performed. Clinical follow-up information was available for all patients.

Results:

Patients with diploid and tetraploid tumors had a 10-year relapse-free survival of 95% and 89%, respectively, compared with 70% and 29% for polyploid and aneuploid tumors, respectively. DNA ploidy analysis was the strongest predictor of survival in multivariate analysis (diploid/tetraploid versus polyploid/aneuploid; relative hazard 9.0) followed by histological grade, including clear cell tumors in the group of poorly differentiated tumors (grade 1–2 versus grade 3 or clear cell; relative hazard 2.7), and FIGO stage (Ib/Ic versus Ia; relative hazard 2.0). In a stratified Kaplan–Meier analysis, patients with grade 1–2, diploid or tetraploid tumors had a 10-year relapse-free survival of 95%, forming a low-risk group. Patients with grade 3 or clear cell, diploid or tetraploid tumors had 10-year relapse-free survival of 86%, forming an intermediate-risk group, while all patients with aneuploid/polyploid tumors formed a high-risk group, with 10-year relapse-free survival of 34%.

Conclusions:

This study points to the importance of including DNA ploidy analysis by image cytometry when selecting patients with early ovarian cancer for adjuvant treatment after surgery.

Key words: DNA ploidy, image cytometry, ovarian neoplasms, prognosis


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Ovarian cancer is the second most common gynecological cancer with an incidence of about 15 per 100 000 in western countries, and is the leading cause of death from gynecological malignancies. Approximately 25% of patients present with disease localized to the ovaries, International Federation of Obstetrics and Gynecology (FIGO) stage I [1]. The prognosis for these women is much better than for women with spread of disease outside the ovaries at time of diagnosis. Reported 5-year survival rates for FIGO stage I range from 70% to 90% [25].

Patients with FIGO stage I ovarian cancer who suffer a relapse after surgery do so because of subclinical metastases at time of surgery, most commonly in the peritoneal cavity, but occasionally in extra-peritoneal locations such as lymph nodes. Identification of patients with such micrometastases is crucial in order to be able to offer additional treatment.

Earlier reports have identified FIGO substage, grade of differentiation, histological type (clear cell carcinoma), dense adhesions, large volume ascites and rupture before or during surgery as independent prognostic factors [4, 6, 7]. The DNA content of tumor cells (DNA ploidy) has been found to be a strong indicator of prognosis in early ovarian cancer [8, 9]. Cells with abnormal DNA content may represent clones that may be expressing multiple mutations. This large-scale genomic instability probably involves cellular changes that affect gene expression and/or function of cell cycle [1012], cell death [13] and/or DNA repair pathways [14]. These changes might well be responsible for the enhanced aggressiveness of the malignant cells.

DNA ploidy analysis can be performed by either flow cytometry or image analysis. For both methods, a suspension of cells is made from a section of the tumor. In flow cytometry all cells in this suspension are used, with no direct visualization of the analyzed cells. With image analysis, the cytologist performs a screening of the cells in the microscope and selects tumor cells for the analysis. Stromal cells, necrotic cells, doublets or cut cells are disregarded. As previously reported [15], image analysis may be more sensitive than flow cytometry in identification of small aneuploid stem-lines. Image analysis also permits better identification of tetraploid tumors, as the presence of debris, doublets and small aggregates may give misleading results when using flow cytometry [16]. Furthermore, with image cytometry, the number of tumor cells with a tetraploid DNA content is quantified against the total number of tumor cells in the sample, while with flow cytometry the total number of cells with a tetraploid DNA content is quantified against the total number of cells in the sample, tumor cells and stromal cells together.

We have performed a retrospective analysis on a large group of patients with early ovarian carcinoma, using a high-resolution image analysis system to evaluate the prognostic significance of DNA ploidy.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
This is a retrospective study performed on tissue samples from 284 patients treated for early ovarian cancer during 1982–1989. Generally, surgery was performed at county hospitals and the patients were admitted to The Norwegian Radium Hospital for evaluation of further treatment. The surgical procedure consisted of a midline incision with thorough inspection of the abdominal cavity, peritoneal washing, hysterectomy, bilateral salpingo-oophorectomy and omentectomy. Lymph node sampling was not performed. In all patients, the tumors were restricted to one or both ovaries with these evaluations and thereby apparently in FIGO stage I. All but 13 patients had adjuvant treatment following surgery: 140 had chemotherapy (119 had six courses of cisplatinum as single drug and 21 received thiotepa for 6 months), 108 had intraperitoneal instillation of 32P, 19 had whole abdominal external radiation and four patients had other types of treatment. Patients who suffered a relapse were readmitted to The Norwegian Radium Hospital and treated with platinum-based chemotherapy if fit for further treatment.

After treatment, all patients were followed up either at local hospitals or at our department until death or 31 December 1998. Follow-up information was also achieved from the National Statistical Bureau, which keeps records of all inhabitants in Norway.

Most of the patients took part in a previously published study on the prognostic significance of flow cytometric evaluated DNA ploidy [9].

Histological classification and grading
All histological sections were reviewed by a single pathologist. Histological classification was performed using the criteria of the World Health Organization (WHO). All tumors were graded as well, moderate or poorly differentiated, except for clear cell carcinomas and mixed tumors with clear cell elements, which were not graded. Intra-observer variation of grading was evaluated by allowing the same pathologist to evaluate grading twice, in a blinded fashion.

DNA cytometry
Paraffin-embedded, formalin-fixed tissue was used for preparation of nuclei suspension. Blocks were selected by the pathologist, who selected the tumor tissue to be used for the preparation. Monolayers were prepared from one or more 50-µm sections using a modification of Hedley’s method [17]. The nuclei were stained with Feulgen–Schiff. We used the Fairfield DNA Ploidy System (Fairfield Imaging Ltd, Nottingham, UK), which consisted of a Zeiss Axioplan microscope equipped with a 40/0.75 objective lens (Zeiss, Jena, Germany), a 546 nm green filter and a black and white high-resolution digital camera (C4742-95; Hamamatsu Photonics K.K., Hamamatsu, Japan) with 1024 x 1024 pixels at 10 bit per pixel for image processing, analysis and classification. The integrated optical density of each nucleus was calculated on the basis of measurements of optical density and area. Background optical density was measured and corrected for each nucleus. Approximately 300 tumor nuclei were measured and stored in galleries in each case, and lymphocytes and plasma cells were included as internal controls when available.

The criteria for classification of histograms were as follows: The tumor was classified as diploid if only one G0/G1 peak (2c) was present, the number of nuclei in the G2 peak (4c) did not exceed 10% of the total number of nuclei and the number of nuclei with a DNA content exceeding 5c did not exceed 1%. A tumor was defined as tetraploid when a peak in the 4c position was present together with a G2 peak in the 8c position or the fraction of nuclei in the tetraploid region (4c) exceeded 10% of the total number of nuclei. A tumor was defined as polyploid when a peak in the 8c position was present together with a G2 peak in the 16c position. The tumor was defined as aneuploid when non-euploid peaks were present or the number of nuclei with a DNA content exceeding 5c/9c, not representing euploid populations, exceeded 1%. The histograms were classified by specially trained personnel without knowledge of the clinical outcome.

Statistical analysis
Differences in proportions were evaluated by the {chi}2 or Fisher’s test, as appropriate. Relapse-free survival was calculated from start of treatment to relapse or 31 December 1998, using the method of Kaplan and Meier. The log rank test was used for univariate analysis and a Cox proportional hazards regression model for multivariate evaluation of relapse-free survival. The hazard proportionality was verified by computing log minus log curves. Candidate variables were accepted if P was <0.2 in univariate analysis. A backwards selection procedure was used. The SPSS statistical package was used for the statistical analysis. Statistical significance was considered as P <0.05.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The median follow-up in surviving patients was 13.2 years (range 10–16.9). At the date of last follow-up, 81 (29%) relapses had occurred, 74 patients had died from disease and seven were alive after a relapse. A total of 203 had not suffered any relapse; of these 175 were still alive while 28 had died from diseases unrelated to ovarian cancer. All patients still alive have been followed up for at least 10 years. The overall 5- and 10-year relapse-free survivals were 78% and 72%, respectively. The overall 5- and 10-year disease-related survivals were 83% and 75%, respectively, and the 5- and 10-year crude survivals were 81% and 68%, respectively.

Estimated 10-year relapse-free survival according to pertinent variables is shown in Table 1. Kaplan–Meier plots of relapse-free survival related to degree of differentiation are shown in Figure 1. Clear cell tumors are not graded in our institution and are therefore displayed as a separate group in Figure 1. Figures 2 and 3 show relapse-free survival related to FIGO stage and DNA ploidy classification by image cytometry, respectively. There was no difference in relapse-free survival between patients with clear cell tumors and poorly differentiated tumors, which is why these patients were categorized together in the multivariate analysis and, for the same reason, patients with FIGO stage Ib and Ic were categorized together. Patients with diploid or tetraploid tumors had a good prognosis with no statistical significant difference in relapse-free survival between the two groups, and were considered together as one group in the multivariate analysis. This group had a 10-year relapse-free survival of 92%. Patients with polyploid or aneuploid tumors had a poor prognosis and were considered as one group in the multivariate analysis. This latter group had a 10-year relapse-free survival of 34%. The 10-year disease-related survival was 95% for patients with diploid/tetraploid tumors and 37% for patients with aneuploid/polyploidy tumors.


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Table 1. Relapse-free survival related to prognostic factors in stage I ovarian carcinoma
 


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Figure 1. Relapse-free survival according to grade of differentiation/clear cell histology.

 


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Figure 2. Relapse-free survival according to FIGO stage.

 


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Figure 3. Relapse-free survival according to DNA ploidy classification by image cytometry.

 
The postoperative adjuvant treatment given to patients in this study did not significantly influence prognosis. The 10-year relapse-free survival for 119 patients receiving cisplatinum was 75%, compared with 69% for 108 patients receiving 32P and 65% for patients receiving another type or no adjuvant treatment (P = 0.68). The 10-year crude survival was 71%, 67% and 65%, respectively.

In the multivariate analysis, DNA ploidy classification, histological grade/type and FIGO stage were of independent prognostic significance for relapse-free and disease-free survival (Table 2).


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Table 2. Significant variables for survival in multivariate analysis
 
A stratified Kaplan–Meier analysis of relapse-free survival was performed based on factors of independent prognostic significance in multivariate analysis (Table 3). FIGO stage did not add to this classification, and was left out. Patients with diploid or tetraploid tumors grade 1–2 tumors formed a low-risk group, with a 10-year relapse-free survival of 95% and a 10-year disease-related survival of 98%. Patients with diploid or tetraploid tumors grade 3 or clear cell tumors formed an intermediate-risk group, with a 10-year relapse-survival of 86% and a 10-year disease-related survival of 88%. All patients with aneuploid or polyploidy tumors made up a high-risk group, with a 10-year relapse-free and disease-related survival of 34% and 37%, respectively. Those with grade 1–2 tumors had a 10-year relapse-free survival of 54% and those with grade 3 or clear cell tumors a 10-year relapse-free survival of only 23%.


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Table 3. Stratified Kaplan–Meyer analysis of relapse-free 10-year survival according to DNA ploidy classification by image cytometry and grade of differentiation/histological type
 
Histological grading had good reproducibility, with a {kappa} value of 0.78 for intra-observer variation.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Histological grading has, in many studies, been found to be the most important predictor of prognosis in stage I ovarian cancer [3, 7, 18, 19], although the evaluation ishampered by inter- and intra-observer variation [20, 21]. In a recently published multinational report on 1545 patients with stage I ovarian cancer, histological grading, FIGO stage, rupture, optimal staging and age were all found to be of independent prognostic significance [7].

DNA ploidy has prognostic significance in early ovarian cancer, as previously shown in several retrospective studies using flow cytometry [8, 9]. Using DNA ploidy analysis with high-resolution image cytometry, we achieved much better discrimination between patients with a good and a poor prognosis than we did in a previous study with flow cytometry on the same patient group [9]. In the flow cytometry study, patients with diploid tumors had a 5-year relapse-free survival of 90% and those with non-diploid tumors a 5-year relapse-free survival of 64%, while patients with diploid/tetraploid tumors in the present study had a 10-year relapse-free survival of 92% and patients with polyploid/aneuploid tumors had a 10-year relapse-free survival of 34%. DNA ploidy analysis using image cytometry is easy to perform and is readily available.

In our study, FIGO stage was of independent prognostic significance, but was a relatively weak factor with a relative hazard of 2.0. In the stratified analysis using grade, DNA ploidy and FIGO stage, stage did not contribute to the risk classification, and was left out. Also, in the European multinational report [7] the difference in 5-year disease-free survival between patients with stage Ia and Ib–c was small, and of marginal significance in the multivariate analysis.

The group of patients with grade 3 or clear cell tumors has a poor prognosis. It is of interest to note that DNA ploidy analysis was able to determine a subgroup with diploid or tetraploid tumors that had a relatively favorable prognosis.

Prognostic indicators are used to select patients for adjuvant treatment. Histological grade and FIGO stage are often used as selection criteria [5, 2224]. Patients with stage IA, grade 1, non-clear cell histology are usually not considered candidates for adjuvant chemotherapy [2224].

In our study, all patients with a tumor in stage I, grade 1–2, with non-clear cell histology and diploid or tetraploid DNA content on image cytometry had a very good prognosis. Even taking into account that one-third of the relapses in this group might have been avoided by the adjuvant treatment given [24], their prognosis is still so good that we find it safe to avoid adjuvant treatment in this group of patients, and instead to offer treatment at the time of relapse to the few patients who will suffer a relapse. This low-risk group represents 46% of the patients in our study, with a 10-year relapse-free survival of 95% and a 10-year disease-related survival of 98%.

The low risk of recurrence for patients with FIGO stage I diploid tumors is confirmed in a prospective randomized study on adjuvant chemotherapy [22] with a relapse in three out of 37 patients with diploid tumors. Patients were randomized either to receive six courses of carboplatin monotherapy or to control. In this study, DNA ploidy analysis was performed with flow cytometry and patients with grade 3 and clear cell histology were also included. Using the more strict criteria for a low-risk group we propose that the risk of a recurrence without adjuvant treatment will be well below 10% for this group of patients. Patients with grade 3 or clear cell tumors classified as diploid or tetraploid by image cytometry had a 10-year disease-free survival of 86%. We consider this group of patients as belonging to an intermediate-risk group that might benefit from adjuvant treatment.

All patients with aneuploid tumors had a markedly increased risk of recurrence and patients with poorly differentiated tumors or tumors with clear cell histology had a moderately increased risk of recurrence. These patients should be considered candidates for adjuvant chemotherapy or studies with new, and hopefully better, treatment modalities.

Most patients in our study had received adjuvant treatment. In accordance with our previous report [25], we found no difference in survival related to the type of adjuvant treatment. However, it should be noted that this study was not designed and powered to evaluate the importance of adjuvant treatment. Most of the earlier studies have failed to show an effect on overall survival from adjuvant therapy [5, 22, 23]. However, the two recently closed European trials (ICON 1 and ACTION) [24] showed an improvement in progression-free survival by 11% and in overall survival by 7% in high-risk patients using chemotherapy compared with follow-up without adjuvant treatment. In the subgroup of patients with an optimal staging procedure, however, the effect of adjuvant chemotherapy on survival seemed to disappear [24]. In a recent study performed on patients who have undergone comprehensive surgical staging [26], the recurrence rate was lowered by 31% by adjuvant treatment with cisplatinum and cyclophosphamide compared with intraperitoneal 32P. However, this difference was not statistically significant (P = 0.075) and data on overall survival have not yet been published. In a publication comprising two trials [23], the relapse rate was reduced by 65% in patients with FIGO stage Ia–b, grade 2–3 disease treated with cisplatin for six cycles compared with no further treatment, and for patients given either cisplatin or 32P a reduction in relapse rate of 61% was seen for patients given cisplatin. No difference in overall survival was seen in either trial. Patients with FIGO stage IA, grade 1 disease were not included in these studies. In this situation, it is important to identify patients with a low risk of relapse and spare these patients from the inconvenience and risks associated with adjuvant chemotherapy; and instead to treat at time of relapse the few patients who will suffer a relapse.

It seems valid to conclude that patients with grade 1–2, diploid or tetraploid tumors have a very low relapse rate, and can be spared adjuvant chemotherapy. Patients in the intermediate- and high-risk groups should be offered adjuvant treatment.

We did not evaluate response to second-line chemotherapy in this study. It is likely that response to second-line treatment was unrelated to ploidy status, as the difference between relapse-free survival and disease-related survival was the same in the diploid/tetraploid group as in the aneuploid/polyploidy group.

The patients in our study underwent thorough intraperitoneal staging, but not lymph node staging. This means that some patients may have had unrecognized metastases to retroperitoneal lymph nodes. Such metastases are most likely to occur in patients with grade 3 or clear cell tumors [27]. The lack of lymph node staging in our study does not influence our conclusions, as the completion of lymph node staging would identify and remove the very few patients with lymph node metastasis that might otherwise fall into the low-risk group.

Younger patients with unilateral tumors may wish to preserve their fertility. The decision to undertake fertility-saving surgery in patients with FIGO stage I tumors has traditionally been based on grade of differentiation [28]. The adjunct of DNA ploidy analysis by image cytometry might considerably increase the safety of such a procedure.

In conclusion, DNA ploidy analysis using image cytometry combined with histological grade and type allows for categorization of patients into risk groups. Patients with ovarian cancer FIGO stage Ia–c, grade 1–2, with non-clear cell histology and diploid or tetraploid DNA content by ploidy analysis with image cytometry have a very low risk of relapse, and can be spared adjuvant treatment.


    Footnotes
 
+ Correspondence to: Dr G. B. Kristensen, Department for Gynecologic Oncology, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway. Tel: +47-22934000; Fax: +47-22934469; E-mail: gunnar.kristensen{at}klinmed.uio.no Back


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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
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