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Flow-cytometric analysis of deoxyribonucleic acid content in advanced ovarian carcinoma: Its importance in long-term survival
Flow-cytometric analysis of deoxyribonucleic acid content in advanced ovarian carcinoma: Its importance in long-term survival Gerardo Zanetta, MD," Gary L. Keeney, MD," Stephen S. Cha, MS," Gist H. Farr, Jr., MD, b Jerry A. Katzmann, PhD, a H. Sam Wieand, PhD,a John H. Edmonson, MD, a and Karl C. Podratz, MD, Phi)" Rochest~ Minnesota, and New Orleans, Louisiana OBJECTIVE: Our purpose was to evaluate the importance of deoxyribonucleic acid content to long-term survival from advanced epithelial ovarian carcinoma. STUDY DESIGN: Clinical and pathologic prognostic factors, including deoxyribonucleic acid content measured by means of flow cytometry, were analyzed for 282 patients. RESULTS: In 80% of the patients, the deoxyribonucleic acid patterns were nondiploid. In univariate analysis stage (p < 0.0001), residual disease (p < 0.0001), deoxyribonucleic acid index (p = 0.01), and deoxyribonucleic acid ploidy (p = 0.02) significantly predicted progression-free survival. In multivariate analysis stage (p < 0.001), residual tumor (p = 0.001), deoxyribonucleic acid ploidy (p = 0.02), and deoxyribonucleic acid index (p = 0.02) retained independent prognostic value. Residual disease and deoxyribonucleic acid content retained independent prognostic value for stage III tumors but not for stage IV tumors. CONCLUSION: Deoxyribonucleic acid analysis with flow cytometry provides prognostic information about long-term progression-free survival from advanced ovarian carcinoma and should be considered in the stratification processes of patients in future clinical trials. This prognostic information appears to be inversely related to tumor burden. (Am J Obstet Gynecol 1996;175:1217-25.)
Key words: Deoxyribonucleic acid ploidy, ovarian carcinoma, prognosis
Ovarian carcinoma is the fifth most frequent cancer among women in the United States and is the most lethal of the gynecologic malignant tumors. The associated 5-year survival rate has improved minimally in recent years despite the use of more aggressive and more sophisticated treatments. 1 Factors such as stage, histologic cell subtype and grade, residual tumor after cytoreductive operation, and age are known to be of prognostic value in this disease. 2' 3 In the past decade several studies4-16have analyzed the prognostic usefulness of deoxyribonucleic acid.(DNA) ploidy measured by means of flow cytometry in ovarian carcinoma. DNA aneuploidy has been correlated with a less favorable prognosis, although there is disagreement regarding its value as an independent progFrom the Mayo Clinic and Mayo Foundationa and the Ochsner Community Clinical Oncology Program.b This study was conducted as a collaborative trial of the North Central Cancer Treatment Group and Mayo Clinic and was supported in part by the Applicazioni Cliniche della Ricerca Oncologica project and United States"Public Health Service grant CA-25224 from the National Cancer Institute, Department of Health and Human Services. Receivedfor publication July 26, 1995; revised Apri115, 1996; accepted May 14, 1996. Reprint requests: Karl C. Podratz, MD, Mayo Clinic, 200First St. SW, Roehest~ M N 55905. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 $5.00+ 0 6/1/74927
nostic factor.13' 16Whether additional prognostic information is derived from determination of DNA index 8' 12~15, 16 and synthesis-phase fraction 6' 11. ts, 16 continues to be debated. Finally, most studies evaluated the prognostic value of DNA content on the basis of short- or mid-term survival; little is known about its possible importance to long-term survival. This study was undertaken to assess the association between DNA status in ovarian carcinoma and survival and to correlate these findings with multiple other clinical and pathologic prognostic factors in a large population. Material and methods
Archival specimens were identified and recruited from patients who had entered one of four clinical trials conducted by the Mayo Clinic or the North Central Cancer Treatment Group (NCCTG). Cases in which the patient underwent a m i n i m u m of 12 months' eligible follow-up and had a sufficient ratio of cancer-to-normal tissue in the available paraffin blocks were analyzed. The resulting study cohort included 323 patients with primary malignant tumors of the ovary; the lesions were in stages III (262 patients) and stage 1V (61 patients). During the study period, from Jan. 1, 1975, through Dec. 31, 1989, 208 patients entered treatment at the Mayo Clinic and 115 at 1217
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Table I. Correlation between ploidy pattern and other clinical or pathologic factors in ovarian carcinoma
Ploidy pattern Factor Age (y) Range Mean Median <50yr (No.) >50 yr (No.) Stage III 1V Histologic type Serous Other FIGO grade 1 and 2 3 Residual disease None Minimal Extensive Massive
Diploid (n = 56)
Nondiploid (n = 226)
Significance*
23-81 57.1 56.5 17 (30%) 39 (70%)
21-86 61.4 62 29 (12.8%) 197 (87.2%)
p= 0.02 (Wilcoxon)
p = 0.001
45 (80%) 11 (20%)
186 (82.3%) 40 (17.7%)
p= 0.74
27 (48%) 29 (52%)
153 (67.7%) 73 (32.3%)
p=0.007
14 (25%) 42 (75%)
42 (18.6%) 184 (81.4%)
p= 0.33
6 24 18 8
I
(11%) (43%) (32%) (14%)
17 90 90 29
(7.5%) (39.8%) (39.8%) (12.8%)
p=0.70
Pearson g2 statistic.
other NCCTG facilities. Most of the patients were recruited from three randomized, prospective, phase III trials assessing the efficacy of platinum-based combination chemotherapy in advanced ovarian carcinoma: (1) cyclophosphamide versus cyclophosphamide plus cisplatin, (2) hexamethylmelamine, cyclophosphamide, doxorubicin hydrochloride (Adriamycin, Adria, Columbus, Ohio), and cisplatin versus cyclophosphamide plus cisplatin, and (3) cyclophosphamide plus cisplatin versus cyclophosphamide plus carboplatin. The fourth clinical trial was designed to assess patient outcome after secondlook laparotomy. All protocols required accurate documentation of residual tumor dimensions or volume before initiation of cytotoxic chemotherapy. Platinum-containing regimens were administered to 297 patients. The other 26 patients received various regimens, the most common being whole-abdominal irradiation, which was used to treat 16 patients. Hematoxylin and eosin-stained slides of the paraffinembedded tumor blocks were retrieved and reviewed by the study pathologist (G.L,K.) to determine the correct histologic characteristics and verify that an adequate percentage (>20%) of tumor ceils was present. Carcinomas were staged according to the classification of the International Federation of Gynecology and Obstetrics (VICO). Nuclear suspensions were prepared from the paraffinembedded tissue blocks according to the technique described by Hedley et al. I7 Three sections 50 pm thick were obtained and deparaffinized with HistoClear (National Diagnostics, Somerville, N.J.), rehydrated with progressively decreasing ethanol concentrations (100%, 95%,
and 70%) for 10 minutes each, and then washed twice in distilled water. The tissue was subsequently incubated for 150 minutes at 37 ° C in 2 ml of 0.5% pepsin solution (P-7012, Sigma, St. Louis), filtered, and centrifuged at 300 revolutions/rain for 15 minutes to obtain the nuclear pellet. The isolated nuclei were sequentially treated according to the technique described by Vindelov et al. 18 with trypsin (T-92523, Sigma) and ribonuclease A (R4875, Sigma) and stained with propidium iodide (P-5264, Sigma) for a m i n i m u m of l hour. The suspension was gently but repeatedly aspirated through a 25-gauge needle before being analyzed. The nuclear content of 10,000 nuclei was measured with a FACScan (Becton Dickinson, Sunnyvale, Calif.) flow cytometer. Cell cycle evaluation of the DNA histograms was performed with a Modfit 5.2 (Verity Software, Topsham, Me.) computerized software program. To define the n u m b e r of nuclei normally associated with the 4n (G2M) fraction of normal ovarian tissue, we analyzed 20 different specimens of normal ovary (divided by age into four groups). The mean percentage of nuclei constituting the G2M fraction was 2.6, and the standard deviation in this normal population was 2.01. An upper normal limit for the 4n peak was defined as the mean + 3 SD of the nonmalignant ovarian tissue (that is, 9%). The mean synthesisphase fraction was 3.8, and the mean coefficient of variation of the gap0-gapl peak was 6.5%. Tumors with only one identifiable gapo-gapl peak were classified as diploid (2n). Tumor samples that contained a statistically significant increase in the 4n peak (>9% of nuclei) and an identifiable 8n population were categorized as tetraploid. Tumor DNA content was classified as DNA aneuploid if a
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separate, identifiable gap0-gapl peak was present and as multiploid if three or more separate gap0-gapl peaks were present. Tumor samples that did not fit into one of these categories were considered unclassifiable. The DNA index was calculated as the ratio of the mean channel n u m b e r of the aneuploid gap0-gap~ peak to the mean channel n u m b e r of the diploid 2n peak. DNA diploid tumors therefore had a DNA index of 1. In multiploid tumors the aneuploid population with the largest DNA index was used to categorize the tumors. All tissue blocks and DNA histograms were analyzed and classified without knowledge of a patient's survival or clinicopathologic features. The patients' ages ranged from 21 to 86 years (median 60 years). The median follow-up interval for the living patients was 8 years (range 1 to 17 years), and 239 deaths were recorded. All statistical analysis was conducted with SAS (Statistical Analysis System) software. 19 Survival curves were generated with the Kaplan-Meier method. 2° Univariate survival comparisons were conducted with the SURVDIFF (SAS) program, and the probability values associated with the log-rank statistics were two-sided. 2~Multivariate analyses were performed with the Cox proportional hazards modelY A backward regression was used to isolate the most important prognostic factors, and variables were eliminated on the basis of the maximal likelihood estimate statistics. This process was terminated when all
maximal likelihood estimate statistics were significant at the 0.05 level.
Results Flow-cytometric DNA content analysis was performed on 323 samples. Six samples were rejected during the histologic review because of an insufficient quantity of tumor cells (<20%), and 17 were rejected because of unclassifiable histograms. Satisfactory results were obtained in 300 cases; however, 18 tumors were confirmed to be borderline at histologic review and accordingly were excluded. Thus 282 tumors (231 stage II! and 51 stage IV) were included in the final analysis. DNA patterns were diploid in 56 (19.8%) and nondiploid in 226 (80.1%) tumors. Nondiploid samples were further subclassified as aneuploid (176 tumors), tetraploid (28 tumors), and multiploid (22 tumors). The distribution of the ploidy patterns and their correlations with other clinical and pathologic factors are summarized in Table I. No statistically significant correlation was observed between ploidy patterns and stage, residual tumor, or grade, but a significant correlation was observed between ploidy pattern and tumor histotype and age. Samples also were categorized irrespective of ploidy pattern according to the DNA index, and different limits (1.3 and 1.5) were used as discriminators between high and low DNA indexes. The correlations were similar to those that were based on ploidy pattern (data not shown). Synthesis-phase fraction
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Years from Entry Fig. 2. Disease-free survival according to DNA index. was measured for 217 tumors (77.0%). Ploidy status, DNA index, and synthesis-phase fractions were highly correlated according to frequency table analyses with ff testing (p < 0.001). For 25 patients with stage III disease, samples of omental metastasis were available for flow cytometric analysis. The DNA content, defined according to ploidy pattern and DNA index, was identical in the primary and metastatic tumors of 18 of the 25 patients (72%), and only minor changes were observed in the tumors of three additional patients (12%). However, for four patients (16%) with primary and metastatic aneuploid tumors, differences in DNA index that exceeded 0.2 were detected between the primary and metastatic sites. When ploidy pattern and DNA index were correlated with progression-free survival, they were significant predictors of clinical outcome. Patients with diploid tumors had a more favorable prognosis than those with nondiploid tumors (p = 0.02) (Fig. 1), and patients whose tumors had a DNA index of <1.3 had a significantly better prognosis than those with a DNA index >1.3 (p = 0.01) (Fig. 2). Patients with multiploid tumors had progression-free and overall survival rates comparable with those of patients with aneuploid tumors. Patients with nondiploid tumors or a DNA index >1.3 also had a poorer overall survival rate, but this difference was not statistically significant. We observed no significant difference in progressionfree and overall survival rate for patients whose tumors had a low or a high synthesis-phase fraction.
In addition to the flow-cytometric factors, stage and residual disease after the primary operation were significant predictors of survival in the univariate analysis (Table II). Separate assessment of stage III and W tumors demonstrated that DNA index (p < 0.01) and DNA ploidy (p = 0.01) were significant predictors of survival for patients with stage III disease (Table III). The observed relative death rates associated with DNA status for patients with stage IV tumors were similar to those for patients with stage III minors, although greater in magnitude. No statistically significant differences were observed in this small subset of patients with stage IV tumors (data not shown). In the multivariate model we compared the relative impact of DNA content (DNA ploidy and DNA index), stage, residual tumor after primary operation, and FIGO grade on progression-free survival. All four factors retained independent prognostic significance (Table IV). It should be noted that the high correlation between DNA index and DNA ploidy complicated this analysis; thus we did not include both variables in the same model. When DNA index and DNA ploidy were compared independently in multivariate analyses, both provided prognostic information about progression-free survival. Patients with DNA aneuploid tumors and a DNA index >1.3 also had poorer overall survival rates, but these differences were not statistically significant. Twenty-two patients had no macroscopic residual disease after the primary operation. After a median follow-up period of 8 years, 2 of the 7 patients whose tumors
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Table II. C o r r e l a t i o n with survival: U n i v a r i a t e analysis for 282 p a t i e n t s with ovarian c a r c i n o m a Factor Stage III IV Residual disease None <2 cm 2-5 cm >5 cm FIGO grade 1 and 2 3 Histologic type Serous Others
No. of patients
5 yr overall survival rate (%) *
231 51
25 10
p = 0.002
17 0
p < 0.0001
23 114 108 37
52 20 22 11
p = 0.001
52 14 9 3
p < 0.0001
56 226
32 20
p = 0.12
25 11
p = 0.08
180 102
23 21
p = 0.20
14 14
p = 0.12
46 236
30 21
p = 0.14
23 12
p = 0.39
56 226
23 22
p = 0.10
24 11
p = 0.02
85 197
23 22
p= 0.13
23 10
p = 0.01
85 132
22 22
p = 0.56
16 12
p = 0.32
Significance
5 yr progv'ession-free survival rate (%) ~
Significance
Age <50 yr >50 yr Ploidy Diploid Nondiploid DNA index <1.3 >1.3 Synthesis-phase fraction; <15% >15%
*Rate for entire group was 22% (median 24 months). J-Rate for entire group was 14% (median 16 months). ~Synthesis-phase fraction was measurable in 217 samples.
h a d a low D N A i n d e x (<1.3) h a d a r e c u r r e n c e , w h e r e a s 11 o f the 15 p a t i e n t s w h o s e t u m o r s h a d a h i g h DNA i n d e x were d e a d o f disease. At s e c o n d - l o o k l a p a r o t o m y 50 p a t i e n t s h a d a pathologically d o c u m e n t e d c o m p l e t e response. T h e Kaplan-Meier 8-year survival estimates after a s e c o n d - l o o k o p e r a t i o n were 62% for t u m o r s with a D N A i n d e x <1.3 a n d 35% for t u m o r s with a D N A i n d e x >1.3 (p = 0.1). W h e n p a t i e n t s with n o e v i d e n c e of residual disease after e i t h e r first or s e c o n d o p e r a t i o n were c o n s i d e r e d together, 65% (15 o f 23) o f t h e p a t i e n t s w h o s e t u m o r s h a d a low D N A i n d e x were alive, w h e r e a s only 33% ( 1 6 / 4 9 ) of t h o s e w h o s e t u m o r s h a d a h i g h D N A i n d e x were alive ( p = 0.03). In this s u b g r o u p of p a t i e n t s t h e d i f f e r e n c e in survival a p p e a r e d to b e c o m e m o r e relev a n t with i n c r e a s i n g l e n g t h o f the follow-up period. Most t u m o r s with a D N A i n d e x <1.3 r e c u r r e d within 3 years, w h e r e a s t u m o r s with a h i g h D N A i n d e x continu e d to r e c u r t h r o u g h o u t t h e follow-up p e r i o d (Figs. 3 a n d 4).
Comment Flow-cytometric analysis o f t h e DNA c o n t e n t in ovarian c a r c i n o m a yields useful i n f o r m a t i o n a b o u t t h e clinical o u t c o m e for patients. All p u b l i s h e d studies r e p o r t a less favorable p r o g n o s i s for D N A a n e n p l o i d t u m o r s t h a n for
DNA diploid tumors. T h e earliest studies r e p o r t e d ploidy status as a useful factor in prognosis, 4"6' 9 w h e r e a s m o r e r e c e n t surveys r e p o r t e d D N A i n d e x as a useful o r possibly s u p e r i o r factor. 8' 12, a~, ~6 O t h e r studies suggested t h a t proliferative activity ( m e a s u r e d as synthesis-phase fraction) has p r o g n o s t i c significance. 8' 12 A l t h o u g h several a u t h o r s have assigned i n d e p e n d e n t p r o g n o s t i c significance to D N A ploidy 4-6' 8. 9 or D N A index, ~' i~, 16 diverging o p i n i o n s o n t h e i n d e p e n d e n t p r o g n o s t i c i m p a c t of these indicators 13 may b e e x p l a i n e d by t h e i n c l u s i o n o f early a n d a d v a n c e d stages in some analyses a n d o f b o r d e r l i n e tum o r s in o t h e r s . We f o u n d t h a t n o n d i p l o i d D N A c o n t e n t a n d h i g h D N A indices were n o t significantly r e l a t e d to u n f a v o r a b l e clinical p r o g n o s t i c factors such as stage IV disease at p r e s e n t a t i o n a n d t h e v o l u m e o f residual t u m o r after p r i m a r y o p e r a t i o n . This o b s e r v a t i o n is i n a g r e e m e n t with m o s t previous r e p o r t s ? ' ~' 9, 13 O n multivariate analysis, D N A i n d e x a n d DNA ploidy r e t a i n e d p r o g n o s t i c significance, as d i d stage a n d residual disease, b u t o t h e r p a t h o l o g i c factors d i d not. T h e s e o b s e r v a t i o n s suggested t h a t D N A c o n t e n t is a n objective i n d i c a t o r o f t h e biologic n a t u r e o f a tumor, t h e i m p o r t a n c e o f w h i c h (in r e l a t i o n to p r o g r e s sion-free survival) is inversely r e l a t e d to t u m o r b u r d e n . In a 1993 study, D N A ploidy was a significant i n d e p e n d e n t p r o g n o s t i c factor t o g e t h e r with t u m o r g r a d e a n d substage
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T a b l e I I I . C o r r e l a t i o n w i t h s u r v i v a l i n s t a g e I I I t u m o r s : U n i v a r i a t e a n a l y s i s f o r 231 p a t i e n t s w i t h ovarian carcinoma
Factor Residual disease None <2 cm 2-5 cm >5 cm FIGO grad e 1 and 2 3 Histologic type Serous Others
No. of patients
5 yr overall survival rate (%) *
Significance
5 yr progression-free survival rate (%)
Significance
20 100 97 14
55 22 23 14
p = 0.008
55 16 10 8
p = 0.008
55 176
42 21
p = 0.04
34 14
p = 0.02
149 82
26 23
p = 0.32
16 17
p = 0.47
33 198
36 23
p = 0.11
29 14
p = 0.23
45 186
26 25
p = 0.09
29 14
p = 0.01
71 160
27 24
p = 0.13
26 13
p < 0.01
69 109
23 27
p = 0.98
20 15
p = 0.42
Age <5 0yr >50 yr Ploidy Diploid Nondiploid DNA i n d e x -<1.3 >1.3 Synthesis-phase fraction + <15% >15%
*Rate for entire g r o u p was 22% (median 24 months). t R a t e for entire g r o u p was 14% ( m e d i a n 16 months). +Synthesis-phase fraction was measurable in 178 samples.
T a b l e IV. M u l t i v a r i a t e a n a l y s i s o f s u r v i v a l i n o v a r i a n c a r c i n o m a
Progression-free
Overall
Hazard rate
95 % Confidence interval
Significance
Hazard rate
95 % Confidence interval
Significance
Nondiploid Stage IV Residual disease
1.5 2.0 2.5
1.0-2.0 1.4-2.7 1.4-4.3
p = 0.02 p = 0.0001 p = 0.001
1.3 1.6 2.3
0.9-1.8 1.1-2.2 1.3-4.1
p = 0.15 p = 0.003 p = 0.003
DNA i n d e x >1.3 Stage IV Residual disease
1.4 2.0 2.4
1.0-1.8 1.4-2.7 1.4-4.3
p = 0.02 p = 0.0001 p = 0.002
1.2 1.6 2.3
0.9-1.5 1.1-2.2 1.3-4.1
p = 0.25 p = 0.004 p = 0.003
Nondiploid Stage IV Residual >2 cm
1.5 1.9 1.3
1.0-2.1 1.4-2.6 1.0-1.7
p = 0.02 p = 0.0001 p = 0.02
1.3 1.5 1.2
0.9-1.8 1.1-2.1 1.0-1.6
p = 0.11 p = 0.008 p = 0.08
DNA i n d e x >1.3 Stage IV Residual >2 cm
1.4 1.9 1.3
1.0-1.9 1.3-2.6 1.0-1.7
p = 0.02 p = 0.0001 p = 0.02
1.2 1.5 1.2
0.9-1.6 1.1-2.1 1.0-1.6
p = 0.18 p = 0.01 p = 0.08
Stage III Nondiploid Residual disease FIGO g r a d i n g
1.6 2.4 1.6
1.1-2.4 1.3-4.5 1.1-2.4
p = 0.017 p = 0.005 p = 0.04
1.3 2.3 1.5
0.9-2.0 1.3-6.3 1.0-2.2
p = 0.12 p = 0.006 p = 0.06
DNA i n d e x >1.3 Residual disease FIGO g r a d i n g
1.4 2.4 1.5
1.0-2.0 1.3-4.4 1.0-2.3
p = 0.03 p = 0.005 p = 0.07
1.2 2.3 1.4
0.9-1.6 1.3-4.3 1.0-2.2
p = 0.26 p = 0.007 p = 0.08
Nondiploid Residual >2 cm FIGO g r a d i n g
' 1.6 1.4 1.6
1.1-2.3 1.0-1.8 1.1-2.4
p = 0.02 p = 0.03 p = 0.03
1.4 1.3 1.5
0.9-2.0 1.0-1.8 1.0-2.2
p = 0.11 p = 0.05 p = 0.06
DNA i n d e x >1.3 Residual >2 cm FIGO g r a d i n g
1.4 1.4 1.5
1.0-2.0 1.0-1.8 1.0-2.3
p = 0.03 p = 0.03 p = 0.05
1.2 1.3 1.4
0.9-1.7 1.0-1.8 1.0-2.2
p = 0.23 p = 0.05 p = 0.07
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Years from Entry Fig. 3. Disease-free survival according to DNA index for 22 patients with no residual disease after primary operation. in early ovarian cancer. 23I n our study, DNA content had decreasing influence on survival as extent of disease increased. Significant dependence was noted among patients who had stage III disease with no macroscopic residual disease, whereas the DNA status was not significant among patients with stage IV disease. Subclassification of patients according to DNA index has defined significantly different survival groups. 12 Two studies suggested that DNA index was superior to ploidy pattern in prediction of outcome. 8' 1~Other investigators have reported the i n d e p e n d e n t significance of this factor in multivariate analysis.16Our data partially confirm these observations. In univariate and multivariate analyses, the prognostic importance of both factors was comparable. DNA index thus is a good predictor of survival, and it is among flow-cytometric factors the least likely to be influenced by technical artifacts, such as cellular debris and doublets, and may be readily measured in fresh, fixed, and paraffin-embedded samples. Some controversy exists about the cutoff limit that yields the most meaningful results; DNA indexes of 1.25, 1.3, and 1.5 have been proposed by different authors. Our results suggest that 1.3 yields the most satisfactory level of discrimination. Determination of the percentage of cells in the synthesis-phase fraction has been designated by some authors as having prognostic value in ovarian carcinoma, 6' 12,24but its usefulness has been questioned by others, a6 In 1990,
Barnabei et al. analyzing paraffin-embedded samples, demonstrated prognostic significance of elevated synthesis-phase fractions in regression analyses that included stage, residual disease, grade, and age. 24In our survey, the synthesis-phase fraction was measurable in 244 samples. Although a less favorable survival rate was associated with tumors with a high synthesis-phase fraction, this difference was not significant. These results are in agreement with previous observations. In particular, Trop~ and Kaern 25 recently found that synthesis-phase fraction was not a reliable determinant in ovarian cancer, particularly when paraffin-embedded tissue was used. This finding may be due in part to the higher risk of heterogeneity caused by technical artifacts and to difficulties in determining the tumor synthesis-phase fraction in histograms in which two or more populations tend to overlap. In the past few years additional studies of the prognostic importance of flow-cytometric DNA analysis have been published. With some notable exceptions most of these studies were based on small populations and included early and advanced tumors with limited follow-up periods. In general, these reports suggested the utility of DNA content as a prognostic factor but were unable to assimilate its relevance into clinical practice. Solid ovarian tumors with more indolent behavior patterns such as ovarian borderline or granulosa cell rumors and endometrial cancer are frequently characterized by
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6
Years from Entry Fig. 4. Disease-free survival according to DNA index for 50 patients with pathologic complete response at second-look operation.
D N A diploid status or low DNA indexes. T h e m o r e favorable o u t c o m e a m o n g p a t i e n t s with these t u m o r s m a y b e a reflection o f c o m p l e t e surgical resectability r a t h e r t h a n t h e effectiveness o f c h e m o t h e r a p y . Data o n l o n g - t e r m survival are t h e r e f o r e o f e x t r e m e i m p o r t a n c e in t h e evalua t i o n o f the i m p o r t a n c e o f D N A c o n t e n t in a d v a n c e d tumors, w h i c h generally c a n n o t b e c o m p l e t e l y cytoreduced. A c c o r d i n g to o u r results, DNA c o n t e n t likewise is o f predictive value for these tumors, even w h e n l o n g - t e r m survival is c o n s i d e r e d . We c o n c l u d e d t h a t flow c y t o m e t r y - d e t e r m i n e d D N A ploidy a n d D N A i n d e x yield statistically significant inform a t i o n a b o u t t h e clinical o u t c o m e for p a t i e n t s with adv a n c e d c a r c i n o m a o f t h e ovary. B o t h factors may b e p r o p o s e d for clinical application. M e a s u r e m e n t s of DNA ploidy p r o v i d e t h e m o s t accurate i n f o r m a t i o n r e g a r d i n g t h e f r e q u e n c y o f a b n o r m a l D N A c o n t e n t in t h e tumors. Assessment o f D N A indexes, however, is less likely to b e i n f l u e n c e d by t e c h n i c a l artifacts a n d fixation. In multivariate analysis D N A c o n t e n t (expressed as DNA i n d e x or D N A ploidy), stage, a n d residual disease r e t a i n statistical significance. O n t h e basis o f o u r o b s e r v a t i o n s a n d t h e results o f p r e v i o u s investigations, DNA c o n t e n t s h o u l d b e c o n s i d e r e d as a factor in the design of f u t u r e trials regarding t h e t r e a t m e n t o f a d v a n c e d ovarian c a r c i n o m a a n d s h o u l d b e i n c l u d e d a m o n g the criteria for t h e stratification o f patients.
REFERENCES
1. Boring CC, Squires TS, Tong T, Montgomery S. Cancer statistics, 1994. CA CaucerJ Clin 1994;44:7-26. 2. Hoskins WJ, McGuire WP, Brady MF, Homesley HD, Creasm a n WT, Berman M, et al. The effect of diameter of largest residual disease on survival after primary cytoreductive surgery in patients with suboptimal residual epithelial ovarian carcinoma. Am J Obstet Gynecol 1994;170:974-9. 3. Swenerton KD, Hislop TG, Spinelli J, LeRiche JC, Yang N, Boyes DA. Ovarian carcinoma: a multivariate analysis of prognostic factors. Obstet Gynecol 1985;65:264-70. 4. Friedlander ML, Hedley DW, Taylor IW, Russell P, Coates AS, Tattersall MH. Influence of cellular DNA content on survival in advanced ovarian cancer. Cancer Res 1984;44:397-400. 5. Rodenburg CJ, Cornelisse cJ, Heintz PA, Hermans J, Fleuren GJ. Tumor ploidy as a major prognostic factor in advanced ovarian cancer. Cancer 1987;59:317-23. 6. Kallioniemi OP, Punnonen R, MattilaJ, Lehtinen M, Koivula T. Prognostic significance of DNA index, multiploidy, and S-phase fraction in ovarian cancer. Cancer 1988;61:334-9. 7: Iversen OE. Prognostic value of the flow cytometric DNA index in human ovarian carcinoma. Cancer 1988;61:971-5. 8. Klemi PJ, Joensuu H, Kiilholma P, Maenpaa J. Clinical significance of abnormal nuclear DNA content in serous ovarian tumors. Cancer 1988;62:2005-10. 9. Friedlander ML, Hedley DW, Swanson C, Russell E Prediction of long-term survival by flow cytometric analysis of cellular DNA content in patients with advanced ovarian cancer. J Clin Oncol 1988;6:282-90. 10. Wils J, van Geuns H, Baak J. Proposal for therapeutic approach based on prognostic factors including morphomettic and flow-cytometric features in stage III-IV ovarian cancer. Cancer 1988;61:1920-5. 11. Kuhn W, Kaufmann M, Feichter GE, Rummel HH, Schmid H, Heberling D. DNA flow cytometry, clinical and morpho-
Volume 175, Number 5 AmJ Obstet Gynecol
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logical parameters as prognostic factors for advanced malignant and borderline ovarian tumors. Gynecol Oncol 1989; 33:360-7. Volm M, Kleine W, Pfleiderer A. Flow-cytometric prognostic factors for the survival of patients with ovarian carcinoma: a 5-year follow-up study. Gynecol Oncol 1989;35:84-9. Erba E, Ubezio P, Pepe S, Vaghi M, Marsoni S, Torri W, et al. Flow cytometric analysis of DNA content in human ovarian cancers. BrJ Cancer 1989;60:45-50. Murray K, Hopwood L, Volk D, WilsonJE Cytofluorometric analysis of the DNA content in ovarian carcinoma and its relationship to patient survival. Cancer 1989;63:2456-60. Klemi PJ, Joensuu H, MaenpaaJ, Kiilholma E Influence of cellular DNA content on survival in ovarian carcinoma. Obstet Gynecol 1989;74:200-4. Brescia RJ, Barakat RA, Beller U, Frederickson G, Suhrland MJ, Dubin N, et al. The prognostic significance of nuclear DNA content in malignant epithelial tumors of the ovary. Cancer 1990;65:141-7. Hedley DW, Friedlander ML, Taylor IW, Rugg CA, Musgrove EA. Method for analysis of cellular DNA content of paraffinembedded pathological material using flow cytometry.J Histochem Cytochem 1983;31:1333-5.
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18. Vindelov LL, Christensen IJ, Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1983;3:323-7. 19. SUGI supplemental library user's guide. 5th ed. Ca W (NC): SAS Institute, 1986:662. 20. Kaplan EL, Meier R Nonparametric estimation from incomplete observations. J Am Star Assoc 1958;58:457-81. 21. Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Nail Cancer Inst 1959;22:719-48. 22. Cox DR. Regression models and life-tables. J R Stat Soc [B] 1972;34:187-220. 23. Vergote IB, Kaern J, Abeler VM, Petterson EO, De Vos LN, Trop4 CG. Analysis of prognostic factors in stage I epithelial ovarian carcinoma: importance of degree of differentiation and deoxyribobnucleic acid ploidy in predicting relapse. Am J Obstet Gynecol 1993;169:40-52. 24. Barnabei VM, Miller DS, Bauer KD, Murad TM, Rademaker AW, LurainJR. Flow cytometric evaluation of epithelial ovarian cancer. Am J Obstet Gynecol 1990;162:1584-90. 25. Trop4 C, KaernJ. DNA ploidy in epithelial ovarian cancer: a new independent prognostic factor? [editorial]. Gynecol Oncol 1994;53:l-4.
I TH £ MOVE?
~ennd u s s ~ ~ a d d r e s s
a t least s i x w e e k s s aheac
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Key words: Deoxyribonucleic acid ploidy, ovarian carcinoma, prognosis
Ovarian carcinoma is the fifth most frequent cancer among women in the United States and is the most lethal of the gynecologic malignant tumors. The associated 5-year survival rate has improved minimally in recent years despite the use of more aggressive and more sophisticated treatments. 1 Factors such as stage, histologic cell subtype and grade, residual tumor after cytoreductive operation, and age are known to be of prognostic value in this disease. 2' 3 In the past decade several studies4-16have analyzed the prognostic usefulness of deoxyribonucleic acid.(DNA) ploidy measured by means of flow cytometry in ovarian carcinoma. DNA aneuploidy has been correlated with a less favorable prognosis, although there is disagreement regarding its value as an independent progFrom the Mayo Clinic and Mayo Foundationa and the Ochsner Community Clinical Oncology Program.b This study was conducted as a collaborative trial of the North Central Cancer Treatment Group and Mayo Clinic and was supported in part by the Applicazioni Cliniche della Ricerca Oncologica project and United States"Public Health Service grant CA-25224 from the National Cancer Institute, Department of Health and Human Services. Receivedfor publication July 26, 1995; revised Apri115, 1996; accepted May 14, 1996. Reprint requests: Karl C. Podratz, MD, Mayo Clinic, 200First St. SW, Roehest~ M N 55905. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 $5.00+ 0 6/1/74927
nostic factor.13' 16Whether additional prognostic information is derived from determination of DNA index 8' 12~15, 16 and synthesis-phase fraction 6' 11. ts, 16 continues to be debated. Finally, most studies evaluated the prognostic value of DNA content on the basis of short- or mid-term survival; little is known about its possible importance to long-term survival. This study was undertaken to assess the association between DNA status in ovarian carcinoma and survival and to correlate these findings with multiple other clinical and pathologic prognostic factors in a large population. Material and methods
Archival specimens were identified and recruited from patients who had entered one of four clinical trials conducted by the Mayo Clinic or the North Central Cancer Treatment Group (NCCTG). Cases in which the patient underwent a m i n i m u m of 12 months' eligible follow-up and had a sufficient ratio of cancer-to-normal tissue in the available paraffin blocks were analyzed. The resulting study cohort included 323 patients with primary malignant tumors of the ovary; the lesions were in stages III (262 patients) and stage 1V (61 patients). During the study period, from Jan. 1, 1975, through Dec. 31, 1989, 208 patients entered treatment at the Mayo Clinic and 115 at 1217
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November 1996 AmJ ObstetGynecol
Zanetta et al.
Table I. Correlation between ploidy pattern and other clinical or pathologic factors in ovarian carcinoma
Ploidy pattern Factor Age (y) Range Mean Median <50yr (No.) >50 yr (No.) Stage III 1V Histologic type Serous Other FIGO grade 1 and 2 3 Residual disease None Minimal Extensive Massive
Diploid (n = 56)
Nondiploid (n = 226)
Significance*
23-81 57.1 56.5 17 (30%) 39 (70%)
21-86 61.4 62 29 (12.8%) 197 (87.2%)
p= 0.02 (Wilcoxon)
p = 0.001
45 (80%) 11 (20%)
186 (82.3%) 40 (17.7%)
p= 0.74
27 (48%) 29 (52%)
153 (67.7%) 73 (32.3%)
p=0.007
14 (25%) 42 (75%)
42 (18.6%) 184 (81.4%)
p= 0.33
6 24 18 8
I
(11%) (43%) (32%) (14%)
17 90 90 29
(7.5%) (39.8%) (39.8%) (12.8%)
p=0.70
Pearson g2 statistic.
other NCCTG facilities. Most of the patients were recruited from three randomized, prospective, phase III trials assessing the efficacy of platinum-based combination chemotherapy in advanced ovarian carcinoma: (1) cyclophosphamide versus cyclophosphamide plus cisplatin, (2) hexamethylmelamine, cyclophosphamide, doxorubicin hydrochloride (Adriamycin, Adria, Columbus, Ohio), and cisplatin versus cyclophosphamide plus cisplatin, and (3) cyclophosphamide plus cisplatin versus cyclophosphamide plus carboplatin. The fourth clinical trial was designed to assess patient outcome after secondlook laparotomy. All protocols required accurate documentation of residual tumor dimensions or volume before initiation of cytotoxic chemotherapy. Platinum-containing regimens were administered to 297 patients. The other 26 patients received various regimens, the most common being whole-abdominal irradiation, which was used to treat 16 patients. Hematoxylin and eosin-stained slides of the paraffinembedded tumor blocks were retrieved and reviewed by the study pathologist (G.L,K.) to determine the correct histologic characteristics and verify that an adequate percentage (>20%) of tumor ceils was present. Carcinomas were staged according to the classification of the International Federation of Gynecology and Obstetrics (VICO). Nuclear suspensions were prepared from the paraffinembedded tissue blocks according to the technique described by Hedley et al. I7 Three sections 50 pm thick were obtained and deparaffinized with HistoClear (National Diagnostics, Somerville, N.J.), rehydrated with progressively decreasing ethanol concentrations (100%, 95%,
and 70%) for 10 minutes each, and then washed twice in distilled water. The tissue was subsequently incubated for 150 minutes at 37 ° C in 2 ml of 0.5% pepsin solution (P-7012, Sigma, St. Louis), filtered, and centrifuged at 300 revolutions/rain for 15 minutes to obtain the nuclear pellet. The isolated nuclei were sequentially treated according to the technique described by Vindelov et al. 18 with trypsin (T-92523, Sigma) and ribonuclease A (R4875, Sigma) and stained with propidium iodide (P-5264, Sigma) for a m i n i m u m of l hour. The suspension was gently but repeatedly aspirated through a 25-gauge needle before being analyzed. The nuclear content of 10,000 nuclei was measured with a FACScan (Becton Dickinson, Sunnyvale, Calif.) flow cytometer. Cell cycle evaluation of the DNA histograms was performed with a Modfit 5.2 (Verity Software, Topsham, Me.) computerized software program. To define the n u m b e r of nuclei normally associated with the 4n (G2M) fraction of normal ovarian tissue, we analyzed 20 different specimens of normal ovary (divided by age into four groups). The mean percentage of nuclei constituting the G2M fraction was 2.6, and the standard deviation in this normal population was 2.01. An upper normal limit for the 4n peak was defined as the mean + 3 SD of the nonmalignant ovarian tissue (that is, 9%). The mean synthesisphase fraction was 3.8, and the mean coefficient of variation of the gap0-gapl peak was 6.5%. Tumors with only one identifiable gapo-gapl peak were classified as diploid (2n). Tumor samples that contained a statistically significant increase in the 4n peak (>9% of nuclei) and an identifiable 8n population were categorized as tetraploid. Tumor DNA content was classified as DNA aneuploid if a
Z a n e t t a et al.
Volume 175, Number 5 AmJ Obstet Gynecol
1219
100 80
--
~
Diploid (N=56) Nondiploid (N=226) Logrank P-Value=0.02
. . . . . . .
u_ 60 c" L_. (O
40
20
"'t._., L''L%---t~..... TL----
1
2
3
L.......
4
5
~._t~
.................
6
.
7
Years from Entry Fig. 1. Disease-free survival according to DNA ploidy.
separate, identifiable gap0-gapl peak was present and as multiploid if three or more separate gap0-gapl peaks were present. Tumor samples that did not fit into one of these categories were considered unclassifiable. The DNA index was calculated as the ratio of the mean channel n u m b e r of the aneuploid gap0-gap~ peak to the mean channel n u m b e r of the diploid 2n peak. DNA diploid tumors therefore had a DNA index of 1. In multiploid tumors the aneuploid population with the largest DNA index was used to categorize the tumors. All tissue blocks and DNA histograms were analyzed and classified without knowledge of a patient's survival or clinicopathologic features. The patients' ages ranged from 21 to 86 years (median 60 years). The median follow-up interval for the living patients was 8 years (range 1 to 17 years), and 239 deaths were recorded. All statistical analysis was conducted with SAS (Statistical Analysis System) software. 19 Survival curves were generated with the Kaplan-Meier method. 2° Univariate survival comparisons were conducted with the SURVDIFF (SAS) program, and the probability values associated with the log-rank statistics were two-sided. 2~Multivariate analyses were performed with the Cox proportional hazards modelY A backward regression was used to isolate the most important prognostic factors, and variables were eliminated on the basis of the maximal likelihood estimate statistics. This process was terminated when all
maximal likelihood estimate statistics were significant at the 0.05 level.
Results Flow-cytometric DNA content analysis was performed on 323 samples. Six samples were rejected during the histologic review because of an insufficient quantity of tumor cells (<20%), and 17 were rejected because of unclassifiable histograms. Satisfactory results were obtained in 300 cases; however, 18 tumors were confirmed to be borderline at histologic review and accordingly were excluded. Thus 282 tumors (231 stage II! and 51 stage IV) were included in the final analysis. DNA patterns were diploid in 56 (19.8%) and nondiploid in 226 (80.1%) tumors. Nondiploid samples were further subclassified as aneuploid (176 tumors), tetraploid (28 tumors), and multiploid (22 tumors). The distribution of the ploidy patterns and their correlations with other clinical and pathologic factors are summarized in Table I. No statistically significant correlation was observed between ploidy patterns and stage, residual tumor, or grade, but a significant correlation was observed between ploidy pattern and tumor histotype and age. Samples also were categorized irrespective of ploidy pattern according to the DNA index, and different limits (1.3 and 1.5) were used as discriminators between high and low DNA indexes. The correlations were similar to those that were based on ploidy pattern (data not shown). Synthesis-phase fraction
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Zanetta et al.
November1996 AmJ ObstetGynecol
100 DNA Index<=1.3 (N=85 ....... DNA Index>l.3 (N=197 Logrank P-Value=0.01 -
80
"'t
\~, ~
-
(D
u_ 6 0 O t-
'
O
a~ 4 0
o~ ~n
20
"t'"'x'""t
. . . . . . . ~'--t. t ...................
0
1
2
3
4
5
6
7
Years from Entry Fig. 2. Disease-free survival according to DNA index. was measured for 217 tumors (77.0%). Ploidy status, DNA index, and synthesis-phase fractions were highly correlated according to frequency table analyses with ff testing (p < 0.001). For 25 patients with stage III disease, samples of omental metastasis were available for flow cytometric analysis. The DNA content, defined according to ploidy pattern and DNA index, was identical in the primary and metastatic tumors of 18 of the 25 patients (72%), and only minor changes were observed in the tumors of three additional patients (12%). However, for four patients (16%) with primary and metastatic aneuploid tumors, differences in DNA index that exceeded 0.2 were detected between the primary and metastatic sites. When ploidy pattern and DNA index were correlated with progression-free survival, they were significant predictors of clinical outcome. Patients with diploid tumors had a more favorable prognosis than those with nondiploid tumors (p = 0.02) (Fig. 1), and patients whose tumors had a DNA index of <1.3 had a significantly better prognosis than those with a DNA index >1.3 (p = 0.01) (Fig. 2). Patients with multiploid tumors had progression-free and overall survival rates comparable with those of patients with aneuploid tumors. Patients with nondiploid tumors or a DNA index >1.3 also had a poorer overall survival rate, but this difference was not statistically significant. We observed no significant difference in progressionfree and overall survival rate for patients whose tumors had a low or a high synthesis-phase fraction.
In addition to the flow-cytometric factors, stage and residual disease after the primary operation were significant predictors of survival in the univariate analysis (Table II). Separate assessment of stage III and W tumors demonstrated that DNA index (p < 0.01) and DNA ploidy (p = 0.01) were significant predictors of survival for patients with stage III disease (Table III). The observed relative death rates associated with DNA status for patients with stage IV tumors were similar to those for patients with stage III minors, although greater in magnitude. No statistically significant differences were observed in this small subset of patients with stage IV tumors (data not shown). In the multivariate model we compared the relative impact of DNA content (DNA ploidy and DNA index), stage, residual tumor after primary operation, and FIGO grade on progression-free survival. All four factors retained independent prognostic significance (Table IV). It should be noted that the high correlation between DNA index and DNA ploidy complicated this analysis; thus we did not include both variables in the same model. When DNA index and DNA ploidy were compared independently in multivariate analyses, both provided prognostic information about progression-free survival. Patients with DNA aneuploid tumors and a DNA index >1.3 also had poorer overall survival rates, but these differences were not statistically significant. Twenty-two patients had no macroscopic residual disease after the primary operation. After a median follow-up period of 8 years, 2 of the 7 patients whose tumors
Volume 175, Number 5 AmJ Obstet Gynecol
Zanetta et al.
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Table II. C o r r e l a t i o n with survival: U n i v a r i a t e analysis for 282 p a t i e n t s with ovarian c a r c i n o m a Factor Stage III IV Residual disease None <2 cm 2-5 cm >5 cm FIGO grade 1 and 2 3 Histologic type Serous Others
No. of patients
5 yr overall survival rate (%) *
231 51
25 10
p = 0.002
17 0
p < 0.0001
23 114 108 37
52 20 22 11
p = 0.001
52 14 9 3
p < 0.0001
56 226
32 20
p = 0.12
25 11
p = 0.08
180 102
23 21
p = 0.20
14 14
p = 0.12
46 236
30 21
p = 0.14
23 12
p = 0.39
56 226
23 22
p = 0.10
24 11
p = 0.02
85 197
23 22
p= 0.13
23 10
p = 0.01
85 132
22 22
p = 0.56
16 12
p = 0.32
Significance
5 yr progv'ession-free survival rate (%) ~
Significance
Age <50 yr >50 yr Ploidy Diploid Nondiploid DNA index <1.3 >1.3 Synthesis-phase fraction; <15% >15%
*Rate for entire group was 22% (median 24 months). J-Rate for entire group was 14% (median 16 months). ~Synthesis-phase fraction was measurable in 217 samples.
h a d a low D N A i n d e x (<1.3) h a d a r e c u r r e n c e , w h e r e a s 11 o f the 15 p a t i e n t s w h o s e t u m o r s h a d a h i g h DNA i n d e x were d e a d o f disease. At s e c o n d - l o o k l a p a r o t o m y 50 p a t i e n t s h a d a pathologically d o c u m e n t e d c o m p l e t e response. T h e Kaplan-Meier 8-year survival estimates after a s e c o n d - l o o k o p e r a t i o n were 62% for t u m o r s with a D N A i n d e x <1.3 a n d 35% for t u m o r s with a D N A i n d e x >1.3 (p = 0.1). W h e n p a t i e n t s with n o e v i d e n c e of residual disease after e i t h e r first or s e c o n d o p e r a t i o n were c o n s i d e r e d together, 65% (15 o f 23) o f t h e p a t i e n t s w h o s e t u m o r s h a d a low D N A i n d e x were alive, w h e r e a s only 33% ( 1 6 / 4 9 ) of t h o s e w h o s e t u m o r s h a d a h i g h D N A i n d e x were alive ( p = 0.03). In this s u b g r o u p of p a t i e n t s t h e d i f f e r e n c e in survival a p p e a r e d to b e c o m e m o r e relev a n t with i n c r e a s i n g l e n g t h o f the follow-up period. Most t u m o r s with a D N A i n d e x <1.3 r e c u r r e d within 3 years, w h e r e a s t u m o r s with a h i g h D N A i n d e x continu e d to r e c u r t h r o u g h o u t t h e follow-up p e r i o d (Figs. 3 a n d 4).
Comment Flow-cytometric analysis o f t h e DNA c o n t e n t in ovarian c a r c i n o m a yields useful i n f o r m a t i o n a b o u t t h e clinical o u t c o m e for patients. All p u b l i s h e d studies r e p o r t a less favorable p r o g n o s i s for D N A a n e n p l o i d t u m o r s t h a n for
DNA diploid tumors. T h e earliest studies r e p o r t e d ploidy status as a useful factor in prognosis, 4"6' 9 w h e r e a s m o r e r e c e n t surveys r e p o r t e d D N A i n d e x as a useful o r possibly s u p e r i o r factor. 8' 12, a~, ~6 O t h e r studies suggested t h a t proliferative activity ( m e a s u r e d as synthesis-phase fraction) has p r o g n o s t i c significance. 8' 12 A l t h o u g h several a u t h o r s have assigned i n d e p e n d e n t p r o g n o s t i c significance to D N A ploidy 4-6' 8. 9 or D N A index, ~' i~, 16 diverging o p i n i o n s o n t h e i n d e p e n d e n t p r o g n o s t i c i m p a c t of these indicators 13 may b e e x p l a i n e d by t h e i n c l u s i o n o f early a n d a d v a n c e d stages in some analyses a n d o f b o r d e r l i n e tum o r s in o t h e r s . We f o u n d t h a t n o n d i p l o i d D N A c o n t e n t a n d h i g h D N A indices were n o t significantly r e l a t e d to u n f a v o r a b l e clinical p r o g n o s t i c factors such as stage IV disease at p r e s e n t a t i o n a n d t h e v o l u m e o f residual t u m o r after p r i m a r y o p e r a t i o n . This o b s e r v a t i o n is i n a g r e e m e n t with m o s t previous r e p o r t s ? ' ~' 9, 13 O n multivariate analysis, D N A i n d e x a n d DNA ploidy r e t a i n e d p r o g n o s t i c significance, as d i d stage a n d residual disease, b u t o t h e r p a t h o l o g i c factors d i d not. T h e s e o b s e r v a t i o n s suggested t h a t D N A c o n t e n t is a n objective i n d i c a t o r o f t h e biologic n a t u r e o f a tumor, t h e i m p o r t a n c e o f w h i c h (in r e l a t i o n to p r o g r e s sion-free survival) is inversely r e l a t e d to t u m o r b u r d e n . In a 1993 study, D N A ploidy was a significant i n d e p e n d e n t p r o g n o s t i c factor t o g e t h e r with t u m o r g r a d e a n d substage
1222
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November 1996 AmJ Obstet Gynecol
T a b l e I I I . C o r r e l a t i o n w i t h s u r v i v a l i n s t a g e I I I t u m o r s : U n i v a r i a t e a n a l y s i s f o r 231 p a t i e n t s w i t h ovarian carcinoma
Factor Residual disease None <2 cm 2-5 cm >5 cm FIGO grad e 1 and 2 3 Histologic type Serous Others
No. of patients
5 yr overall survival rate (%) *
Significance
5 yr progression-free survival rate (%)
Significance
20 100 97 14
55 22 23 14
p = 0.008
55 16 10 8
p = 0.008
55 176
42 21
p = 0.04
34 14
p = 0.02
149 82
26 23
p = 0.32
16 17
p = 0.47
33 198
36 23
p = 0.11
29 14
p = 0.23
45 186
26 25
p = 0.09
29 14
p = 0.01
71 160
27 24
p = 0.13
26 13
p < 0.01
69 109
23 27
p = 0.98
20 15
p = 0.42
Age <5 0yr >50 yr Ploidy Diploid Nondiploid DNA i n d e x -<1.3 >1.3 Synthesis-phase fraction + <15% >15%
*Rate for entire g r o u p was 22% (median 24 months). t R a t e for entire g r o u p was 14% ( m e d i a n 16 months). +Synthesis-phase fraction was measurable in 178 samples.
T a b l e IV. M u l t i v a r i a t e a n a l y s i s o f s u r v i v a l i n o v a r i a n c a r c i n o m a
Progression-free
Overall
Hazard rate
95 % Confidence interval
Significance
Hazard rate
95 % Confidence interval
Significance
Nondiploid Stage IV Residual disease
1.5 2.0 2.5
1.0-2.0 1.4-2.7 1.4-4.3
p = 0.02 p = 0.0001 p = 0.001
1.3 1.6 2.3
0.9-1.8 1.1-2.2 1.3-4.1
p = 0.15 p = 0.003 p = 0.003
DNA i n d e x >1.3 Stage IV Residual disease
1.4 2.0 2.4
1.0-1.8 1.4-2.7 1.4-4.3
p = 0.02 p = 0.0001 p = 0.002
1.2 1.6 2.3
0.9-1.5 1.1-2.2 1.3-4.1
p = 0.25 p = 0.004 p = 0.003
Nondiploid Stage IV Residual >2 cm
1.5 1.9 1.3
1.0-2.1 1.4-2.6 1.0-1.7
p = 0.02 p = 0.0001 p = 0.02
1.3 1.5 1.2
0.9-1.8 1.1-2.1 1.0-1.6
p = 0.11 p = 0.008 p = 0.08
DNA i n d e x >1.3 Stage IV Residual >2 cm
1.4 1.9 1.3
1.0-1.9 1.3-2.6 1.0-1.7
p = 0.02 p = 0.0001 p = 0.02
1.2 1.5 1.2
0.9-1.6 1.1-2.1 1.0-1.6
p = 0.18 p = 0.01 p = 0.08
Stage III Nondiploid Residual disease FIGO g r a d i n g
1.6 2.4 1.6
1.1-2.4 1.3-4.5 1.1-2.4
p = 0.017 p = 0.005 p = 0.04
1.3 2.3 1.5
0.9-2.0 1.3-6.3 1.0-2.2
p = 0.12 p = 0.006 p = 0.06
DNA i n d e x >1.3 Residual disease FIGO g r a d i n g
1.4 2.4 1.5
1.0-2.0 1.3-4.4 1.0-2.3
p = 0.03 p = 0.005 p = 0.07
1.2 2.3 1.4
0.9-1.6 1.3-4.3 1.0-2.2
p = 0.26 p = 0.007 p = 0.08
Nondiploid Residual >2 cm FIGO g r a d i n g
' 1.6 1.4 1.6
1.1-2.3 1.0-1.8 1.1-2.4
p = 0.02 p = 0.03 p = 0.03
1.4 1.3 1.5
0.9-2.0 1.0-1.8 1.0-2.2
p = 0.11 p = 0.05 p = 0.06
DNA i n d e x >1.3 Residual >2 cm FIGO g r a d i n g
1.4 1.4 1.5
1.0-2.0 1.0-1.8 1.0-2.3
p = 0.03 p = 0.03 p = 0.05
1.2 1.3 1.4
0.9-1.7 1.0-1.8 1.0-2.2
p = 0.23 p = 0.05 p = 0.07
Volume 175, Number Am J Obstet Gynecol
Z a n e t t a et al.
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Years from Entry Fig. 3. Disease-free survival according to DNA index for 22 patients with no residual disease after primary operation. in early ovarian cancer. 23I n our study, DNA content had decreasing influence on survival as extent of disease increased. Significant dependence was noted among patients who had stage III disease with no macroscopic residual disease, whereas the DNA status was not significant among patients with stage IV disease. Subclassification of patients according to DNA index has defined significantly different survival groups. 12 Two studies suggested that DNA index was superior to ploidy pattern in prediction of outcome. 8' 1~Other investigators have reported the i n d e p e n d e n t significance of this factor in multivariate analysis.16Our data partially confirm these observations. In univariate and multivariate analyses, the prognostic importance of both factors was comparable. DNA index thus is a good predictor of survival, and it is among flow-cytometric factors the least likely to be influenced by technical artifacts, such as cellular debris and doublets, and may be readily measured in fresh, fixed, and paraffin-embedded samples. Some controversy exists about the cutoff limit that yields the most meaningful results; DNA indexes of 1.25, 1.3, and 1.5 have been proposed by different authors. Our results suggest that 1.3 yields the most satisfactory level of discrimination. Determination of the percentage of cells in the synthesis-phase fraction has been designated by some authors as having prognostic value in ovarian carcinoma, 6' 12,24but its usefulness has been questioned by others, a6 In 1990,
Barnabei et al. analyzing paraffin-embedded samples, demonstrated prognostic significance of elevated synthesis-phase fractions in regression analyses that included stage, residual disease, grade, and age. 24In our survey, the synthesis-phase fraction was measurable in 244 samples. Although a less favorable survival rate was associated with tumors with a high synthesis-phase fraction, this difference was not significant. These results are in agreement with previous observations. In particular, Trop~ and Kaern 25 recently found that synthesis-phase fraction was not a reliable determinant in ovarian cancer, particularly when paraffin-embedded tissue was used. This finding may be due in part to the higher risk of heterogeneity caused by technical artifacts and to difficulties in determining the tumor synthesis-phase fraction in histograms in which two or more populations tend to overlap. In the past few years additional studies of the prognostic importance of flow-cytometric DNA analysis have been published. With some notable exceptions most of these studies were based on small populations and included early and advanced tumors with limited follow-up periods. In general, these reports suggested the utility of DNA content as a prognostic factor but were unable to assimilate its relevance into clinical practice. Solid ovarian tumors with more indolent behavior patterns such as ovarian borderline or granulosa cell rumors and endometrial cancer are frequently characterized by
1224
Zanetta et al.
November 1996 Am J Obstet Gynecol
100
I ........
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Years from Entry Fig. 4. Disease-free survival according to DNA index for 50 patients with pathologic complete response at second-look operation.
D N A diploid status or low DNA indexes. T h e m o r e favorable o u t c o m e a m o n g p a t i e n t s with these t u m o r s m a y b e a reflection o f c o m p l e t e surgical resectability r a t h e r t h a n t h e effectiveness o f c h e m o t h e r a p y . Data o n l o n g - t e r m survival are t h e r e f o r e o f e x t r e m e i m p o r t a n c e in t h e evalua t i o n o f the i m p o r t a n c e o f D N A c o n t e n t in a d v a n c e d tumors, w h i c h generally c a n n o t b e c o m p l e t e l y cytoreduced. A c c o r d i n g to o u r results, DNA c o n t e n t likewise is o f predictive value for these tumors, even w h e n l o n g - t e r m survival is c o n s i d e r e d . We c o n c l u d e d t h a t flow c y t o m e t r y - d e t e r m i n e d D N A ploidy a n d D N A i n d e x yield statistically significant inform a t i o n a b o u t t h e clinical o u t c o m e for p a t i e n t s with adv a n c e d c a r c i n o m a o f t h e ovary. B o t h factors may b e p r o p o s e d for clinical application. M e a s u r e m e n t s of DNA ploidy p r o v i d e t h e m o s t accurate i n f o r m a t i o n r e g a r d i n g t h e f r e q u e n c y o f a b n o r m a l D N A c o n t e n t in t h e tumors. Assessment o f D N A indexes, however, is less likely to b e i n f l u e n c e d by t e c h n i c a l artifacts a n d fixation. In multivariate analysis D N A c o n t e n t (expressed as DNA i n d e x or D N A ploidy), stage, a n d residual disease r e t a i n statistical significance. O n t h e basis o f o u r o b s e r v a t i o n s a n d t h e results o f p r e v i o u s investigations, DNA c o n t e n t s h o u l d b e c o n s i d e r e d as a factor in the design of f u t u r e trials regarding t h e t r e a t m e n t o f a d v a n c e d ovarian c a r c i n o m a a n d s h o u l d b e i n c l u d e d a m o n g the criteria for t h e stratification o f patients.
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1. Boring CC, Squires TS, Tong T, Montgomery S. Cancer statistics, 1994. CA CaucerJ Clin 1994;44:7-26. 2. Hoskins WJ, McGuire WP, Brady MF, Homesley HD, Creasm a n WT, Berman M, et al. The effect of diameter of largest residual disease on survival after primary cytoreductive surgery in patients with suboptimal residual epithelial ovarian carcinoma. Am J Obstet Gynecol 1994;170:974-9. 3. Swenerton KD, Hislop TG, Spinelli J, LeRiche JC, Yang N, Boyes DA. Ovarian carcinoma: a multivariate analysis of prognostic factors. Obstet Gynecol 1985;65:264-70. 4. Friedlander ML, Hedley DW, Taylor IW, Russell P, Coates AS, Tattersall MH. Influence of cellular DNA content on survival in advanced ovarian cancer. Cancer Res 1984;44:397-400. 5. Rodenburg CJ, Cornelisse cJ, Heintz PA, Hermans J, Fleuren GJ. Tumor ploidy as a major prognostic factor in advanced ovarian cancer. Cancer 1987;59:317-23. 6. Kallioniemi OP, Punnonen R, MattilaJ, Lehtinen M, Koivula T. Prognostic significance of DNA index, multiploidy, and S-phase fraction in ovarian cancer. Cancer 1988;61:334-9. 7: Iversen OE. Prognostic value of the flow cytometric DNA index in human ovarian carcinoma. Cancer 1988;61:971-5. 8. Klemi PJ, Joensuu H, Kiilholma P, Maenpaa J. Clinical significance of abnormal nuclear DNA content in serous ovarian tumors. Cancer 1988;62:2005-10. 9. Friedlander ML, Hedley DW, Swanson C, Russell E Prediction of long-term survival by flow cytometric analysis of cellular DNA content in patients with advanced ovarian cancer. J Clin Oncol 1988;6:282-90. 10. Wils J, van Geuns H, Baak J. Proposal for therapeutic approach based on prognostic factors including morphomettic and flow-cytometric features in stage III-IV ovarian cancer. Cancer 1988;61:1920-5. 11. Kuhn W, Kaufmann M, Feichter GE, Rummel HH, Schmid H, Heberling D. DNA flow cytometry, clinical and morpho-
Volume 175, Number 5 AmJ Obstet Gynecol
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18. Vindelov LL, Christensen IJ, Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1983;3:323-7. 19. SUGI supplemental library user's guide. 5th ed. Ca W (NC): SAS Institute, 1986:662. 20. Kaplan EL, Meier R Nonparametric estimation from incomplete observations. J Am Star Assoc 1958;58:457-81. 21. Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Nail Cancer Inst 1959;22:719-48. 22. Cox DR. Regression models and life-tables. J R Stat Soc [B] 1972;34:187-220. 23. Vergote IB, Kaern J, Abeler VM, Petterson EO, De Vos LN, Trop4 CG. Analysis of prognostic factors in stage I epithelial ovarian carcinoma: importance of degree of differentiation and deoxyribobnucleic acid ploidy in predicting relapse. Am J Obstet Gynecol 1993;169:40-52. 24. Barnabei VM, Miller DS, Bauer KD, Murad TM, Rademaker AW, LurainJR. Flow cytometric evaluation of epithelial ovarian cancer. Am J Obstet Gynecol 1990;162:1584-90. 25. Trop4 C, KaernJ. DNA ploidy in epithelial ovarian cancer: a new independent prognostic factor? [editorial]. Gynecol Oncol 1994;53:l-4.
I TH £ MOVE?
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