Flow cytometric deoxyribonucleic acid index: A prognostic factor in endometrial carcinoma

Flow cytometric deoxyribonucleic acid index: A prognostic factor in endometrial carcinoma Ole E. Iversen, M.D. Bergen, Norway In a prospective study, flow cytometric deoxyribonucleic acid analyses were performed on cell samples from benign and malignant tumors of the endometrium and benign endometrium. No ploidy aberrations were found in normal endometrium. Of 52 cases with adenocarcinomas, 38 (73%) were diploid and 14 (27%) aneuploid. Both the frequency and the degree of aneuploidy were correlated to histologic tumor grade but not to other prognostic variables such as International Federation of Gynecologists and Obstetricians stage, depth of myometrial invasion, or patient age. Patients with aneuploid tumors had a higher recurrence rate and shorter disease-free intervals as compared to those with diploid tumors. Similarly, death rates were higher and the median survival was shorter in the aneuploid group. Flow cytometric ploidy determination may therefore serve as an important prognostic parameter. (AM J OBSTET GYNECOL 1986;155:770-6.)

Key words: Endometrial carcinoma, flow cytometry, deoxyribonucleic acid, prognosis An increased incidence of endometrial carcinoma has been reported over the past years. It has become the most frequent gynecologic tumor in some countries. With the prolonged lifie expectancy of women it will also be of increasing clinical importance. Although a favorable prognosis may be expected in most cases, some factors (for example, stage, tumor grade, and degree of myometrial invasion) are known to be predictive of the aggressiveness of the disease to some extent. More precise predictors of outcome would be of considerable value to individualize therapy. Chromosomal instability leading to structural or numerical aberrations are recognized as an early feature of malignant transformation. Extensive cytogenetic studies have been carried out in a variety of tumors,' but the procedure is impractical for clinical purposes. Deoxyribonucleic acid (DNA) measurements by absorption cytometry were found to be of prognostic value in endometrial carcinomas. 2 · 3 The method is, however, too time consuming for routine use. The more recently developed flow cytometric technique has the advantages of high speed and resolution with the potential of being clinically useful. Some reports on its applicability and prognostic value in general have been presented,<-6 but the information on endometrial carcinoma is scarce. 7

From the Department of Obstetrics and Gynecology and the Gade Institute, Department of Pathology, Haukeland Sykehus, University of Bergen. The study was supported by the Norwegian Cancer Society. Received for publication February 24, 1986; revised june 2, 1986; accepted june 4, 1986. Reprint requests: Ole E. Iversen, Kvinneklinikken, 5016 Haukeland Sykehus, Norway.

770

The aim of this study was first to measure the cellular DNA content in fresh specimens of endometrial carcinomas and to compare the DNA distribution patterns to known prognostic factors; the second part of the study was to analyze the clinical outcome with regard to the DNA ploidy status of the tumor. The study was coordinated with an ongoing investigation of steroid receptors in endometrial and ovarian carcinomas. Material and methods

A prospective study was started in january 1981, covering all patients with endometrial carcinoma referred to the gynecologic department of the University Clinic, which serves as the regional hospital in the county of Hordaland. Concomitantly, nonneoplastic endometrial samples were obtained from the upper 2 to 3 mm of the uterine cavity after hysterectomy for leiomyomas or bleeding disturbances. The sampling was done from macroscopically normal tissue and confirmed by microscopic examination. From 57 consecutive cases of known or suspected endometrial carcinoma, specimens were taken fresh in the operation room from a site regarded to be representative of the lesion, after removal of the uterus. In six of the patients the sample was taken from a metastasis, because it was not possible to remove the uterus. Necrotic tissue was avoided. The sample was divided into three parts: one for flow cytometry, one for steroid receptor analysis (to be reported separately), and one for histologic examination. When the control microscopy revealed that the sampling site was not adequate, the case was rejected from the study. This included five patients for whom microscopy of adjacent tissue revealed benign polyps (n = 2), normal endometrium

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80

= 1,7"/.

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% 40

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Fig. 1. DNA histograms obtained from (a) normal endometrium at the sixteenth day of the cycle, (b) a diploid endometrial carcinoma, (c) ascites from a patient with aneuploid tumor, and (d) the aneuploid endometrial carcinoma (DI = L7). The proportion of cells with S-phase DNA content was 1.7% (a), 10.4% (b), and 22.2% (d).

(2), and mainly necrotic tissue (1), leaving 52 cases of endometrial carcinoma for the study of clinical and histopathologic correlations. In two of the cases specimens were also obtained from metastatic tissue at a later relapse. A suspension of tumor cells was obtained by repeated syringing in physiologic saline solution. Thereafter the cells were spun down, fixed, and stored in 96% ethanol at - 20° C until assayed. Flow cytometric analyses. Before DNA analysis the ethanol was removed by centrifugation (2000 rpm for 5 minutes), and the resuspended cells washed once in saline solution. Cytoplasm was removed by incubation with 0.5% pepsin (Rikshospitalets Apotek, The National Hospital, Oslo) dissolved in 0.2% hydrochloric acid at 37° C for 30 minutes (for details, see Farsund 8 ). The nuclei were washed in 0.9% saline solution and 3 drops of ribonuclease (Sigma, l mg/ml in distilled water) were added to the pellet. DNA was stained specifically with ethidium bromide and mithramycin (10 mg of ethidium bromide in l L of Tris-buffer at pH 7.5 mixed with 10 mg of mithramycin in 0.4 L of Trisbuffer at pH 7.5). Measurements were performed with an ICP 22 flow cytometer (Ortho Diagnostic Instruments) attached to a multichannel analyzer (Tectronix 2102, 512 channels). The data were presented as DNA histograms (Fig. 1). From these histograms the proportions of cells in DNA synthesis and G2 -phase were calculated by use of a planimetric method. 9 When the DNA histograms contained more than one identifiable cell population, the tumors were classified as aneuploid, while the others were diploid. The degree of DNA disturbances (aneuploidy) was given by the DNA-index

0

Primery

Metesteses

Fig. 2. Percent distribution of ploidy in 46 primary and eight secondary tumors (p < 0.025).

(Dl), defined as the proportion of DNA in the aneuploid tumor G,-cells (peak channel) to the DNA content of the diploid cells in the sample. Trout red blood cells with a known relative DNA content to normal endometrial cells were added to the samples before enzymatic treatment and staining in order to serve as an internal DNA standard. The ploidy assessments were done without knowledge of the histologic report and before the clinical follow-up data were available. Clinical data. The disease stage was assessed according to the criteria of the International Federation of Gynecologists and Obstetricians ( FIGO). As this staging system does not take operative and histopathologic findings into account, a reclassification based on this information was also done using the same stage definitions. This will be referred to as the surgical stages. The microscopic examination and grading of the tumors were all done by one experienced pathologist, based on the specimens from the adjacent tissue. These data will be presented in detail elsewhere (Utaaker E, Iversen OE, Skaarland E, unpublished observations). No patient received preoperative radiation or hormonal treatment, and no patient received postoperative adjuvant hormonal or cytotoxic treatment. Cases with deep myometrial invasion had either postoperative radium applied to the vaginal vault (Mallet), or when this was not feasible, external pelvic irradiation was given. Relapses and primary disseminated disease were treated with medroxyprogesterone acetate, and tamoxifen was used as second-line endocrine therapy. Statistical analysis. The Fisher's exact test was used for analysis of the observed frequencies of ploidy status related to clinical and histologic variables, and t tests and Wilcoxon's two-sample test were used for comparison of grouped numberical data. All testings were two sided. Cox proportional hazards analysis in a computerized program (BMPD2L) was used to evaluate the relative effect of prognostic variables on survival.

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Fig. 3. A, Distribution of tumor ploidy to FIGO disease stage in 52 cases, and B, the surgical stages based on operative and microscopic findings in 49 patients of endometrial carcinoma.

Results The DNA histograms were in general of good resolution (coefficient of variation, 2.0% to 4.5%) and based on measurements of 10' -10' cells (Fig. 1). Some specimens were subjected to repeated analyses before acceptable histograms were obtained, and a few were found to have persistently wide distribution (coefficient of variation, 7% to 8%). Thirty-six of the tumors were subjected to repeated analyses (range, two to eight). Storage of cell suspensions for >3 years did not alter the histograms significantly. In all of the 17 normal endometria and in two benign endometrial polyps, a unimodal diploid DNA content was found. Of the 52 malignant tumors, 38 (73%) were diploid and 14 (27%) were aneuploid. None of the tumors were found to be hypodiploid. With the exception of a single case with two aneuploid populations (DI, 1.65 and 2.34, respectively) all the aneuploid tumors had a modal DNA content in the diploid to tetraploid range (1 < DI < 2). Five (63%) of the eight specimens from metastases were aneuploid (Fig. 2) compared to 10 (22%) of the 46 primary tumors (p < 0.025). The two tumor recurrences had ploidy identical to the cor-

responding primary tumors obtained at hysterectomy (one diploid, one aneuploid). Ascitic fluid was available from two patients with aneuploid tumors, and the DI of the tumor cells in ascites corresponded to the value in the tumor specimen (Fig. 1, c and d). In the aneuploid tumors two separate cell populations were always found. Control analysis with the internal biologic DNA standard identified the lower DNA peak in the histogram as the diploid one in every case. When trout red blood cells were used as an internal standard of DNA content, both the normal endometria and the diploid cells in the tumor samples had a relative DNA content 5% to 7% above human leukocytes (Iversen OE, Laerum OD, unpublished observations), that is, their stainability differed slightly. In consequence, only cells of the same origin as the tumors, namely, normal endometrial cells, were used for calculation of the degree of aneuploidy expressed by the DNA index. Because of overlap of populations, the fraction of cells with S-phase DNA content could not always be calculated. When calculation was possible, aneuploid tumors were found to have an average fraction of 22.7 (±7.3%) of cells with S-phase DNA content (Table I) compared to 9.7 (±3.7%) for the diploid tumors (p < 0.0001, t test). Normal endometria had lower values than the diploid tumors. Stage. The frequency of aneuploidy in FICO Stages I (31%) and II (17%) was not significantly different (Table I, Fig. 3). Only one patient had more advanced disease on the preoperative, clinical staging. In the surgical stages in 49 evaluable patients, eight of 39 (21%) Stage I tumors were aneuploid as compared to five of 10 Stage II to IV tumors. The difference was not significannt (p < 0.1). The FICO stage corresponded to the surgical stage in only 33 cases (67%). Discrepancies were mainly found in FICO Stage II, with no cervical infiltration in the hysterectomy specimens (n = 10), and in cases of FICO Stages I and II in which metastases were discovered at laparotomy (n = 6). The ploidy status was also studied in relation to the depth of myometrial invasion. No correlation was found when the groups with no infiltration, with infiltration less than half of the myometrium, and with infiltration more than half were compared. Histologic tumor grade. The frequency of aneuploidy (Fig. 4, A; Table II) was found to increase with the loss of tumor differentiation, from three of 30 (10%) in grade I to seven of nine (78%) in grade III tumors (p < 0.001). It should be observed, however, that both diploidy and aneuploidy were seen in tumors with every degree of differentiation. The degree of DNA aberrations in the aneuploid tumors also increased from a median D I of 1.14 in the aneu plaid grade I tumors to 1.47 and 1.69 in the grade II and III tumors, respectively (Fig. 4, B). The observed dif-

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1.80

30

160

25

20

Dl

140

No . of

. t s 15 pet1en 10

1.20

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II

GRADE

GRADE

Fig. 4. A, Distribution of ploidy status to tumor grade in 52 cases of endometrial carcinomas. B, The DNA index (DI) of each of the aneuploid tumors within each grade. The mean values are indicated by horizontal lines.

Table I. Distribution of ploidy according to tumor site, FIGO and surgical stage of disease, age of patient, and the fraction of cells with S-phase DNA content Stage(%) Primary tumors (n = 46)

(n l39)

22 78

62 38

69

(%)

Aneuploid Diploid p value

FICO Metastases (%) (n = 8)

<0.025

I

Surgical II-IV (n = 13)

(n l39)

15 85

21 79

31

NS

ference of median DI in grade I and III tumors was significant (p < 0.025), while the differences when comparing grade I and II and grade II and III was of borderline significance (Table II). Age ·and menopausal status. Patients with diploid tumors had a mean age ( ± SD) of 64.0 ± 11.0 years compared to 67.1 ± 10.0 for the aneuploid group. The mean age for all the 52 patients was 64.7 ± 10.9 years. Aneuploidy was found in 13 of 45 (29%) postmenopausal and one of seven (14%) of the premenopausal patients. The difference was not significant. Clinical course. Follow-up data were available for all patients for a median of 24 months (range, 2 to 46). Eleven patients died from the disease in the observation period (Table III, Fig. 5). Seven of the 14 (50%) in the aneuploid group died, and they had a median survival of 17 months. Four of the 38 patients (11 %) in the diploid group died, with a median survival of 27.5 months (p < 0.005). One patient died from unrelated disease. Among the 45 patients considered to be without residual disease after initial surgery, seven relapses

I O.o7

II-IV (n = 10)

Age of patient (yr) (mean ± SD)

S-phase DNA content

50 50

67.1 ± 10.0 64.0 ± 11.0

22.7 ± 7.3 9.7 ± 3.7 <0.0001

NS

(%)

were seen (Table III), three of nine (33%) in the aneuploid group with a median disease-free interval of 10 months and four of 36 (11 %) in the diploid group with a median disease-free interval of 24 months (NS). The Cox proportional hazards model indicated ploidy to be the best prognostic variable for survival before grade. When controlled for ploidy, grade lost prognostic significance.

Comment The present study strongly indicates that the presence of aneuploidy in endometrial carcinomas predicts a more aggressive disease. Previous flow cytometric and cytogenetic studies on these carcinomas have included only a few cases, and clinical follow-up results were not presented. However, two studies by absorption cytometry reached similar conclusions regarding survival.'· 3 The death rate among patients with aneuploid tumors reported here was significantly higher as compared to those with diploid tumors. The higher relapse rate observed in the aneuploid group may reach statistical sig-

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25

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months Fig. 5 Cumulative proportion of patients surviving with aneuploid (n = 14) and diploid (n = 38) endometrial carcinomas. Median observation, 24 months.

nificance in prolonged or enlarged studies. The shorter median disease-free interval of patients with aneuploid tumors was also notable. Similarly, those who died from aneuploid tumors had a shorter median survival (Table III, Fig. 5). Variations in treatment cannot account for the differences, since no such distinctions were made between subgroups of patients. These results must be seen in view of the relatively short observation period and the limited number of patients with recurrent disease and of patients who died. The pattern observed resembles observations on tumors in the breast," ovary, 10 uterine cervix, 11 colon, and the urinary bladder.4·5 On the other hand, in brain tumors and leukemia diploidy was associated with a worse prognosis.'· 5 The results of the Cox analysis must be interpreted with caution as it was not possible to verify the proportional hazards assumption in this limited study. The information obtained from grading of tumors seems to be included in ploidy, since grade lost prognostic value in the model when ploidy was controlled for. The observation may not be surprising in view of the strong association of ploidy to tumor grade (Table II). It should be noted, however, that aneuploidy was seen in tumors of all degrees of differentiation. Treatment decisions today are usually based on the disease stage ( FIGO) and the degree of tumor differentiation. In endometrial carcinomas, the FIGO staging assessed before operation has previously been shown to be at variance with operative findings in one third of the patients. 12 This was also observed in the present study. When possible, therefore, surgical staging should be taken into account in studies comparing different treatment strategies. Furthermore, steroid receptor measurements have recently been shown to be of prognostic value in endometrial carcinomas, 13 and this is also in accordance

with preliminary observations for our patient group. This finding was also shown to be valid for ovarian carcinoma in our hospital. 14 Hence, steroid receptor measurements and flow cytometric ploidy assessments seem to be promising new parameters for better prognostic assessment of patients. Further studies along these lines are in progress. The frequency of aneuploidy in various malignant tumors differs considerably.'·' From cytogenetic studies chromosomal abnormalities have been shown in nearly all tumors examined, 1 but often not to an extent that is detectable by cellular DNA measurements. In a recent review of chromosomal and absorption cytometric reports/ one third of endometrial carcinomas were considered aneuploid. This is in good accordance with the 27% in our study. Compared to the higher incidence of aneuploidy reported in ovarian (two thirds)'· 10 and cervical carcinomas," the lower proportion of aneuploidy in endometrial carcinomas may reflect the generally better prognosis of these tumors. The metastases had a significantly higher frequency of aneuploidy as compared to the group of primary tumors (Table 1), which suggests that aneuploid tumors are more likely to metastasize. However, the ploidy level of a given tumor seems to be stable throughout the course of the disease.' This was also the case in this study when both the primary tumor and later recurrences as well as ascites were examined. In earlier chromosome studies only a few of the examined tumors could be evaluated for technical reasons. 7 In contrast, by flow cytometry it was possible to assess the ploidy status in all the samples examined in this study. Hypodiploid stemlines were not found, in contrast to the frequent reports of such tumors by chromosome and absorption cytometric investigations. 7 The lower resolution and potential technical biases com-

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Table II. Distribution of aneuploidy and DNA index (DI) to tumor grade Aneuploid*

Grade I Grade II Grade III Total

%

Dlt:f:

10

1.14 1.47 1.65 1.50

n

n

30 13 9

4 7

31

52

14

27

3

78

*By Fischer's exact test, p values are as follows: grades II II, <0.1; grades II/III, <0.05; grades IIlli, <0.00 1. tMedian DI of the aneuploid tumors within each grade. tBy Wilcoxon two-sample test, p values are as follows: grades II II, <0.1; grades II/III, <0.1; grades IIlli, <0.025.

Table III. Ploidy status and disease course Recurrence rate n

Aneuploid (n = 14) Diploid (n = 38) p value

I

3/9

4/36

0.13

% 33 II

Deaths Disease-free interval (mo) (median)

Survival of those who died (mo) (median)

10

17 27.5 0.16*

24 0.11*

n

7

4

l

%

50

<0.005

II

*Wilcoxon two-sample test. bined with limited number of cells examined by those methods may explain some of the difference. Flow cytometric DNA measurements have earlier been evaluated as a tool for automatic cancer screening of jet-wash samples but were found unsuitable, because of a high frequency of false positive and negative cases. 15 In view of the later reported low incidence of aneuploidy in endometrial carcinomas, only one third of the malignant cases could be expected to be identified by single-parameter DNA analysis. However, these investigators worked with an unacceptably low resolution of the measurements. The fraction of cells with S-phase DNA content was found to be highest in the aneuploid tumors (Table 1), in accordance with reports on ovarian and mammary carcinomas.'· 6 Because this parameter can not always be assessed, the DNA index may become a more precise and valuable indicator in the future. Differences in stainability of the DNA in leukocytes and endometrial gland cells with known equal amounts of DNA up to 20% have been reported previously by absorption cytometry. 16· 17 A smaller (5% to 7%) but significant difference was found in this study also (unpublished observations). Therefore normal endometrial cells should be the DNA reference standard for endometrial carcinomas in order to prevent an arbitrary overestimation of aneuploidy in the near diploid region. Similar staining differences have been reported on mature sperm cells, which are significantly below the haploid DNA value unless special preparation techniques are used to ease the access of stain to the condensed DNA molecule. 1' Still, only a proportion of the DNA will be stained. Variation in size and shape may

also explain differences encountered. For a variety of organs such control studies are still incomplete, and some caution should be executed when comparisons of aneuploidy in different tumors are made. Disease stage. The distribution of ploidy by clinical stage could not be evaluated in the previous reports on flow cytometric investigations (see Iversen and Laerum 7 ), mainly because they were done on small numbers of patients. Moberger et al.,' using absorption photometry, found no association between ploidy and clinical (FIGO) stage, while in the extensive study by Atkin," the stages were not reported. The majority of patients reported here were in FIGO stages I and II, and no significant correlation of ploidy to stage was found, nor was the ploidy correlated to the depth of myometrial invasion. The reclassification into surgical stages based on operative and microscopic findings are considered to reflect better the biology and aggressiveness of the malignant tumor. The FIGO classification corresponded to operative findings in only two thirds of the patients. The association of ploidy in surgical Stage I versus Stages II through IV approached statistical significance (p < 0.1 ). These observations point to limitations in the prognostic value of the FIGO stages. The prognostic information of aneuploidy seems to be independent of and additional to that obtained by the FIGO disease staging. Tumor grade. Cytogenetic studies of endometrial carcinomas have been performed on limited numbers of tumors (for recent survey, see Iverson and Laerum 7 ) and the data were not conclusive regarding a possible association of ploidy status to tumor differentiation. Atkin, 2 using absorption photometry, reported a sig-

776 Iversen

nificantly higher proportion of high-ploidy tumors in cancers of low diifferentiation (grade III) when an arbitrary level of DNA equivalent to 63 chromosomes (DI = 1.37) was used for separation. No such relationship was found in a recent report although a high number of grade III tumors were aneuploid. 3 The two studies are not directly comparable, because the criteria of aneuploidy were different. Flow cytometric investigations have so far not included information on ploidy and tumor differentiation. The assessment of tumor grade is partly subjective with interpersonal variations, 19 while flow cytometric DNA measurements are objective and more reproducible. In the series reported here, grade III tumors were significantly more often aneuploid than both grade I and grade II tumors. In addition, the degree of aneuploidy, as expressed by the median DI of the aneuploid tumors within each grade, increased significantly with the loss of differentiation (Table II). One may speculate whether there is a continuous increase in the degree of DNA aberration with the growth of the individual tumor, or whether the DI remains unaltered from the initial malignant transformation. Aneuploidy has been reported in some premalignant lesions of the uterine cervix and some colon polyps identified to have a higher potential for malignant transformation. From the time of clinical detection, a late stage in the progression of the tumor, the amount of information is limited. Based on available data 5 in addition to our own results on endometrial and ovarian carcinomas, variation in the DI for a particular tumor and its metastases seems to be a rare incident. It is therefore reasonable to conclude that the ploidy status of a given tumor is usually an inherent property, which is not subjected to major variations with the progression of the disease. The DI may therefore be a marker for the aggressiveness of adenocarcinomas of the endometrium. The assistance of Knut Korsbrekke with the Cox analysis is acknowledged. REFERENCES l. Sandberg AA. The chromosomes in human cancer and leukemia. New York: Elsevier, 1980. 2. Atkin NB. Prognostic significance of ploidy level in human tumors. I. Carcinoma of the uterus. JNCI 1976;56: 909-10.

October 1986 Am J Obstet Gynecol

3. Moberger B, Auer G, Forsslund G, Moberger G. The prognostic significance of DNA measurements in endometrial carcinoma. Cytometry 1985 ;5 :430-6. 4. Barlogie B, Raber MN, Schumann J, eta!. Flow cytometry in clinical cancer research. Cancer Res 1984;43:3982-97. 5. Friedlander ML, Hedley DW, Taylor IW. Clinical and biological significance of aneuploidy in human tumours. J Clin Pathol 1984;37:961-74. 6. McGuire WL, Dressler LG. Emerging impact of low cytometry in predicting recurrence and survival in breast cancer patients. JNCI 1985;75:405-10. 7. Iversen OE, Laerum OD. Ploidy disturbances in endometrial and ovarian carcinomas: a review. Anal Quant Cytol Histol 1985;7:327-36. 8. Farsund T. Selective sampling of cells for morphological and quantitative cytology of bladder epithelium. J Urol 1982; 128:267-71. 9. Baisch H, G!ilhde W, Linden WA. Mathematical analysis of ICP-data to determine the fraction of cells in the various phases of cell cycle. In: Haanen CAM, Hillen HFP, Wessels JMC, eds. Pulse cytophotometry. Ghent, Belgium: European Press Medicon, 1975:68-75. 10. Friedlander ML, Taylor IW, Russell P, Musgrove EA, Hedley DW, Tattersall MHN. Ploidy as a prognostic factor in ovarian cancer, lnt J Gynecol Pathol 1983;2:55-63. 11. Jakobsen A. Prognostic impact of ploidy level in carcinoma of the cervix. Am J Clin Oncol 1984;7:475-80. 12. Onsrud M, Aalders J, Abeler V, Taylor P. Endometrial carcinoma with cervical involvement (Stage II): prognostic factors and value of combined radiological-surgical treatment. Gynecol Oncol 1982; 13:76-86. 13. Creasman WT, Soper JT, McCarty KS Jr., McCarty KS Sr., Hinshaw W, Clarke-Pearson DL. Influence of cytoplasmic steroid receptor content on prognosis of early stage endometrial carcinoma. AM J 0BSTET GvNECOL 1985; 151:922-32. 14. Iversen OE, Skaarland E, Utaaker E. Steroid receptor content in ovarian tumors: survival of patients with ovarian carcinoma related to steroid receptor content. Gynecol Oncol 1986;23:65-76. 15. Sprenger E, Hilgarth M. Vogt-Schaden M. The diagnosis of endometrial carcinoma by means of jet wash and DNA flow-through cytophotometry. Pathol Res Pract 1978; 162: 263-8. 16. Atkin NB, Richards BM, Ross AJ. The deoxyribonucleic acid content of carcinoma of the uterus: an assessment of its possible significance in relation to histopathology and clinical course, based on data from 165 cases. Br J Cancer 1959; 13:773-87. 17. Wagner D, Richart RM, Terner JY. Deoxyribonucleic acid content of presumed precursors of endometrial carcinoma. Cancer 1967;20:2067-77. 18. Zante J, Schumann J, G!ilhde W, Hacker U. DNA-fluorometry of mammalian sperm. In: Lutz D, ed. Pulsecytophotometry. vol 3. Ghent, Belgium: European Press, 1978,401-7. 19. Baak JPA, Lindeman J. Overdiep SH. Langley FA. Disagreement of histopathological diagnosis of different pathologists in ovarian tumors, with some theoretical considerations. Eur J Obstet Gynecol Reprod Bioi 1982; 13: 51-5.