Prognostic Value of p53 and Proliferating Cell Nuclear Antigen Expression in Endometrial Carcinoma

GYNECOLOGIC ONCOLOGY ARTICLE NO.

62, 192–198 (1996)

0214

Prognostic Value of p53 and Proliferating Cell Nuclear Antigen Expression in Endometrial Carcinoma NICHOLAS W. HAMEL, M.D., THOMAS J. SEBO, M.D., PH.D., TIMOTHY O. WILSON, M.D., GARY L. KEENEY, M.D., PATRICK C. ROCHE, PH.D., VERA J. SUMAN, PH.D., THERESA C. HU, M.S., AND KARL C. PODRATZ, M.D., PH.D.1 Department of Obstetrics and Gynecology, Division of Anatomic Pathology, Division of Hematopathology, and Section of Biostatistics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905 Received May 8, 1995

The degree of expression of p53 and proliferating cell nuclear antigen (PCNA) was measured in archival samples from 221 patients managed surgically for endometrial carcinoma between 1979 and 1983. With use of primary antibodies to the p53 protein (DO7) and PCNA (PC10), immunoperoxidase nuclear staining of paraffin-embedded tissue was performed. The computerized CAS200 Image Analysis System was used to determine the percentage of nuclear area stained. There was no evidence to conclude that progression-free survival differed with respect to PCNA expression. In contrast, intense p53 expression (66% or more nuclear area stained) was significantly associated with compromised progression-free survival both in the analysis of all stages (P õ 0.001) and in the subset of patients with stage I disease (P õ 0.001). Intense expression of p53 was significantly associated with other prognostic indicators, including stage, grade, depth of myometrial invasion, histologic subtype, cytologic findings, DNA ploidy, and HER-2/neu expression. Multivariate analysis identified four independent prognostic factors for progression-free survival in endometrial carcinoma: intense p53 expression, histologic subtype, DNA ploidy status, and HER-2/neu expression. When none of these four independent factors are present, the 4-year progression-free survival is 96%. In contrast, it is 63% when one or more of these factors are present (P õ 0.001) and 40% when two or more factors are present (P õ 0.001). q 1996 Academic Press, Inc.

According to 1994 estimates, 31,000 new cases of endometrial carcinoma were detected and 5900 women died of this disease [1]. Although most (90%) endometrial carcinomas are diagnosed in stages I and II, most of the deaths in absolute numbers occur in these stages [2–4]. These observations potentially reflect suboptimal therapies or the clinician’s inability to identify patients with high-risk tumor biology or a combination of both. Traditionally, prognostic facPresented at the 26th Annual Meeting of the Society of Gynecologic Oncologists, San Francisco, CA, February 19–22, 1995. 1 To whom reprint requests should be addressed at Mayo Clinic, 200 First Street SW, Rochester, MN 55905.

Gyn 4386

/

6d0f$$$581

MATERIALS AND METHODS

A retrospective cohort analysis was performed of patients receiving primary surgical management for endometrial carcinoma at the Mayo Clinic between 1979 and 1983. During this period, 388 patients received primary surgical extirpation and postoperative adjunctive therapy as indicated based on the traditional surgical–pathologic factors. Sufficient archival carcinomatous tissue measuring at least 1 cm2 was available in 221 paraffin blocks. Hematoxylin–eosin-stained sections of the tumor-containing paraffin blocks were reviewed earlier by Hetzel et al. [10] to identify the most representative block. Clinical and surgical staging, histologic assessment including grade and subtype, DNA flow cytometric analysis, and assessment of HER-2/neu staining were conducted as previously reported [7, 10, 23]. The immunohistochemical staining technique used a modification of the avidin–biotin method reported by Hsu et al. [36]. The paraffin blocks were cut in 5-mm sections and mounted on silanized glass slides. The slides were dewaxed

192

0090-8258/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

AID

tors such as stage, grade, depth of myometrial invasion, histologic subtype, presence of positive cytologic results, and nodal metastasis have been used to determine optimal therapy [2–7]. Recently, efforts have focused on attempting to correlate cytokinetic or molecular events more accurately with the malignant potential of cancers. Specifically, several laboratories have evaluated factors that regulate gene expression, such as the HER-2/neu oncogene [8–11] and the p53 tumor suppressor protein [12–20]. Expression of steroid receptors and colony-stimulating factor receptors has been examined [21, 22], and indicators of cell proliferation, such as DNA ploidy, S-phase fraction, DNA index, proliferative index, Ki-67, and proliferating cell nuclear antigen (PCNA), have also been evaluated [7, 23–35]. In this study, we explore the prognostic value of p53 and PCNA expression in endometrial carcinoma and their correlation with the traditional phenotypic prognostic factors.

07-19-96 18:31:45

goa

AP: Gyn Onc

193

p53 AND PCNA EXPRESSION IN ENDOMETRIAL CARCINOMA

in two changes of xylene and rehydrated through graded ethanol solutions. Endogenous peroxidase activity was blocked by treatment with 0.6% peroxide/methanol for 20 min at room temperature. Nonspecific binding sites were then blocked by incubation in 5% goat serum/PBS/Tween 20 for 10 min at room temperature. One set of slides was incubated in the presence of a primary antibody to PCNA (PC10 diluted 1:500 in 1% normal goat serum/PBS/Tween 20) to measure PCNA expression. Another set of slides was incubated with a monoclonal antibody (DO7) to the p53 antigen diluted 1:100 in the same solution. The slides were then rinsed and treated with biotinylated goat anti-mouse IgG diluted 1:200 in 1% normal human AB serum/PBS/Tween 20 for 30 min at room temperature. After another rinsing, the slides were treated with peroxidase-labeled streptavidin diluted 1:500 in 1% normal human AB serum/PBS/Tween 20 for 30 min at room temperature, exposed to a Tris–imidazole buffer (pH 7.4), treated with diaminobenzidine for 15 min, rinsed, counterstained in 0.2% methyl green solution for 10 min, rinsed in absolute ethanol, cleared in xylene, and sealed with a coverslip. Staining was quantitated on a CAS200 Image Analysis System with a software program provided by the manufacturer. Cell nuclei were sensed by two separate image acquisition channels of the CAS200 camera at 500 and 620 nm. At 620 nm, all of the nuclei are sensed, but only the DABpositive nuclei are sensed at 500 nm because methyl green absorbs minimal light at this wavelength. Measurements are made of the total nuclear area from the 620-nm image. The proliferating index or percentage nuclear staining (% NS) was calculated by dividing the summed total DAB-positive nuclear areas (detected at 500 nm) by the total nuclear areas (detected at 650 nm). Approximately 8 to 12 images were analyzed for each slide, focusing on the regions that demonstrated positive staining. The Kaplan–Meier product limit method was used to estimate the progression-free survival distribution. The log-rank test was used to assess which factors were univariately associated with progression-free survival [37, 38]. Multivariate modeling using Cox’s proportional hazard models was performed to obtain a subset of independent predictors of progression-free survival [39]. RESULTS

The median age at operation among the 221 patients was 64 years (range, 25 to 96 years). At last contact, 176 patients had not had recurrence and were alive without disease, 2 were alive without disease after treatment of recurrence, 2 were alive with disease, 13 were dead with no evidence of disease, and 28 were dead of disease. Of the 180 patients who were alive at last contact, the median duration of followup was 3.7 years (range, 1.1 to 6.1 years). Of the 221 pa-

AID

Gyn 4386

/

6d0f$$$581

07-19-96 18:31:45

TABLE 1 Distribution of Stage, Grade, Depth of Invasion, and Histologic Subtype in 221 Primary Endometrial Carcinomas No.

%

Stage I II IIIA IVA IVB

176 14 15 14 2

79.6 6.3 6.8 6.3 0.9

Grade 1 2 3 4

50 130 31 10

22.6 58.8 14.0 4.5

Histologic subtype Endometrioid Endometrioid with squamous differentiation Adenosquamous Serous papillary Clear cell Undifferentiated

155 38 13 8 4 3

70.1 17.2 5.9 3.6 1.8 1.4

Depth of invasion Endometrium £1/3 myometrium 1/3–2/3 myometrium ú2/3 myometrium Penetration of serosa

24 129 31 21 16

10.9 58.4 14.0 9.5 7.2

tients, 32 had a recurrence or progression. Of these 32 patients, 15 initially had a diagnosis of stage I disease. The relative distribution of each of the traditional prognostic factors within this group of patients is shown in Table 1. Each of these traditional phenotypic risk factors as well as ploidy status and HER-2/neu expression was univariately associated with progression-free survival (Table 2). There was no evidence to suggest that progression-free survival differs with respect to the level of PCNA expression. Three different levels of PCNA staining were evaluated: õ33% NS, 33 to 65% NS, and §66% NS. Figure 1 shows the progression-free survival curves for each of these subgroups (P Å 0.369). In contrast, p53 expression was associated with compromised progression-free survival. When various levels of nuclear staining reflecting p53 expression were evaluated, a significant difference between the group with §66% NS and the remaining two groups (õ33% NS and 33 to 65% NS) was observed (Fig. 2). A discriminating value of §66% NS, defining intense nuclear staining, was subsequently used to detect existing associations between p53 expression and other prognostic factors. Intensity of p53 expression was found to differ with respect to stage, histologic grade, depth of myometrial invasion, histologic subtype, ploidy, and degree of HER-2/neu staining (Table 2).

goa

AP: Gyn Onc

194

HAMEL ET AL.

TABLE 2 Correlation between p53 Expression and Clinical and Pathologic Variables with Corresponding Progression-Free Survival in 221 Patients with Endometrial Carcinoma p53 expression §66%

No. of patients

4-year PFSa

Stage I and II III and IV

190 31

Grade 1 and 2 3 and 4

P value

õ66%

No.

%

P value

88.3% 53.6%

õ0.001

179 19

11 12

5.8 38.7

õ0.001

180 41

89.4% 58.1%

õ0.001

173 25

7 16

3.9 39

õ0.001

184 37

86.3% 69%

õ0.002

169 29

15 8

8.2 21.6

0.033

Endometrioid Yes No

193 28

89.7% 41.7%

õ0.001

183 15

10 13

5.2 46.4

õ0.001

Diploid Yes No

178 43

88.8% 61.5%

õ0.001

167 31

11 12

6.2 27.9

õ0.001

HER-2/neu staining None Weak Strong

61 129 31

95% 83.8% 59.4%

õ0.001

59 115 24

2 14 7

3.3 10.9 22.6

0.017

198 (89.6%) 23 (10.4%)

88.9% 34.2%

õ0.001

Outer wall £2/3 myometrial invasion ú2/3 myometrial invasion or penetration of serosa

p53 õ66% §66% a

PFS, progression-free surival.

A series of Cox’s proportional hazard models were generated with use of a backward elimination process and a forward selection process. Intense p53 staining (§66% NS) was evaluated along with six other prognostic factors listed

in the previous paragraph. Cytology was not included because 64 of the patients did not have cytology performed. With multivariate analysis, four independent prognostic factors based on progression-free survival were identified: in-

FIG. 1. Progression-free survival according to proliferating cell nuclear antigen (PCNA) staining intensity in 221 patients with endometrial carcinoma (P Å 0.369).

FIG. 2. Progression-free survival according to p53 staining intensity in 221 patients with endometrial carcinoma.

AID

Gyn 4386

/

6d0f$$$581

07-19-96 18:31:45

goa

AP: Gyn Onc

p53 AND PCNA EXPRESSION IN ENDOMETRIAL CARCINOMA

TABLE 3 Independent Predictors of Progression-Free Survival Determined by Multivariate Analysis

1. 2. 3. 4.

Intense p53 expression Nondiploid HER-2/neu expression Endometrioid histologic subtype

DISCUSSION

Adjusted risk ratio

Risk limitsa

P value

3.45b 2.53c 2.22d 4.32e

1.5 –7.95 1.21–5.3 1.24–3.99 1.92–9.7

0.004 0.014 0.007 0.0004

a

Risk limit is the 95% confidence interval for the risk ratio. Relative to p53 expression õ66% stained. c Relative to diploid tumor. d Relative to the next higher level of staining. e Relative to nonendometrioid histologic subtype. b

tense p53 expression (yes, no), endometrioid histologic subtype (yes, no), diploid (yes, no), and HER-2/neu expression (none and weak or strong) (Table 3). Figure 3 displays the progression-free survival curves when none, or at least one of the four factors (intense p53 expression, nonendometrioid subtype, nondiploid tumor, or strong HER-2/neu expression), were present. Thirty-three of the 221 patients had two or more risk factors present and had a progression-free survival rate of 40%. The 4-year progression-free survival rate was 63% when one or more of these factors were present but 96% when none were identified (P õ 0.001). A significant association existed between the stage of disease and whether one or more of these factors was present (P õ 0.001). When none of the four factors were present, only 5 of 141 patients (4%) had advanced disease (stage III or IV). However, when one or more factors were present, 26 of 80 patients (33%) had advanced disease. Furthermore, 19 of 33 patients (58%) with two or more factors present had advanced disease. Similarly, a significant association existed between surgical stage and whether one or more of the three nonphenotypic factors (intense p53 expression, strong HER-2/neu staining, and nondiploid tumors) were present (Table 4). Because most patients present with surgical stage I disease, the value of p53 expression as a prognostic factor in this select subgroup of patients was assessed. Univariate analysis demonstrated that intense p53 expression (§66% NS) was significantly associated with a less favorable progression-free survival (P Å 0.002) (Fig. 4). Other factors significantly associated with a decreased progression-free survival included higher grade (3 or 4) (P Å 0.041), nonendometrioid subtypes (P õ 0.001), nondiploid DNA status (P õ 0.001), and strong HER-2/neu staining (P Å 0.002). Multivariate analysis was not considered because only 15 patients progressed in this subgroup.

AID

Gyn 4386

/

6d0f$$$581

195

07-19-96 18:31:45

With the continued advancements in technologies capable of assessing molecular events, the identification of specific genotypic characteristics should afford sufficient discrimination of unfavorable tumor biologic factors and thereby allow recognition of patients at risk for adverse outcomes and the institution of more appropriate primary therapy. In this series of patients managed surgically and selectively treated with adjuvant therapy according to the traditional prognostic factors, 32 patients had recurrences, including 15 initially diagnosed with surgical stage I disease. We examined the discriminating value of p53 and PCNA expression for predicting treatment failures in patients who had hysterectomy and selective postoperative adjuvant therapy for endometrial carcinoma. PCNA, or cyclin, is a 35-kDa accessory protein to DNA polymerase that is present in the cell nucleus during the Sphase [27]. Quantification of the protein with immunohistochemical techniques is thought to provide a measure of cell proliferation. Some investigators report a good correlation between PCNA expression and other measures of cell proliferation such as thymidine labeling index and the S-phase fraction determined by flow cytometry [28, 29]. A few studies have examined the role of PCNA as a prognostic factor in endometrial carcinoma and have found conflicting results [40–42]. Other studies have suggested indirectly that PCNA may play a role in predicting survival. Some investigators have found a strong correlation between PCNA and Ki-67, another marker of cell proliferation which has been found to correlate significantly with other poor prognostic factors of endometrial cancer such as grade and depth of myometrial invasion [30–32, 43]. Others have not found correlation between PCNA and Ki-67 [33, 34]. In this series of patients with endometrial carcinoma, there was no evidence to suggest that progression-free survival differs with respect to the degree of PCNA expression.

FIG. 3. Progression-free survival in 221 patients with endometrial carcinoma according to number of unfavorable independent risk factors (listed in Table 3) present (none, one, and two or more).

goa

AP: Gyn Onc

196

HAMEL ET AL.

TABLE 4 Progression-Free Survival and Distribution of Stage in Regard to Number of Unfavorable Independent Prognostic Factors Present in 221 Patients with Endometrial Carcinoma Stage I/II (n Å 190)

Factors presentb None (n Å 141) One (n Å 47) Two or more (n Å 33)

No.

% with stage I/II

No.

% with stage III/IV

Four-year PFSa

136 40 14

96 85 42

5 7 19

4 15 58

96% 79% 40%

94 75 57

9 13 9

6 25 43

95% 70% 40%

Only p53, HER-2/neu, and DNA ploidy considered 139 None (n Å 148) 39 One (n Å 52) 12 Two or more (n Å 21) a b

III/IV (n Å 31)

PFS, progression-free survival. Factors included intense p53 expression, strong HER-2/neu staining, nondiploid tumors, and nonendometrioid subtypes.

Hence, further analysis of PCNA and its association with other prognostic factors was not deemed justifiable. In contrast, intense p53 expression was significantly associated with decreased progression-free survival. Previous studies addressing the associations between p53 expression and other prognostic factors have reported conflicting results. Our analysis demonstrated a significant correlation between intense p53 expression and each of the unfavorable prognostic factors evaluated, including ploidy status, HER-2/neu expression, and the phenotypic factors of stage, grade, myometrial penetration, and histologic subtype. To date, three studies have compared p53 expression and other factors with use of multivariate analysis and have reported conflicting results regarding p53 as an independent predictor of survival. Lukes et al. [44] assessed 100 primary endometrial carcinomas and demonstrated that p53 expression and other traditional factors were individually predictive

FIG. 4. Progression-free survival in 176 patients with surgical stage I endometrial carcinoma according to p53 staining intensity.

AID

Gyn 4386

/

6d0f$$$581

07-19-96 18:31:45

of persistent or recurrent disease. However, only stage, grade, ploidy, and myometrial invasion retained significance when subjected to multivariate analysis. Analyzing a larger group of patients (221), Ito et al. [45] demonstrated that p53 overexpression was an independent predictor of survival when compared with clinical stage, tumor grade, and age of the patient. Similar observations have recently been reported by Pisani et al. [46]. Multivariate analysis in our study revealed that p53 expression, ploidy status, HER-2/neu expression, and histologic subtype were independent predictors of progressionfree survival. These observations differ from those of Lukes et al. [44], but their patient population was smaller and their method for assessing p53 expression or overexpression differed from ours. They did not use the Image Analysis System to evaluate p53 expression. The potential advantages of using an image analyzer for analysis of staining include the incorporation of objective assessments and standardization of discriminating levels. Rather than assigning a positive or negative value, the computer generates a numerical value between 1 and 100% which can then be evaluated in either a categorical or a continuous fashion. The image analyzer also offers the potential of affording a standard method of analysis which can be reproduced from one institution to another. Progression-free survival was also assessed based on the number of independent prognostic factors present. Having a larger number of risk factors is associated with an increased frequency of advanced (stages III and IV) disease. This association was also present when only the three factors of strong p53 expression, strong HER-2/neu expression, and ploidy status were considered. Because this information should be obtainable from a small tissue sample (that is, endometrial

goa

AP: Gyn Onc

p53 AND PCNA EXPRESSION IN ENDOMETRIAL CARCINOMA

biopsy), an estimate of potential stage and progression-free survival might be available to the clinician before instituting definitive therapy and would allow triaging of high-risk patients to the gynecologic oncologist. Analysis of p53 expression, HER-2/neu expression, and ploidy status is being performed on specimens obtained at dilatation and curettage at our institution to address this issue. Analysis of the subset of patients with stage I disease showed that intense p53 expression, high grade (3 or 4), nonendometrioid subtypes, nondiploid status, and strong HER-2/neu staining were univariably associated with decreased progression-free survival. Mutation of the p53 gene is the most common genetic lesion found in human cancers and is thought to play a role in cancers of the colon, breast, lung, pancreas, stomach, bladder, and ovary [15–17]. Numerous studies have suggested that mutation of the p53 gene also may have a significant role in the pathogenesis of endometrial carcinoma [47– 49]. The percentage of mutated p53 discovered in these studies has ranged from 9.5 to 32% of the cancers analyzed. Our study suggests that overexpression of p53 is one of several molecular or cytokinetic indicators of the malignant potential for endometrial carcinoma. Our data support previous findings that suggest an association between p53 gene expression and adverse outcomes. As additional genotypic information accumulates, the predictability regarding advanced disease and adverse outcome may be rendered from a pretreatment endometrial biopsy, thereby affording the patient a more optimal primary treatment plan and more appropriate triaging to the gynecologic oncologist. REFERENCES 1. Boring, C. C., Squires, T. S., Tong, T., and Montgomery, S. Cancer statistics, 1994, Ca Cancer J. Clin. 44, 7–26 (1994). 2. DiSaia, P. J., and Creasman, W. T. Management of endometrial adenocarcinoma stage I with surgical staging followed by tailored adjuvant radiation therapy, Clin. Obstet. Gynecol. 13, 751–765 (1986). 3. Malkasian, G. D., Jr., McDonald, T. W., and Pratt, J. H. Carcinoma of the endometrium: Mayo Clinic experience, Mayo Clin. Proc. 52, 175– 180 (1977). 4. Malkasian, G. D., Jr. Carcinoma of the endometrium: Effect of stage and grade on survival, Cancer 41, 996–1001 (1978). 5. Genest, P., Drouin, P, Gerig, L., Girard, A., Stewart, D., and Prefontaine, M. Prognostic factors in early carcinoma of the endometrium, Am. J. Clin. Oncol. 10, 71–77 (1987). 6. Imachi, M., Tsukamoto, N., Matsuyama, T., and Nakano, H. Peritoneal cytology in patients with endometrial carcinoma, Gynecol. Oncol. 30, 76–86 (1988). 7. Wilson, T. O., Podratz, K. C., Gaffey, T. A., Malkasian, G. D., Jr., O’Brien, P. C., and Naessens, J. M. Evaluation of unfavorable histologic subtypes in endometrial adenocarcinoma, Am. J. Obstet. Gynecol. 162, 418–423 (1990). 8. Berchuck, A., Rodriguez, G., Kinney, R. B., Soper, J. T., Dodge, R. K., Clarke-Pearson, D. L., and Bast, R. C., Jr. Overexpression of HER-2/neu in endometrial cancer is associated with advanced stage disease, Am. J. Obstet. Gynecol. 164, 15–21 (1991).

AID

Gyn 4386

/

6d0f$$$581

07-19-96 18:31:45

197

9. Bigsby, R. M., Li, A. X., Bomalaski, J., Stehman, F. B., Look, K. Y., and Sutton, G. P. Immunohistochemical study of HER-2/neu, epidermal growth factor receptor, and steroid receptor expression in normal and malignant endometrium, Obstet. Gynecol. 79, 95–100 (1992). 10. Hetzel, D. J., Wilson, T. O., Keeney, G. L., Roche, P. C., Cha, S. S., and Podratz, K. C. HER-2/neu expression: A major prognostic factor in endometrial cancer, Gynecol. Oncol. 47, 179–185 (1992). 11. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., and McGuire, W. L. Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene, Science 235, 177–182 (1987). 12. Steinmeyer, K., Maacke, H., and Deppert, W. Cell cycle control by p53 in normal (3T3) and chemically transformed (Meth A) mouse cells. I. Regulation of p53 expression, Oncogene 5, 1691–1699 (1990). 13. Kastan, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig, R. W. Participation of p53 protein in the cellular response to DNA damage, Cancer Res. 51, 6304–6311 (1991). 14. Chen, P. L., Chen, Y. M., Bookstein, R., and Lee, W. H. Genetic mechanisms of tumor suppression by the human p 53 gene, Science 250, 1576–1580 (1990). 15. Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. p53 mutations in human cancers, Science 253, 49–53 (1991). 16. Chang, K., Ding, I., Kern, F. G., and Willingham, M. C. Immunohistochemical analysis of p53 and HER-2/neu proteins in human tumors, J. Histochem. Cytochem. 39, 1281–1287 (1991). 17. Bartek, J., Bartkova, J., Vojtesek, B., Staskova, Z., Lukas, J., Rejthar, A., Kovarik, J., Midgley, C. A., Gannon, J. V., and Lane, D. P. Aberrant expression of the p53 oncoprotein is a common feature of a wide spectrum of human malignancies, Oncogene 6, 1699–1703 (1991). 18. Kraiss, S., Spiess, S., Reihsaus, E., and Montenarh, M. Correlation of metabolic stability and altered quaternary structure of oncoprotein p53 with cell transformation, Exp. Cell Res. 192, 157–164 (1991). 19. Okamoto, A., Sameshima, Y., Yamada, Y., Teshima, S., Terashima, Y., Terada, M., and Yokota, J. Allelic loss on chromosome 17p and p53 mutations in human endometrial carcinoma of the uterus, Cancer Res. 51, 5632–5635 (1991). 20. Kohler, M. F., Berchuck, A., Davidoff, A. M., Humphrey, P. A., Dodge, R. K., Iglehart, J. D., Soper, J. T., Clarke-Pearson, D. L., Bast, R. C., Jr., and Marks, J. R. Overexpression and mutation of p53 in endometrial carcinoma, Cancer Res. 52, 1622–1627 (1992). 21. Leiserowitz, G. S., Harris, S. A., Subramaniam, M., Keeney, G. L., Podratz, K. C., and Spelsberg, T. C. The proto-oncogene c-fms is overexpressed in endometrial cancer, Gynecol. Oncol. 49, 190–196 (1993). 22. Creasman, W. T., Soper, J. T., McCarty, K. S., Jr., McCarty, K. S., Sr., Hinshaw, W., and Clarke-Pearson, D. L. Influence of cytoplasmic steroid receptor content on prognosis of early stage endometrial carcinoma, Am. J. Obstet. Gynecol. 151, 922–932 (1985). 23. Britton, L. C., Wilson, T. O., Gaffey, T. A., Lieber, M. M., Wieand, H. S., and Podratz, K. C. Flow cytometric DNA analysis of stage I endometrial carcinoma, Gynecol. Oncol. 34, 317–322 (1989). 24. Stendahl, U., Strang, P., Wagenius, G., Bergstrom, R., and Tribukait, B. Prognostic significance of proliferation in endometrial adenocarcinomas: A multivariate analysis of clinical and flow cytometric variables, Int. J. Gynecol. Pathol. 10, 271–284 (1991). 25. Dyas, C. H., Simmons, T. K., Ellis, C. N., Austin, J. M., Jr., Partridge, E. E., Jr., Kilgore, L. C., Reading, C., Nelson, K., and Blakemore, W. S. Effect of deoxyribonucleic acid ploidy status on survival of patients with carcinoma of the endometrium, Surg. Gynecol. Obstet. 174, 133–136 (1992). 26. Podratz, K. C., Wilson, T. O., Gaffey, T. A., Cha, S. S., and Katzmann,

goa

AP: Gyn Onc

198

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

HAMEL ET AL.

J. A. Deoxyribonucleic acid analysis facilitates the pretreatment identification of high-risk endometrial cancer patients, Am. J. Obstet. Gynecol. 168, 1206–1213 (1993). Bravo, R., Frank, R., Blundell, P. A., and Macdonald-Bravo, H. Cyclin/ PCNA is the auxiliary protein of DNA polymerase-delta, Nature 326, 515–517 (1987). Dawson, A. E., Norton, J. A., and Weinberg, D. S. Comparative assessment of proliferation and DNA content in breast carcinoma by image analysis and flow cytometry, Am. J. Pathol. 136, 1115–1124 (1990). Battersby, S., and Anderson, T. J. Correlation of proliferative activity in breast tissue using PCNA/cyclin, Hum. Pathol. 21, 781 (1990). [Letter to Editor] Landberg, G., and Roos, G. Expression of proliferating cell nuclear antigen (PCNA) and Ki-67 antigen in human malignant hematopoietic cells, Acta Oncol. 30, 917–921 (1991). Dervan, P. A., Magee, H. M., Buckley, C., and Carney, D. N. Proliferating cell nuclear antigen counts in formalin-fixed paraffin-embedded tissue correlate with Ki-67 in fresh tissue, Am. J. Clin. Pathol. 97(Suppl. 1), S21–S28 (1992). Kamel, O. W., LeBrun, D. P., Davis, R. E., Berry, G. J., and Warnke, R. A. Growth fraction estimation of malignant lymphomas in formalinfixed paraffin-embedded tissue using anti-PCNA/cyclin 19A2. Correlation with Ki-67 labeling, Am. J. Pathol. 138, 1471–1477 (1991). Leonardi, E., Girlando, S., Serio, G., Mauri, F. A., Perrone, G., Scampini, S., Dalla Palma, P., and Barbareschi, M. PCNA and Ki67 expression in breast carcinoma: Correlations with clinical and biological variables, J. Clin. Pathol. 45, 416–419 (1992). Hall, P. A., Levison, D. A., Woods, A. L., Yu, C. C., Kellock, D. B., Watkins, J. A., Barnes, D. M., Gillett, C. E., Camplejohn, R., Dover, R., Waseem, N. H., and Lane, D. P. Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: An index of cell proliferation with evidence of deregulated expression in some neoplasms, J. Pathol. 162, 285–294 (1990). Hartman, L. C., Sebo, T. J., Kamel, N. A., Podratz, K. C., Cha, S. S., Wieand, H. S., Keeney, G. L., and Roche, P. C. Proliferating cell nuclear antigen in epithelial ovarian cancer: Relation to results at second-look laparotomy and survival, Gynecol. Oncol. 47, 191–195 (1992). Hsu, S. M., Raine, L., and Fanger, H. Use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem. 29, 577–580 (1981). Peto, R., and Peto, J. Asymptotically efficient rank invariant procedures (with discussion), J. R. Stat. Soc. A 135, 185–207 (1972).

AID

Gyn 4386

/

6d0f$$$581

07-19-96 18:31:45

38. Gehan, E. A. A generalized Wilcoxon test for comparing arbitrarily singly-censored samples, Biometrika 52, 203–223 (1965). 39. Cox, D. R. Regression models and life-tables (with discussion), J. R. Stat. Soc. B 34, 187–220 (1972). 40. Khalifa, M. A., Mannel, R. S., Haraway, S. D., Walker, J., and Min, K. W. Expression of EGFR, HER-2/neu, P53, and PCNA in endometrioid, serous papillary, and clear cell endometrial adenocarcinomas, Gynecol. Oncol. 53, 84–92 (1994). 41. Yu, C. C., Wilkinson, N., Brito, M. J., Buckley, C. H., Fox, H., and Levison, D. A. Patterns of immunohistochemical staining for proliferating cell nuclear antigen and p53 in benign and neoplastic human endometrium, Histopathology 23, 367–371 (1993). 42. Schofield, J. B., Mansi, J., Camplejohn, R. S., Lane, D. P., and Fisher, C. Proliferating cell nuclear antigen and S phase fraction in endometrial stromal sarcoma, J. Clin. Pathol. 45, 664–667 (1992). 43. Yabushita, H., Masuda, T., Sawaguchi, K., Noguchi, M., and Nakanishi, M. Growth potential of endometrial cancers assessed by a Ki-67 Ag/ DNA dual-color flow-cytometric assay, Gynecol. Oncol. 44, 263–267 (1992). 44. Lukes, A. S., Kohler, M. F., Pieper, C. F., Kerns, B. J., Bentley, R., Rodriguez, G. C., Soper, J. T., Clarke-Pearson, D. L., Bast, R. C., Jr., and Berchuck, A. Multivariable analysis of DNA ploidy, p53, and HER-2/neu as prognostic factors in endometrial cancer, Cancer 73, 2380–2385 (1994). 45. Ito, K., Watanabe, K., Nasim, S., Sasano, H., Sato, S., Yajima, A., Silverberg, S. G., and Garrett, C. T. Prognostic significance of p53 overexpression in endometrial cancer, Cancer Res. 54, 4667–4670 (1994). 46. Pisani, A. L., Barbuto, D. A., Chen, D., Ramos, L., Lagasse, L. D., and Karlan, B. Y. HER-2/neu, p53, and DNA analyses as prognosticators for survival in endometrial carcinoma. Obstet. Gynecol. 85, 729– 734 (1995). 47. Enomoto, T., Fujita, M., Inoue, M., Rice, J. M., Nakajima, R., Tanizawa, O., and Nomura, T. Alterations of the p53 tumor suppressor gene and its association with activation of the c-K-ras-2 protooncogene in premalignant and malignant lesions of the human uterine endometrium, Cancer Res. 53, 1883–1888 (1993). 48. Risinger, J. I., Dent, G. A., Ignar-Trowbridge, D., McLachlan, J. A., Tsao, M. S., Senterman, M., and Boyd, J. p53 gene mutations in human endometrial carcinoma, Mol. Carcinog. 5, 250–253 (1992). 49. Wachtel, S. S., Wachtel, G., Shulman, L. P., Phillips, O., Miller, B., and Photopulos, G. Identification of p53 mutations in endometrial adenocarcinoma by polymerase chain reaction–single-strand conformation polymorphism, J. Soc. Gynecol. Invest. 1, 234–237 (1994).

goa

AP: Gyn Onc