- Email: [email protected]
neu overexpression
European Journal of Obstetrics & Gynecology and Reproductive Biology 98 (2001) 103±108
P53 overexpression predicts endometrial carcinoma recurrence better than HER-2/neu overexpression Pluvio J. Coronadoa,*, Jose A. Vidarta, Jose A. Lopez-asenjob, Maria Faseroa, Vicente Furio-baceteb, Javier Magrinac, Manuel Escuderoa a
Department of Obstetrics and Gynecology, Hospital ClõÂnico San Carlos, Madrid, Spain b Department of Pathology, Hospital ClõÂnico San Carlos, Madrid, Spain c Department of Gynecology Surgery, Mayo Clinic Scottsdale, Arizona, USA
Received 8 March 2000; received in revised form 24 October 2000; accepted 18 November 2000
Abstract Objective: to investigate the prognostic value of p53 and HER-2/neu overexpression in endometrial cancer. Study Design: p53 and HER2/neu immunostaining was performed in 114 paraf®n-embedded specimens of endometrial cancer diagnosed and treated between 1990 and 1997. Nuclear p53 and membrane HER-2/neu immunostaining were used. Results: p53 and HER-2/neu overexpression was observed in 17 cases (14.9%) and in 19 cases (16.7%), respectively. In univariate analysis p53 (P < 0:001) and HER-2/neu (P 0:018) overexpression had a positive correlation with a high risk of recurrence. In multivariate analysis, age (P < 0:001), FIGO stage (P < 0:001), differentiation (P 0:013), non-endometrioid subtypes (P < 0:001) and p53 overexpression (P < 0:001), but not HER-2/neu overexpression, were independent prognostic indicators of recurrence. Simultaneous p53 and HER-2/neu overexpression made worse the prognostic (P < 0:001). Conclusions: p53 overexpression was an independent predictor of recurrent disease in endometrial cancer. HER-2/neu overexpression had a more limited effect but enhance the effect of p53. # 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: p53; HER-2/neu; Endometrial cancer; Prognostic factors; Survival; Immunohistochemistry
1. Introduction Endometrial cancer is the most common neoplasm of the female genital tract [1]. Traditionally prognostic factors such as FIGO stage, tumor grade, depth of myometrial invasion, histologic subtype, positive peritoneal cytology and nodal metastases have been used to detect patients with high risk of recurrence [2±7]. Nevertheless, some patients have recurrences despite good prognostic factors. The emergence and maintenance of cancer is complex. Abnormalities involving oncogenes and tumor suppresser genes play a central role [8]. The p53 tumor suppresser gene is located on the short arm of chromosome 17 and encodes a 53 kD phosphoprotein which is considered a tumor suppresser [9]. The relation between p53 abnormalities and prognosis in endometrial carcinoma is not clear. Some studies have shown a correlation between p53 overexpression and decreased survival [10,11] but others have not [12].
* Corresponding author. Tel.: 34-91-330-3526; fax: 34-91-330-3526. E-mail address: [email protected] (P.J. Coronado).
HER-2/neu oncogene (c-erbB-2), located on chromosome 17 but on its long arm, encodes a 185 kD glycoprotein that is very similar to EGF-R [13]. Studies about HER-2/neu protein expression in endometrial carcinoma have also reached different conclusions. Hamel et al. [10] considered HER-2/neu overexpression an independent prognostic factor but another recent study reported no association [14]. The aim of this study was to evaluate the independent prognostic value of p53 and HER-2/neu overexpression in endometrial carcinoma and their correlation with traditional phenotypic prognostic factors. 2. Materials and methods One hundred and fourteen patients with primary endometrial adenocarcinoma were included in this study. All patients underwent exploratory laparotomy, total abdominal hysterectomy and selective lymphadenectomy at Hospital ClõÂnico San Carlos between 1990 and 1997. Cytological washings of the pelvis were obtained in all patients. None of them received preoperative pelvic radiation or chemotherapy.
0301-2115/01/$ ± see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 0 ) 0 0 5 6 2 - 5
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P.J. Coronado et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 98 (2001) 103±108
Postoperative radiotherapy was used according to the treating radiation oncologist based on the traditional surgicalpathologic factors. Postoperative chemotherapy was not used. 2.1. Immunohistochemical staining A single 5 mm section from paraf®n-embedded specimens was routinely deparaf®ned and incubated in methanol with 0.3% H2O2 to block endogenous peroxidase activity. The specimens were embedded in buffer-citrate 0 0 1 H and incubated in microwave to 750 W for 7 min. After cooled and washed in PBS (pH 7.6), the ®rst antibody was used. We used two antibodies: the monoclonal antibody DO7 (Novocastra Laboratory, Newcastle, UK), which recognizes an epitope in human p53, at optimal dilution in Tris±albumin (1/50); and the monoclonal antibody anti-c-erbB-2 (Medac) that recognizes the intracellular domain of p185 protein at optimal dilution in Tris±albumin (1/40). Both antibodies were incubated overnight in humidi®ed chamber at 2±88C. After treatment with biotinated anti-mouse inmunoglobulin for 30 min at room temperature, the sections were incubated with peroxidase-conjugated streptavidin for 30 min at room temperature. A ®nal wash was followed by samples developed with 3 mg of 3,30 -diaminobencidine and 15 ml of hydrogen peroxide in Tris±HCl buffered at pH 7.6. The slides were then rinsed for 10 min in tap water, then they were stained with hematoxylin and coverslips were mounted. Positive and negative control antibodies were used to ensure tissue viability and exclude the possibility of nonspeci®c staining. Two pathologists scored all slides. Staining cell numbers were scored as none, weak (, <30%), moderate (, 30±70%) and intense (, >70%). Overexpression was de®ned as intense staining. In the case of HER2/neu, only membrane immunostaining was evaluated. The w2 test or Ficher's exact test was used to evaluate the correlation between p53 and HER-2/neu overexpression with traditional prognostic factors. Kaplan±Meier's method was used to estimate the progression-free survival distribution [15]. Log rank's test was used to calculate the statistical signi®cation of the curves. Cox's method was used to assess which factors were univariately associated with recurrence. Multivariate modeling using Cox's proportional hazard models was performed to obtain a subset of independent predictors of progression-free survival [16]. 3. Results The average age of the patients was 65 years (range 37±85). At a median follow up of 69 months (range 14±107) 85 patients were alive with no disease, 14 were dead from disease, 12 dead with no evidence of disease and three dead with recurrence but not due to disease. Seventeen patients experienced recurrence: 15 in the vagina or pelvis,
Table 1 Distribution of age, stage, grade, depth of invasion and histologic subtype in 114 primary endometrial adenocarcinomas N
(%)
Age 65 years <65 years
59 55
52 48
FIGO stage I II III IV
86 6 21 1
75 5.3 18 0.9
Histologic subtype Endometrioid Clear cell Serous papillary Adenosquamous
87 16 7 4
75 14 6.1 3.5
Myometrial invasion None 50% >50%
9 56 49
7.9 49 43
Grade G1 G2 G3
35 66 13
31 58 11
one in the supraclavicular nodes and one in the liver. The mean time to recurrence was 26 months (range 6±64). The distribution of traditional prognostic factors is shown in Table 1. p53 overexpression was present in 17 of 114 cases (15%) and was observed only in tumor cells as nuclear staining. The p53 overexpression was associated with non-endometrioid histologic subtypes (P 0:026) and deep myometrial invasion (P < 0:001) (Table 2). Moreover, p53 overexpression was more frequent in FIGO stages III±IV and in poorly differentiated tumors, but without reaching statistical signi®cance (Table 2). HER-2/neu membrane overexpression was observed in 19 of 94 cases (17%). HER-2/neu overexpression was more frequent in advanced FIGO stages, in non-endometrioid subtypes, in tumors with deep myometrial invasion and in less differentiated carcinomas. No statistical signi®cation was found when correlated with these classical prognostic factors (Table 2). Nine of the 17 patients who had recurrence demonstrated p53 overexpression (53%), while it was only observed in 8 out of 97 patients with no recurrence (8.2%). No relationship was found between p53 overexpression and site of recurrence. Kaplan±Meier's curves showed a signi®cant decrease of progression-free survival in patients with p53 overexpression (Fig. 1). That signi®cant decrease of survival was observed both in early stages (P < 0:001) and advanced stages (P < 0:001). HER-2/neu overexpression was observed in 6 of 17 patients (35%) with recurrence and in 13 of 97 (13%) patients with no evidence of recurrence. No relationship
P.J. Coronado et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 98 (2001) 103±108
105
Table 2 Relationship between p53 and HER-2/neu and traditional prognostic factors in endometrial carcinoma Overexpression p53 (%) Overexpression FIGO stage I and II III and IV
P value
17/114 (18)
Overexpression HER-2/neu (%)
P value
19/114 (17)
12/92 (14) 5/22 (22)
0.315
14/92 (15) 5/22 (23)
0.523
Histologic subtype Endometrioid No endometrioid
9/87 (10) 8/27 (30)
0.026
13/87 (15) 6/27 (22)
0.384
Myometrial invasion 50% >50%
3/65 (4.6) 14/49 (29)
<0.001
9/65 (14) 10/49 (20)
0.447
14/101 (14) 3/13 (23)
0.408
16/101 (16) 3/13 (23)
0.452
Grade G1±G2 G3
was found between HER-2/neu overexpression and site of recurrence. Progression-free survival curves re¯ected a signi®cant decrease of survival for patients with HER-2/neu overexpression (Fig. 2). That signi®cant decrease of survival
was observed in early stages (P 0:008), but not in advanced stages (P 0:308). When a combined overexpression of both genes was considered, an important decrease of survival was observed.
Fig. 1. Relationship between p53 overexpression and progression-free survival.
Fig. 2. Relationship between HER-2/neu overexpression and progression-free survival.
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P.J. Coronado et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 98 (2001) 103±108
Fig. 3. Combined p53 and HER-2/neu overexpression.
The four patients with simultaneous p53 and HER-2/ neu overexpression had a dramatically lower survival rate (Fig. 3). Each of the traditional phenotypic risk factors as well as p53 and HER-2/neu overexpression was univariately associated with progression-free survival. As expected, classical indicators of prognosis including FIGO stages III±IV, grade 3, deep myometrial invasion, non-endometrioid histologic subtypes and older age were signi®cantly associated with poor outcome (Table 3). In order to determine whether p53 and HER-2/neu overexpression had validity as independent prognostic factors, a multivariate analysis according to Cox's proportional hazard model, including the prognostic factors listed previously was carried out. FIGO stage III±IV, grade 3, nonendometrioid subtypes, age of patient, and p53 overexpression were considered independent prognostic factors for
progression-free survival. HER-2/neu overexpression was not an independent prognostic factor (Table 4). 4. Comment One of the objectives of molecular tumor investigation is to identify biologic prognostic indicators to be used in addition to the traditional morphologic factors to further delineate the outcome of endometrial cancer patients. This study evaluated the prognostic signi®cance of p53 and HER2/neu overexpression in relation to disease recurrence. Previous studies have reported a statistically signi®cant association between p53 overexpression and some of the traditional prognostic factors for endometrial carcinomas such as advanced stage [10,17±19], poorly differentiated tumors [10,11,17,19], deep myometrial invasion [10,20] and
Table 3 Correlation of p53 overexpression and clinical pathologic variables with corresponding progression-free survival in 114 patients with endometrial carcinoma
Age p53 HER-2/neu FIGO stage Histologic subtype endometrioid Grade Myometrial invasion
Linear increment of 1 year Overexpression No overexpression Overexpression No overexpression III and VII I and II Yes No G3 G1±G2 >50% 50%
RR
CI 95%
P value
1.09 8.7 1 3.7 1 9.5 1 0.12 1 4.4 1 6.9 1
1.03±1.2 3.4±22.8
<0.001 <0.001
1.4±10.2
0.018
3.5±25.8
<0.001
0.04±0.3
<0.001
1.6±12.6
0.013
2±24
<0.001
P.J. Coronado et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 98 (2001) 103±108
107
Table 4 Independent predictors of progression-free survival determined by multivariate analysis
p53 HER-2/neu FIGO stage Grade Histologic subtype Age
Overexpression No overexpression Overexpression No overexpression III and IV I and II G3 G1±G2 Endometrioid No-endometrioid Linear increment of 1 year
non-endometrioid histological types [10,17,21±24]. We found a statistical association between p53 overexpression and non-endometrioid histologic subtypes and deep myometrial invasion. We observed a higher p53 overexpression in patients with advanced FIGO stage and high grade (G3) tumors, although those differences were not signi®cant. We found, like others [12,17,19,25±27], an incomplete association with the surgical-pathological prognostic factors. Those ®ndings may re¯ect either a wide variety of p53 altered forms in endometrial cancers or different results due to the type of antibody used and the observers subjectivity. Nevertheless, p53 overexpression appears to be a feature of tumor aggressiveness. Several studies [10,11,29±30] have identi®ed p53 nuclear overexpression as an independent prognostic factor of recurrence in endometrial cancer. However, some authors found no correlation between p53 overexpression and poor prognosis either in univariate [12] or multivariate [14,20,31,32] analysis, and even Soong et al. [28] observed a better prognosis when a cytoplasmatic immunoreactivity appeared. In our study, p53 overexpression was an independent factor of poor prognosis. Those ®ndings suggest that determination of p53 in endometrial cancer could identify high-risk patients of recurrence and moreover, it con®rms the predictive value of p53 overexpression in endometrial adenocarcinomas. Previous studies have shown an association of HER-2/ neu overexpression with advanced stage, non-endometrioid histology [33] and depth of myometrial invasion [34], while other have not [36,37]. In this study, HER-2/neu overexpression was noted more frequently in association with advanced stage, non-endometrioid subtypes, high grade and deep myometrial invasion, but the differences were not statistically signi®cant. This is in agreement with Gassel et al. [14] who found no statistical association between HER-2/neu overexpression and classical prognostic factors as in 222 patients with endometrial carcinoma. In the present study, HER-2/neu overexpression was predictive of recurrent disease in univariate analysis, but not in the multivariate analysis. Others [14,35] have reached similar conclusions; however, some studies have established HER2/neu overexpression as an independent prognostic factor
Adjusted RR
CI 95%
P value
9.72 1 1.49 1 11.09 1 2.98 1 0.27 1 1.08
2.9±32.9
<0.001
0.3±6.5
0.595
3.6±34.5
<0.001
0.9±9.3
0.076
0.1±0.8
0.017
1.01±1.2
0.024
[10,32,33,37]. In our series, the prognostic value of HER-2/ neu overexpression was higher in early stages than in advanced stages. It suggests that HER-2/neu overexpression could select high-risk patients of recurrence in initial stages of the disease and loss power of prediction in advanced stages. We observed a higher trend to recurrent disease in patients with simultaneous overexpression of both genes. Others have found similar result [10]. Studies of breast cancer are also consistent with these observations [38]. Those ®ndings would indicate that multiple simultaneous gene alterations highly worsen the prognosis of the disease, although the presence of p53 overexpression seems to be a more powerful indicator of recurrence than HER-2/neu. More studies are necessary to determinate the positive interaction of both genes in the prognostic of the endometrial cancer. In conclusion, p53 overexpression is an important prognostic factor in endometrial carcinoma while HER-2/neu overexpression has a more limited role in the recurrence of these cancers. Simultaneous p53 and HER-2/neu overexpression results in a higher risk of recurrence than overexpression of each gene independently. Acknowledgements This study was ®nanced through grants from F.I.S 95/ 0415 and B.A.E. 97/5415, Ministerio de Sanidad y Consumo, Madrid, Spain. References [1] Bruke TW, Tortolero-Luna G, Malpica A, et al. Endometrial hyperplasia and endometrial cancer. Obstet Gynecol Clin North Am 1996;23:411±56. [2] Wolfson AH, Sightler SE, Markoe AM, et al. The prognostic significance of surgical staging for carcinoma of the endometrium. Gynecol Oncol 1992;45:142±46. [3] DiSaia PJ, Creasman WT, Boronow RC, Blessing JA. Risk factor and recurrence patterns in stage I endometrial cancer. Am J Obstet Gynecol 1985;51:1009±15.
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[4] Malkasian Jr. GD. Carcinoma of the endometrium: effect of stage and grade on survival. Cancer 1978;41:996±1001. [5] Genest P, Drouin P, Gerig L, Girard A, Stewart A, Prefontaine M. Prognostic factors in early carcinoma of endometrium. Am J Clin Oncol 1987;10:71±7. [6] Imachi M, Tsukamoto N, Matsuyama T, Nakano H. Peritoneal cytology in patients with endometrial carcinoma. Gynecol Oncol 1988;30:76±86. [7] Wilson TO, Podratz KC, Gaffey TA, Malkasian Jr. GD, O'Brien PC, Naessens JM. Evaluation of unfavorable histologic subtypes in endometrial adenocarcinoma. Am J Obstet Gynecol 1990;162: 418±23. [8] Weinberg RA. Tumor suppresor genes. Science 1991;254:1138±46. [9] Levine AJ, Momand J, Finlay CA. The p53 tumor supressor gene. Nature 1991;135:453±6. [10] Hamel NW, Sebo TJ, Wilson TO, et al. Prognostic value of p53 and proliferating cell nuclear antigen expression in endometrial carcinoma. Gynecol Oncol 1996;62:192±8. [11] Ito K, Watanabe K, Nasim S, et al. Prognostic significance of p53 overexpression in endometrial cancer. Cancer Res 1994;54:4667±70. [12] Coppola D, Fu L, Nicosia SV, Kounelis S, Jones M. Prognostic significance of p53, bcl-2, vimentin and S100 protein-positive Langerhans cells in endometrial carcinoma. Hum Pathol 1998;29:455±62. [13] Bargmann CI, Hung MC, Weinberg RA. The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature 1986;319:226±30. [14] Gassel AM, Backe J, Krebs S, SchoÈn S, Caffier H, MuÈller-Hermelink HK. Endometrial carcinoma: inmunohistochemically detected proliferation index is a prognosticator of long term outcome. J Clin Pathol 1998;51:25±9. [15] Kaplan J, Meier P. Non parametric stimation from incomplete observation. J Am Stat Assoc 1984;38:511±35. [16] Cox DR. Regression models and life-tables. J R Stat Soc 1972;B34:187±220. [17] Koler MF, Berchuck A, Davidoff AM, et al. Overexpression and mutation of p53 in endometrial carcinoma. J Cancer Res 1992;52:1622±27. [18] Olson SH, Finstad CL, Harlap S, Kurian L, Saigo PE, Barakat RR. A case-case analysis of factors related to overexpression of p53 in endometrial cancer following breast cancer. Cancer Epidemiol Biomarkers Prev 1997;6:815±7. [19] Ambros RA, Vigna PA, Figge J, et al. Observations on tumor and metastatic suppressor gene status in endometrial carcinoma with particular emphasis on p53. Cancer 1994;73:1686±92. [20] Inoue M, Akira O, Fujita M, et al. Clinicopathological characteristics of p53 overexpression in endometrial cancers. Int J Cancer 1994;58:14±9. [21] Lax SF, Pizer ES, Ronnett BM, Kurman R. Clear cell carcinoma of the endometrium is characterized by a distinctive profile of p53, Ki67, estrogen and progesterone receptor expression. Hum Pathol 1998;29:551±8. [22] Kovalev S, Marchenko ND, Gugliotta BG, Chalas E, Chumas J, Moll UM. Loss of p53 function in uterine papillary serous carcinoma. Hum Pathol 1998;29:613±9.
[23] Kuwashima Y, Toshitaka U, Kishi K, Shiromizu K, Matsuzawa M, Takayama S. Proliferative and apoptotic status in endometrial adenocarcinoma. Int J Gynecol Pathol 1995;14:45±9. [24] King SA, Adas AA, LiVolsi VA, et al. Expression and mutation analysis of the p53 gene in uterine papillary serous carcinoma. Cancer 1995;75:2701±5. [25] Koshiyama M, Konishi I, Wang DP, et al. Immunohistochemical analysis of p53 protein overexpression in endometrial carcinomas: inverse correlation with sex steroid receptor status. Virchows Archiv A Pathol Anat 1993;423:265±71. [26] Enomoto T, Fujita M, Inoue M, et al. Alterations of the p53 tumor suppressor gene and its association with activation of the c-K-ras 2 protooncogene in premalignant and lesion of the human uterine endometrium. Cancer Res 1993;53:1883±8. [27] Kohlberger P, Gitsch G, Loesch A, et al. p53 Protein overexpression in early stage endometrial cancer. Gynecol Oncol 1996;62:213±7. [28] Soong R, Knowles S, Williams KE, Hammond IG, Wysocki SJ, Iacopetta BJ. Overexpression of p53 protein is an independent prognostic indicator in human endometrial carcinoma. Br J Cancer 1996;74:562±7. [29] Powell B, Soong R, Grieu F, Knowles S, Hammond I, Iacopetta B. p53 Protein overexpression is a prognostic indicator of poor survival in stage I endometrial carcinoma. Int J Oncol 1999;14:175±9. [30] Pisani AL, Barbuto DA, Chen D, Ramos L, Lagasse LD, Karlan BY. HER-2/neu, p53 and DNA analyses as prognosticators for survival in endometrial carcinoma. Obstet Gynecol 1995;85:729±34. [31] Reinartz JJ, George E, Lindgren BR, Niechans GA. Expressions of p53, transforming growth factor alpha, epidermal growth factor receptor and c-erbB-2 in endometrial carcinoma and correlation with survival and known predictors of survival. Hum Pathol 1994;161:1247±52. [32] Lukes AS, Kohler MF, Pieper CF, et al. Multivariable analysis of DNA ploidy, p53 and HER-2/neu as prognostic factors in endometrial cancer. Cancer 1994;73:2380±5. [33] Berchuck A, Rodriguez G, Kinney RB, et al. Overexpression of HER-2/neu in endometrial cancer is associated with advanced stage disease. Am J Obstet Gynecol 1991;164:15±21. [34] Khalifa MA, Mannel RS, Haraway SD, Walker J, Min KW. Expression of EGFR, HER-2/neu, p53 and PCNA in endometrioid, serous papillary and clear cell endometrial adenocarcinomas. Gynecol Oncol 1994;53:84±92. [35] Wang D, Konishi I, Koshiyama M, et al. Expression of c-erbB-2 protein and epidermal growth factor receptor in endometrial carcinomas. Cancer 1993;72:2628±37. [36] Bigsby RM, Li AX, Bomalaski J, Stehman FB, Look KY, Sutton GP. Immunohistochemical study of HER-2/neu, epidermal growth factor receptor and steroid receptor expression in normal and malignant endometrium. Obstet Gynecol 1992;79:95±100. [37] Hetzel DJ, Wilson TO, Keeney GL, Roche PC, Cha SS, Podratz KC. HER-2/neu expression: a major prognostic factor in endometrial cancer. Gynecol Oncol 1992;7:179±85. [38] Marks JR, Humphrey PA, Wu K, et al. Overexpression of p53 and HER-2/neu proteins as prognostic markers in early stage breast cancer. Ann Surg 1994;219:332±41.
P53 overexpression predicts endometrial carcinoma recurrence better than HER-2/neu overexpression Pluvio J. Coronadoa,*, Jose A. Vidarta, Jose A. Lopez-asenjob, Maria Faseroa, Vicente Furio-baceteb, Javier Magrinac, Manuel Escuderoa a
Department of Obstetrics and Gynecology, Hospital ClõÂnico San Carlos, Madrid, Spain b Department of Pathology, Hospital ClõÂnico San Carlos, Madrid, Spain c Department of Gynecology Surgery, Mayo Clinic Scottsdale, Arizona, USA
Received 8 March 2000; received in revised form 24 October 2000; accepted 18 November 2000
Abstract Objective: to investigate the prognostic value of p53 and HER-2/neu overexpression in endometrial cancer. Study Design: p53 and HER2/neu immunostaining was performed in 114 paraf®n-embedded specimens of endometrial cancer diagnosed and treated between 1990 and 1997. Nuclear p53 and membrane HER-2/neu immunostaining were used. Results: p53 and HER-2/neu overexpression was observed in 17 cases (14.9%) and in 19 cases (16.7%), respectively. In univariate analysis p53 (P < 0:001) and HER-2/neu (P 0:018) overexpression had a positive correlation with a high risk of recurrence. In multivariate analysis, age (P < 0:001), FIGO stage (P < 0:001), differentiation (P 0:013), non-endometrioid subtypes (P < 0:001) and p53 overexpression (P < 0:001), but not HER-2/neu overexpression, were independent prognostic indicators of recurrence. Simultaneous p53 and HER-2/neu overexpression made worse the prognostic (P < 0:001). Conclusions: p53 overexpression was an independent predictor of recurrent disease in endometrial cancer. HER-2/neu overexpression had a more limited effect but enhance the effect of p53. # 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: p53; HER-2/neu; Endometrial cancer; Prognostic factors; Survival; Immunohistochemistry
1. Introduction Endometrial cancer is the most common neoplasm of the female genital tract [1]. Traditionally prognostic factors such as FIGO stage, tumor grade, depth of myometrial invasion, histologic subtype, positive peritoneal cytology and nodal metastases have been used to detect patients with high risk of recurrence [2±7]. Nevertheless, some patients have recurrences despite good prognostic factors. The emergence and maintenance of cancer is complex. Abnormalities involving oncogenes and tumor suppresser genes play a central role [8]. The p53 tumor suppresser gene is located on the short arm of chromosome 17 and encodes a 53 kD phosphoprotein which is considered a tumor suppresser [9]. The relation between p53 abnormalities and prognosis in endometrial carcinoma is not clear. Some studies have shown a correlation between p53 overexpression and decreased survival [10,11] but others have not [12].
* Corresponding author. Tel.: 34-91-330-3526; fax: 34-91-330-3526. E-mail address: [email protected] (P.J. Coronado).
HER-2/neu oncogene (c-erbB-2), located on chromosome 17 but on its long arm, encodes a 185 kD glycoprotein that is very similar to EGF-R [13]. Studies about HER-2/neu protein expression in endometrial carcinoma have also reached different conclusions. Hamel et al. [10] considered HER-2/neu overexpression an independent prognostic factor but another recent study reported no association [14]. The aim of this study was to evaluate the independent prognostic value of p53 and HER-2/neu overexpression in endometrial carcinoma and their correlation with traditional phenotypic prognostic factors. 2. Materials and methods One hundred and fourteen patients with primary endometrial adenocarcinoma were included in this study. All patients underwent exploratory laparotomy, total abdominal hysterectomy and selective lymphadenectomy at Hospital ClõÂnico San Carlos between 1990 and 1997. Cytological washings of the pelvis were obtained in all patients. None of them received preoperative pelvic radiation or chemotherapy.
0301-2115/01/$ ± see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 0 ) 0 0 5 6 2 - 5
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P.J. Coronado et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 98 (2001) 103±108
Postoperative radiotherapy was used according to the treating radiation oncologist based on the traditional surgicalpathologic factors. Postoperative chemotherapy was not used. 2.1. Immunohistochemical staining A single 5 mm section from paraf®n-embedded specimens was routinely deparaf®ned and incubated in methanol with 0.3% H2O2 to block endogenous peroxidase activity. The specimens were embedded in buffer-citrate 0 0 1 H and incubated in microwave to 750 W for 7 min. After cooled and washed in PBS (pH 7.6), the ®rst antibody was used. We used two antibodies: the monoclonal antibody DO7 (Novocastra Laboratory, Newcastle, UK), which recognizes an epitope in human p53, at optimal dilution in Tris±albumin (1/50); and the monoclonal antibody anti-c-erbB-2 (Medac) that recognizes the intracellular domain of p185 protein at optimal dilution in Tris±albumin (1/40). Both antibodies were incubated overnight in humidi®ed chamber at 2±88C. After treatment with biotinated anti-mouse inmunoglobulin for 30 min at room temperature, the sections were incubated with peroxidase-conjugated streptavidin for 30 min at room temperature. A ®nal wash was followed by samples developed with 3 mg of 3,30 -diaminobencidine and 15 ml of hydrogen peroxide in Tris±HCl buffered at pH 7.6. The slides were then rinsed for 10 min in tap water, then they were stained with hematoxylin and coverslips were mounted. Positive and negative control antibodies were used to ensure tissue viability and exclude the possibility of nonspeci®c staining. Two pathologists scored all slides. Staining cell numbers were scored as none, weak (, <30%), moderate (, 30±70%) and intense (, >70%). Overexpression was de®ned as intense staining. In the case of HER2/neu, only membrane immunostaining was evaluated. The w2 test or Ficher's exact test was used to evaluate the correlation between p53 and HER-2/neu overexpression with traditional prognostic factors. Kaplan±Meier's method was used to estimate the progression-free survival distribution [15]. Log rank's test was used to calculate the statistical signi®cation of the curves. Cox's method was used to assess which factors were univariately associated with recurrence. Multivariate modeling using Cox's proportional hazard models was performed to obtain a subset of independent predictors of progression-free survival [16]. 3. Results The average age of the patients was 65 years (range 37±85). At a median follow up of 69 months (range 14±107) 85 patients were alive with no disease, 14 were dead from disease, 12 dead with no evidence of disease and three dead with recurrence but not due to disease. Seventeen patients experienced recurrence: 15 in the vagina or pelvis,
Table 1 Distribution of age, stage, grade, depth of invasion and histologic subtype in 114 primary endometrial adenocarcinomas N
(%)
Age 65 years <65 years
59 55
52 48
FIGO stage I II III IV
86 6 21 1
75 5.3 18 0.9
Histologic subtype Endometrioid Clear cell Serous papillary Adenosquamous
87 16 7 4
75 14 6.1 3.5
Myometrial invasion None 50% >50%
9 56 49
7.9 49 43
Grade G1 G2 G3
35 66 13
31 58 11
one in the supraclavicular nodes and one in the liver. The mean time to recurrence was 26 months (range 6±64). The distribution of traditional prognostic factors is shown in Table 1. p53 overexpression was present in 17 of 114 cases (15%) and was observed only in tumor cells as nuclear staining. The p53 overexpression was associated with non-endometrioid histologic subtypes (P 0:026) and deep myometrial invasion (P < 0:001) (Table 2). Moreover, p53 overexpression was more frequent in FIGO stages III±IV and in poorly differentiated tumors, but without reaching statistical signi®cance (Table 2). HER-2/neu membrane overexpression was observed in 19 of 94 cases (17%). HER-2/neu overexpression was more frequent in advanced FIGO stages, in non-endometrioid subtypes, in tumors with deep myometrial invasion and in less differentiated carcinomas. No statistical signi®cation was found when correlated with these classical prognostic factors (Table 2). Nine of the 17 patients who had recurrence demonstrated p53 overexpression (53%), while it was only observed in 8 out of 97 patients with no recurrence (8.2%). No relationship was found between p53 overexpression and site of recurrence. Kaplan±Meier's curves showed a signi®cant decrease of progression-free survival in patients with p53 overexpression (Fig. 1). That signi®cant decrease of survival was observed both in early stages (P < 0:001) and advanced stages (P < 0:001). HER-2/neu overexpression was observed in 6 of 17 patients (35%) with recurrence and in 13 of 97 (13%) patients with no evidence of recurrence. No relationship
P.J. Coronado et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 98 (2001) 103±108
105
Table 2 Relationship between p53 and HER-2/neu and traditional prognostic factors in endometrial carcinoma Overexpression p53 (%) Overexpression FIGO stage I and II III and IV
P value
17/114 (18)
Overexpression HER-2/neu (%)
P value
19/114 (17)
12/92 (14) 5/22 (22)
0.315
14/92 (15) 5/22 (23)
0.523
Histologic subtype Endometrioid No endometrioid
9/87 (10) 8/27 (30)
0.026
13/87 (15) 6/27 (22)
0.384
Myometrial invasion 50% >50%
3/65 (4.6) 14/49 (29)
<0.001
9/65 (14) 10/49 (20)
0.447
14/101 (14) 3/13 (23)
0.408
16/101 (16) 3/13 (23)
0.452
Grade G1±G2 G3
was found between HER-2/neu overexpression and site of recurrence. Progression-free survival curves re¯ected a signi®cant decrease of survival for patients with HER-2/neu overexpression (Fig. 2). That signi®cant decrease of survival
was observed in early stages (P 0:008), but not in advanced stages (P 0:308). When a combined overexpression of both genes was considered, an important decrease of survival was observed.
Fig. 1. Relationship between p53 overexpression and progression-free survival.
Fig. 2. Relationship between HER-2/neu overexpression and progression-free survival.
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Fig. 3. Combined p53 and HER-2/neu overexpression.
The four patients with simultaneous p53 and HER-2/ neu overexpression had a dramatically lower survival rate (Fig. 3). Each of the traditional phenotypic risk factors as well as p53 and HER-2/neu overexpression was univariately associated with progression-free survival. As expected, classical indicators of prognosis including FIGO stages III±IV, grade 3, deep myometrial invasion, non-endometrioid histologic subtypes and older age were signi®cantly associated with poor outcome (Table 3). In order to determine whether p53 and HER-2/neu overexpression had validity as independent prognostic factors, a multivariate analysis according to Cox's proportional hazard model, including the prognostic factors listed previously was carried out. FIGO stage III±IV, grade 3, nonendometrioid subtypes, age of patient, and p53 overexpression were considered independent prognostic factors for
progression-free survival. HER-2/neu overexpression was not an independent prognostic factor (Table 4). 4. Comment One of the objectives of molecular tumor investigation is to identify biologic prognostic indicators to be used in addition to the traditional morphologic factors to further delineate the outcome of endometrial cancer patients. This study evaluated the prognostic signi®cance of p53 and HER2/neu overexpression in relation to disease recurrence. Previous studies have reported a statistically signi®cant association between p53 overexpression and some of the traditional prognostic factors for endometrial carcinomas such as advanced stage [10,17±19], poorly differentiated tumors [10,11,17,19], deep myometrial invasion [10,20] and
Table 3 Correlation of p53 overexpression and clinical pathologic variables with corresponding progression-free survival in 114 patients with endometrial carcinoma
Age p53 HER-2/neu FIGO stage Histologic subtype endometrioid Grade Myometrial invasion
Linear increment of 1 year Overexpression No overexpression Overexpression No overexpression III and VII I and II Yes No G3 G1±G2 >50% 50%
RR
CI 95%
P value
1.09 8.7 1 3.7 1 9.5 1 0.12 1 4.4 1 6.9 1
1.03±1.2 3.4±22.8
<0.001 <0.001
1.4±10.2
0.018
3.5±25.8
<0.001
0.04±0.3
<0.001
1.6±12.6
0.013
2±24
<0.001
P.J. Coronado et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 98 (2001) 103±108
107
Table 4 Independent predictors of progression-free survival determined by multivariate analysis
p53 HER-2/neu FIGO stage Grade Histologic subtype Age
Overexpression No overexpression Overexpression No overexpression III and IV I and II G3 G1±G2 Endometrioid No-endometrioid Linear increment of 1 year
non-endometrioid histological types [10,17,21±24]. We found a statistical association between p53 overexpression and non-endometrioid histologic subtypes and deep myometrial invasion. We observed a higher p53 overexpression in patients with advanced FIGO stage and high grade (G3) tumors, although those differences were not signi®cant. We found, like others [12,17,19,25±27], an incomplete association with the surgical-pathological prognostic factors. Those ®ndings may re¯ect either a wide variety of p53 altered forms in endometrial cancers or different results due to the type of antibody used and the observers subjectivity. Nevertheless, p53 overexpression appears to be a feature of tumor aggressiveness. Several studies [10,11,29±30] have identi®ed p53 nuclear overexpression as an independent prognostic factor of recurrence in endometrial cancer. However, some authors found no correlation between p53 overexpression and poor prognosis either in univariate [12] or multivariate [14,20,31,32] analysis, and even Soong et al. [28] observed a better prognosis when a cytoplasmatic immunoreactivity appeared. In our study, p53 overexpression was an independent factor of poor prognosis. Those ®ndings suggest that determination of p53 in endometrial cancer could identify high-risk patients of recurrence and moreover, it con®rms the predictive value of p53 overexpression in endometrial adenocarcinomas. Previous studies have shown an association of HER-2/ neu overexpression with advanced stage, non-endometrioid histology [33] and depth of myometrial invasion [34], while other have not [36,37]. In this study, HER-2/neu overexpression was noted more frequently in association with advanced stage, non-endometrioid subtypes, high grade and deep myometrial invasion, but the differences were not statistically signi®cant. This is in agreement with Gassel et al. [14] who found no statistical association between HER-2/neu overexpression and classical prognostic factors as in 222 patients with endometrial carcinoma. In the present study, HER-2/neu overexpression was predictive of recurrent disease in univariate analysis, but not in the multivariate analysis. Others [14,35] have reached similar conclusions; however, some studies have established HER2/neu overexpression as an independent prognostic factor
Adjusted RR
CI 95%
P value
9.72 1 1.49 1 11.09 1 2.98 1 0.27 1 1.08
2.9±32.9
<0.001
0.3±6.5
0.595
3.6±34.5
<0.001
0.9±9.3
0.076
0.1±0.8
0.017
1.01±1.2
0.024
[10,32,33,37]. In our series, the prognostic value of HER-2/ neu overexpression was higher in early stages than in advanced stages. It suggests that HER-2/neu overexpression could select high-risk patients of recurrence in initial stages of the disease and loss power of prediction in advanced stages. We observed a higher trend to recurrent disease in patients with simultaneous overexpression of both genes. Others have found similar result [10]. Studies of breast cancer are also consistent with these observations [38]. Those ®ndings would indicate that multiple simultaneous gene alterations highly worsen the prognosis of the disease, although the presence of p53 overexpression seems to be a more powerful indicator of recurrence than HER-2/neu. More studies are necessary to determinate the positive interaction of both genes in the prognostic of the endometrial cancer. In conclusion, p53 overexpression is an important prognostic factor in endometrial carcinoma while HER-2/neu overexpression has a more limited role in the recurrence of these cancers. Simultaneous p53 and HER-2/neu overexpression results in a higher risk of recurrence than overexpression of each gene independently. Acknowledgements This study was ®nanced through grants from F.I.S 95/ 0415 and B.A.E. 97/5415, Ministerio de Sanidad y Consumo, Madrid, Spain. References [1] Bruke TW, Tortolero-Luna G, Malpica A, et al. Endometrial hyperplasia and endometrial cancer. Obstet Gynecol Clin North Am 1996;23:411±56. [2] Wolfson AH, Sightler SE, Markoe AM, et al. The prognostic significance of surgical staging for carcinoma of the endometrium. Gynecol Oncol 1992;45:142±46. [3] DiSaia PJ, Creasman WT, Boronow RC, Blessing JA. Risk factor and recurrence patterns in stage I endometrial cancer. Am J Obstet Gynecol 1985;51:1009±15.
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