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Keywords:

  • endometrial cancer;
  • EphA2;
  • estrogen receptor;
  • progesterone receptor;
  • Ki-67

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

BACKGROUND:

EphA2 is a tyrosine kinase receptor in the ephrin family that is implicated in oncogenesis and angiogenesis. The objective of the current investigation was to study the role of EphA2 in endometrial cancer and its relation to steroid hormone receptor expression.

METHODS:

EphA2, estrogen receptor (ER), progesterone receptor (PR), and Ki-67 expression levels were evaluated using immunohistochemistry in 139 endometrioid endometrial carcinoma (EEC) samples and in 10 benign endometrial samples. Samples were scored by 2 investigators who were blinded to clinical outcome. The results were correlated with clinicopathologic characteristics using univariate and multivariate analysis. A P value <.05 was considered statistically significant.

RESULTS:

High expression of EphA2 was detected in 48% of EEC samples versus 10% of benign samples. EphA2 overexpression was associated significantly with high disease stage (P = .04), high tumor grade (P = .003), increased depth of myometrial invasion (P = .05), low ER expression (P = .01), low PR expression (P = .006), and high Ki-67 expression (P = .04). Low ER and PR expression levels were associated with high tumor grade, positive lymph nodes, high Ki-67 expression, and high EphA2 expression. On univariate analysis of all patients, high EphA2 expression was associated significantly with shorter disease-specific survival (DSS) (P < .001). On multivariate analysis, age (P < .001), high disease stage (P = .002), and high EphA2 expression (P = .04) were independent predictors of poor DSS.

CONCLUSIONS:

EphA2 overexpression was associated with aggressive phenotypic features in EEC and was associated inversely with ER and PR expression. Thus, EphA2 may be an important therapeutic target, especially in patients with hormone receptor-negative endometrial carcinoma. Cancer 2009. © 2009 American Cancer Society.

Endometrial cancer is the most common cancer of the genital tract in women and accounted for 40,100 new cases in 2008.1 Eighty percent of endometrial cancers are of endometrioid histology.2 Although the majority of patients (range, 70%-80%) present with early stage disease and have a 5-year survival rate of approximately 80%, an estimated 7470 women will die from their disease in 2008.1, 2 Thus, a better understanding of the underlying molecular mechanisms is needed to plan effective therapeutic strategies, especially for women with advanced and recurrent endometrial cancer.

Receptor tyrosine kinases play an important role in diverse molecular functions, such as cellular proliferation, differentiation, and migration.3, 4 The Eph receptors are the largest family of tyrosine kinases and are divided into 2 subclasses based on interaction with their ligands, ephrin-A and ephrin-B.5 There is growing evidence that several Eph receptors play critical roles in cancer development and progression.6 EphA2 is a transmembrane protein that is found primarily on adult human epithelial cells, unlike other Eph kinases, which are expressed mainly during embryogenesis.5, 6 Although the specific role for EphA2 in normal epithelium is not fully known, EphA2 may regulate cell growth, invasion, angiogenesis, and survival of cancer cells.6, 7 EphA2 overexpression has been demonstrated in several solid tumors, including melanoma and breast, prostate, lung, and ovarian carcinomas.7-10 However, the clinical relevance of EphA2 expression in endometrial cancer is unknown.

In the current study, we sought to examine the role of EphA2 expression in patients with endometrial carcinoma. In addition, we investigated the relation of EphA2 with known prognostic factors in endometrial cancer, including the expression of steroid hormone receptors and markers of cellular proliferation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

Samples for Immunostaining

After we received Institutional Review Board approval for the study, archived formalin-fixed, paraffin-embedded samples were obtained from 139 patients with endometrioid endometrial carcinoma (EEC) who underwent surgery at the University of Texas M. D. Anderson Cancer Center and Methodist Hospital between 2000 and 2004 and who had adequate tissue available for immunohistochemical evaluation. All patients were staged surgically based on the International Federation of Gynecology and Obstetrics (FIGO) staging system. None of the patients received preoperative chemotherapy or radiation. Endometrial samples from 10 women who underwent hysterectomy for benign indications were included as controls for the expression of the study proteins.

Immunohistochemical Staining

The methods for immunohistochemical staining of paraffin-embedded samples have been described previously.10, 11 Briefly, formalin-fixed, paraffin-embedded samples were sectioned at 4 μm and stained with hematoxylin and eosin (H&E) for tumor confirmation. Sections adjacent to the H&E staining were used for immunohistochemical staining. Sections were deparaffinized and then probed with either a monoclonal EphA2 antibody (MedImmune, Gaithersburg, Md), mouse antihuman estrogen receptor (ER) α (dilution, 1:10) mouse antihuman progesterone receptor (PR) (dilution, 1:75); and mouse antihuman Ki-67 (dilution, 1:100; DakoCytomation, Carpinteria, Calif). Next, the slides were rinsed with phosphate-buffered saline, incubated with a secondary antibody for 30 minutes, then incubated with a ready-to-use avidin-biotin complex method reagent for 5 to 15 minutes, and counterstained with a 1:10 dilution of Mayer hematoxylin for 35 to 60 seconds.

Semiquantitative Analysis of Immunostaining

All samples were reviewed by 2 investigators (A.A.K. and D.C.) who were blinded to the clinical outcome of patients. Semiquantitative assessment of immunohistochemical expression was performed as described previously11 by assessing the percentage of stained tumor cells and staining intensity. Briefly, the percentage of positively stained cells was rated as follows: 0 points, 0% to 5% positively stained cells; 1 point, 6% to 50% positively stained cells; 2 points, 51% to 75% positively stained cells; 3 points, >75% positively stained cells. The staining intensity was rated in the following manner: 2 points, weak intensity; 3 points, moderate intensity; 4 points, strong intensity. The product of the scores for intensity and the percentage of positive cells were used to calculate an overall score index (SI) ranging from 0 to 3. Tumors were categorized into 3 groups based on the SI: negative or weak expression (SI, 0-4), moderate expression (SI, 5-8), and strong expression (SI, 9-12). For Ki-67 expression, the number of positively stained cells in 5 high-power fields in the areas of greatest proliferation were calculated, and the percentage of positive cells per field was calculated.

For statistical analyses, tumors were dichotomized into 2 groups based on the level of immunostaining as follows: for EphA2, low expression (negative, weak, or moderate staining; SI, 0-8) and high expression (strong staining; SI, 9-12); for ER and PR, low expression (negative or weak staining; SI, 0-4) and high expression (moderate and strong staining; SI, 5-12); for Ki-67, low expression (≤30% Ki-67-positive cells) and high expression (>30% Ki-67-positive cells). For all immunohistochemical analyses, the independent scores from both investigators were consolidated into a final score, which is reported in this study. Any differences in the scores were adjudicated after a discussion between the 2 investigators.

Clinicopathologic Analysis

All patients underwent surgical exploration and primary surgical staging as initial treatment. The extent of the surgical staging was based on the preoperative endometrial biopsy, frozen-section pathology, and the surgeon's clinical judgment. The treating gynecologic oncologist determined the adjuvant therapy. The pathologic diagnosis was verified by the pathology reports. A gynecologic pathologist (D.C.) reviewed all H&E slides to confirm the histopathologic diagnosis and tumor grade. On the basis of FIGO stage, patients were divided into 2 groups, low stage (FIGO stages I and II, n = 108 patients) and high stage (FIGO stages III and IV, n = 31 patients). A clinical remission was defined as no evidence of disease based on physical examination and/or imaging studies. Disease-specific survival (DSS) was defined as the time from diagnosis to the date of death or the date of last contact.

Statistical Analysis

Chi-square or Fisher exact tests were used, as appropriate, to test for the association in the proportions across levels of a single covariate factor and expression of EphA2, ER, PR, and Ki-67. Patients who were alive at last follow-up or who died from causes other than uterine cancer were censored at the date of last follow-up. DSS was estimated using the Kaplan-Meier product-limit method. A 2-sided log-rank test was used to test for differences between survival curves. DSS was assessed using both univariate and multivariate Cox proportional hazards regression. A P value <.05 on 2-tailed testing was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

Demographic Factors

To determine the clinical significance of EphA2 expression in human endometrial cancers, we examined 139 samples that were stained for EphA2, ER, PR, and Ki-67 expression. The demographic features of the patients are listed in Table 1. The mean age of patients was 63 years (range, 27-91 years). Seventy-eight percent of patients had stage I or II disease, all tumors were of endometrioid histology, and only 18% of tumors were poorly differentiated. The median follow-up for patients in this study was 24.9 months.

Table 1. Demographic Features of Patients With Endometrioid Endometrial Cancer
VariableNo. of Patients (%), N=139
Mean age [range], y63 [27-91]
Menopausal status 
 Premenopausal24 (17)
 Postmenopausal112 (81)
 Unknown3 (2)
Stage 
 Low (I/II)108 (78)
 High (III/IV)31 (22)
Grade 
 135 (25)
 279 (57)
 325 (18)
Depth of myometrial invasion, % 
 ≤50100 (72)
 >5039 (28)
Lymph node status 
 Negative74 (53)
 Positive49 (35)
 Not done16 (12)

EphA2 Expression in Human Endometrioid Endometrial Cancer Samples

Representative photomicrographs illustrating negative, low, and high expression of EphA2 are presented in Figure 1. In addition, 10 samples of benign endometrium were stained for EphA2 expression. The majority of the benign samples (90%) demonstrated negative or weak expression of EphA2. Among the invasive EECs, 72 tumors (52%) had low or weak EphA2 expression, and 67 tumors (48%) overexpressed EphA2. In 2 samples, EphA2 expression could not be assessed because of poor sample quality.

thumbnail image

Figure 1. These are representative images of invasive endometrioid endometrial cancers after immunohistochemical staining for the ephrin A2 tyrosine kinase receptor (EphA2), estrogen receptor (ER), progesterone receptor (PR), and Ki-67 (original magnification, ×200).

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Association of EphA2 Expression With Clinical and Pathologic Variables

On the basis of the finding that EphA2 is overexpressed in a large proportion of EECs, next, we analyzed the association of EphA2 expression with known prognostic variables. The correlations of EphA2 overexpression with various clinical and pathologic variables are listed in Table 2. EphA2 was overexpressed in 65% of patients with high-stage disease compared with 43% of patients with low-stage disease (P = .04). It is noteworthy that high levels of EphA2 expression also were associated with higher grade tumors (grade 3, 76%; P = .003) and with increasing depth of myometrial invasion (P = .047). There was no significant difference in EphA2 expression among patients with negative, positive, or unknown lymph node status.

Table 2. Association of Ephrin A2 Tyrosine Kinase Receptor Overexpression With Clinicopathologic Variables in Endometrioid Endometrial Cancer (N=139)
VariableEphA2 Expression, No. of Patients*P
LowHigh
  • EphA2 indicates the ephrin A2 tyrosine kinase receptor; ER, estrogen receptor; PR, progesterone receptor.

  • *

    Missing numbers denote samples that were damaged and could not be evaluated.

  • Low expression of ER and PR denotes negative, weak, or moderate expression (staining intensity score index [SI], 0-8); high expression of ER and PR denotes strong expression (SI, 9-12).

  • Low expression of Ki-67 denotes ≤30% positive cells, and high expression denotes >30% positive cells.

Stage   
 Low (I/II)6147.04
 High (III/IV)1019 
Grade   
 Low (1 or 2)6547.003
 High (3)619 
Depth of myometrial invasion, %   
 ≤505642.047
 >501524 
Lymph node status   
 Negative4034.7
 Positive2524 
 Not done68 
ER expression   
 Low2841.01
 High4325 
PR expression   
 Low2337.006
 High4829 
Ki-67 expression   
 Low4833.04
 High2333 

Because the presence of steroid hormone receptor expression is a good prognostic indicator in patients with endometrial cancer and may guide therapeutic strategies, next, we evaluated the expression of ER and PR in our cohort. High expression of ER and PR (Fig. 1) was demonstrated in 46% and 55% of samples, respectively. When we analyzed the expression of EphA2 in relation to steroid hormone receptor expression, EphA2 overexpression was demonstrated in 59% of tumors with low ER expression compared with 32% of tumors with high ER expression (P = .01). A similar observed was made with PR: Sixty-two percent of tumors with low PR expression had high EphA2 expression compared with 38% of tumors with high PR expression, a highly significant difference (P = .006). Finally, we studied the relation of EphA2 expression with the degree of cellular proliferation as represented by the percentage of tumor cells that stained positive for Ki-67 (Fig. 1). Tumors with a higher fraction of proliferating cells demonstrated a higher level of EphA2 expression (59%) compared with tumors that had lower levels of Ki-67 (41%; P = .04).

Clinical Outcome Based on EphA2 Expression

Before we tested the prognostic significance of EphA2 expression, we performed univariate analyses of traditional clinical variables for DSS (Table 3). Age was analyzed as a continuous variable, and advancing age was associated significantly with an increased risk of death from EEC (P = .002). High stage (P = .01), high tumor grade (P = .001), and depth of myometrial invasion (P = .003) all were associated with a shorter DSS, as expected. It is noteworthy that lymph node status was not a significant determinant of DSS. It also is important to note that there were 5 deaths among 12 patients who had unknown lymph node status; all of those patients demonstrated high EphA2 expression in their tumors. Among the other variables that we tested, low ER expression (P = .001), low PR expression (P = .002), and high Ki-67 expression (P < .001) were associated significantly with significantly shorter DSS among patients with EEC. The median DSS for patients who had tumors that overexpressed EphA2, compared with patients who had tumors with low EphA2 expression, was highly significant (P < .001) (Fig. 2). In an analysis that was restricted to tumors with low ER, patients who had tumors with EphA2 overexpression had a median DSS of 43 months compared with patients who had tumors with low ER and low EphA2 expression (median DSS, 80 months; P = .01).

Table 3. Univariate Survival Analysis of Prognostic Variables for Disease-specific Survival in Patients With Endometrioid Endometrial Cancer (N=139)
VariableMedian Survival, moHR (95% CI)P
LowHigh
  • HR indicates hazard ratio; CI, confidence interval; NR, not reached; FIGO, International Federation of Gynecology and Obstetrics; ER, estrogen receptor; PR, progesterone receptor; EphA2, the ephrin A2 tyrosine kinase receptor.

  • *

    For the clinical variables, the groups are subclassified as low and high as follows: stage, low (I/II) and high (III/IV); FIGO grade, low (1 or 2) and high (3); histology, low (endometrioid) and high (nonendometrioid); depth of myometrial invasion: low (≤50% myometrial thickness) and high (>50% myometrial thickness); lymph node status: low (negative) and high (positive). The criteria used for low and high expression of immunohistochemical variables are the same as those used in Table 2.

Age, per yNRNR1.05 (1.02-1.09).002
Stage*NRNR2.67 (1.21-5.87).01
FIGO grade*NR254.44 (2.11-9.35).001
Depth of myometrial invasion*NRNR3.18 (1.49-6.78).003
Lymph node status*NRNR0.7 (0.27-1.81).4
ER93NR0.22 (0.09-0.56).001
PR93NR0.27 (0.12-0.63).002
Ki-67NR934.19 (1.78-9.86).001
EphA2NRNR4.66 (1.87-11.59)<.001
thumbnail image

Figure 2. These Kaplan-Meier curves illustrate the effect of study markers on disease-specific survival in patients with endometrioid endometrial cancer. EphA2 indicates the ephrin A2 tyrosine kinase receptor; ER, estrogen receptor; PR, progesterone receptor.

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On the basis of the findings that EphA2 overexpression is associated with aggressive features in patients with EEC, next, we assessed whether there was an independent association between EphA2 expression and DSS. By using a Cox proportional hazards model, we performed a multivariate survival analysis (Table 4). By using a model that included age, disease stage, tumor grade, depth of invasion, lymph node status, and markers (including EphA2, ER, PR, and Ki-67), we observed that increasing age (P < .001), high stage (P = .002), and EphA2 overexpression (P = .04) were independent predictors of shorter DSS. In this model, patients who had tumors that overexpressed EphA2 had a 3-fold increased risk of death compared with patients who had tumors with low EphA2 expression. Because positive lymph node status is a known predictor of poor prognosis for patients with endometrial cancer, we reanalyzed our cohort with the exclusion of the 16 patients who had unknown lymph node status. Among patients with known lymph node status, there remained a significant association of high EphA2 expression with shorter DSS (P = .002). On multivariate analysis in this subgroup of patients, once again, high EphA2 expression was associated independently with shorter DSS (hazard ratio, 3.0; 95% confidence interval, 1.05-8.56; P = .04).

Table 4. Multivariate Survival Analysis Using Cox Proportional Hazards Model (N=139)
VariableHR (95% CI)P
  1. HR indicates hazard ratio; CI, confidence interval; ER, estrogen receptor; PR, progesterone receptor, EphA2, the ephrin A2 tyrosine kinase receptor.

Age, per y1.09 (1.04-1.14)<.001
High-stage (III/IV)5.64 (1.89-16.8).002
High-grade (grade 3)1.78 (0.62-5.13).3
Depth of invasion > ½1.96 (0.79-4.84).14
Positive lymph nodes0.56 (0.19-1.63).3
High ER expression0.4 (0.12-1.3).12
High PR expression0.66 (0.2-2.12).48
High Ki-67 expression2.07 (0.69-6.25).2
High EphA2 expression3.05 (1.05-8.87).04

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

The major findings of the current study are that a large proportion of EECs overexpress EphA2 compared with weak or negative expression in the majority of benign endometrial samples. EphA2 overexpression was associated significantly with several aggressive clinical variables and was associated inversely with the expression of steroid hormone receptors. Significantly, EphA2 overexpression was an independent predictor of shorter DSS in these patients. Together, these results indicate that EphA2 may be an attractive therapeutic target, especially in patients with steroid receptor-negative endometrial cancer.

Our work adds to the growing evidence that EphA2 may play an important role in the progression and development of several malignancies.5, 6 EphA2 is a receptor tyrosine kinase that is observed at low levels on nontransformed epithelial cells.12 Although the role of EphA2 in normal epithelia is not fully understood, the cellular consequences of ligand binding include negative regulation of cell growth, migration, and invasion.7, 13, 14 EphA2 overexpression has been reported in many cancers, including melanoma and breast, prostate, lung, and ovarian carcinomas.10 Lu and colleagues reported that EphA2-transfected, nontransformed cells demonstrate increased growth in vitro and form larger and more aggressive tumors in vivo.15 Moreover, EphA2 was identified as an independent prognostic factor for survival in patients with ovarian cancer, because >75% of tumors over express this oncoprotein.10 In our cohort, tumors that overexpressed EphA2 were associated with deep myometrial invasion. Recently, overexpression of EphA2 was associated with elevated levels of several matrix metalloproteinases (MMPs), including MMP-2, MMP-9, and membrane type 1 MMP, all of which facilitate migration and invasion of tumor cells.16 It is noteworthy that EphA2 expression was associated with a poor clinical outcome independent of lymph node status. In our study, EphA2 overexpression was noted in tumors with higher levels of proliferation. These findings are supported by other studies regarding the role of EphA2 in tumor cell growth, angiogenesis, and differentiation.17, 18

Obesity and other hyperestrogenic states are known risk factors for the development of endometrial cancer.2 EECs, which account for >80% of all endometrial cancers, are associated with an estrogen-driven model of carcinogenesis in which unopposed estrogen stimulation leads to malignant transformation of benign endometrium.19 In general, high levels of ER and PR are correlated directly with lower tumor grade, less myometrial invasion, lower incidence of lymph node metastases, and a good clinical outcome.20, 21 In our study, low expression of both steroid hormone receptors was associated significantly with a shorter DSS. There is some evidence to suggest that there is close interplay between EphA2 and ER.22 For example, EphA2 levels in breast cancer cells are related inversely to ER expression.22 Previous studies also have demonstrated that EphA2 overexpression increases the malignant characteristics of ER-positive breast cancer cells.15 Moreover, these growth-promoting effects of EphA2 occur in the absence of estrogen, rendering these cells resistant to tamoxifen. The inhibition of EphA2 using an antibody can reverse these effects, making these cells sensitive to tamoxifen once again.15 Our results extend these findings and indicate further that the subset of patients who had tumors with low ER expression and high EphA2 expression had a worse prognosis than the patients who had tumors with low EphA2 expression. These data suggest that EphA2 overexpression may contribute to the increased growth and invasiveness of ER-deficient cells; however, additional mechanistic studies are required.

In patients with recurrent or advanced endometrial cancers, hormone therapies commonly are used, and the best response rates are observed in patients who have well differentiated, steroid hormone receptor-positive tumors.23 Thus, these findings may have therapeutic implications for patients who have poorly differentiated endometrial cancers that are steroid hormone deficient.

Efforts to target EphA2 are being pursued actively in our laboratory using a variety of different approaches. One approach using an agonistic antibody against EphA2 in conjunction with paclitaxel inhibited tumor growth and improved survival in mice with advanced ovarian cancer.24 Another novel approach for targeting EphA2 using small interfering RNA (siRNA) incorporated into neutral liposomes recently was described in an orthotopic ovarian cancer model.25 Intraperitoneal administration of liposomal EphA2-siRNA complexes along with paclitaxel resulted in significant reduction in tumor growth compared with nonsilencing siRNA and paclitaxel treatment.25 Thus, EphA2-directed approaches in conjunction with chemotherapy appear promising and may provide alternative treatment options for patients with advanced endometrial cancer.

In summary, to the best of our knowledge, this is the first study to investigate the clinical relevance of EphA2 in EEC. Tumors that display EphA2 overexpression are associated with features that portend a poor prognosis, including high disease stage, high tumor grade, deep myometrial invasion, a high proliferative index, and low expression of steroid hormone receptors. It is noteworthy that, along with age and advanced disease stage, EphA2 overexpression is an important independent predictor of shorter DSS in patients with EEC. These findings support the evaluation of EphA2-targeted therapies in patients who have advanced or recurrent endometrial cancer.

Conflict of Interest Disclosures

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

Portions of this work were supported by the Betty Ann Asche-Murray Fellowship Award to A.A.K.; Training Grant T32 CA101642 from the National Cancer Institute, Department of Health and Human Services, National Institutes of Health to W.M.M, Y.G.L., and W.A.S.; The University of Texas M. D. Anderson Cancer Center Specialized Programs of Research Excellence in Ovarian Cancer (grant P50 CA083639) and Uterine Cancer (grant P50 CA098258); a Marcus Foundation grant; and the Betty Ann Asche Murray Distinguished Professorship to A.K.S.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References