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2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group
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Kemal Gungorduk a , Ibrahim Egemen Ertas b, *, Ahmet Sahbaz c , Seyfettin Ozvural a , Yagmur Sarica d , Aykut Ozdemir a , Sevil Sayhan e, Mehmet Gokcu a , Bulent Yilmaz b , Muzaffer Sanci a , Sevinc Inan d , Mehmet Harma c, Yusuf Yildirim a a
Department of Gynecologic Oncology, Tepecik Education and Research Hospital, Izmir, Turkey Department of Obstetrics and Gynecology, Tepecik Education and Research Hospital, Izmir, Turkey c Department of Obstetrics and Gynecology, Bulent Ecevit University, School of Medicine, Zonguldak, Turkey d Department of Histology and Embryology, Celal Bayar University, School of Medicine, Manisa, Turkey e Department of Pathology, Tepecik Education and Research Hospital, Izmir, Turkey b
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A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 January 2014 Received in revised form 11 May 2014 Accepted 28 May 2014
Objective: To analyze the expression patterns of extracellular signal-regulated kinase (ERK1/2) and phosphorylated (p)-AKT in the tissues of non-pathologic endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer using indirect immunohistochemistry, and also to investigate the effect of ERK1/2 and p-AKT expression patterns on prognosis in endometrioid adenocancer. Study design: Immunolocalization of ERK1/2 and p-AKT was examined in six different types of endometrial tissues: proliferative endometrium (PE; n = 10, 11.2%), secretuar endometrium (SE; n = 10, 11.2%), simple hyperplasia (SH; n = 15, 16.9%), complex hyperplasia (CH; n = 3, 3.4%) and atypical complex hyperplasia (ACH; n = 10, 11.2%), which were obtained from endometrial biopsies, curettage materials, and hysterectomy specimens and classified as the benign group; and both early stage endometrioid (n = 21, 23.6%) and advanced stage endometrioid adenocancer (AC; n = 20, 22.5%), which were obtained from complete surgical staging materials and classified as the malignant group. All specimens were fixed in 10% formalin and processed using routine paraffin protocols. Immunostaining intensities were evaluated as negative or weak (assigned as low expression) and moderate or strong (assigned as high expression). Results: In the malignant group, 23 of 41 patients (56.1%) had high ERK1/2 and p-AKT expression, whereas only two of 48 patients in the benign group (4.2%) had high ERK1/2 and p-AKT expression (P < 0.0001 and P < 0.0001, respectively). p-AKT expression was significantly higher in women with positive lymph nodes (OR 9.0; 95% CI: 1.2–100.0; P = 0.03). Higher expression of p-AKT was significantly associated with poor progression-free survival (PFS) and overall survival (OS). In contrast, ERK1/2 expression was not associated with PFS or OS.Conclusions ERK1/2 and p-AKT can be useful in the differential diagnosis of benign vs. malignant endometrial lesions, as well as early vs. advanced stage endometrioid endometrial adenocancer. Additionally, higher p-AKT expression could be used as a marker of poor prognosis in the management of patients with endometrioid endometrial adenocancer. ã 2014 Published by Elsevier Ireland Ltd.
Keywords: Normal endometrium Endometrial hyperplasia Endometrioid endometrial adenocancer ERK1/2 p-AKT Immunolocalization Prognosis
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* Corresponding author at: Department of Obstetrics and Gynecology, Tepecik Education and Research Hospital, Gaziler Street, 35120 Izmir, Turkey. Tel.: +90 5057404206; fax: +90 2324494949. E-mail addresses: [email protected], [email protected] (I.E. Ertas).
Introduction
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Endometrioid endometrial adenocancer (AC) is the most common gynecological malignancy in the western world, the fourth most common cancer among women in developed countries and the eighth most common cause of cancer death in the United States [1].
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http://dx.doi.org/10.1016/j.ejogrb.2014.05.040 0301-2115/ ã 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Gungorduk K, et al.. Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.05.040
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Endometrial hyperplasia is considered to be a precursor lesion of type I endometrioid AC and is linked with prolonged and unopposed estrogen stimulation of the endometrium. Atypical hyperplasia is associated with a higher risk of progression to endometrial cancer [2]. Over the last 10 years, intracellular molecular pathways, such as the extracellular signal-regulated kinase (ERK) 1/2 and phosphatidylinositol 3-kinase (PI3K)-AKT pathways, have been highlighted as attractive pathways for the investigation of carcinogenesis and cancer therapy targets [3,4]. However, the significance of these major signaling pathways in endometrial carcinogenesis as well as on the stage of disease has not been adequately addressed and remains unclear. ERK1/2 is a pro-survival factor belonging to an important subfamily of mitogen-activated protein (MAP) kinases that contributes to the regulation of mammalian somatic cell proliferation by controlling both cell growth and cell cycle progression. In normal cells, ERK1/2 induction is the key event for G1-to-S-phase progression and is related to the activation of positive regulators of the cell cycle and inactivation of antiproliferative genes [5]. Disruption of the ERK pathway is common in cancer, and a number of reports link ERK1/2 expression with certain types of drug resistance [6]. Akt is a 57 kDa serine/threonine kinase and central mediator of the PI3K pathway, with numerous downstream target molecules that mainly influence cell survival and apoptosis [4]. Akt can be activated byestrogen, insulin and various growth factors [7]. Evidence suggests that Akt is associated with the survival, proliferation, migration and infiltration of many kinds of cancer cells [8]. The goal of this study was to investigate the expression patterns of ERK1/2 and phosphorylated (p)-AKT in tissues of non-pathologic endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial AC using indirect immunohistochemistry, and also to investigate the effect of ERK1/2 and p-AKT expression patterns on prognosis in endometrial AC.
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Materials and methods
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In the present study, immunolocalization of ERK1/2 and p-AKT was examined using indirect immunohistochemistry in six different types of endometrial tissues: proliferative endometrium (PE; n = 10, 11.2%), secretuar endometrium (SE; n = 10, 11.2%), simple hyperplasia (SH; n = 15, 16.9%), complex hyperplasia (CH; n = 3, 3.4%) and atypical complex hyperplasia (ACH; n = 10, 11.2%), which were obtained from endometrial biopsies, curettage materials and hysterectomy specimens, and both early stage (International Federation of Gynecology and Obstetrics (FIGO) stage I and II) endometrioid endometrial AC (n = 21, 23.6%) and advanced stage (FIGO stage III and IV) endometrioid endometrial AC (n = 20, 22.5%), which were obtained from complete surgical staging materials; all tissues were obtained between January 1998 and December 2012. The study design was approved by the ethical committee of the Izmir Tepecik Education and Research Hospital. Proliferative and secretuar endometrium was defined according to the Noyes criteria [9]. Endometrial hyperplasia was classified according to a modified system as simple hyperplasia (SH), complex hyperplasia (CH) and atypical complex hyperplasia (ACH) [10]. Tumors were staged surgically according to the FIGO 2009 revised surgical staging system and grouped as either early stage (I and II) or advanced stage (III and IV) tumors. The histological grades of the tumors were also reviewed according to the FIGO system. Previous or concurrent cancer was not present in any of the patients. All patients with malignant disease underwent complete surgical staging, including total abdominal or radical hysterectomy, bilateral salpingo-oophorectomy, infracolic omentectomy, and systematic pelvic and para-aortic lymphadenectomy. All formalin-fixed, paraffin-embedded tissue blocks (n = 89) were retrieved from the Department of Pathology, Tepecik
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
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Table 1 ERK 1/2 expression in different endometrial tissue types. Tissue type
Benign group Proliferative endometrium Secretuar endometrium Simple hyperplasia Complex hyperplasia Atypical complex hyperplasia Malignant group Early stage endometrioid adenocancer Advanced stage endometrioid adenocancer
ERK1/2 expression Low expression
High expression
Negative
Moderate Strong
8 7 10 1 3
(80.0) (70.0) (66.7) (33.3) (30.0)
2 (9.5) –
Weak (20) (30) (33.3) (66.7) (50.0)
– – – – 2 (20.0)
15 (71.4)
3 (14.3)
1 (4.8)
1 (5.0)
5 (25.0)
14 (70.0)
2 3 5 2 5
– – – – –
All values are expressed as n (%).
Education and Research Hospital, and were evaluated at the Research Laboratory of Histology and Embryology, Celal Bayar University. The tissue samples were fixed in 10% formalin solution for 24 h, dehydrated in a graded ethanol series, cleared in xylene, embedded in paraffin wax and sectioned with a 5 mm thickness. The tissue blocks were chosen carefully after histologic assessment of sections stained with hematoxylin and eosin (HE; hematoxylin acc. to Gill III, cat. no. 1.05174). Additional sections from the same tissues were prepared for indirect immunohistochemical staining. Deparaffinization was achieved by incubating the sections at 60 C overnight, followed by incubating in xylene and rehydrating with a series of ethanol solutions. Sections were washed with distilled water and phosphate-buffered saline (PBS) for 10 min and then treated with 0.1% trypsin. They were delineated with a Dako PAP pen and incubated in a solution of 3% H2O2 for 5 min to inhibit endogenous peroxidase activity. After washing in PBS, sections were incubated with non-immune serum for 1 h, and then the antibody solution was removed without washing the sections. Sections were incubated with the following primary antibodies for 18 h overnight at 4 C in a humidity chamber: anti-ERK-1/2 (phosphorylated ERK-1/2) (SC-101761, 1:100 dilution, Santa Cruz Biotechnology Inc.) and anti-pAKT-1/2/3 (phosphorylated protein kinase B-1/2/3) (SC-135561, 1:100 dilution, Santa Cruz Biotechnology Inc.). An avidin-biotin-peroxidase system (Histostain Plus Bulk Kit, Invitrogen1 2nd generation LAB-SA Detection System, cat. no. 85-904, broad spectrum, Invitrogen, Camarillo, CA, USA) was used as a secondary kit. Sections were washed three times for 5 min each with PBS and then incubated with biotinylated secondary antibody followed by streptavidin conjugated to horseradish peroxidase in PBS for 30 min each. DAB (diaminobenzidine Table 2 Phosphorylated (p)-AKT expression in different endometrial tissue types. Tissue type
Benign group Proliferative endometrium Secretuar endometrium Simple hyperplasia Complex hyperplasia Atypical complex hyperplasia Malignant group Early stage endometrioid adenocancer Advanced stage endometrioid adenocancer
p-AKT expression Low expression
High expression
Negative
Moderate Strong
Weak (30) (40) (13.3) (33.3) (20.0)
– – – 1 (33.3) 1 (10.0)
– – – – –
5 (23.8) 13 (61.9)
3 (14.2)
–
1 (5.0)
19 (95.0)
7 6 13 1 7
–
(70) (60) (86.7) (33.3) (70.0)
3 4 2 1 2
–
All values are expressed as n (%).
Please cite this article in press as: Gungorduk K, et al.. Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.05.040
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chromogen, DAB Kit, Invitrogen, cat. no. 00-2014) was applied to the sections, which were then counterstained with Mayer’s hematoxylin, rehydrated in a series of ethanol solutions, and covered with Entellan1 by clearing in xylene. One section was reserved for control staining without antibody application in order to test whether or not the immunoreactivity was specific. Two observers, blinded to the pathologic information, observed the immunostained sections using a BX 40 bright-field microscope (Olympus, Tokyo, Japan) with a 200 objective and evaluated the immunohistochemical staining by scoring the percentage of cells with positive nuclei and cytoplasms within tumors independently. No statistically significant inter-observer difference was found. The intensity of immunostaining was scored as 0 (–),1 (+), 2 (++) or 3 (+++) (negative, weak, moderate and strong, respectively). For survival analysis, patients with negative or weak expression were assigned as
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low expression, and those with moderate or strong expression were assigned as high expression. Additionally, PE, SE, SH and ACH were classified as the benign group, and early and advanced stage endometrioid endometrial AC as the malignant group. For the malignant group, standart treatments were completed with adjuvant radiation (brachytherapy external beam) alone or adjuvant radiation + chemotherapy based on stage of the disease. Moreover, patients returned for a follow-up evaluation every 3 months for the first 2 years, every 6 months for the next 3 years, and annually thereafter. Computed tomography or magnetic resonance imaging was performed annually. Survival data were analyzed in December 2013. Data are presented as means standard deviations, medians (range), or percentages. The normality of continuous variable distributions was assessed using the Kolmogorov–Smirnov test.
Fig. 1. Immunohistochemical staining of ERK1/2 and p-AKT in cases of normal endometrium, simple hyperplasia, complex hyperplasia and atypical complex hyperplasia (200, original magnification). PE: proliferative endometrium; SE: secretuar endometrium; SH: simple hyperplasia; CH: complex hyperplasia; ACH: atypical complex hyperplasia.
Please cite this article in press as: Gungorduk K, et al.. Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.05.040
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Fig. 2. Immunohistochemical staining of ERK1/2 and p-AKT in cases of early and advanced stage endometrioid endometrial adenocancer (200, original magnification). EEAC: early endometrioid adenocancer; AEAC: advanced endometrioid adenocancer.
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The chi-squared test was used to analyze categorical variables, Student’s t-test was used for normally distributed variables, and the Mann–Whitney U-test was used for non-normally distributed variables. Relative risks (RRs) with 95% confidence intervals (CIs) were calculated. Factors identified as potential risk factors in unadjusted analyses (P < 0.05) were used to create a logistic regression model with lymph node metastasis as the dependent variable. The survival analysis was based on the Kaplan–Meier method, and the results were compared using the log-rank test. Progression-free survival (PFS) was defined as the time elapsed from the date of primary surgery to that of detection of recurrence or the latest observation. Overall survival (OS) was defined as the time elapsed from the date of primary surgery to that of death or the latest observation. Cox regression analysis was used to determine factors affecting survival, and the results were presented as hazard ratios (HRs) with 95% CIs. All statistical analyses were performed using MedCalc software version 11.5 for Windows, Mariakerke, Belgium. A P-value of <0.05 was considered significant. Results This study was performed on archival material consisting of 89 endometrial tissues. The mean age of the patients was 46.5 4.1 years for PE, 40.3 6.0 for SE, 45.2 5.2 for SH, 51.3 9.2 for CH, 56.7 9.7 for ACH, 58.3 11.7 for early stage endometrioid endometrial AC and 60.4 11.6 for advanced stage endometrioid endometrial AC. The FIGO stages of the AC cases were as follows:
12 cases with stage IA, eight with stage IB, one with stage II, five with stage IIIA, nine with stage IIIC1, four with stage IIIC2, one with stage IVA and one with stage IVB. The FIGO histological grades of 41 tumors were classified as follows: grade I in 14 EAC cases (34.1%), grade II in 14 (34.1%) and grade III in 13 (31.7%). The ERK1/2 and p-AKT immunohistochemical staining scores for normal, hyperplastic and malign endometrium are shown in Tables 1 and 2. In the malignant group, 23 of 41 patients (56.1%) showed high ERK1/2 and p-AKT expression (++ and +++), whereas only two of 48 patients in the benign group (4.2%) showed high ERK1/2 and p-AKT expression (P < 0.0001 and P < 0.0001, respectively). Figs. 1 and 2 demonstrates the expression of ERK1/2 and p-AKT in six different endometrial tissues. Logistic regression showed a relationship between p-AKT expression and lymph node metastasis. Expression of p-AKT was significantly higher in women with positive lymph nodes (OR 9.0; 95% CI 1.2–100.0; P = 0.03). By contrast, such a relationship was not found for ERK1/2 expression (OR 1.1; 95% CI 0.3–3.7; P = 0.83). Follow-up data were available for all patients in the malignant group. The median duration of follow-up was 52 months (range 16–168 months). Six patients (14.6%) died from their disease during follow-up. Results of Kaplan–Meier analyses for PFS and OS are shown in Table 3. Advanced FIGO stages (III and IV), higher expression of p-AKT (++/+++) (Fig. 3a), higher grade, positive lymphovascular invasion, myometrial invasion 1/2 and tumor size 2 cm were adverse prognostic factors for PFS. Moreover, advanced FIGO stages (III and IV), higher expression of p-AKT
Table 3 Results of univariate analyses of progression free and overall survival of patients with endometrioid endometrial adenocancer. Variable
Age (<60 vs. 60 years) Stage (I/II vs. III/IV) Grade (I vs. II vs. III) ERK1/2 expression (low vs. high) p-AKT expression (low vs. high) Myometrial invasion (<1/2 vs. 1/2) Lymphovascular invasion Tumor size (<2 cm vs. 2 cm) Menopause
Progression free survival
Overall survival
Hazard ratio
95%CI
P
Hazard ratio
95% CI
P
0.8 0.4 2.0 0.7 0.3 0.4 0.4 0.5 0.8
0.4–1.5 0.1–0.8 1.2–3.3 0.3–1.4 0.1–0.8 0.2–0.9 0.2–0.9 0.2–1.0 0.3–1.8
0.50 0.002 0.003 0.36 0.0009 0.01 0.01 0.04 0.62
0.8 0.4 1.9 0.6 0.4 0.5 0.5 0.6 0.7
0.4–1.6 0.1–0.9 1.1–3.1 0.3–1.3 0.1–0.8 0.2–1.0 0.2–1.0 0.3–1.1 0.3–1.6
0.56 0.004 0.007 0.24 0.001 0.04 0.04 0.10 0.46
Please cite this article in press as: Gungorduk K, et al.. Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.05.040
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Fig. 3. (a and b) Progression-free and overall survival curves according to p-AKT expression; (c and d) progression-free and overall survival curves according to ERK1/2 expression.
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(Fig. 3b), higher grade, positive lymphovascular invasion and myometrial invasion 1/2 were significantly associated with poor OS. However, ERK1/2 expression was not associated with PFS or OS (Fig. 3c and d). Table 4 Correlation between phosphorylated (p)-AKT expression and clinicopathological factors in patients with endometrioid endometrial adenocancer. Low (negative/ weak) (n = 18) Age (years) <60 60 Stage Early (I/II) Advanced (III/ IV) Grade I II III Myometrial invasion <1/2 1/2 LVI Negative Positive Tumor size (cm) <2 2 Menopause Yes No
High (moderate/ strong) (n = 23)
P value
0.21 11 (61.1) 7 (38.9)
Odds ratio (95% CI)
1.5 (0.8–3.0)
9 (39.1) 14 (60.9) <0.0001 –
18 (100) –
3 (13) 20 (87) <0.0001 –
12 (66.7) 6 (33.3) –
2 (8.7) 8 (34.8) 13 (56.5) <0.0001 3.7 (1.5–9.0)
14 (77.8) 4 (22.2)
4 (17.4) 19 (82.6)
10 (55.6) 8 (44.4)
5 (21.7) 18 (78.3)
9 (50) 9 (50)
8 (34.8) 15 (65.2)
14 (77.8) 4 (22.2)
21 (91.3) 2 (8.7)
0.04
1.7 (1.0–3.0)
0.35
1.3 (0.7–2.2)
0.37
LVI: lymphovascular invasion. All values are expressed as n (%).
1.1 (0.8–1.5)
We also assessed the correlation between p-AKT expression and clinicopathological characteristics (Table 4) using the chi-squared test. There were correlations of p-AKT expression with FIGO stage, depth of myometrial invasion, pathological grade and lymphovascular invasion.
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In the present study, we found advanced FIGO stage, higher expression of p-AKT, higher grade, positive lymphovascular invasion, tumor size 2 cm and myometrial invasion 1/2 to be adverse prognostic factors for PFS in endometrioid endometrial AC. We additionally found that advanced stage (III/IV), higher expression of p-AKT, higher grade, positive lymphovascular invasion, and myometrial invasion 1/2 had statistically significant associations with poor OS, but we did not find any association between ERK1/2 expression and PFS or OS. Several mutations have been implicated in endometrial carcinogenesis, the most well-known being PTEN and KRAS mutations [11]. Mutation of PTEN results in activation of the PI3K pathway, and mutation of KRAS causes continuous activation of ERK –MAPK pathways [12]. The activated MAPK pathway results in phosphorylation of ERK1/2. Normally, activated ERK1/2 is important in cell survival, differentiation and proliferation [5,13,14]. In the present study, ERK1/2 expression was found to be higher in endometrial cancer when compared with normal, non-atypical hyperplastic and atypical hyperplastic endometrium. Wang et al. [15] also reported higher ERK expression in endometrial cancer than in normal endometrium. They also found significantly higher ERK expression in advanced stage endometrium cancer. In contrast, in a study by Mizumuto et al. [16], higher ERK1/2 expression was found to be significantly associated with favorable prognosis in patients with endometrial cancer. In the present
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study, even though we found higher ERK1/2 expression in endometrial cancer, we did not find any statistically significant correlation between PFS or OS in endometrial cancer and ERK1/2 expression. Akt, a serine/threonine kinase, is activated by PI3K and plays an important role in cell survival and apoptosis inhibition [4,17]. IGF-I, insulin, estrogen, TNF and platelet-derived growth factors are growth factors, cytokines and steroids that activate PI3K and thereby activate Akt [7,18–22]. Akt expression plays an important role in cancer prognosis by inhibiting cell adhesion and increasing invasiveness [23]. Sun et al. [24] and Vilgelm et al. [25] reported that the activated Akt pathway also activates estrogen receptoralpha and creates a hyperestrogenic environment, thereby increasing neoplastic transformation. Increased expression of Akt in endometrial cancer was reported in two previous studies [25,26]. Pant et al. [27] showed that inhibition of Akt signaling resulted in increased apoptosis in Ishikawa cells and a reduction in tumor size in nude mice. Zhao et al. [28] reported that PI3K/Akt and ERK1/2 play roles in endometrial cancer proliferation and operate independently. They also showed that stromal cell-derived factor (SDF)-1a, an estrogen-induced chemokine, induces cell growth by activating Akt and ERK1/2 pathways in two types of endometrial cancer cells (Ishikawa and HEC-1A cell lines) in a dose-dependent manner [28]. In a study by Mori et al. [26] from 2007, pathologic specimens from 63 endometrial cancer patients were evaluated for p-AKT expression. They scored p-AKT expression in the same way as in our study: no, weak, moderate or strong. Although they did not detect a statistically significant difference in p-AKT expression between early and advanced stage endometrial cancer, in the present study, we did detect significantly higher p-AKT expression in advanced stage endometrial cancer compared with early stage endometrial cancer. Mori et al. also failed to find any correlation between p-AKT expression and lymph node metastasis, myometrial invasion depth or grade. In contrast, we found a relationship between p-AKT expression and lymph node metastasis. Higher p-AKT expression was detected in lymph node-positive women. We also found higher p-AKT expression in the myometrial invasion 1/2 group than in the myometrial invasion 1/2 group. Although Mori et al. [26] reported a correlation between low p-AKT expression and low relapse-free survival rates in endometrial cancer, we found decreased survival rates in patients with higher p-AKT expression in the present study. Consistent with the present study, Abe et al. [29] reported poor prognosis in endometrial cancer patients with higher p-AKT expression. While they did not find any difference in total p-AKT expression among normal endometrium epithelium, hyperplasia and endometrial cancer, we found significantly higher p-AKT expression in endometrial cancer than in benign pathologies such as proliferative endometrium, secretuar endometrium and hyperplastic tissues. In summary, although, the potential limitations of our study are its retrospective nature and small sample size for each groups, some important conclusions can be drawn. First, we found higher p-AKT expression to be a marker of poor prognosis in endometrial adenocancer patients, in that higher p-AKT expression was found to be associated with poorer OS and PFS. Second, p-AKT is an important key point in endometrial carcinogenesis and a promising prognostic marker in the management of patients with endometrioid endometrial AC. Further clinical trials including large samples are required to evaluate its role as a prognostic marker.
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Conflict of interest
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The authors declare no conflicts of interest.
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group
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Kemal Gungorduk a , Ibrahim Egemen Ertas b, *, Ahmet Sahbaz c , Seyfettin Ozvural a , Yagmur Sarica d , Aykut Ozdemir a , Sevil Sayhan e, Mehmet Gokcu a , Bulent Yilmaz b , Muzaffer Sanci a , Sevinc Inan d , Mehmet Harma c, Yusuf Yildirim a a
Department of Gynecologic Oncology, Tepecik Education and Research Hospital, Izmir, Turkey Department of Obstetrics and Gynecology, Tepecik Education and Research Hospital, Izmir, Turkey c Department of Obstetrics and Gynecology, Bulent Ecevit University, School of Medicine, Zonguldak, Turkey d Department of Histology and Embryology, Celal Bayar University, School of Medicine, Manisa, Turkey e Department of Pathology, Tepecik Education and Research Hospital, Izmir, Turkey b
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A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 January 2014 Received in revised form 11 May 2014 Accepted 28 May 2014
Objective: To analyze the expression patterns of extracellular signal-regulated kinase (ERK1/2) and phosphorylated (p)-AKT in the tissues of non-pathologic endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer using indirect immunohistochemistry, and also to investigate the effect of ERK1/2 and p-AKT expression patterns on prognosis in endometrioid adenocancer. Study design: Immunolocalization of ERK1/2 and p-AKT was examined in six different types of endometrial tissues: proliferative endometrium (PE; n = 10, 11.2%), secretuar endometrium (SE; n = 10, 11.2%), simple hyperplasia (SH; n = 15, 16.9%), complex hyperplasia (CH; n = 3, 3.4%) and atypical complex hyperplasia (ACH; n = 10, 11.2%), which were obtained from endometrial biopsies, curettage materials, and hysterectomy specimens and classified as the benign group; and both early stage endometrioid (n = 21, 23.6%) and advanced stage endometrioid adenocancer (AC; n = 20, 22.5%), which were obtained from complete surgical staging materials and classified as the malignant group. All specimens were fixed in 10% formalin and processed using routine paraffin protocols. Immunostaining intensities were evaluated as negative or weak (assigned as low expression) and moderate or strong (assigned as high expression). Results: In the malignant group, 23 of 41 patients (56.1%) had high ERK1/2 and p-AKT expression, whereas only two of 48 patients in the benign group (4.2%) had high ERK1/2 and p-AKT expression (P < 0.0001 and P < 0.0001, respectively). p-AKT expression was significantly higher in women with positive lymph nodes (OR 9.0; 95% CI: 1.2–100.0; P = 0.03). Higher expression of p-AKT was significantly associated with poor progression-free survival (PFS) and overall survival (OS). In contrast, ERK1/2 expression was not associated with PFS or OS.Conclusions ERK1/2 and p-AKT can be useful in the differential diagnosis of benign vs. malignant endometrial lesions, as well as early vs. advanced stage endometrioid endometrial adenocancer. Additionally, higher p-AKT expression could be used as a marker of poor prognosis in the management of patients with endometrioid endometrial adenocancer. ã 2014 Published by Elsevier Ireland Ltd.
Keywords: Normal endometrium Endometrial hyperplasia Endometrioid endometrial adenocancer ERK1/2 p-AKT Immunolocalization Prognosis
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* Corresponding author at: Department of Obstetrics and Gynecology, Tepecik Education and Research Hospital, Gaziler Street, 35120 Izmir, Turkey. Tel.: +90 5057404206; fax: +90 2324494949. E-mail addresses: [email protected], [email protected] (I.E. Ertas).
Introduction
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Endometrioid endometrial adenocancer (AC) is the most common gynecological malignancy in the western world, the fourth most common cancer among women in developed countries and the eighth most common cause of cancer death in the United States [1].
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http://dx.doi.org/10.1016/j.ejogrb.2014.05.040 0301-2115/ ã 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Gungorduk K, et al.. Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.05.040
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Endometrial hyperplasia is considered to be a precursor lesion of type I endometrioid AC and is linked with prolonged and unopposed estrogen stimulation of the endometrium. Atypical hyperplasia is associated with a higher risk of progression to endometrial cancer [2]. Over the last 10 years, intracellular molecular pathways, such as the extracellular signal-regulated kinase (ERK) 1/2 and phosphatidylinositol 3-kinase (PI3K)-AKT pathways, have been highlighted as attractive pathways for the investigation of carcinogenesis and cancer therapy targets [3,4]. However, the significance of these major signaling pathways in endometrial carcinogenesis as well as on the stage of disease has not been adequately addressed and remains unclear. ERK1/2 is a pro-survival factor belonging to an important subfamily of mitogen-activated protein (MAP) kinases that contributes to the regulation of mammalian somatic cell proliferation by controlling both cell growth and cell cycle progression. In normal cells, ERK1/2 induction is the key event for G1-to-S-phase progression and is related to the activation of positive regulators of the cell cycle and inactivation of antiproliferative genes [5]. Disruption of the ERK pathway is common in cancer, and a number of reports link ERK1/2 expression with certain types of drug resistance [6]. Akt is a 57 kDa serine/threonine kinase and central mediator of the PI3K pathway, with numerous downstream target molecules that mainly influence cell survival and apoptosis [4]. Akt can be activated byestrogen, insulin and various growth factors [7]. Evidence suggests that Akt is associated with the survival, proliferation, migration and infiltration of many kinds of cancer cells [8]. The goal of this study was to investigate the expression patterns of ERK1/2 and phosphorylated (p)-AKT in tissues of non-pathologic endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial AC using indirect immunohistochemistry, and also to investigate the effect of ERK1/2 and p-AKT expression patterns on prognosis in endometrial AC.
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Materials and methods
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In the present study, immunolocalization of ERK1/2 and p-AKT was examined using indirect immunohistochemistry in six different types of endometrial tissues: proliferative endometrium (PE; n = 10, 11.2%), secretuar endometrium (SE; n = 10, 11.2%), simple hyperplasia (SH; n = 15, 16.9%), complex hyperplasia (CH; n = 3, 3.4%) and atypical complex hyperplasia (ACH; n = 10, 11.2%), which were obtained from endometrial biopsies, curettage materials and hysterectomy specimens, and both early stage (International Federation of Gynecology and Obstetrics (FIGO) stage I and II) endometrioid endometrial AC (n = 21, 23.6%) and advanced stage (FIGO stage III and IV) endometrioid endometrial AC (n = 20, 22.5%), which were obtained from complete surgical staging materials; all tissues were obtained between January 1998 and December 2012. The study design was approved by the ethical committee of the Izmir Tepecik Education and Research Hospital. Proliferative and secretuar endometrium was defined according to the Noyes criteria [9]. Endometrial hyperplasia was classified according to a modified system as simple hyperplasia (SH), complex hyperplasia (CH) and atypical complex hyperplasia (ACH) [10]. Tumors were staged surgically according to the FIGO 2009 revised surgical staging system and grouped as either early stage (I and II) or advanced stage (III and IV) tumors. The histological grades of the tumors were also reviewed according to the FIGO system. Previous or concurrent cancer was not present in any of the patients. All patients with malignant disease underwent complete surgical staging, including total abdominal or radical hysterectomy, bilateral salpingo-oophorectomy, infracolic omentectomy, and systematic pelvic and para-aortic lymphadenectomy. All formalin-fixed, paraffin-embedded tissue blocks (n = 89) were retrieved from the Department of Pathology, Tepecik
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Table 1 ERK 1/2 expression in different endometrial tissue types. Tissue type
Benign group Proliferative endometrium Secretuar endometrium Simple hyperplasia Complex hyperplasia Atypical complex hyperplasia Malignant group Early stage endometrioid adenocancer Advanced stage endometrioid adenocancer
ERK1/2 expression Low expression
High expression
Negative
Moderate Strong
8 7 10 1 3
(80.0) (70.0) (66.7) (33.3) (30.0)
2 (9.5) –
Weak (20) (30) (33.3) (66.7) (50.0)
– – – – 2 (20.0)
15 (71.4)
3 (14.3)
1 (4.8)
1 (5.0)
5 (25.0)
14 (70.0)
2 3 5 2 5
– – – – –
All values are expressed as n (%).
Education and Research Hospital, and were evaluated at the Research Laboratory of Histology and Embryology, Celal Bayar University. The tissue samples were fixed in 10% formalin solution for 24 h, dehydrated in a graded ethanol series, cleared in xylene, embedded in paraffin wax and sectioned with a 5 mm thickness. The tissue blocks were chosen carefully after histologic assessment of sections stained with hematoxylin and eosin (HE; hematoxylin acc. to Gill III, cat. no. 1.05174). Additional sections from the same tissues were prepared for indirect immunohistochemical staining. Deparaffinization was achieved by incubating the sections at 60 C overnight, followed by incubating in xylene and rehydrating with a series of ethanol solutions. Sections were washed with distilled water and phosphate-buffered saline (PBS) for 10 min and then treated with 0.1% trypsin. They were delineated with a Dako PAP pen and incubated in a solution of 3% H2O2 for 5 min to inhibit endogenous peroxidase activity. After washing in PBS, sections were incubated with non-immune serum for 1 h, and then the antibody solution was removed without washing the sections. Sections were incubated with the following primary antibodies for 18 h overnight at 4 C in a humidity chamber: anti-ERK-1/2 (phosphorylated ERK-1/2) (SC-101761, 1:100 dilution, Santa Cruz Biotechnology Inc.) and anti-pAKT-1/2/3 (phosphorylated protein kinase B-1/2/3) (SC-135561, 1:100 dilution, Santa Cruz Biotechnology Inc.). An avidin-biotin-peroxidase system (Histostain Plus Bulk Kit, Invitrogen1 2nd generation LAB-SA Detection System, cat. no. 85-904, broad spectrum, Invitrogen, Camarillo, CA, USA) was used as a secondary kit. Sections were washed three times for 5 min each with PBS and then incubated with biotinylated secondary antibody followed by streptavidin conjugated to horseradish peroxidase in PBS for 30 min each. DAB (diaminobenzidine Table 2 Phosphorylated (p)-AKT expression in different endometrial tissue types. Tissue type
Benign group Proliferative endometrium Secretuar endometrium Simple hyperplasia Complex hyperplasia Atypical complex hyperplasia Malignant group Early stage endometrioid adenocancer Advanced stage endometrioid adenocancer
p-AKT expression Low expression
High expression
Negative
Moderate Strong
Weak (30) (40) (13.3) (33.3) (20.0)
– – – 1 (33.3) 1 (10.0)
– – – – –
5 (23.8) 13 (61.9)
3 (14.2)
–
1 (5.0)
19 (95.0)
7 6 13 1 7
–
(70) (60) (86.7) (33.3) (70.0)
3 4 2 1 2
–
All values are expressed as n (%).
Please cite this article in press as: Gungorduk K, et al.. Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.05.040
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chromogen, DAB Kit, Invitrogen, cat. no. 00-2014) was applied to the sections, which were then counterstained with Mayer’s hematoxylin, rehydrated in a series of ethanol solutions, and covered with Entellan1 by clearing in xylene. One section was reserved for control staining without antibody application in order to test whether or not the immunoreactivity was specific. Two observers, blinded to the pathologic information, observed the immunostained sections using a BX 40 bright-field microscope (Olympus, Tokyo, Japan) with a 200 objective and evaluated the immunohistochemical staining by scoring the percentage of cells with positive nuclei and cytoplasms within tumors independently. No statistically significant inter-observer difference was found. The intensity of immunostaining was scored as 0 (–),1 (+), 2 (++) or 3 (+++) (negative, weak, moderate and strong, respectively). For survival analysis, patients with negative or weak expression were assigned as
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low expression, and those with moderate or strong expression were assigned as high expression. Additionally, PE, SE, SH and ACH were classified as the benign group, and early and advanced stage endometrioid endometrial AC as the malignant group. For the malignant group, standart treatments were completed with adjuvant radiation (brachytherapy external beam) alone or adjuvant radiation + chemotherapy based on stage of the disease. Moreover, patients returned for a follow-up evaluation every 3 months for the first 2 years, every 6 months for the next 3 years, and annually thereafter. Computed tomography or magnetic resonance imaging was performed annually. Survival data were analyzed in December 2013. Data are presented as means standard deviations, medians (range), or percentages. The normality of continuous variable distributions was assessed using the Kolmogorov–Smirnov test.
Fig. 1. Immunohistochemical staining of ERK1/2 and p-AKT in cases of normal endometrium, simple hyperplasia, complex hyperplasia and atypical complex hyperplasia (200, original magnification). PE: proliferative endometrium; SE: secretuar endometrium; SH: simple hyperplasia; CH: complex hyperplasia; ACH: atypical complex hyperplasia.
Please cite this article in press as: Gungorduk K, et al.. Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.05.040
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Fig. 2. Immunohistochemical staining of ERK1/2 and p-AKT in cases of early and advanced stage endometrioid endometrial adenocancer (200, original magnification). EEAC: early endometrioid adenocancer; AEAC: advanced endometrioid adenocancer.
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The chi-squared test was used to analyze categorical variables, Student’s t-test was used for normally distributed variables, and the Mann–Whitney U-test was used for non-normally distributed variables. Relative risks (RRs) with 95% confidence intervals (CIs) were calculated. Factors identified as potential risk factors in unadjusted analyses (P < 0.05) were used to create a logistic regression model with lymph node metastasis as the dependent variable. The survival analysis was based on the Kaplan–Meier method, and the results were compared using the log-rank test. Progression-free survival (PFS) was defined as the time elapsed from the date of primary surgery to that of detection of recurrence or the latest observation. Overall survival (OS) was defined as the time elapsed from the date of primary surgery to that of death or the latest observation. Cox regression analysis was used to determine factors affecting survival, and the results were presented as hazard ratios (HRs) with 95% CIs. All statistical analyses were performed using MedCalc software version 11.5 for Windows, Mariakerke, Belgium. A P-value of <0.05 was considered significant. Results This study was performed on archival material consisting of 89 endometrial tissues. The mean age of the patients was 46.5 4.1 years for PE, 40.3 6.0 for SE, 45.2 5.2 for SH, 51.3 9.2 for CH, 56.7 9.7 for ACH, 58.3 11.7 for early stage endometrioid endometrial AC and 60.4 11.6 for advanced stage endometrioid endometrial AC. The FIGO stages of the AC cases were as follows:
12 cases with stage IA, eight with stage IB, one with stage II, five with stage IIIA, nine with stage IIIC1, four with stage IIIC2, one with stage IVA and one with stage IVB. The FIGO histological grades of 41 tumors were classified as follows: grade I in 14 EAC cases (34.1%), grade II in 14 (34.1%) and grade III in 13 (31.7%). The ERK1/2 and p-AKT immunohistochemical staining scores for normal, hyperplastic and malign endometrium are shown in Tables 1 and 2. In the malignant group, 23 of 41 patients (56.1%) showed high ERK1/2 and p-AKT expression (++ and +++), whereas only two of 48 patients in the benign group (4.2%) showed high ERK1/2 and p-AKT expression (P < 0.0001 and P < 0.0001, respectively). Figs. 1 and 2 demonstrates the expression of ERK1/2 and p-AKT in six different endometrial tissues. Logistic regression showed a relationship between p-AKT expression and lymph node metastasis. Expression of p-AKT was significantly higher in women with positive lymph nodes (OR 9.0; 95% CI 1.2–100.0; P = 0.03). By contrast, such a relationship was not found for ERK1/2 expression (OR 1.1; 95% CI 0.3–3.7; P = 0.83). Follow-up data were available for all patients in the malignant group. The median duration of follow-up was 52 months (range 16–168 months). Six patients (14.6%) died from their disease during follow-up. Results of Kaplan–Meier analyses for PFS and OS are shown in Table 3. Advanced FIGO stages (III and IV), higher expression of p-AKT (++/+++) (Fig. 3a), higher grade, positive lymphovascular invasion, myometrial invasion 1/2 and tumor size 2 cm were adverse prognostic factors for PFS. Moreover, advanced FIGO stages (III and IV), higher expression of p-AKT
Table 3 Results of univariate analyses of progression free and overall survival of patients with endometrioid endometrial adenocancer. Variable
Age (<60 vs. 60 years) Stage (I/II vs. III/IV) Grade (I vs. II vs. III) ERK1/2 expression (low vs. high) p-AKT expression (low vs. high) Myometrial invasion (<1/2 vs. 1/2) Lymphovascular invasion Tumor size (<2 cm vs. 2 cm) Menopause
Progression free survival
Overall survival
Hazard ratio
95%CI
P
Hazard ratio
95% CI
P
0.8 0.4 2.0 0.7 0.3 0.4 0.4 0.5 0.8
0.4–1.5 0.1–0.8 1.2–3.3 0.3–1.4 0.1–0.8 0.2–0.9 0.2–0.9 0.2–1.0 0.3–1.8
0.50 0.002 0.003 0.36 0.0009 0.01 0.01 0.04 0.62
0.8 0.4 1.9 0.6 0.4 0.5 0.5 0.6 0.7
0.4–1.6 0.1–0.9 1.1–3.1 0.3–1.3 0.1–0.8 0.2–1.0 0.2–1.0 0.3–1.1 0.3–1.6
0.56 0.004 0.007 0.24 0.001 0.04 0.04 0.10 0.46
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Fig. 3. (a and b) Progression-free and overall survival curves according to p-AKT expression; (c and d) progression-free and overall survival curves according to ERK1/2 expression.
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(Fig. 3b), higher grade, positive lymphovascular invasion and myometrial invasion 1/2 were significantly associated with poor OS. However, ERK1/2 expression was not associated with PFS or OS (Fig. 3c and d). Table 4 Correlation between phosphorylated (p)-AKT expression and clinicopathological factors in patients with endometrioid endometrial adenocancer. Low (negative/ weak) (n = 18) Age (years) <60 60 Stage Early (I/II) Advanced (III/ IV) Grade I II III Myometrial invasion <1/2 1/2 LVI Negative Positive Tumor size (cm) <2 2 Menopause Yes No
High (moderate/ strong) (n = 23)
P value
0.21 11 (61.1) 7 (38.9)
Odds ratio (95% CI)
1.5 (0.8–3.0)
9 (39.1) 14 (60.9) <0.0001 –
18 (100) –
3 (13) 20 (87) <0.0001 –
12 (66.7) 6 (33.3) –
2 (8.7) 8 (34.8) 13 (56.5) <0.0001 3.7 (1.5–9.0)
14 (77.8) 4 (22.2)
4 (17.4) 19 (82.6)
10 (55.6) 8 (44.4)
5 (21.7) 18 (78.3)
9 (50) 9 (50)
8 (34.8) 15 (65.2)
14 (77.8) 4 (22.2)
21 (91.3) 2 (8.7)
0.04
1.7 (1.0–3.0)
0.35
1.3 (0.7–2.2)
0.37
LVI: lymphovascular invasion. All values are expressed as n (%).
1.1 (0.8–1.5)
We also assessed the correlation between p-AKT expression and clinicopathological characteristics (Table 4) using the chi-squared test. There were correlations of p-AKT expression with FIGO stage, depth of myometrial invasion, pathological grade and lymphovascular invasion.
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In the present study, we found advanced FIGO stage, higher expression of p-AKT, higher grade, positive lymphovascular invasion, tumor size 2 cm and myometrial invasion 1/2 to be adverse prognostic factors for PFS in endometrioid endometrial AC. We additionally found that advanced stage (III/IV), higher expression of p-AKT, higher grade, positive lymphovascular invasion, and myometrial invasion 1/2 had statistically significant associations with poor OS, but we did not find any association between ERK1/2 expression and PFS or OS. Several mutations have been implicated in endometrial carcinogenesis, the most well-known being PTEN and KRAS mutations [11]. Mutation of PTEN results in activation of the PI3K pathway, and mutation of KRAS causes continuous activation of ERK –MAPK pathways [12]. The activated MAPK pathway results in phosphorylation of ERK1/2. Normally, activated ERK1/2 is important in cell survival, differentiation and proliferation [5,13,14]. In the present study, ERK1/2 expression was found to be higher in endometrial cancer when compared with normal, non-atypical hyperplastic and atypical hyperplastic endometrium. Wang et al. [15] also reported higher ERK expression in endometrial cancer than in normal endometrium. They also found significantly higher ERK expression in advanced stage endometrium cancer. In contrast, in a study by Mizumuto et al. [16], higher ERK1/2 expression was found to be significantly associated with favorable prognosis in patients with endometrial cancer. In the present
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study, even though we found higher ERK1/2 expression in endometrial cancer, we did not find any statistically significant correlation between PFS or OS in endometrial cancer and ERK1/2 expression. Akt, a serine/threonine kinase, is activated by PI3K and plays an important role in cell survival and apoptosis inhibition [4,17]. IGF-I, insulin, estrogen, TNF and platelet-derived growth factors are growth factors, cytokines and steroids that activate PI3K and thereby activate Akt [7,18–22]. Akt expression plays an important role in cancer prognosis by inhibiting cell adhesion and increasing invasiveness [23]. Sun et al. [24] and Vilgelm et al. [25] reported that the activated Akt pathway also activates estrogen receptoralpha and creates a hyperestrogenic environment, thereby increasing neoplastic transformation. Increased expression of Akt in endometrial cancer was reported in two previous studies [25,26]. Pant et al. [27] showed that inhibition of Akt signaling resulted in increased apoptosis in Ishikawa cells and a reduction in tumor size in nude mice. Zhao et al. [28] reported that PI3K/Akt and ERK1/2 play roles in endometrial cancer proliferation and operate independently. They also showed that stromal cell-derived factor (SDF)-1a, an estrogen-induced chemokine, induces cell growth by activating Akt and ERK1/2 pathways in two types of endometrial cancer cells (Ishikawa and HEC-1A cell lines) in a dose-dependent manner [28]. In a study by Mori et al. [26] from 2007, pathologic specimens from 63 endometrial cancer patients were evaluated for p-AKT expression. They scored p-AKT expression in the same way as in our study: no, weak, moderate or strong. Although they did not detect a statistically significant difference in p-AKT expression between early and advanced stage endometrial cancer, in the present study, we did detect significantly higher p-AKT expression in advanced stage endometrial cancer compared with early stage endometrial cancer. Mori et al. also failed to find any correlation between p-AKT expression and lymph node metastasis, myometrial invasion depth or grade. In contrast, we found a relationship between p-AKT expression and lymph node metastasis. Higher p-AKT expression was detected in lymph node-positive women. We also found higher p-AKT expression in the myometrial invasion 1/2 group than in the myometrial invasion 1/2 group. Although Mori et al. [26] reported a correlation between low p-AKT expression and low relapse-free survival rates in endometrial cancer, we found decreased survival rates in patients with higher p-AKT expression in the present study. Consistent with the present study, Abe et al. [29] reported poor prognosis in endometrial cancer patients with higher p-AKT expression. While they did not find any difference in total p-AKT expression among normal endometrium epithelium, hyperplasia and endometrial cancer, we found significantly higher p-AKT expression in endometrial cancer than in benign pathologies such as proliferative endometrium, secretuar endometrium and hyperplastic tissues. In summary, although, the potential limitations of our study are its retrospective nature and small sample size for each groups, some important conclusions can be drawn. First, we found higher p-AKT expression to be a marker of poor prognosis in endometrial adenocancer patients, in that higher p-AKT expression was found to be associated with poorer OS and PFS. Second, p-AKT is an important key point in endometrial carcinogenesis and a promising prognostic marker in the management of patients with endometrioid endometrial AC. Further clinical trials including large samples are required to evaluate its role as a prognostic marker.
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Conflict of interest
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The authors declare no conflicts of interest.
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Please cite this article in press as: Gungorduk K, et al.. Immunolocalization of ERK1/2 and p-AKT in normal endometrium, endometrial hyperplasia, and early and advanced stage endometrioid endometrial adenocancer and their prognostic significance in malignant group. Eur J Obstet Gynecol (2014), http://dx.doi.org/10.1016/j.ejogrb.2014.05.040
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