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Triple-high expression of phosphatase and tensin homolog (PTEN), estrogen receptor (ER) and progesterone receptor (PR) may predict favorable prognosis for patients with Type I endometrial carcinoma

Endometrial carcinoma (EC) is the most common malignant tumors in female derived from the endometrial epithelium. Several previous studies have described estrogen receptors (ER), progesterone Receptor (PR) and phosphatase and tensin homolog (PTEN) are associated with clinicopathological factors and prognosis in EC patients. However, during EC patients follow-up, we found that some EC patients with down-regulation of PTEN, but up-regulation of ER or PR , and some EC patients with down-regulation of ER or PR, but up-regulation of PTEN also had a poor prognosis. Therefore, to reveal the prognosis of EC patients with different phenotypes based on PTEN, ER and PR expression, 120 cases formalin-fixed paraffin-embedded EC tissues and 543 cases uterine corpus endometrial carcinoma (UCEC) patients from the cancer genome atlas (TCGA) UCEC datasets were analyzed. Results showed that EC tissues can be classified to PTENLERLPRL, PTENHERLPRL, PTENHERHPRH, PTENLERHPRH, PTENHERHPRL, PTENHERLPRH, and PTENLERHPRL phenotypes basing on IHC analysis. Additionally, EC patients with PTENLERLPRL showed high malignancy, while patients with PTENHERHPRH showed low malignancy. Therefore, combined detection of PTEN, ER, PR may help identify a small subset of EC with more aggressive behavior and may aid in risk stratification.


Introduction
Endometrial carcinoma (EC) is the most common malignant tumors in female derived from the endometrial epithelium. Recently, the EC incidence is increased, while the 5-year survival rate is decreased [1]. The etiology of EC is not yet clear. Experimental and epidemiologic evidence showed that the risk factors of EC including BMI≥25kg/m 2 , hypertension, diabetes, smoking, taking tamoxifen, family history of Ivyspring International Publisher cancer and non-pregnant women [2,3]. EC can be broadly classified into two types. Majority (~80%) of EC patients are of Type I endometrioid histology, up to 15% are Type II EC patients, primarily serous carcinomas [3][4][5]. Most of Type I EC patients present with low-grade and early-stage disease and have a favorable prognosis. According to the previous study, 74-91% of International Federation of Gynecology and Obstetrics (FIGO) stages I-II type I EC patients have a 5-year OS time [6]. However, 10-20% of early stages I-II and 50-70% of advanced stage III-IV type I EC patients will recur after primary treatment [7]. Previous studies have revealed that clinicopathological parameters such as histological grade, FIGO clinical stage, myometrial tumor invasion, tumor size, lymph node metastasis, lymphovascular space invasion, and the patients' age and race has prognostic effect in type I EC patients [6,8,9]. However, these factors are usually obtained postoperatively and have proven to be insufficient to predict recurrence and estimate survival time. Therefore, it is necessary to identify more effective prognostic predictors to identify preoperative high-risk type I EC patients.
Several previous studies have described estrogen receptors (ER), progesterone Receptor (PR) and phosphatase and tensin homolog (PTEN) are associated with clinicopathological factors and prognosis in EC patients [10][11][12][13][14][15][16]. Both of ER and PR are members of the nuclear receptor family that release relevant related ligand-activated transcription factors to regulate cell gene expression through activation and suppression of transcription [17][18][19]. Low expression or deletion of ER and PR associated with increased malignancy, invasion, and non-hormone-dependent tumor transformation [20]. The absence of PR and ER indicates poor prognosis, poor histologic type, higher histological grade, higher frequency of lymph node metastasis, and higher clinical staging at diagnosis [18,19]. PTEN located on chromosome 10q23.3, is known as a vital tumor suppressor gene. Recently, researchers found that PTEN has low expression during the development and progression of EC, and implies poor prognosis, higher histological grade and clinical staging, and shorter survival [21,22]. Especially, the mutation rate of PTEN in EC is about 34% ~ 55%, which is higher than the mutation rate of K-ras and P53 [23,24]. However, during follow-up of EC patients, we found that some EC patients with down-regulation of PTEN, but up-regulation of ER or PR, and some EC patients with down-regulation of ER or PR, but up-regulation of PTEN also had a poor prognosis. Therefore, to reveal the prognosis of EC patients with different phenotypes based on PTEN, ER and PR expression, 120 cases formalin-fixed paraffin-embedded EC tissues and 543 cases UCEC patients from TCGA-UCEC datasets were analyzed.

Immunohistochemistry (IHC)
Tissue sections (4-μM) were prepared from formalin-fixed paraffin embedded tissue blocks and then subjected to stain with hematoxylin and eosin as our previously described [27][28][29]. The results were assessed by two pathologists to demonstrate the presence of tumor and the proportion of tumor cells in each section. Tissue sections were subjected to incubate in 0.3% Hydrogen peroxide solution for 10 min at room temperature to block the endogenous peroxidase activity, and then washed by Phosphate Buffered Saline (PBS) solution. Antigenic epitopes were next retrieved by heating for 2 min in 10 mmol/L citrate buffer (pH 6.0). The slides were then first incubated with antibodies against PTEN (ZSGB-BIO, China), ER (ZSGB-BIO, China), PR (ZSGB-BIO, China), Ki-67 (Thermofisher,USA), p53 (Thermofisher,USA), CEA (Thermofisher,USA) and CA125 (Thermofisher,USA) for 30 min at room temperature. Next, the sections were washed with PBS for three times and followed by a goat anti-rabbit and mouse IgG-HRP (Kit-0015, Maixin Biotech, Fuzhou, China) secondary antibody for one hour at room temperature at 1:500 dilutions. The slides were visualized using DAB Detection Kit (Enhanced Polymer) (Kit-0015, Maixin Biotech, Fuzhou, China) and chromogenic reaction was controlled under a microscope (Nikon). After immunostaining, sections were immersed into hematoxylin for nuclear staining, then dehydrated through gradient concentrations of ethanol, cleared with xylene, and covered with neutral balsam.

Score of immunohistochemical sections
The score of immunohistochemical sections were assessed by two pathologists in a blinded fashion to the clinical status of the patients, as our previously reported [27][28][29]. The immunoreactive area (percentage of positive staining cells) and intensity scores of PTEN, ER and PR were evaluated. In brief, according to the immunoreactive area, 0-5% scored 0, 6-25% scored 1, 26-50% scored 2, 51~75% scored 3 and more than 75% scored 4. If the scores was 0~2, the section was defined as low expression, and if the final scores was 3~6 was defined as high expression. Two specialists who were blinded to the clinical status of the patients evaluated the staining independently.

mRNA expression
All available mRNA expression data were collected from 543 UCEC tumors and 23 adjacent non-EC tissues in TCGA (https://cancergenome.nih .gov/). According to the average value of mRNA expression of each gene, EC patients were divided into 8 phenotypes ( , according to high and low PTEN, PGR and ESR1 mRNA expression.

Statistical analysis
Statistical analysis was performed using SPSS 19.0 Software (SPSS, Chicago, IL, USA). Chi-square test or Fisher's exact test was employed for analysis the differences of categorical variables. For survival analysis, overall survival (OS) or disease-free survival (DFS) was calculated using Kaplan-Meier method and evaluated by log-rank test, as our previously reported [30,31]. Multivariate analysis was based on the Cox proportional hazard regression model. A p value <0.05 was considered with statistical significance. datasets and found that PTEN mRNA expression was down-regulated in EC tumor tissues compared with adjacent normal tissues (ANT) (Fig. 1A). Prognostic factors of EC include the presence of ER and PR. We also found that the mRNA expression of PGR encoding PR, but not ESR1 encoding ER, down-regulated in EC tissues compared with ANT in TCGA-UCEC datasets (Fig. 1A). Furthermore, correlation analysis showed that there was a significant correlation among PTEN, PGR and ESR1 mRNA expression (Fig. 1B), and they all associated with the prognosis of EC (Fig. 1C). This was also consistent with the results reported in most previous studies [18,19,21,22]. To further reveal the relationship between differential expression of PTEN, ER and PR, and EC prognosis, EC patients were divided into 8 phenotypes (PTEN L ESR1 L PGR L ,

Classification of EC tissues based on PTEN, ER and PR expression
and PTEN H ESR1 L PGR H ), according to high (H) and low (L) PTEN, PGR and ESR1 mRNA expression (Fig. 1D). Additionally, we collected 120 formalin-fixed paraffin-embedded EC tissues and examined PTEN, ER and PR expression by IHC analysis (Fig. E)

EC patients with triple-high expression of PTEN, ER and PR showed a lower degree of malignancy and proliferative activity
To reveal the proliferative activity of EC patients with different phenotypes, Ki-67 and p53 were detected by IHC analysis (Fig. 2A). Results showed that Ki-67 was low expressed in EC patients with PTEN H ER L PR L and PTEN H ER H PR H phenotype, while high expressed in EC patients with PTEN L ER L PR L phenotype (Fig. 2B). Indeed, based on TCGA-UCEC datasets, we also found that EC patients with PTEN L ESR1 L PGR L phenotype had high expression of Ki67 mRNA, while patients with PTEN H ESR1 H PGR H phenotype had low expression of Ki67 mRNA (Fig.  2C). Simultaneously, we also found p53 was low expressed in EC patients with PTEN H ER H PR H phenotype, and TP53 (encoding p53) mRNA was low expressed in EC patients with PTEN H ESR1 H PGR H phenotype (Fig. 2D, 2E) (Fig. 2F). These results suggest that EC patients with triple-high expression of PTEN, ER and PR showed a lower degree of malignancy and proliferative activity.

EC patients with triple-high expression of PTEN, ER and PR showed a lower expression of CA125
Carcinoma-associated antigens are considered to be useful markers for the detection of recurrent disease in EC patients [37]. Herein, we detected CA125 and CEA expression in EC tissues by IHC analysis (Fig. 3A) (Fig. 3F). These studies suggest that there are differences in CEA and CA125 expression in EC patients with different phenotypes. In particular, EC patients with triple-high expression of PTEN, ER and PR showed low expression of CA125, this was positively correlated with CEA.

Triple-high expression of PTEN, ER and PR may predict favorable prognosis in EC patients
Subsequently, we analyzed the relationship between patients with different EC phenotypes and prognosis, and found that EC patients with  (Fig. 4B). These studies suggest that triple-high expression of PTEN, ER and PR may predict favorable prognosis in EC patients.

Discussion
EC is a type of female reproductive malignant tumor, the incidence of which is generally 20~30%. Exploring effective molecular prognosis targets for EC patients has become a hot topic in current research. In 1991, Raju KS, et al -3β) [55] can serve as a poor prognostic marker for EC patients. Herein, we also found that decrease of PTEN, PGR and ESR1 mRNA expression were associated with poor prognosis. However, it was not accurate during follow-up. Especially, we found that some EC patients with decrease of PTEN, but up-regulation of ER or PR, and some EC patients with decrease of ER or PR, but up-regulation of PTEN also have a poor prognosis (data not shown).  These characteristics can also be observed when analyzing PTEN, PGR and ESR1 mRNA expression using the TCGA database. Indeed, we found that EC patients can be divided into different phenotypes based on PTEN, ER and PR expression. EC patients PTEN is a tumor suppressor gene with double phosphatase activity discovered in 1997. It is a homologous gene of phosphatase and tensin which often associated with the deletion of chromosome allele 10q site [23]. PTEN plays an important role in the inhibition of tumorigenesis by regulating PTEN/PI3K/AKT, PTEN/FAK/P130cas, PTEN/ ERK, p53/MDM2, and FRAP/mTOR signaling pathways. These signal pathways through its lipid phosphatase and protein phosphatase activity, induce apoptosis, block cell cycle, to inhibit tumor cell invasion and metastasis and tumor angiogenesis [13,14]. At present, abnormal expression of PTEN in EC patients has been confirmed by most researchers. In the previous study, we also found that the loss rate of PTEN in EC patients was 58.8% (60/102), and the loss expression of PTEN was closely related to the grade of histological in EC patients, suggesting that the absent expression of PTEN plays an important role on EC occurrence and development [56,57]. Additionally, the expression of ER and PR may be related to the increase of malignancy, the increase of invasion and the transformation of non-hormone-dependent tumors in EC patients [10][11][12][13][14][15][16] Therefore, combined detection of PTEN, ER, PR may help identify a small subset of EC with more aggressive behavior and may aid in risk stratification.
In summary, our findings indicate that EC patients with triple-high expression of PTEN, ER and PR showed a lower degree of malignancy and proliferative activity and predicted a favorable prognosis. However, the physiological and pathological features of EC patients with different phenotypes basing on PTEN, ER and PR expression still require further investigation.