The prognostic value of estrogen receptor beta and proline-, glutamic acid- and leucine-rich protein 1 (PELP1) expression in ovarian cancer

Paraffin embedded ovarian tumor tissue from primary surgery was used from 63 patients with EOC FIGO stage I-IV, receiving cytoreductive surgery at the Department of General Gynaecology and Gynaecological Oncology, Medical University of Vienna, Austria, between 1996 and 2001. In a second step, a validation set was used comprising 86 EOC patients of only serous histology, receiving cytoreductive surgery at the Medical University of Vienna, Austria, between 2004 and 2010. An informed consent according to the criteria of the Medical University of Vienna was obtained from all patients. Approval for this study was obtained by the institutional review board of the Medical University of Vienna (IRB-no.:266/2010). Patients were treated according to standards of the present institution with upfront surgery and adjuvant platinum–based chemotherapy. Surgical staging according to FIGO guidelines was performed, including hysterectomy, bilateral salpingo-oophorectomy, pelvic and/or paraaortic lymphadenectomy, appendectomy, omentectomy and cytoreductive procedure in order to resect all gross tumor. All patients with tumor stages FIGO Ic to III and all patients with clear cell carcinoma received a platinum-based chemotherapy. Patients wishing to preserve fertility and tumor stage FIGO Ia were treated with conservative surgery (unilateral salpingo-oophorectomy) and full surgical staging including washings, omentectomy, appendectomy, node biopsies and a thorough abdominal exploration with biopsies of all suspicious areas. Residual tumor load was defined as negative, if macroscopically absent. Post-therapeutically all patients were followed up four times annually, including pelvic examination, abdominal ultrasound examination, and serum tumor marker evaluation. Overall survival was the time interval between diagnosis and death. Overall observation time was the time interval between diagnosis and last contact, defined as death from the disease or last follow-up. Recurrent disease was defined as at least a twofold increase in the nadir serum CA-125 level after first-line chemotherapy or radiological diagnosis. Patients without recurrence, cancer progression or death were censored at the time of last follow-up. Experienced gynecological oncologists and an experienced pathologist performed the clinical and histopathological evaluation and the evaluation of response to first-line treatment.

Paraffin embedded tissue-blocks were processed using standardized procedures. Tissue microarrays (TMAs) was assembled by taking three core needle ‘biopsies’ from defined tumor regions in the preexisting paraffin-embedded tissue blocks, using techniques and an apparatus developed by Beecher Instruments Inc., Micro-Array Technology (Sun Prairie, WI, USA). Separate TMAs were constructed for the test and validation set. Immunohistochemistry (IHC) procedures were performed at room temperature. Samples were deparaffinized, rehydrated and treated with 3% H2O2 for 10 minutes to quench endogenous peroxidase. For ERbeta antigen heat retrieval was performed using heat-induced epitope retrieval in DakoCytomation Target Retrieval Solution (No. S1700, DAKO, Denmark), and for ERalpha and PELP1 heat-induced epitope retrieval was performed using citrate buffer (Citra-BioGenex No. HK 087-5K). The sections were incubated at 4°C overnight with primary antibodies (ERalpha, 1:50, clone 1D5, mouse IgG1, Dako, Denmark; ERbeta1, 1:20, clone PPG5/10, mouse IgG2a, Dako, Denmark; PELP1, 1:500, polyclonal rabbit, No. IHC-00013, Bethyl Laboratories, USA). As positive controls, FFPE sections of ER positive human breast adenocarcinoma were used. Negative control mouse and rabbit isotypes were used as negative controls. Slides were incubated for 25 minutes with a biotinylated secondary antibody (Link, No. K0673, Dako, Denmark), followed by incubation with Streptavidin Peroxidase-HRP (25 Minutes, No. K 0673, Dako, Denmark). The slides were stained with diamino-benzidine (DAB Chromogen 1:50 in DAB Substrate Buffer, K0673, Dako, Denmark) for 2 minutes. For counterstaining the slides were dipped into hematoxylin for 25 seconds. Intra-nuclear distribution of PELP1 was determined by immunofluorescence staining. The fluorescence labeled secondary antibody, goat anti-rabbit (1:5000; Invitrogen, AlexaFluor® 488 fragment of goat anti-rabbit IgG (H+L)) was used besides DAPI for nuclear counterstaining.

The intensity patterns and nuclear positivity of staining were analyzed by two independent co-workers, including a gynecological pathologist (RH), applying a semi-quantitative scale of ImmunoReactive Score (IRS) calculated by multiplication of the number of positively labeled cells (4 percentage groups) with the intensity of the staining reaction (3 grades) [19]. The intensity of reaction was scored as negative (intensity IRS 0–2, no reaction, and IRS 3–4 showing a very weak reaction of staining with <10% positive cells) or positive (IRS 6–8 with a moderate reaction and 10 to 50% positive cells; IRS 9–12 with a strong reaction and 51% to 80% positive cells). Statistical analyses were performed using SPSS software version 19 (IBM Corporation, Armonk, New York, United States). P-values <0.05 were considered statistically significant. Associations between ERalpha-expression, ERbeta-expression, PELP1-expression, and clinicopathological parameters were calculated using Pearson’s χ ² or Fisher’s exact tests as appropriate. Impact of the potential new prognostic factors on disease-free survival (DFS) and overall survival (OS) was determined by univariate and multiple Cox proportional-Hazards model analyses, whereby in the test set, only the patients receiving chemotherapy according to standardized protocols were included in these analyses (n=50). In the validation set, only patients receiving chemotherapy were included. Survival analyses were performed for all three parameters and the combination pattern ERbeta+/PELP1+ versus ERbeta+/Pelp1-, ERbeta-/PELP1+ and ERbeta-/Pelp1- tumors as well as for the combination pattern ERalpha+/PELP1+ versus ERalpha+/Pelp1-, ERalpha-/PELP1+ and ERalpha-/Pelp1- tumors.

The characteristics of the patients included in the study show a typical heterogeneous ovarian cancer population and are depicted in Table 1. Mean age of the EOC patients at time of cytoreductive surgery was 58.3 years (±13.9years). The mean observation period was 57.8 months. Within the observation period, 36 patients died (57.1%) and 36 patients (57.1%) experienced tumor recurrence. 50 patients (79.4%) received carboplatin-paclitaxel based standard chemotherapy. Out of these 50 patients, 46 women (73.0%) showed initial response to chemotherapy and a total of 34 women died (68.0%). The characteristics of the validation set are likewise presented in Table 1. In this validation set the mean observation time was 51.3 months and a total of 45 (52.3%) patients died.

Of the 63 cancer tissues, 48 (76.2%) of patients were found to express nuclear staining of PELP1. Out of the 48 patients classified as PELP1 positive, a moderate PELP1 expression was found in 24 (38.1%) tissues and a strong expression was observed in 23 (36.5%) of the samples. Representative immunohistochemical examples of PELP1 positive and negative stainings in ovarian cancer tissue are shown in Figure 1A-D. Using immunofluorescence, intra-nuclear distribution of PELP1 was analyzed. As depicted in Figure 2, an intensive PELP1 staining within the nucleoli of the tumor cells can be observed. Further examination of histological subtypes revealed expression of PELP1 in all included subtypes of ovarian cancer. All undifferentiated carcinomas were PELP1 positive, as was the majority of serous and endometrioid tumors (85.7% and 86.7%, respectively), whereas the majority of mucinous tumors (62.5%) showed no nuclear PELP1 expression (p=0.02, Fisher’s exact). No significant difference of PELP1 expression regarding age (<55 years vs > 55 years), FIGO stage, grade or residual tumor load after cytoreductive surgery could be observed.

In a next step, we analyzed the coexpression of PELP1 together with ERalpha and ERbeta. A significant association between PELP1 expression and nuclear ERbeta staining was found (p=0.01, Fisher’s exact), whereas no significant association between PELP1 and nuclear ERalpha staining was observed (p=0.3, Fisher’s exact). Expression of PELP1 and both ERs was observed in 10 patients (15.8%).

Table 2A shows the association between PELP1, ER-alpha, ERbeta and survival. In a univariate analysis, ovarian cancer patients with PELP1 expressing tumor tissue had a better OS and DFS (p = 0.04, p = 0.004; respectively) compared to patients without PELP1 expression. ERbeta and ERalpha had no significant univariate influence on survival. Interestingly, the coexpression of PELP1 and ERbeta turned out to be the most relevant prognostic factor in univariate survival analysis (DFS: p=0.03, OS: p=0.02). In a univariate analysis, the clinicopathological parameters FIGO-stage (p < 0.001 and p < 0.001) and residual tumor (p < 0.001 and p < 0.001) were associated with DFS and OS, respectively.

In a next step, we added the coexpression of PELP1 and ERbeta and all clinicopathological parameters in a Cox proportional-hazards regression model. Multiple Cox regression analysis revealed that ERbeta+/PELP1+ tumors had the strongest independent impact on survival, with a significantly longer DFS (HR 0.3 [0.1-0.7], p = 0.004) and OS (HR 0.3 [0.1-0.7], p = 0.005).

To provide more profound and histotype specific survival data we have additionally performed a validation analysis in a validation set of 86 EOC with only serous histology. In univariate and multivariate analysis, ERbeta+/PELP1+ expression had no significant influence on survival in this validation set of only serous histology. Survival data is presented in Table 2B.