Pre-treatment Serum Osteoprotegerin, and P53 Protein Levels and Urine Telomerase as prognostic factors affecting survival in Egyptian Bladder Cancer Patients

Rania Bakry, MD1, Mohamed I. El-Sayed, MD2*, Hisham M. Hamza, MD3, Khaled H. Hassan, MSc4

Departments of Clinical Pathology1, Radiotherapy2, and Surgical Oncology3, South Egypt Cancer Institute (SECI), Assiut University, and Department of Biochemistry4, .Faculty of Pharmacy (Boys), AI-Azhar University, Egypt

Corresponding author: Mohamed I. El-Sayed, MD, Department radiotherapy, South * .Cancer Institute (SECI), Assiut University, Egypt Abstract

Background: Bladder cancer constitutes 30% of all cancer patients, for whom a .non-invasive marker is, required for the follow-up and diagnosis and follow up

Aim: Evaluation of the potential prognostic significance of serum osteoprotegerin .(OPG), and pS3 protein and urine telomerase in patients with bladder cancer

Methods: For all patients, serum levels of OPG and pS3 protein were determined using ELISA and urine telomerase by PCR ELISA technique. Patients were then assigned into group1 (cystectomy and adjuvant radiotherapy) and group2 (TUR and .(chemoradiation

Results: Serum OPG and p53 levels and urine telomerase were significantly higher in bladder cancer patients than in healthy individuals. (p < 0.0001). High serum OPG was associated with significantly lower OAS and DFS rates (p=0.001), and was correlated with advanced T stages (p<0.0001), high grades (p<0.0001) and presence of disease relapse (p=0.001).Serum p53 and urine telomerase did not .show prognostic significance

Conclusions: Serum OPG level may be used as a diagnostic tool and a prognostic variable in patients with muscle invasive bladder cancer. Future trials are needed to .elucidate its therapeutic role in those patients

.Key words: Tumour markers, Bladder cancer, Overall survival

:Introduction

Bladder cancer constitutes 30% of all cancer patients attended at NCI in Egypt [1], and has diverse biological characteristics identified by molecular alterations that results in uncontrolled cell proliferation, and metastasis with the result that affecting the patient's prognosis[2]. Surveillance is required for patients with noninvasive bladder cancer. Currently this follow up is done by cystoscopy (which is an invasive procedure), and urine cytology, every 3 months during the first two years, every 6 months during third and fourth years and every year thereafter, until local recurrence .[is detected [3

A non-invasive marker is, therefore, required for the follow-up and diagnosis of bladder cancer, with early detection may decrease costs and decrease morbidity and mortality [4]. Osteoprotogerin, that is a cytokine of the tumor necrosis factor receptor super-family and inhibits osteoclastogenesis [5, 6], may have clinical usefulness as a .[prognostic marker for patients with bladder carcinoma [7

Destruction of tumor cells by immune system is prevented by development of resistance to TRAIL (TNF-related apoptosis inducing ligand)- induced toxicity through expression of functional receptors or overexpression of decoy receptors on tumor cell membrane [8]. Osteoprotogerin, being a soluble decoy receptor for TRAIL it protects tumor cells from TRAIL mediated apoptosis as it binds to and inhibit the activity of TRAIL, and as a result it may contribute to tumor progression [7]. The tumor suppressor p53 plays a key role in regulating cell cycle progression and apoptosis under genotoxic conditions. The p53 gene mutations are the most common genetic defect in human cancers [9]. Mutated p53 protein often results in a prolonged half-life compared with the wild-type p53. It accumulates in the cell nuclei and can be detected by IHC [10]. Telomeres are regions located at the end of human chromosomes. Their function is to stabilize and protect chromosomes [11, 12]. With each cell cycle, the ends of the telomeres shorten, until a critical length is reached after which cell division leads to breakdown of the telomere. Telomerase is a ribonucleoprotein enzyme that adds telomere repeats to maintain telomere length. Telomerase is inactivated in normal human epithelial tissue, but is reactivated in neoplasia. The Telomeric Repeat Amplification Protocol (TRAP) assay measures enzymatic activity of telomerase [11]. The aim of this study was to evaluate the role of pretreatment serum OPG and p53 levels and urine telomerase as prognostic factors in patients with muscle invasive bladder cancer.

:Patients and methods

This prospective study was carried out in radiation therapy and clinical pathology departments, SECI, Assiut University, in period between June 01, 2007 and June 01, 2014, and included patients with non metastatic muscle-invasive bladder cancer (clinical stage T2-T4 N0 M0). The study protocol was approved by the local institutional review board at South Egypt Cancer Institute, Assiut University. Each patient, was subjected to evaluation by history taking and physical examination, routine laboratory investigations, abdomino-pelvic CT scans with contrast, chest X- ray, and if indicated, bone scan. Histopathologic diagnosis was obtained from the bladder mass by cystoscopy and biopsy. According to billharzial cytology and World Health Organization System (2004) for tumor grading, was subdivided as Non- Bilharzial Low Grade Bladder Cancer (NBLG), Bilharzial Low Grade Bladder Cancer (BLG), Non-Bilharzial High Grade Bladder Cancer (NBHG), Bilharzial High Grade Bladder Cancer (BHG).

All patients and healthy individuals were subjected to laboratory assay as follows; Serum was isolated from blood samples, and used for OPG and P53 detection by ELISA using an available kit (Ray Biotech, Inc. USA) and (Bender Med-systems .USA) respectively on Evolis machine by Biorad USA

Urine samples obtained from the patients and healthy individuals were collected for Telomerase activity measurement by PCR-ELISA technique by thermal cycler PTC 100, Germany and Evolis Bio-rad, USA) using telomerase repeat amplification protocol (TRAP) according to the kit purchased from Roche Germany. Telomeric repeats (TTAGGG) was added to the 3' end of the biotin labelled synthetic P1-TS primer. These elongation products, as well as the internal standard (IS) in the same reaction vessel, were amplified by PCR using the primers P1-TS and P2. . The PCR products were split into two aliquots sample and internal standard. Denaturated and hybridized products were added to the streptavidin coated plates; with digoxygenin (DIG) labelled detection probes (P3 T, P3 IS for the sample and internal standard, respectively). These probes are complementary to telomeric repeat sequence. The immobilized amplicons were detected using anti-digoxigenin antibody conjugated to horseradish peroxidase (anti-DIG-HRP) and the sensitive peroxidase substrate TMB (tetramethyl benzidine). Absorbance at 450 mm was determined and using the .formula provided by the manufactures to determine the telomerase activity

Patients were then non-randomly assigned into 2 groups. Group 1 included patients who underwent radical cystectomy (anterior pelvic exentration in female patients) and postoperative radiation therapy using 2 dimensional planning and 3- field technique with radiation dose of 50 Gy/25 fractions over 5 weeks using 15 MV photons. Group 2 included patients who underwent tri-modality therapy (Complete TUR, and concurrent chemo-radiation). Radiation therapy was initiated 4 - 6 weeks after TUR using a 3-dimentional planning in 2 phases. In phase I, clinical target volume (CTV) included urinary bladder, proximal urethra (and prostate in male patients) and regional lymph nodes (hypogastric, external iliac and obturator nodes). Planning target volume (PTV) included CTV plus a 1 cm margin was treated with total dose of 46 Gy in 23 fractions, 5 fractions per week. In phase II, PTV included whole bladder plus 2 cm margins and received additional total dose of 20 Gy in 10 fractions with15 MV photons. Chemotherapy, Gemcitabine was given at 30 mg/m2 by 30 minute intravenous infusion before radiation therapy sessions twice weekly. Follow-up examinations were performed routinely every three months after treatment. Abdomino-pelvic CT scan, chest radiography and for group 2 patients, cystoscopy were performed annually.

Statistical methods The study cutoff point was June, 01, 2014. Overall and disease free survival rates were estimated by the Kaplan-Meier method using the Graphed Prism program. The log-rank test was used to examine differences in OAS and DFS rates. Comparison of mean values of OPG in different groups was done using one way ANOVA. A p value of less than 0.05 was considered statistically significant.

Middle East J Cancer 2011; 2 :Results

Patients’ characteristics are listed in Table 1. The median age of patients was 60 years. The majority of patients were males (55 patients; 84.6% with male to female ratio of 5.5:1), presented with T3 disease stage (35 patients; 54%), TCC pathologic type (49 patients; 75.5%), and high grade disease (35 patients; 54%). Twenty nine patients (44.5%), showed a positive history of bilharziasis. and received post- operative radiotherapy (63 patients; 49 of them were males). Radical cystectomy followed by adjuvant radiation therapy was done in the vast majority of patients (71%), while tri-modality therapy was done in 29%. The median follow up from the date of enrollment was 30 months and ranged from 3 to 60 months. :Table 1: Patients' characteristics

Variable (%) No (Age (median years 60 Sex

Females (15.4) 10 males (84.6) 55 T Stage

T2 disease (33.9) 22

T3 disease (53.8) 35

T4 disease (12.3) 8 Pathological type

TCC (75.4) 49

SCC (24.6) 16 Bilharziasis and grade

NB LG (29.2) 19

B LG (16.9) 11

NB HG (26.2) 17

B HG (27.7) 18 (Laboratory tumor markers (mean ± SD

(OPG (pg/ml 211.5±80.8

(P53 (u/ml 27.3±11.6

(Telomerase (u/gm protein 13.9±6.2 Treatment modality

Total cystectomy and postoperative radiotherapy (70.8) 46

(Trimodality therapy (TUR+Chemoradiation (29.2) 19 Total (100) 65

Laboratory biomarkers were assessed in cancer patients and healthy individuals (table 2). There were statistically significant higher mean values of serum OPG, and p53 and urine .(telomerase in bladder cancer patients than those in healthy volunteers (p<0.0001 Table 2: Laboratory biomarkers in cancer patients and healthy individuals

Variable (Normal persons (n=15 (Bladder cancer patients (n=65 P value

Mean ±SD Mean ±SD (Serum OPG (pg/ml 15.7 62.3± 214.9±80.4 0.0001> (Serum P53 (μ/ml 11.4±1.9 27.3±11.5 0.0001> (Urine Telomerase(μg/assay 3.4±0.63 13.9±6.1 0.0001>

After a median follow up of 30 months, 3-year OAS and DFS rates were 59% and 58% respectively. There were no significant differences between OAS and DFS rates according to age, sex, pathologic type, treatment modality, serum p53, and urine telomerase (Tables 3 & 4). On the other hand, there were significant differences in three-year OAS and DFS rates according to T stage (p<0.0001), histologic grade and bilharzial status (p=0.032 and 0.015 respectively) and serum OPG (p=0.001). Table 3: Univariate analysis of prognostic .factors affecting OAS and DFS rates

Variable year OAS rate-3 P value year DFS rate-3 P value Age 0.28 0.18

years 60 > 66.6% 66.6%

≥years 60 52.7% 50.9% Gender 0.24 0.21

Female 78.8% 78.8%

Male 56.0% 54.4% Bilharziasis and grade 0.032 0.015

NB LG 84.2% 84.2%

B LG 68.2% 68.2%

NB HG 52.9% 52.9%

B HG 42.9% 28.6% T stage 0.0001> 0.0001>

T2 95.5% 95.5%

T3 53.8% 50.1%

T4 *25% *25% Pathological type 0.19 0.26

TCC 65.7% 61.1%

SCC 48.6% 48.6% Treatment modality 0.72 0.89 RC and postop. RT 60.9% 59.3%

TUR and CRT 54% 54% Two year survival rate*

Table 4: Cox Regression stepwise OAS and DFS analyses according to laboratory markers Variable OAS rate DFS rate

OPG P=0.001, HR: 1.01; 95% CI: 1-1.01 P=0.001, HR: 1.01; 95% CI: 1-1.01 P53 P=0.97 P=0.82 Telomerase P=0.61 P=0.72

Table 5 shows that only 4 patients (6%) developed local recurrence, 7 patients (11%) developed distant metastases, 4 of them in bones, two in lungs and one in bones and liver and one patient (1.5%) developed mixed local and distant (in bones and lungs) .metastases

Table 5: Disease relapse

Disease relapse NO % Local recurrence 4 6.2 Distant metastases 7 10.8 Mixed relapse 1 1.5 Total 12 18.5

Table 6, shows correlation of serum OPG level and prognostic variables. Patients with high serum OPG were correlated only with advanced T stage (p<0.0001), high .(histologic grade (p<0.0001), and presence of disease relapse (p=0.001 Table 6: Correlation of serum OPG level and different prognostic variables Variable OPG Mean ± SD According bilharz. & grade NB-LG 128.4±25.3 B-LG 163.2±17.6 NB-HG 244.1±20.9 B-HG 310.3±49.9 P value 0.0001> According T stage T2 157.1±48.6 T3 231.4±77.1 T4 301.8±50.9 P value 0.0001> According to Histo-pathological type TCC 220.1±83.1 SCC 199.1±71.5 P value 0.37 According to Age years 60 > 196.2±84.8 ≥years 60 230.0±74.4 P value 0.092 According to gender Female 206.5±67.7 Male 216.5±82.9 P value 0.72 According to disease relapse Relapse 285±60.8 No relapse 199.1±76.1 P value 0.001

Figures 1-4 list the OAS and DFS data according to histologic grade, disease stage, and serum level of OPG.

:Discussion

Identification of biomarkers may improve the screening and diagnosis of bladder cancer, and determine the prognosis. Ideal biomarkers should be noninvasive; rapid; easy to obtain, use, and interpret; inexpensive; and, accurate [13, 14]. A large number of molecular markers that have prognostic value have been determined in reported studies of molecular biology and genetics, [10] including serum OPG, and p53 and urine telomerase. In the present study, it was found that serum OPG and p53 antibodies levels and urine telomerase were statistically significant higher (p<0.0001) in patients with bladder carcinoma than in healthy individuals. This is in agreement with Mizutani et al., [7] who reported that the mean serum OPG concentration in patients with bladder carcinoma was approximately 3 times greater than the mean concentration in healthy volunteers. Shimada et al., [15] stated that surveillance of S-p53 Abs may be useful in detecting various types of malignant tumors, including bladder cancer. Telomerase is activated in cancer cells, but not in normal somatic cells; therefore, its detection can be a diagnostic marker for cancer [16]. Urothelial tumors of all grades express telomerase activity in voided urine [17]. With a median follow up of 30 months, the 3- year OAS, and DFS rates were 59% and 58% respectively in the present study. Survival rates, in the current study, are comparable to those found in the reported studies [18, 19]. The postoperative radiotherapy (in cystectomy group), and the trimodality therapy ( i.e. TUR, and chemoradiation, in bladder preservation group) resulted in relatively low relapse rate in the present study (18.5%). Zaghloul et al., [20] showed that adjuvant radiation therapy improve local control rate, and that the therapeutic benefit of postoperative irradiation was consistent for all tumor types, histological grades, and pathological .stages for both the disease-free survival and local control Among the studied variables in the current study, only T stage, bilharzial infestation & histologic grade, and serum OPG biomarker significantly influenced both OAS (p<0.0001, 0.032 and 0.001 respectively) and DFS (p<0.0001, 0.015 and 0.001 respectively) rates. Many studies analyzed the prognostic factors in patients with bladder cancer treated with radical cystectomy. The pathological stage has been considered by many authors to be the main prognostic factors in these patients [21- .[27

Unlike to OPG in the present study, serum p53 and urine telomerase did not significantly affect survival rates. Regarding p53 status as a prognostic factor in muscle-invasive bladder cancer, the results were contradictory [10]. However, Schmitz-Dräger et al.[28] reviewed all published research on the association of p53 positivity and prognosis of patients with bladder cancer in a meta-analysis. In the majority of trials (5 out of 7) in muscle-invasive bladder cancer, p53 was not regarded as an independent prognostic marker of disease progression. The present study is matched with this meta-analysis. It was also stated that, although urinary telomerase could be a potentially useful urinary tumor marker, its use for diagnosis in symptomatic patients or its impact during surveillance is still unknown. Moreover, there will need to be normalization and standardization of the assays before they can .[become useful in clinical practice [16 The use of serum OPG measurements as a prognostic marker has been investigated and, elevated levels of serum OPG are found to be associated with poor prognosis [7, 29-31]. Among patients with muscle-invasive bladder carcinoma, the 5-year disease- specific survival rate was found to be higher for those with low serum OPG levels than for those with high serum OPG levels. This finding suggests that OPG concentration may also be a significant prognostic factor for patients with muscle- invasive bladder carcinoma, with low serum OPG levels being considered a favorable prognostic indicator. Serum OPG levels in patients with bladder carcinoma were analyzed according to patient's age, and gender, disease stage, tumor grade, pathologic type, and occurrence of disease relapse in the present study. Patients with high serum OPG were correlated with advanced T stage, high histologic grade (p<0.0001), and presence of relapse (p=0.001). This is in agreement with Mizutani et al., [7] and Holen and Shipman [32], who found that patients with high serum OPG were associated with advanced disease stage, high grade and high 5-year disease specific survival rate. A limitation of the present study is that the current analysis is restricted since patient selection for .treatment in both treatment groups was not randomized In spit of the advent of effective chemotherapy and modern radiation therapy techniques, treatment outcome of bladder cancer is unsatisfactory as local recurrence and distant metastases are major problems. Therefore, treatment protocols inhibiting OPG production may increase TRAIL-mediated toxicity of malignant cells and thus may improve treatment results [7]. This necessitates future trials in larger patient populations with longer follow up to clarify also the prognostic role of OPG in different disease stages, and pathologic types and grades.

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