Abstract
Aim: The aim of this study was to develop multiplex-PCR assays for the detection of circulating tumor cells in peripheral blood and urine samples of patients with bladder cancer. Materials and Methods: Peripheral blood and urine samples were collected from 208 patients (169 patients and 39 healthy volunteers). After RNA extraction and cDNA synthesis, the samples were analyzed for the expression of cytokeratin 19 (CK19), CK20 and epidermal growth factor receptor (EGFR) mRNA in blood and for SURVIVIN, human telomerase reverse transcriptase (hTERT), cytokeratin 20 (CK20) mRNA in urine, using multiplex-PCR assays. Results: EGFR and CK20 alone or in combination as well as all urine markers correlated well with histological grade. hTERT correlated well with primary tumor size T≥3. Patients with positive urine markers had significantly worse progression-free survival. Conclusion: Multiplex-PCR assays can be a useful tool for staging and monitoring purposes in patients with bladder cancer.
Bladder cancer is the most common malignancy of the urinary tract, the eleventh most commonly diagnosed cancer and a leading cause of cancer morbidity and mortality (1). The standard procedure for diagnosing bladder cancer includes cystoscopy combined with cytological examination (2). Cystoscopy is an invasive, high-cost method and is considered the gold standard for detecting and monitoring bladder tumors, with a sensitivity of 70%, whereas cytology is a low-sensitive operator-dependent method (3, 4). There is a need for sensitive and specific non-invasive urinary markers that might reduce both cystoscopy and cost. Several markers are commercially available but none of them alone is sensitive and specific (5-7). An ideal bladder cancer marker should have high sensitivity and specificity, it should be non-invasive and easy to interpret (8).
The presence of circulating tumor cells (CTCs) in the peripheral blood was first reported by Ashworth in 1869 (9). Malignant cells are detached from primary tumors and become invasive. These cells may then intravasate into the blood or lymphatic circulation and subsequently extravasate from the circulation and establish a secondary tumor in another organ far from the primary tumor (10, 11). The detection of CTCs has been well-demonstrated in breast, lung, colon, prostate, bladder, melanoma and other malignancies (12-17). Although CTC detection can contribute to tumor diagnosis and identify patients with advanced bladder cancer, such assays cannot be used as initial screening diagnostic tests due to their low sensitivity (17). Polymerase chain reaction (PCR)-based methods are commonly used rapid and low-cost methods for the detection of CTCs (18).
Cytokeratins (CKs) are intermediate filaments expressed in epithelial cells. CK20 is more highly expressed in urothelial tumors compared with normal transitional epithelium and can, thus, be considered as a marker of urothelial differentation (19). Epidermal growth factor receptor (EGFR) is regarded as a specific marker of CTCs that is mainly correlated with advanced clinical stages (20). SURVIVIN mRNA detection using reverse transcriptase-polymerase chain reaction (RT-PCR) can be used as an important adjunct method for cystoscopy in early screening and postoperative monitoring of bladder cancer (21, 22). In addition, urinary human telomerase reverse transcriptase (hTERT) activity is a good marker for the early diagnosis of bladder tumors in symptomatic patients (23), having the same specificity as urinary bladder cytology but higher sensitivity (24), while the combination of hTERT with cytology increases sensitivity to 95% (25).
The aim of this study was the development of a multiplex-PCR assay for the detection of CTCs in patients with bladder cancer using primers for CK19, CK20, EGFR in peripheral blood, and SURVIVIN, hTERT and CK20 in urine samples, and to assess its potential use for clinical applications.
Materials and Methods
Study participants. A total of 208 patients (177 men and 31 women) from one tertiary University Hospital, prior to surgical or any other therapeutic intervention were enrolled in the study. They were categorized into three groups. Group one included patients newly-diagnosed with bladder cancer (n=105), group two included patients with history of bladder cancer (n=64), and group three included healthy volunteers (n=39). People without clinical or laboratory suspicion, without a known history of malignancy or who were operated for another reason entirely irrelevant (e.g. inguinal hernia), considered as healthy volunteers. Patients were excluded from the study if they had another histological type of cancer, had received neoadjuvant or adjunant chemotherapy, and had a medical history of the same or another type of cancer.
In the group of patients with a history of bladder cancer, less than five years had passed from the last occurrence, without any recurrence noted in the interim. Samples in this group were collected before their admission to hospital either for regular follow-up or early workup due to suspicion of recurrence. Clinical staging was based on pathological findings at the time of transurethral resection, examination under anesthesia, cross-section imaging of the abdomen and pelvis with either computed tomography or magnetic resonance imaging, and chest X-ray. Transurethral resection and radical cystectomy specimens were graded and staged according to TNM classification. Patients were also categorized based on the American Joint Committee on Cancer (AJCC) staging.
The clinical and demographic characteristics of the patients are shown in Table I. All participants provided written informed consent to this research protocol and this study was conducted under the approval of the local Ethics Committee (Approval number of scientific council: E.S. 55/07-04-2011) and conforms to the Declaration of Helsinki.
Primer design. Primers for the selected markers were designed using FastPCR software (http://primerdigital.com/fastpcr.html). The sequences of the five primer pairs were additionally tested on NCBI Nucleotide Blast (http://blast.ncbi.nlm.nih.gov/blast) to confirm that they were specific for each target. Glyceraldehyde-3-phosphate dehydro-genase (GAPDH) was used to evaluate the performance of cDNA synthesis (housekeeping gene). The sequences of the assay primers are presented in Table II.
RNA isolation. Six milliliters of blood were drawn from each participant, using a venous catheter, into 3 ml EDTA-containing vacutainers. Fifty milliliters of spontaneously voided urine (second void of the day) was taken from each patient before they received any treatment or underwent surgery.
Clinical and demographic characteristics for the three study groups.
Blood and urine samples were processed within 3 h of collection. Erythrocyte Lysis Buffer (ELB) (155 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA, pH of 7.4) was used to lyse all erythrocytes by osmosis and enrich the samples in nucleated leucocytes. ELB (7.5 ml) was added to each sample and the samples were kept on ice for 45 minutes, with occasional mixing by inversion and they were then centrifuged at 400g for 10 min at 4°C. The supernatant was discarded and if red blood cells still remained in the sample, 5 ml of ELB were added to the pellet, the cells were resuspended, kept on ice for an additional 10 min and the centrifugation step was repeated. Urine samples were centrifuged at 2000 × g for 10 min. The cell pellet was homogenized in 1 ml Tri-Reagent RT-111 (MRC Inc., Cincinnati, OH,USA). Total cellular RNA was then extracted, according to the manufacturer's instructions. Diethylpyrocarbonate water (DEPC-treated H2O) was used for RNA pellet dilution. Total RNA concentration and quality were determined by ultraviolet spectophotometry (measurements at 260 nm, 280 nm).
cDNA synthesis. cDNA was synthesized using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). A mixture containing 2 μg total RNA (blood samples) or 1 μg total RNA (urine samples), with 500 μg oligo dT18 primer (Fermentas St.Leon-Rot, Germany), 0.5 mM deoxynucleotides (HT Biotechnology LTD., Cambridge, UK) and DEPC-treated water up to 12 μl, was heated at 65°C for 5 min and then chilled on ice for an additional 5 min. A second mixture containing RT buffer (50 mM Tris-HCl/pH 8.3, 75 mM KCl, 3 mM MgCl2), 10 mM dithiothreitol, 20 units RNase OUT Recombinant Ribonuclease Inhibitor (Invitrogen), was added to the first mixture and then incubated at 37°C for 2 min. In the next step, 150 units of M-MLV RT was added up to a final volume of 20 μl, and the incubation continued at 37°C for 50 min and the reaction was completed by an inactivation step at 70°C for 15 min. The resulting complementary DNA was then used for multiplex PCR reaction.
Sequences of the assay primers.
PCR conditions. All PCR reactions were performed using Qiagen Multiplex PCR Kit (Qiagen, Hilden, Germany) in a final reaction volume of 25 μl. The reaction mixture contained 1X Qiagen Multiplex PCR Mix (HotStarTaq DNA pol, Multiplex PCR Buffer with 6 mM MgCl2/pH 8.7, dNTP mix), 0.125 μM CK19 primers, 0.125 μM CK20 primers, 0.4 μM EGFR primers, 0.3 μM SURVIVIN primers, 0,3 μM hTERT primers and DEPC-treated water up to 23 μl. Complementary DNA (2 μl) was added to the reaction volume. Cycling conditions were 95°C for 15 min (1 cycle), 94°C for 30 s, 57°C for 90 s, 72°C for 60 s (36 cycles), and 72°C for 10 min (1 cycle).
For GAPDH detection, the PCR reaction was performed using Qiagen Taq PCR Kit (Qiagen) in a final reaction volume of 15 μl. The reaction mixture contained 1× Qiagen PCR Buffer (2.5 units Taq DNA pol, PCR Buffer with 1.5 mM MgCl2, 200 μM dNTPs mix), 0.66 μM GAPDH primers and DEPC-treated water up to 14 μl. of Complementary DNA (1 μl) was added to the reaction volume. Cycling conditions were the same as for multiplex PCR.
PCR products were analyzed by electrophoresis in a 2% agarose gel (ethidium bromide stained) and were then captured under UV light in KODAK EDAS 290 Imaging System (CareStream Health, Rochester, NY, USA).
We proceed to a qualitative analysis. Before developing multiplex PCR, first we perfomed single PCR for each gene using bladder cancer tissue as a positive control and blood and urine from healthy volunteers as negative control, normalizing the PCR so that there is no expression of any of these genes in the healthy controls. Blood and urine samples with detectable GAPDH band together with the positive marker product were considered positive for any gene expression. Samples were considered negative when GAPDH-band were identified alone. In cases of missing GAPDH signal, blood and urine samples were excluded from analysis.
Statistical analysis. Quantitative variables are expressed as mean values±standard deviation (SD)/median values (interquartile range). Qualitative variables are expressed as absolute and relative frequencies. For comparisons of proportions, chi-square and Fisher's exact tests were used. Analysis of variance (ANOVA) was used for the comparison of mean age between the study groups. Kaplan–Meier survival estimates for recurrence and disease progression were graphed over the follow-up period for the study groups. Recurrence-free survival was defined as the time from diagnosis of bladder cancer to the date of the first bladder recurrence (same or lower disease stage or grade). Progression-free survival was defined as the time from diagnosis of bladder cancer to the date that higher disease grade or stage were detected. Log-rank tests were used for the comparison of survival curves. Sensitivity, specificity, negative and positive predictive values were calculated to estimate the discriminative ability of study markers between the group with history of bladder cancer and controls (no cancer) as well as between the newly diagnosed group and controls. All reported p-values are two-tailed. Statistical significance was set at p<0.05 and analyses were conducted using the Statistical Package for the Social Sciences Predictive Analysis Software (SPSS PASW statistics software version 19.0; IBM SPSS Statistics, IBM Corporation,. Chicago, IL, USA).
Results
The proportion of men was 85.7%, 90.6% and 74.4% in the newly-diagnosed group, in patients with history of bladder cancer and in healthy volunteers, respectively (p=0.077).
Twenty-three patients (21.9%) from the newly-diagnosed group underwent radical cystectomy and the corresponding proportion was 12.5% for the group with history of bladder cancer (p=0.125). None of the patients had distal metastasis, whereas positive nodes were found in 7.6% of the newly-diagnosed group and 3.1% of the group with history of bladder cancer (p=0.322). Of the newly-diagnosed group, 17.2% were categorized as having disease of anatomic stage III or IV (AJCC) and the corresponding proportion was 4.7% for the group with history of bladder cancer (p=0.017). Recurrence was recorded in 20 patients (19.2%) from the newly-diagnosed group and in 26 patients (40.6%) from the group with history of bladder cancer (p=0.002), while disease progression was recorded in 12 of the patients with newly-diagnosed bladder cancer (11.8%) and in 11 (18.0%) with history of bladder cancer (p>0.050). The mean follow-up period was 26.9 months (SD=11.9), with a median of 32 months (interquartile range from 15 to 37 months).
Expression of all study markers and their combinations in blood and urine in the three study groups.
Expression of EGFR, CK19, CK20 in peripheral blood. The detection of all markers and their respective combinations in blood and urine samples in the three study groups is presented in Table III.
With regard to blood measurements, EGFR-positive detections were more frequent in the group of patients with newly-diagnosed bladder cancer compared to the group with a history of bladder cancer and to healthy volunteers. CK19 detections were more frequent in the newly-diagnosed group compared to the control group, whereas CK20-positive detections were less frequent in the control group compared to the two patient groups. The combination of positive detection for both EGFR and CK19 was more frequent in the newly-diagnosed group compared to the group of patients with history of bladder cancer, while the combination of both EGFR and CK20 was more frequent in newly diagnosed as compared to group with history with bladder cancer and the control group. Triple-positive detections for EGFR plus CK19 plus CK20 were more frequent in the newly-diagnosed group compared to the group of patients with a history of bladder cancer. EGFR–CK19-positive detections were rare in control group compared with newly-diagnosed and patients with history of bladder cancer. Furthermore, the combined positive detections of EGFR and CK20, CK19 and CK20, and EGFR with CK19 and CK20 were rare in the control group as compared with patient groups.
Expression of hTERT, CK20 and SURVIVIN in urine. Urine detection of SURVIVIN, CK20, the combined positive detections of SURVIVIN plus CK20, SURVIVIN-hTERT-positive, SURVIVIN-CK20-positive, hTERT-CK20-positive and SURVIVIN-hTERT-CK20-positive were frequent in the newly-diagnosed group and the group of patients with history of bladder cancer, and rare in the control group (Table III).
Sensitivity, specificity, negative and positive predictive value of markers used for discriminating newly-diagnosed and control groups. Most of the markers and their respective combinations in blood revealed high specificity and predictive values (Table IV). EGFR detection showed low sensitivity, while the sensitivity of CK19 and CK20 detection was 58.7% and 53.9%, respectively. The highest sensitivity was found for the combined positive detections of EGFR and CK19 (65.4%), CK19 and CK20 (73.1%), and EGFR-CK19-CK20-positive (76%).
Urinary markers and their combinations had high positive predictive values ranging from 91.7% to 100% and high specificity rates ranging from 94.9% to 100%. The sensitivity was low, the highest being detected for combined positive detection of hTERT-CK20-positive (35.9%), and SURVIVIN-hTERT-CK20-positive (36.2%).
Sensitivity, specificity, negative and positive predictive values of marker detections in patients with history of bladder cancer and control group. Most of the markers and their respective combinations in blood showed high sensitivity and negative predictive values (Table V). The highest sensitivity rates concerning blood measures were found for CK20 (65.6%), EGFR and CK20 (68.8%), CK19 and CK20 (73.4%), and EGFR-CK19-CK20-positive (75.0%).
Urinary markers also presented high specificity and positive predictive values, while the highest sensitivity rates were found for CK20, SURVIVIN-CK20-positive, hTERT-CK20-positive and SURVIVIN-hTERT-CK20-positive.
Association with progression-free survival. Patients in the newly-diagnosed bladder cancer group with positive urine SURVIVIN detection had worse progression-free survival compared to those that were negative for SURVIVIN (log-rank test, p=0.011) (Figure 1). Specifically, three of the newly-diagnosed patients with negative SURVIVIN (8.8%) had disease progression and the corresponding proportion was 26.9% in cases that SURVIVIN was detected. The positive detection of urinary CK20 was also associated with worse progression free-survival in newly diagnosed patients (log-rank test, p=0.050) (Figure 2).
Worse progression free-survival was found in the newly-diagnosed group with combined positivity for the detection of urinary SURVIVIN and hTERT (log-rank test, p=0.048), urinary SURVIVIN and CK20 (log-rank test, p=0.011), urinary hTERT and CK20 (log rank test, p=0.048), urinary SURVIVIN with hTERT and CK20 (log-rank test, p=0.048), urinary SURVIVIN and hTERT (log-rank test, p=0.029), and urinary SURVIVIN and CK20 (log-rank test, p=0.050). In addition, recurrence-free rates were worse in the cases of newly diagnosed patients with combined positivity for urinary SURVIVIN and hTERT (log-rank test, p=0.050), urinary hTERT and CK20 (log-rank test, p=0.050), and urinary SURVIVIN plus hTERT plus CK20 (log-rank test, p=0.048).
Correlation of positive detections with various clinicopathological parameters of patients with bladder cancer. In the newly-diagnosed group, EGFR was more frequently expressed in patients with histological grade 3 or carcinoma in situ compared to those with histological grade <3 (31.4% vs. 15.1%, p=0.049). In this group, the positive detection of CK20 in blood was correlated with histological grade 3 or carcinoma in situ as compared to those with histological grade <3 (68.6% vs. 39.6%, p=0.003). Similarly, the combined positive detection in blood of EGFR and CK20 (72.5% vs. 41.5%, p=0.001) and EGFR-CK19-CK20-positive (84.3% vs. 67.9%, p=0.050) correlated with histological grade 3 or carcinoma in situ.
Furthermore, in newly-diagnosed patients, positive hTERT detection in urine was associated with primary tumor size ≥3 (that is T3 or T4) compared to those with tumor size <3 (that is T1 or T2) (28.6% vs. 7.7%, p=0.032) and with AJCC stage 2 or greater, compared to stage <2 (50.0% vs. 6.3%, p=0.028). Similarly, the combined positive detection of SURVIVIN and hTERT (50.0% vs. 6.3%, p=0.028), hTERT and CK20 (50.0% vs. 6.3%, p=0.028), and SURVIVIN with hTERT and CK20 (50.0% vs. 6.3%, p=0.028) were associated with AJCC stage 2 or greater.
In addition, all marker detections in urine samples and their respective combinations were associated with histological grade 3 or carcinoma in situ (for SURVIVIN 31.4% vs. 11.1%, p=0.011; for hTERT: 19.6% vs. 1.9%, p=0.003; and for CK20: 54.9% vs. 13.0%, p<0.001).
In the blood samples of the group of patients with history of bladder cancer, the detection of CK20 (80.0% vs. 52.9%, p=0.023) and the combined positive detection of EGFR and CK20 (83.3% vs. 55.9%, p=0.018), CK19 and CK20 (86.7% vs. 61.8%, p=0.024), and EGFR with CK19 and CK20 (90.0% vs. 61.8%, p=0.009) correlated with histological grade 3 or carcinoma in situ as compared to those with histological grade <3, respectively.
Discussion
Although the European Organization for Research and Treatment of Cancer (EORTC) Genito-Urinary Cancer Group has developed a scoring system and risk tables (26), more intensive research is required in the field of CTCs in order to improve the predictive accuracy of the currently existing risk tables (27, 28). The goal would be not only to use these molecular markers as a screening test in populations at higher risk for bladder cancer (29, 30), but to also achieve a better surveillance (31-34) and perhaps a better understanding over the biological behavior of the disease, especially when there is interobserver variability in classification of stage T1 versus Ta tumors and tumor grading in both 1997 and 2004 classifications. In such difficult cases, where an additional review by an experienced genitourinary pathologist is recommended, an objective, easy-to-perform and highly specific assay would be of great importance.
Percentage sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of study markers for the discrimination between newly-diagnosed patients with bladder cancer and controls.
Percentage sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of study markers for the discrimination between the group with history of bladder cancer and controls.
Kaplan-Meier estimates for disease progression in the newly-diagnosed group according to the expression of SURVIVIN.
Rink et al. showed significantly worse overall progression-free survival and cancer-specific survival in CTC-positive compared to CTC-negative patients (35). Msaouel and Koutsilieris further performed a systematic review and meta-analysis of the value of CTC detection in bladder and urothelial cancer, highlighting the potential clinical role of CTCs as markers of advanced bladder cancer (17). It has also been shown that CTC status may provide prognostic information in the face of negative imaging (36). In the case of urothelial carcinoma, it could provide clinicians with valuable information that might be applied to preoperative management algorithms. Furthermore, CTC status could potentially identify patients who would be most appropriate for neoadjuvant chemotherapy or clinical trial protocols.
Kaplan–Meier estimates for disease progression in the group of patients with newly-diagnosed bladder cancer according to the detection of CYTOKERATINE 20 (CK20).
The advantages of multi-marker use for CTC detection in patients with colorectal cancer (CRC) have already been described (15, 18, 37, 38). Many previous studies have shown that the use of more than one marker independently increases the sensitivity and specificity of CTC detection and thus the correlation with disease stage in patients with CRC (39-43). Implementation of multiplex RT-PCR, which allows for examination of multiple marker expression in a single reaction, could be advantageous, in contrast with the application of separate PCR reactions for each marker, which can be time-consuming and costly. Moreover, the use of multiplex RT-PCR on peripheral blood and urine samples represents an easily applied, non-invasive technique for the detection of CTCs in patients with cancer (44).
The aim of the present study was to develop and then evaluate the clinical significance of a multiplex PCR-based detection of CTCs using primers for CK19, CK20 and EGFR (in blood samples) and SURVIVIN, hTERT and CK20 (in urine samples) and to assess clinical applications of these markers in patients with bladder cancer. With a substantial follow-up period, we were able to estimate several clinical parameters (sensitivity, specificity, positive predictive value, negative predictive value, progression-free survival) and assess their prognostic relevance.
Our data highlight the known correlation between primary tumor (T stage) and high grade (grade 3 or carcinoma in situ) with worst prognosis regarding recurrence and progression in patients with bladder cancer. Beyond these, there seems to be a statistically significant correlation of those clinical parameters with the some of the evaluated mRNA markers.
There is considerable heterogenicity in tumor markers used in several studies. From the markers we used, EGFR and CK20 appeared to be the most commonly used markers in many studies. Positive EGFR detection in blood samples demonstrated high specificity (and positive predictive values) for diagnosis of bladder cancer, both in the newly-diagnosed group and in patients with history of bladder cancer. CK20 had relatively the highest sensitivity rates in both groups, both in blood and urine samples. These results seem to agree with those from relevant studies (17). On the other hand, EGFR demonstrated low diagnostic sensitivity rates compared with all other markers tested. Urine markers demonstrated also high specificity and positive predictive values, both in the newly diagnosed group and in patients with history of bladder cancer. Therefore CTCs detection may be useful in confirming the cancer diagnosis.
Positive SURVIVIN detection in urine of newly-diagnosed patients, revealed worse prognosis. The positive detection of CK20 in urine samples was also associated with worse progression free-survival in newly-diagnosed patients. In the same group, EGFR, as well as CK2O detection in blood samples (and their combination in group of patients with history of bladder cancer) was more frequently expressed in patients with high tumor grade (histological grade 3 or carcinoma in situ). Furthermore, in newly-diagnosed, positive hTERT detections correlated more with primary tumor size (T) ≥3 and with AJCC stage 3 or greater as compared with stage <3. In addition, all marker detections in urine samples and their respective combined positivity correlated with high-grade cancer (histological grade 3 or carcinoma in situ), in the newly-diagnosed patients. There is thus a considerable connection with the conclusion of Rink et al., that that the presence of CTC may be predictive for early systemic disease (35).
Multiplex RT-PCR assay can provide useful information concerning bladder cancer stage, grading and progression-free and recurrence-free survival. The combination of the mRNA markers in a single reaction could be beneficial in the early detection of high-grade/clinically relevant disease and monitoring of bladder cancer patients (mainly those with high possibility to progress or recurrence), as it has previously been documented in other malignancies (38, 43, 45-49).
This study does have several limitations. The overall number of patients needs to be greater, therefore, the results must be interpreted with caution. The usage of CTCs seems promising, but for the moment is mainly study-based and no large trials have been performed yet; therefore, no clinically usable urinary markers have been identified. Relatively larger-scale studies and longer follow-up surveys would prove the clinical significance and prognostic importance of multiplex PCR-based detection of CTCs using EGFR, CK19 and CK20 in blood, hTERT, CK20 and SURVIVIN in urine and mainly their combinations in patients with bladder cancer. Despite the relatively low number of patients, to our knowledge, it is one of the largest studies to date in patients with bladder cancer. There is also a need for large quantitative studies, which will allow us to evaluate the optimal cut-off CTC count for increased sensitivity and specificity.
Another minor limitation is probably that of the “healthy” control group. They may have had occult bladder cancer which could somewhat confound interpretation of the results. In bladder cancer, is not easy to find volunteers for cystoscopy. People without clinical or laboratory suspicion, without a known history of malignancy or who are operated on for another entirely irrelevant reason (e.g. inguinal hernia), can probably be considered as control group. For a small proportion (n=6) of the control group (viz incidents audited even with transurethral resection), despite the clinical suspicion (random ultrasound scan, mostly for abdominal pain, which showed a possible bladder tumor), the final diagnosis (even with cystoscopy and random biopsies) was negative. Although not be considered exactly as healthy volunteers at the beginning, they proved to be healthy.
The future of marker development seems bright and new techniques are emerging. Beyond finding good markers, the financial cost–effectiveness will be an important issue and that has not been sufficiently studied yet. Currently, no single marker can guide us in surveillance and lower the frequency of urethrocystoscopy. Whether use of a set of markers will be the answer will have to be studied.
Footnotes
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↵* These Authors contributed equally to this study.
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This article is freely accessible online.
- Received July 28, 2014.
- Revision received September 12, 2014.
- Accepted September 18, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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