Introduction

Urinary bladder cancer (UBC) ranks lower in a total number of cancer-related deaths than it does in incidence. Most lesions are non-muscle-invasive UBC (NMIUBC) which has a high propensity (60–70 %) for recurrence but rarely progress to muscle-invasive disease which results in a very high prevalence of tumors in developed countries [1]. Numerous institutions normally use screening cystoscopy, voided urinary cytology (VUC) and random bladder biopsies in an attempt for initial diagnosis of UBC. It is well known that painless hematuria, the most prevalent symptom, is found in only 4–10 % of cases of UBC [2]. However, up to 25 % of patients with UBC may not have hematuria, even when they have a known bladder tumor [3]. Early diagnosis of the UBC and careful follow-up for recurrences after conservative treatment are the major challenges of contemporary urology. Unification of markers into surveillance protocol needs to be investigated and, perhaps, presents the most attractive potential initial application in which they could be considered replacements for cystoscopy. So, new markers for screening, initial diagnosis, and surveillance for recurrent lesions, with the ultimate aim to alter clinical management of patients, are the need of the hour [4]. In the last few years, various tumor markers [5] have been studied for UBC. Recently, some studies have highlighted the role of a group of long (>400 bp) non-protein-coding RNAs (ncRNAs) in carcinogenesis and suggested that this class of genes might be used as biomarkers in cancer [6]. Urothelial carcinoma associated 1 (UCA1) is a novel ncRNA gene which belongs to the human endogenous retrovirus H (HERV-H) family [7] and could be involved in the cell cycle progression as explained in a recent study in BLZ-211 transitional cell carcinoma (TCC) cell line [8]. UCA1 was analyzed first in the tissue and urine in 2006 and was found that it might play a pivotal role in UBC progression and embryonic development. Wang et al. analyzed the UCA1 and its performance as a urine marker by reverse transcriptase-polymerase chain reaction in the urine from TCC patients, and they found that it may well be used as a new non-invasive marker for the follow-up of patients with TCC of the bladder. Clinical test showed that UCA1 assay was highly specific (91.8 %) and very sensitive (80.9 %) in the diagnosis of UBC and was especially valuable for NMI G2–G3 patients (sensitivity 91.1 %). The low expression rate in other cancer types, especially in renal cancers, makes UCA1 more discriminative in the diagnosis of TCC [9]. Recently, another study by Zhang et al. showed that UCA1 was highly specific (92.4 %) and quite sensitive (84.4 %) in the diagnosis of UBC [10].

Very limited data are available concerning the significance of UCA1 in predicting its role in the early diagnosis in both new-onset and recurrent bladder tumors. Taking the hypothesis that UCA1 can be used as a functional marker of early diagnosis in both new-onset and recurrent bladder tumors, the objective of the current study was designed to assess the clinical utility of UCA1 as a diagnostic non-invasive biomarker with the help of real-time PCR in detection of UBC and to compare it with cytology as the conventional non-invasive marker in TCC from a clinically mixed population of patients.

Materials and methods

Experimental design and patients’ characteristics

The present study was a prospective case series study, in which healthy controls and patients with urinary tract benign disease and TCC cases of the urinary bladder were included after signing voluntary informed consent. The ethical approval was obtained from institutional review board before the recruitment of subjects in this study. A total of 191 (163 males and 28 females) subjects were enrolled in this study. Subjects of the study were as follows: healthy control participants (group I, n = 28); patients diagnosed with non-cancerous urinary tract disease such as benign prostate hyperplasia, urinary tract infection, and urethral stricture (group II, n = 46); and TCC patients of the urinary bladder (group III, n = 117). Group III had three subgroups which included patients with (a) primary non-muscle-invasive UBC (n = 28), (b) primary muscle-invasive UBC (n = 41), and (c) recurrent cases of UBC (n = 48). Table 1 summarizes the demographics of controls and cases.

Table 1 Characteristics of cases and controls
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The post-surgical disease stage was classified according to the revised tumor–node–metastasis (TNM) staging system (UICC 2002) [11]. The urinary bladder tumors were graded according to the World Health Organization grading system [12]. The cases with stages T1–T2 were categorized as early stage and, above this (i.e., T3–T4), were categorized as advance stage. Patients with a history of bowel interposition, surgery, and other malignancies like squamous cell and adenomatous carcinoma of the urinary bladder and patients with any concurrent malignancy or disease like tuberculosis, diabetes mellitus, hepatitis B or C infection, or HIV infection were excluded from the study.

Sample collection and processing

A single and naturally voided midstream urine sample was obtained from all subjects. Approximately 50 cm3 of urine sample was collected, which was put on ice immediately and centrifuged as soon as possible (not later than an hour interval) at 3,000 rpm, 4 °C, for 7 min. The pellet was used for isolation of total RNA. We also measured UCA1 in a UBC cell line (T24) that was obtained from the National Centre for Cell Science (Pune, India). The cell line was maintained in McCoy’s 5A medium containing 10 % fetal bovine serum.

Cytology

The urine cytology was analyzed from fresh urine according to the standard protocol. All the urine cytology results were interpreted by a single observer. The cytopathologist was not aware of the patient’s disease status. We reported the specimens as positive when malignant cells were identified on cytology.

RNA isolation and cDNA synthesis and real-time PCR for UCA1

T24 cells were switched to serum-free medium for 48 h and then collected and lysed by freezing and thawing three times in 500 μL phosphate-buffered saline. Total RNA was isolated from cell lysate of T24 cells using Tri Reagent (Ambion, TX, USA), and an exfoliated cell RNA purification kit (Norgen Biotek, Canada) was used for isolation of total RNA from exfoliated cells in urine. The quality of RNA bands on FA gel was measured using the 2100 Bio-Analyzer (Agilent Technologies, USA). QuantiTect® Reverse Transcription Reagent (Qiagen) was used to synthesize the first-strand cDNA according to the manufacturer’s instruction.

The list of forward and reverse primers is presented as follows. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as endogenous control having a sequence of forward primer 5′-GAAGGTGAAGGTCGGAGT-3′ with reverse primer 5′-GAAGATGGTGATGGGATTTC-3′. For UCA1, we used the oligo sequence of forward 5′-TAAAGCCATGCCCATCAGACAGC-3′ and reverse: 5′-GGGATGGCCATTTGGAAGGAGTG-3′. The PCR protocol for 40 cycles was established with the help of EXPRESS One-Step SYBR® Green kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction with a total of 25 μL reaction mixture. Every sample was run in duplicate, and the mean was calculated for data analysis. Cycle threshold (Ct) values were obtained using the real-time PCR melting curve plot. Values for GAPDH and UCA1 expression were correlated to their controls using the 2−ΔΔCt calculation method [13].

Statistical analysis

Data were summarized as mean ± SD. Groups were compared using Student’s t test and ANOVA followed by Bonferroni post hoc test. Discrete variables were compared by chi-square (χ 2) test. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic accuracy, and likelihood ratio (LR) were the validity measures and were assessed by receiver operating characteristic (ROC) curve analysis [14]. All tests and calculations were done with the help of Microsoft Excel (version 2007, Microsoft Corp, Redmond, WA, USA), SPSS software (version 15). A two-tailed (α = 2) probability, p < 0.05, was considered statistically significant.

Results

Basic characteristics

The expression of UCA1 was found to be significantly higher in the cancer group than in the non-cancer group as measured by real-time PCR (t = 11.2, p < 0.001), and upraised level of UCA1 was also found in T24 cell line and in urine samples. The UCA1 expression was found to be significantly different (p = 0.025) between healthy controls and non-malignant patients. Our results also revealed that the expression of UCA1 was not significantly associated with age, sex and smoking habit (p > 0.05). The mean Ct value of UCA1 of the T24 cells was higher (26.70 ± 1.15) than the mean Ct value of UCA1 of TCC cases (33.15 ± 3.16).

Association of UCA1 with various clinicopathological parameters

The relative mean fold expression of UCA1 was found to have 32.9-fold higher expressions in TCC cases than in controls. The clinical characteristics of the TCC patients and controls are shown in Table 1. We found a significant association for all study subgroups versus controls (p < 0.001). The differences among the subgroups were also significant (p < 0.05) in TCC cases as shown in Fig. 1a. UCA1 messenger RNA (mRNA) expression showed a significant (p < 0.05) association with stage and grade, while it did not show any association with nodal status and cytology (p > 0.05) as shown in Fig. 1b; however, the expression of UCA1 shows an increased positivity in TCC cases in comparison with the VUC. A significant difference of UCA1 mRNA expression was found between primary cases (n = 69) and recurrent cancer patients (n = 48) in comparison to the controls (p < 0.001).

Fig. 1
figure 1

Expression of UCA1 by real-time PCR in patients with bladder cancer according to clinicopathological parameters. A  Box plots show the expression in TCC study subgroups versus controls. B The bar diagram shows fold change. *p < 0.05, a difference is found to be statistically significant in respect to stage and grades

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Comparison of UCA1 and urinary cytology in UBC

Out of 117 cancer cases, 78 (66.6 %) were found to be cytology positive and 93 (79.5 %) were found to be UCA1 positive. The UCA1 levels as detected in the urine obtained from cancer and non-cancerous subjects were constructed on a ROC curve as shown in Fig. 2. The upregulated UCA1 level shows a sensitivity of 79.49 % (95 % confidence interval 71.03–86.39 %) and a specificity of 79.73 % (95 % confidence interval 68.78–88.19 %) having 86.11 % PPV, 71.08 % NPV, and 3.92 LR with a diagnostic accuracy of 93.3 % (p < 0.001), whereas the sensitivity, specificity, PPV, NPV, LR, and diagnostic accuracy of urinary cytology are 66.67, 95.95, 96.30, 64.55, 16.44, and 78.3 %, respectively (p < 0.001). The area under the ROC curve for UCA1 was 0.863 (standard error ±0.027), whereas for cytology, it is 0.813 (standard error ±0.031). The groupwise comparison of sensitivity between VUC and urinary UCA1 is described in Table 2. Primary non-muscle-invasive TCC cases had a lower sensitivity of VUC (46.43 %) in comparison to the UCA1 (82 %); however, in recurrent cases, UCA1 had lower sensitivity (70.73 %) than the VUC (81.48 %). The sensitivity of VUC and UCA1 expression was further evaluated according to grade and stage of the disease, and we found that UCA1 was more sensitive for diagnosis of patients with early stage in comparison to the sensitivity by VUC. Similarly, we found a better sensitivity of UCA1 in a combination of VUC for both high- and low-grade TCC cases as compared to VUC alone. We considered negative when either of the markers showed negative results and found a positive and significant correlation between UCA1 and VUC (Pearson correlation coefficient r = 0.5, p < 0.05). The combination of these urinary markers represents better sensitivity (100 %) and specificity (55.56 %) in the diagnosis of UBC.

Fig. 2
figure 2

ROC curve of UCA1 expression of UCA1 by real-time PCR, which predicts the presence of bladder cancer in terms of sensitivity and specificity

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Table 2 Comparison of sensitivity of UCA1 and cytology in urinary bladder cancer
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Conclusion

Recent years have witnessed enormous progress in identifying molecular markers that led to the development of cancer management. Invigorated efforts are needed for identification and evaluation of new biomarkers for early detection, monitoring, and prognosis of UBC. In this approach, we analyzed the mRNA expression of UCA1 in exfoliated cells in the urine of TCC cases of UBC. In the present study, UCA1 mRNA expression in TCC exfoliated cells was not significantly correlated with the clinical characteristics such as patient’s age, sex, nodal status, and smoking habits (p > 0.05). This is in agreement with most of the published literature. In the detection of UBC, 46 positive control subjects had a sensitivity of 6.52 % and a specificity of 95.95 % for VUC. This is due to those patients with symptoms of urinary calculi and chronic inflammation with catheterization which may lead to false diagnosis of UBC. To confirm the presence of UCA1 in UBC, we evaluated the expression of UCA1 in an established, high-grade TCC cell line (T24). Initially, a study by Wang et al. [9] describes that the UCA1 has low expression rate in other cancer types including renal cancer, whereas a high expression in exfoliated cell of urine in the patients with TCCUB makes UCA1 more discriminative in the diagnosis of TCC. Our results showed that the urine of UBC patients had a higher expression of UCA1 mRNA and it had a significant association with grade and stage (Fig. 3); however, in patients of other urological disorders, UCA1 mRNA expression was very low (sensitivity 23.91 %). The likelihood ratio of UCA1 was 3.92, and correlation was 0.5 with VUC. These indicate that it may be a useful urinary biomarker as an adjunct to urine cytology in order to improve the diagnosis of UBC. The overall sensitivity and specificity of UCA1 mRNA expression in exfoliated cells of urine using real-time PCR were 79.49 and 79.73 %, respectively. The sensitivity found in our study was almost equal to the previous study, and the specificity of UCA1 was found to be lower than that of previous studies [9, 10, 15], in which they found the UCA1 expression in urine samples of UBC patients, original or recrudescent, and its sensitivity and specificity were 80.9 and 91.8, 84.4 and 92.4, and 88.52 and 91.89 %, respectively. It was also found that the difference was significant between the sensitivity of urinary UCA1 and VUC for primary non-muscle TCC cases as shown in Table 2, but in case of recurrent cases, VUC had better sensitivity than UCA1, which is because the diagnosis of recurrent NMI cases from VUC may be better as compared to UCA1 (data not given) and this increases the sensitivity of VUC in overall recurrent cases. In our study, there was an apparent trend toward increased UCA1 levels from G1 to G3 cancer.

Fig. 3
figure 3

Box plots show UCA1 association in A grade and B T stage of TCC cases

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The sensitivity of UCA1 assay was observed in high-grade (G2–G3) TCC cases, which is consistent with the findings of the previous studies [9, 10]. Most importantly, we found that UCA1 had better sensitivity in patients of early stage as compared to cytology. However, cytology showed higher sensitivity as compared to UCA1 for patients with advance-stage TCC (Table 2). In our study, UCA1 expression was higher in high-grade TCC cases in comparison to low-grade cases, and there was a significant (p < 0.01) difference between high- and low-grade tumors as shown in Fig. 2a. This was in agreement with the previously reported literature [9, 10]. Regarding the relationship of UCA1 expression in urine with tumor stage, available literature is equivocal. In our study, expression of UCA1 was higher in advance-stage tumors in comparison to early-stage tumors and there was a significant (p < 0.01) difference between early and advance stages as shown in Fig. 2b, while in previous studies, it was not evidenced. Our results indicate that, without invasive procedure, urinary UCA1 gives higher positive results in comparison to cytology, particularly in non-muscle-invasive low-grade disease.

The study concludes that UCA1 mRNA is a better non-invasive biomarker for diagnosis of non-muscle-invasive low-grade UBC, as compared to the VUC. It may be used in combination with cytology for non-invasive diagnosis of UBC and may increase the interval between monitoring cystoscopy and replacing cystoscopies altogether. Although these molecular assays may not yet be clinically available outside the research setting, further evaluation on a larger patient population is warranted before its use may be recommended for routine clinical purpose for the benefit of both the patients and the clinicians.