Evaluation of hTERT, KRT7, and survivin in urine for noninvasive detection of bladder cancer using real-time PCR.

BACKGROUND: Transitional cell carcinoma (TCC) of the bladder is the second most common genitourinary malignancy. Because of the low sensitivity of urinary cytology and the invasiveness and expense of frequent cystoscopies for the detection of low-grade superficial lesions, we aim to establish a sensitive molecular approach to detect bladder cancer noninvasively. METHODS: Voided urine samples were collected from 80 patients with bladder cancer at the time of diagnosis, in addition to 30 patients with non-bladder cancer urological diseases and 20 healthy volunteers. The level of hTERT, KRT7, and survivin (SVV) mRNAs were analyzed using a qRT-PCR assay. RESULTS: The optimal threshold values for hTERT, KRT7, and SVV in urine were calculated by ROC curves analysis. The overall sensitivity was 81.3%, 91.3%, and 68.8% for hTERT, KRT7, and SVV, respectively, which were significantly higher than urine cytology (22.2%, p < 0.001). A higher positive ratio was obtained using multi-marker detection in comparison to single marker detection. The combined use of markers increased the sensitivity of cytology from 22.2 to 100%. In contrast with the urine cytology method, the sensitivity of these biomarkers was not correlated with the grades and stages of the bladder tumors. CONCLUSIONS: Our data indicate that urinary hTERT, KRT7, and SVV have superior sensitivities over cytology. The combined use of these markers offers a powerful potential assay and promising tool for a sensitive, noninvasive, and highly specific diagnostic method and follow-up of low-grade TCC of the bladder.

Telomerase Activity Analysis In Urine Sediment for Bladder Cancer.

Bladder cancer with an incidence of 15 cases per 100,000 persons in the global population is the most common tumor of the urinary tract. Imaging techniques, cytoscopy, and cytology are not sufficiently accurate to detect early stage tumors, and the need for new diagnostic markers is still an urgency. Among the biomarkers most recently proposed to improve diagnostic accuracy and especially sensitivity, increasing attention has been focused on the role of the ribonucleoprotein, telomerase. Previous studies have shown that the quantitative telomerase repeat amplification protocol (TRAP) assay performed in voided urine is an important noninvasive tool for the diagnosis of bladder tumors since it has very high sensitivity and specificity, even for early stage and low-grade tumors. Telomerase activity in urine determined by TRAP seems to be marker of great potential, even more advantageous in cost-benefit terms when used in selected symptomatic patients or professionally high-risk subgroups. Here we report the real-time PCR protocol to detect telomerase activity in urine sediment for bladder cancer.

An institutional experience evaluating hTERT immunostaining in 100 consecutive ThinPrep urine specimens.

INTRODUCTION: Studies have shown that expression of human telomerase reverse transcriptase (hTERT) in mature urothelial cells indicates an increased risk of urothelial carcinoma. We evaluated the utility of immunocytochemistry with a commercially available anti-hTERT antibody (SCD-A7) in 100 consecutive urine cytology specimens using ThinPrep processing. MATERIALS AND METHODS: ThinPrep slides prepared from 100 consecutive urine specimens were stained using anti-hTERT antibody (SCD-A7) after staining optimization had been successfully completed. Patient demographics, cytology diagnoses, histologic follow-up data, and anti-hTERT staining results were recorded. RESULTS: The cytology diagnoses included 7 cases of high-grade urothelial carcinoma (HGUC), 2 cases suspicious for HGUC (SHGUC), 24 cases of atypical urothelial cells (AUCs), and 67 cases negative for HGUC (NHGUC). Of 92 samples, 68 (74%) were positive and 24 (26%) were negative for anti-hTERT staining. Although 31 of 32 specimens (97%) with a diagnosis of AUCs and greater showed positive staining, 37 of 60 NHGUC cases (62%) were also positive for anti-hTERT. Although the HGUC and suspicious for HGUC cases were more likely to show strong staining (6 of 9; 67%), 7 AUC (32%) and 8 NHGUC (22%) cases also demonstrated strong staining. Eight samples (8%) were unsatisfactory for interpretation. Anti-hTERT staining of nonurothelial cells was seen in 77 of 92 samples (84%). CONCLUSIONS: Interpretation of anti-hTERT immunocytochemical staining of ThinPrep material is challenging owing to obscuring of nonurothelial cell staining and difficulty discerning individual urothelial cell cytomorphology when the cells are stained. The significance of the large number of anti-hTERT-positive but cytology-negative cases in our study is uncertain.

Target-Driven Nanozyme Growth in TiO2 Nanochannels for Improving Selectivity in Electrochemical Biosensing.

Nanozymes have been used in colorimetric and electrochemical sensing because of their low cost and high stability. However, the wide applications of nanozymes in sensing devices are largely limited due to their poor selectivity. In this study, unlike traditional methods using prepared nanozymes for target detection, we designed a target-driven nanozyme growth strategy in TiO2 nanochannels to detect analytes. Using telomerase as an example, the established recognition event was used to expand the photocatalytic activity of TiO2 to visible-light region, thus triggering Prussian blue nanoparticle (PBNP) growth in visible light. Benefiting from the peroxidase (POD)-like activity of PBNPs, the uncharged 3,5,3',5'-tetramethylbenzidine (TMB) is oxidized to positively charged oxTMB, which induces significant ionic transport changes in nanochannels, and thus in turn provides information about telomerase activity. Such a nanozyme-triggered sensing system exhibited excellent performance in telomerase detection in urine specimens from patients with bladder cancer. This innovative target-driven signal generation strategy might provide a new method for applying nanozymes in developing sensitive, rapid, and accurate biological sensing systems.

A portable dual-mode sensor based on a TiO2 nanotube membrane for the evaluation of telomerase activity.

A portable dual-mode sensing platform based on a self-standing TiO2 nanotube membrane is developed for simultaneously performing both qualitative analysis by the naked eye and quantitative analysis by ionic current. This dual-mode diagnosis strategy exhibits a high performance in telomerase detection in urine specimens from patients with bladder cancer.

Non-invasive diagnosis of bladder cancer by detecting telomerase activity in human urine using hybridization chain reaction and dynamic light scattering.

Cystoscopy and histology are the gold standards for detection of bladder cancer. However, these methods are highly subjective, expensive, and invasive. We have developed a non-invasive method for the diagnosis of bladder cancer by detecting telomerase activity in human urine. Telomerase substrate (TS) primer is elongated with repeating sequences of (TTAGGG)n in the presence of telomerase. The elongated primer can trigger hybridization chain reaction between two hairpins H1 and H2, result in the aggregation of AuNPs due to the hybridization between the tail sequence on H1 (or H2) and DNA-AuNPs probe, and accompany with the increase of hydrodynamic diameter of AuNPs, which can be measured with dynamic light scattering (DLS). The biosensor displayed a detection limit of 4 MCF-7 cells (a signal-to-noise ratio of 3) and a dynamic range of 10-1000 cells. Moreover, only urine specimens from bladder cancer patients induced a significant change in the average hydrodynamic diameter, indicating its specificity for the non-invasive diagnosis of bladder cancer.

Strand displacement amplification-coupled dynamic light scattering method to detect urinary telomerase for non-invasive detection of bladder cancer.

Despite huge successes achieved by strand displacement amplification (SDA) and gold nanoparticles (AuNPs) in biomolecules sensing, the strategy of combination of SDA and AuNPs-based dynamic light scattering (DLS) for a biomolecule sensing is unexplored. Here we developed a non-invasive, SDA-based DLS method for the diagnosis of bladder cancer by detecting telomerase activity in human urine. In the presence of telomerase, the telomerase substrate (TS) primer was elongated with repeating sequences of (TTAGGG)n, and the resulting product triggers SDA between the hairpin deoxyribonucleic acid (DNA) and the Primer. The SDA product can be recognized by the oligonucleotide-modified AuNPs probes, resulting in DLS measurable AuNPs aggregation. The assay displayed a detection limit of 3 MCF-7 cells with a signal-to-noise ratio of 3 in a dynamic range of 5-1000 cells. The method was simple, reliable and has been successfully applied in the detection of telomerase in urine with good accuracy, selectivity and reproducibility. Moreover, only urine samples from bladder cancer patients induced a significant change in the average hydrodynamic diameter, indicating practical applicability of the method for the non-invasive diagnosis of bladder cancer.

Highly sensitive ratiometric fluorescent paper sensor for the urine assay of cancer.

Telomerase, as a valuable biomarker, is an important target in cancer diagnosis. Here, we report a ratiometric fluorescent probe for telomerase activity assay in urine and bladder cancer diagnoses based on the color change of Rox-DNA functionalized quantum dots (QDs). The green fluorescence of the QDs was sensitive to H2O2, but the red fluorescence of Rox showed no change. An HRP-mimicking hemin/G-quadruplex, which was formed with the help of telomerase activity, catalyzed H2O2 into H2O and O2. This quadruplex effectively avoided H2O2 interference with green fluorescence. In the presence of H2O2, the detected color changed from red to yellow-green by increasing the telomerase concentration. The detection limit (LOD) was 10 cells, and response time was within 60 min. More importantly, a paper sensor was developed based on this probe and used for the assay of telomerase activity in urine samples. The results were highly sensitive and reproducible, and visual semi-quantitative detection was realized using the naked eye.

Dual-Mode Au Nanoprobe Based on Surface Enhancement Raman Scattering and Colorimetry for Sensitive Determination of Telomerase Activity Both in Cell Extracts and in the Urine of Patients.

Telomerase is a valuable biomarker, which is highly correlated to cancer diseases. However, the single-mode probe for telomerase detection cannot satisfy the challenge of detection of telomerase activity rapidly, simply with high selectivity, sensitivity, and accuracy both in preliminary diagnosis and in point of care (POC) testing. Therefore, there is an urgent need to develop a robust approach with controllable assembly and high accuracy to consider both the simplification of preliminary diagnosis and POC testing and the quantification requirement for early clinical diagnosis and treatment. Herein, a novel dual-mode Au NPs probe was developed for determination of telomerase activity with controllable assembly and aggregation statement based on surface enhancement Raman scattering (SERS) and colorimetry. In this strategy, an Au dimer-based probe with high uniformity was assembled successfully, telomerase activity was reflected according to the color variations of solution and the Raman intensity of Raman reporter. Taking advantage of the uniformity of Au dimers and the combination of colorimetry and SERS techniques, our strategy determined the telomerase activity with high accuracy, sensitivity, and wide range. The established probe possessed of high selectivity, sensitivity, and accuracy, which was approved as a reliable, intuitional, and convenient approach for detecting telomerase activity.

Evaluation of Sienna Cancer Diagnostics hTERT Antibody on 500 Consecutive Urinary Tract Specimens.

OBJECTIVES: Telomerase activity can be detected in up to 90% of urothelial carcinomas (UC). Telomerase activity can also be detected in urinary tract cytology (UTC) specimens and indicate an increased risk of UC. We evaluated the performance of a commercially available antibody that putatively binds the telomerase reverse transcriptase (hTERT) subunit on 500 UTC specimens. STUDY DESIGN: Unstained CytospinTM preparations were created from residual urine specimens and were stained using the anti-hTERT antibody (SCD-A7). Two algorithms were developed for concatenating the hTERT result and cytologic diagnosis: a "no indeterminates algorithm," in which a negative cytology and positive hTERT result are considered positive, and a "high-specificity algorithm," in which a negative cytology and positive hTERT result are considered indeterminate (and thus negative for comparison to the gold standard). RESULTS: The "no indeterminates algorithm" and "high-specificity algorithm" yielded a sensitivity of 60.6 and 52.1%, a specificity of 70.4 and 90.7%, a positive predictive value of 39.1 and 63.8%, and a negative predictive value of 85.0 and 85.8%, respectively. CONCLUSIONS: A positive hTERT result may identify a subset of patients with an increased risk of high-grade UC (HGUC) who may otherwise not be closely followed, while a negative hTERT immunocytochemistry result is associated with a reduction in risk for HGUC.

Non-invasive prediction of recurrence in bladder cancer by detecting somatic TERT promoter mutations in urine.

BACKGROUND: Urothelial bladder cancer (UBC) is characterised by a high risk of recurrence. Patient monitoring is currently based on iterative cystoscopy and on urine cytology with low sensitivity in non-muscle-invasive bladder cancer (NMIBC). Telomerase reverse transcriptase (TERT) is frequently reactivated in UBC by promoter mutations. METHODS: We studied whether detection of TERT mutation in urine could be a predictor of UBC recurrence and compared this to cytology/cystoscopy for patient follow-up. A total of 348 patients treated by transurethral bladder resection for UBC were included together with 167 control patients. RESULTS: Overall sensitivity was 80.5% and specificity 89.8%, and was not greatly impacted by inflammation or infection. TERT remaining positive after initial surgery was associated with residual carcinoma in situ. TERT in urine was a reliable and dynamic predictor of recurrence in NMIBC (P<0.0001). In univariate analysis, TERT positive-status after initial surgery increased risk of recurrence by 5.34-fold (P=0.0004). TERT positive-status was still associated with recurrence in the subset of patients with negative cystoscopy (P=0.034). CONCLUSIONS: TERT mutations in urine might be helpful for early detection of recurrence in UBC, especially in NMIBC.

FGFR3, TERT and OTX1 as a Urinary Biomarker Combination for Surveillance of Patients with Bladder Cancer in a Large Prospective Multicenter Study.

PURPOSE: Patients with nonmuscle invasive bladder cancer are followed with frequent cystoscopies. In this study FGFR3, TERT and OTX1 were investigated as a diagnostic urinary marker combination during followup of patients with primary nonmuscle invasive bladder cancer. MATERIALS AND METHODS: In this international, multicenter, prospective study 977 patients with nonmuscle invasive bladder cancer were included. A total of 2,496 urine samples were collected prior to cystoscopy during regular visits. Sensitivity was estimated to detect concomitant recurrences. Kaplan-Meier curves were used to estimate the development of future recurrences after urinalysis and a negative cystoscopy. RESULTS: Sensitivity of the assay combination for recurrence detection was 57% in patients with primary low grade, nonmuscle invasive bladder cancer. However, sensitivity was 83% for recurrences that were pT1 or muscle invasive bladder cancer. Of the cases 2% progressed to muscle invasive bladder cancer. Sensitivity for recurrence detection in patients with primary high grade disease was 72% and 7% of them had progression to muscle invasive bladder cancer. When no concomitant tumor was found by cystoscopy, positive urine samples were more frequently followed by a recurrence over time compared to a negative urine sample (58% vs 36%, p <0.001). High stage recurrences were identified within 1 year after a positive urine test and a negative cystoscopy. CONCLUSIONS: Recurrences in patients with primary nonmuscle invasive bladder cancer can be detected by a combination of urine assays. This study supports the value of urinalysis as an alternative diagnostic tool in patients presenting with low grade tumors and as a means to identify high stage tumors earlier.

A sensitive, label-free electrochemical detection of telomerase activity without modification or immobilization.

Telomerase has become one of the most typical tumor marker because it is closely related to cancers. In this paper, a simple label-free electrochemical detection of telomerase activity by using methylene blue (MB) as a G-quadruplex binding probe was proposed, avoiding commonly used complex label procedures, nano-probe synthesis, complicated electrode modification, probe immobilization or signal amplification. In the presence of telomerase substrate (TS) primer, the binding of MB on primer was weak. When repeats of (TTAGGG) were extended on the TS primer under the action of telomerase, they formed multiple G-quadruplexes with the help of K+. As a result, a large amount of MB bounded to multiple G-quadruplexes because they have more strong interaction with G-quadruplexes than TS primer. As a result, the diffusion current of MB decreased sharply, which was strongly dependent on the telomerase activity. The DPV current change has a linear correlation with the logarithm of HeLa cell number in the range of 10-10,000 cells, with the detection limit of 3 cells. The high sensitivity was due to the formed multiple G-quadruplexes. Using indium tin oxide (ITO) as working electrode without modification ensured the good reproducibility of the method. The method was also simple, rapid, and has been successfully applied in the telomerase activity detection in urine with good selectivity and reproducibility, which is significant for cancer diagnosis, anticancer drugs screening, and cancer therapy evaluation.

A highly sensitive and facile graphene oxide-based nucleic acid probe: Label-free detection of telomerase activity in cancer patient's urine using AIEgens.

Molecular beacon (MB)-based sensing platforms that consist of a fluorogen-quencher pair play an important role in medical and biological researches. However, the synthesis of both fluorogen and quencher in the nucleic acid probes will increase the burden of organic synthesis works and induce the difficulties for precisely controlling the relative distance between fluorogen and quencher, which may lead to false-positive and false-negative results. In this work, initially we report a single labeled MB (FAM-MB, with carboxyfluorescein as fluorogen and without quencher) thus simplifies MBs with the aid of graphene oxide (GO) to detect telomerase activity. To further simplify this structure, namely label-free strategy, we design a facile, sensitive and selective platform using a label-free beacon (AIE-MB, without fluorogen and quencher), based on aggregation-induced emission fluorogen (silole-R). Upon the addition of telomerase, AIE-MB induced comb-like DNA structure leads to high aggregation of silole-R and thus exhibits strong fluorescence emission. By exploitation of this, we can detect telomerase with superior sensitivity and demonstrate their applications in bladder cancer diagnosis. Compared to single-labeled FAM-MB based telomerase activity assay, the label-free AIE-MB induced method could perform the sensitive detection with high signal-to-background ratio.

A simple, fast, label-free colorimetric method for detection of telomerase activity in urine by using hemin-graphene conjugates.

Telomerase, a widely accepted cancer biomarker for early cancer diagnostics, is considered as an important therapeutic target. To now, it is still a challenging subject to develop a simple and sensitive strategy for telomerase activity detection. Herein, we reported a simple colorimetric strategy for label-free quantification of human telomerase activity in urine by using hemin-graphene nanomaterial (H-GNs). H-GNs possessed tailored dispersibility in the high salt concentration and highly active biomimetic oxidation catalyst property. In this strategy, H-GNs were adjusted to coagulate to appropriate degree by carefully selecting the contained NaCl amount in the presence of original TS primer. The supernatant of the solution contained few H-GNs and showed light blue color. Under the action of telomerase, TS primer was elongated with repeating sequences of (TTAGGG)n. These negatively charged DNA enhanced individual H-GNs electrostatic repulsion and resisted salt-induced H-GNs coagulation. As a result, the supernate of the corresponding solution containing more dispersed H-GNs and showed dark blue color after chromogenic reaction. Thus, telomerase activity could be quasi-quantified by naked eye and precise quantified by UV spectrometer. The proposed method has the linear range from 100 to 2300 HeLa cells/mL and the detection limit was 60 cells/mL. It has been successfully applied to detect telomerase activity in real urine samples. Obtained results were in good agreement with the clinical diagnosis. Therefore, this colorimetric approach affords simplicity, sensitivity and reliability in telomerase activity detection.

Label-Free Detection of Telomerase Activity in Urine Using Telomerase-Responsive Porous Anodic Alumina Nanochannels.

Telomerase is closely related to cancers, which makes it one of the most widely known tumor marker. Recently, many methods have been reported for telomerase activity measurement in which complex label procedures were commonly used. In this paper, a label-free method for detection of telomerase activity in urine based on steric hindrance changes induced by confinement geometry in the porous anodic alumina (PAA) nanochannels was proposed. Telomerase substrate (TS) primer was first assembled on the inside wall of PAA nanochannels by Schiff reaction under mild conditions. Then, under the action of telomerase, TS primer was amplified and extended to repeating G-rich sequences (TTAGGG)x, which formed multiplex G-quadruplex in the presence of potassium ions (K(+)). This configurational change led to the increment of steric hindrance in the nanochannels, resulting in the decrement of anodic current of potassium ferricyanide (K3[Fe(CN)6]). Compared with previously reported methods based on PAA nanochannels (usually one G-quadruplex formed), multiplex repeating G-quadruplex formed on one TS primer in this work. As a result, large current drop (∼3.6 µA, 36%) was obtained, which gave facility to improve the detection sensitivity. The decreased ratio of anodic current has a linear correlation with the logarithm of HeLa cell number in the range of 10-5000 cells, with the detection limit of seven cells. The method is simple, reliable, and has been successfully applied in the detection of telomerase in urine with good accuracy, selectivity and reproducibility. In addition, the method is nondestructive test compared to blood analysis and pathology tests, which is significant for cancer discovery, development, and prognosis.

Facile Probe Design: Fluorescent Amphiphilic Nucleic Acid Probes without Quencher Providing Telomerase Activity Imaging Inside Living Cells.

Nowadays, the probe with fluorophore but no quencher is promising for its simple preparation, environmental friendliness, and wide application scope. This study designs a new amphiphilic nucleic acid probe (ANAP) based on aggregation-caused quenching (ACQ) effect without any quencher. Upon binding with targets, the dispersion of hydrophobic part (conjugated fluorene, CF) in ANAP is enhanced as a signal-on model for proteins, nucleic acids, and small molecules detection or the aggregation of CF is enhanced as a signal-off model for ion detection. Meanwhile, because of the high specificity of ANAP, a one-step method is developed powerfully for monitoring the telomerase activity not only from the cell extracts but also from 50 clinic urine samples (positive results from 45 patients with bladder cancer and negative results from 5 healthy people). ANAPs can also readily enter into cells and exhibit a good performance for distinguishing natural tumor cells from the tumor cells pretreated by telomerase-related drugs or normal cells. In contrast to our previous results ( Anal. Chem. 2015 , 87 , 3890 - 3894 ), the present CF is a monomer which is just the structure unit of the previous fluorescent polymer. Since the accurate molecular structure and high DNA/CF ratio of the present CF, these advanced experiments obtain an easier preparation of probes, an improved sensitivity and specificity, and broader detectable targets.

Quencher group induced high specificity detection of telomerase in clear and bloody urines by AIEgens.

Telomerase is a widely used tumor biomarker for early cancer diagnosis. On the basis of the combined use of aggregation-induced emission (AIE) fluorogens and quencher, a quencher group induced high specificity strategy for detection of telomerase activity from cell extracts and cancer patients' urine specimens was creatively developed. In the absence of telomerase, fluorescence background is extremely low due to the short distance between quencher and AIE dye. In the addition of telomerase, fluorescence enhances significantly. The telomerase activity in the E-J, MCF-7, and HeLa extracts equivalent to 5-10 000 cells can be detected by this method in ∼1 h. Furthermore, the distinguishing of telomerase extracted from 38 cancer and 15 normal urine specimens confirms the reliability and practicality of this protocol. In contrast to our previous results (Anal. Chem. 2015, 87, 6822-6827), these advanced experiments obtain more remarkable specificity.

Real-Time, Quantitative Lighting-up Detection of Telomerase in Urines of Bladder Cancer Patients by AIEgens.

As a biomarker for early cancer diagnosis, telomerase are one of the promising targets for cancer therapeutics. Inspired by the fluorescent emission principle of aggregation-induced emission fluorogens, we creatively designed an AIE-based turn-on method to detect telomerase activity from cell extracts. A positively charged fluorogen (TPE-Z) is not fluorescent when freely diffused in solution. The fluorescence of TPE-Z is enhanced with the elongation of the DNA strand which could light up telomere elongation process. By exploitation of it, we can detect telomerase activity from different cell lines (E-J, HeLa, MCF-7, and HLF) with high sensitivity and specificity. Moreover, our method is successfully employed to demonstrate the applications in bladder cancer diagnosis (41 urine specimens from bladder cancer patients and 15 urine specimens from normal people are detected). The AIE-based method provides a simple one-pot technique for quantification and monitoring of the telomerase activity and shows great potential for future use in clinical tests.