In vivo assessment of bladder cancer with diffuse reflectance and fluorescence spectroscopy: A comparative study.

OBJECTIVES: The aim of this work is to assess the performance of multimodal spectroscopic approach combined with single core optical fiber for detection of bladder cancer during surgery in vivo. METHODS: Multimodal approach combines diffuse reflectance spectroscopy (DRS), fluorescence spectroscopy in the visible (405 nm excitation) and near-infrared (NIR) (690 nm excitation) ranges, and high-wavenumber Raman spectroscopy. All four spectroscopic methods were combined in a single setup. For 21 patients with suspected bladder cancer or during control cystoscopy optical spectra of bladder cancer, healthy bladder wall tissue and/or scars were measured. Classification of cancerous and healthy bladder tissue was performed using machine learning methods. RESULTS: Statistically significant differences in relative total haemoglobin content, oxygenation, scattering, and visible fluorescence intensity were found between tumor and normal tissues. The combination of DRS and visible fluorescence spectroscopy allowed detecting cancerous tissue with sensitivity and specificity of 78% and 91%, respectively. The addition of features extracted from NIR fluorescence and Raman spectra did not improve the quality of classification. CONCLUSIONS: This study demonstrates that multimodal spectroscopic approach allows increasing sensitivity and specificity of bladder cancer detection in vivo. The developed approach does not require special probes and can be used with single-core optical fibers applied for laser surgery.

Development of a Sensitive Digital Droplet PCR Screening Assay for the Detection of GPR126 Non-Coding Mutations in Bladder Cancer Urine Liquid Biopsies.

Recent whole-genome sequencing studies identified two novel recurrent mutations in the enhancer region of GPR126 in urothelial bladder cancer (UBC) tumor samples. This mutational hotspot is the second most common after the TERT promoter in UBC. The aim of the study was to develop a digital droplet PCR screening assay for the simultaneous detection of GPR126 mutations in a single tube. Its performance combined with TERT promoter mutation analysis was evaluated in urine of healthy volunteers (n = 50) and patients with cystitis (n = 22) and UBC (n = 70). The developed assay was validated using DNA constructs carrying the studied variants. None of the mutations were detected in control and cystitis group samples. GPR126 mutations were observed in the urine of 25/70 UBC patients (area under the ROC curve (AUC) of 0.679; mutant allele fraction (MAF) of 21.61 [8.30-44.52] %); TERT mutations-in 40/70 (AUC of 0.786; MAF = 28.29 [19.03-38.08] %); ≥1 mutation-in 47/70 (AUC of 0.836)). The simultaneous presence of GPR126 and TERT mutations was observed in 18/70 cases, with no difference in MAFs for the paired samples (31.96 [14.78-47.49] % vs. 27.13 [17.00-37.62] %, p = 0.349, respectively). The combined analysis of these common non-coding mutations in urine allows the sensitive and non-invasive detection of UBC.

Urine TERT promoter mutations-based tumor DNA detection in patients with bladder cancer: A pilot study.

Telomerase reverse transcriptase (TERT) promoter mutations are the most frequent genetic events in bladder cancer (BC). The aim of the present pilot study was to evaluate the diagnostic potential of urine TERT promoter mutations-based liquid biopsy in patients with an ongoing oncological process, as well as in post-resection patients at risk of BC recurrence. A total of 60 patients were enrolled, of whom 27 patients had histologically proven BC; 23 had no signs of BC (control group); and 10 patients underwent transurethral malignancy resection 3-6 months prior to urine donation ('second look' group). Urine TERT promoter mutations were detected using Droplet Digital PCR. Receiver operating characteristic curve analysis revealed significant diagnostic power of the present approach (area under the curve: -0.768). At the cut-off value of tumor DNA fraction 0.34%, the sensitivity and specificity were 55.56 and 100%, respectively. In the positive samples, tumor DNA fraction varied significantly from 0.59 to 48.77%. In the 'second look' group, tumor DNA was detected in 4/10 patients, highlighting the possibility of BC recurrence with its fraction ranging only from 0.90 to 6.61%. Therefore, urine TERT promoter mutations-based liquid biopsy appears to be a promising tool for BC diagnosis and surveillance. The main study will include recruitment of additional patients, extension of the mutation panel, prolonged follow-up of the post-resection patients, as well as screening of industrial workers exposed to specific carcinogens.

Activating Telomerase TERT Promoter Mutations and Their Application for the Detection of Bladder Cancer.

This review summarizes state-of-the-art knowledge in early-generation and novel urine biomarkers targeting the telomerase pathway for the detection and follow-up of bladder cancer (BC). The limitations of the assays detecting telomerase reactivation are discussed and the potential of transcription-activating mutations in the promoter of the TERT gene detected in the urine as promising simple non-invasive BC biomarkers is highlighted. Studies have shown good sensitivity and specificity of the urinary TERT promoter mutations in case-control studies and, more recently, in a pilot prospective cohort study, where the marker was detected up to 10 years prior to clinical diagnosis. However, large prospective cohort studies and intervention studies are required to fully validate their robustness and assess their clinical utility. Furthermore, it may be interesting to evaluate whether the clinical performance of urinary TERT promoter mutations could increase when combined with other simple urinary biomarkers. Finally, different approaches for assessment of TERT promoter mutations in urine samples are presented together with technical challenges, thus highlighting the need of careful technological validation and standardization of laboratory methods prior to translation into clinical practice.

Tissue ACE phenotyping in prostate cancer.

Epithelial cells of prostate express significant level of ACE and, as a result, seminal fluid has 50-fold more ACE than plasma. The substitution of highly specialized prostate epithelial cells by tumor cells results in dramatic decrease in ACE production in prostate tissues. We performed detailed characterization of ACE status in prostate tissues from patients with benign prostate hyperplasia (BPH) and prostate cancer (PC) using new approach- ACE phenotyping, that includes evaluation of: 1) ACE activity with two substrates (HHL and ZPHL); 2) the ratio of the rates of their hydrolysis (ZPHL/HHL ratio); 3) the ratio of immunoreactive ACE protein to ACE activity; 4) the pattern of mAbs binding to different epitopes on ACE - ACE conformational fingerprint - to reveal conformational changes in prostate ACE due to prostate pathology. ACE activity dramatically decreased and the ratio of immunoreactive ACE protein to ACE activity increased in PC tissues. The catalytic parameter, ZPHL/HHL ratio, increased in prostate tissues from all patients with PC, but was did not change for most |BPH patients. Nevertheless, prostate tissues of several patients diagnosed with BPH based on histology, also demonstrated decreased ACE activity and increased immunoreactive ACE protein/ACE activity and ZPHL/HHL ratios, that could be considered as more early indicators of prostate cancer development than routine histology. Thus, ACE phenotyping of prostate biopsies has a potential to be an effective approach for early diagnostics of prostate cancer or at least for differential diagnostics of BPH and PC.