Feliksas Jankevičius

Considerations on implementing diagnostic markers into clinical decision making in bladder cancer

Bladder cancer is a common disease that is often detected late and has a high rate of recurrence and progression. Cystoscopy is the main tool in detection and surveillance of bladder cancer but is invasive and can miss some cancers. Cytology is frequently utilized but suffers from a poor sensitivity. There are several commercially available urine-based tumor markers currently available but their use is not recommended by guideline panels. Markers such as the Urovysion FISH assay and the NMP22 BladderChek test are approved for surveillance and detection in patients with hematuria. The added benefit of these markers and other commercially available markers (e.g. Ucyt+, BTA stat) has not been well investigated though it appears these markers are insufficiently sensitive to replace cystoscopy. Additional studies are needed to determine the clinical scenarios where bladder markers are best utilized (screening, surveillance, early detection, evaluating cytologic atypia) and what impact they should have on clinical decision making. Furthermore, a variety of issues and barriers can affect the movement of clinical tests from research to clinical practice. This article addresses some of the challenges facing research and medical communities in the delivery and integration of markers for bladder cancer diagnosis. Moreover, we attempt to outline criteria for the clinical utility of new bladder cancer diagnostic markers.

Promoter Hypermethylation in Tumour Suppressor Genes Shows Association with Stage, Grade and Invasiveness of Bladder Cancer

Aims: Superficial bladder cancer is a highly recurrent disease, with progression to muscle invasiveness occurring in 15–30% of cases. Promoter hypermethylation in a panel of tumour suppressor genes involved in cell cycle control, apoptosis and DNA repair was analyzed in superficial bladder tumours in order to evaluate the suitability of epigenetic biomarkers for an earlier prediction of the aggressive course of the disease. Method: Promoter hypermethylation in p16, RARβ, RASSF1A, DAPK, and MGMT genes was analyzed in 58 cases with superficial bladder cancer and 2 cases with benign urological disease using methylation-specific PCR. Results: Promoter hypermethylation was frequently detected in RARβ, RASSF1A and DAPK genes, and 62% of bladder tumours exhibited hypermethylation in at least one gene. The overall frequency of hypermethylation and the number of genes involved increased with tumour stage, grade and muscle invasiveness. Aberrant methylation of RASSF1A and RARβwas predominant (p < 0.05) in muscle-invasive tumours and high-grade tumours, respectively. Cases with concurrent hypermethylation in DAPK, p16 and RARβ genes were moresusceptible to relapse. Conclusion: The results suggest analysis of promoter hypermethylation as a valuable biomarker for prognosis of the aggressive course of disease in bladder cancer.

Epigenetic markers of prostate cancer in plasma circulating DNA

Epigenetic differences are a common feature of many diseases, including cancer, and disease-associated changes have even been detected in bodily fluids. DNA modification studies in circulating DNA (cirDNA) may lead to the development of specific non-invasive biomarkers. To test this hypothesis, we investigated cirDNA modifications in prostate cancer patients with locally confined disease (n = 19), in patients with benign prostate hyperplasias (n = 20) and in men without any known prostate disease (n = 20). This initial discovery screen identified 39 disease-associated changes in cirDNA modification, and seven of these were validated using the sodium bisulfite-based mapping of modified cytosines in both the discovery cohort and an independent 38-patient validation cohort. In particular, we showed that the DNA modification of regions adjacent to the gene encoding ring finger protein 219 distinguished prostate cancer from benign hyperplasias with good sensitivity (61%) and specificity (71%). We also showed that repetitive sequences detected in this study were meaningful, as they indicated a highly statistically significant loss of DNA at the pericentromeric region of chromosome 10 in prostate cancer patients (p = 1.8 × 10(-6)). Based on these strong univariate results, we applied machine-learning techniques to develop a multi-locus biomarker that correctly distinguished prostate cancer samples from unaffected controls with 72% accuracy. Lastly, we used systems biology techniques to integrate our data with publicly available DNA modification and transcriptomic data from primary prostate tumors, thereby prioritizing genes for further studies. These data suggest that cirDNA epigenomics are promising source for non-invasive biomarkers.

The utility of urine-circulating miRNAs for detection of prostate cancer.

Background:In this paper, the utility of urine-circulating microRNAs (miRNAs) as the potential biomarker of prostate cancer (PCa), the second most prevalent male cancer worldwide, was evaluated.Methods:Cancerous (N=56) and non-cancerous (N=16) prostate tissues were analysed on TaqMan Low Density Array, with the initial screening of 754 miRNAs in a subset of the samples. The abundance of selected miRNAs was analysed in urine specimens from two independent cohorts of patients with PCa (N=215 overall), benign prostatic hyperplasia (BPH; N=23), and asymptomatic controls (ASC; N=62) by means of quantitative reverse transcription PCR.Results:Over 100 miRNAs were found deregulated in PCa as compared with non-cancerous prostate tissue. After thorough validation, four miRNAs were selected for the analysis in urine specimens. The abundance of miR-148a and miR-375 in urine was identified as specific biomarkers of PCa in both cohorts. Combined analysis of urine-circulating miR-148a and miR-375 was highly sensitive and specific for PCa in both cohorts (AUC=0.79 and 0.84) and strongly improved the diagnostic power of the PSA test (AUC=0.85, cohort PCa1), including the grey diagnostic zone (AUC=0.90).Conclusions:Quantitative measurement of urine-circulating miR-148a and miR-375 can serve as the non-invasive tool for sensitive and specific detection of PCa.

Non‐linear optical microscopy of kidney tumours

The unregulated cancer cell growth leads to strong alterations in morphology and composition of the tissue. The combination of coherent anti-Stokes Raman scattering, two-photon excited fluorescence and second harmonic generation enables a high resolution imaging with strong information on tissue composition and can then provide useful information for tumour diagnosis. Here we present the potential of multimodal non-linear microscopy for imaging of renal tumours. Using cryosections of human oncocytoma and carcinoma, the method gave a detailed insight in cancer morphology and composition, enabling to discern between normal kidney tissue, tumour and necrosis. Several features significant for the diagnosis were clearly visualised without use of any staining. Translation of this method in clinical pathology will greatly improve speed and quality of the analyses.

Detection of miRNAs in urine of prostate cancer patients.

BACKGROUND AND AIM Prostate cancer (PCa) is the second most prevalent oncologic disease among men worldwide. Expression of various transcripts, including miRNAs, is markedly deregulated in cancerous prostate tissue. This study aimed at identifying a PCa-specific expression profile of miRNAs for subsequent use in noninvasive diagnostics. MATERIALS AND METHODS MiRNA expression was profiled in 13 PCa tissues using human miRNA microarrays. Highly expressed miRNAs were selected for the analysis in urine of patients with PCa (N=143) and benign prostate hyperplasia (BPH; N=23) by means of real time PCR, while miRNAs showing the expression differences in relation to clinical variables were further analyzed in 52 PCa and 12 noncancerous prostate tissues (NPT) on TaqMan Low Density Arrays (TLDA). RESULTS Analysis of miRNA expression in prostate tissue linked miR-95 to aggressive form of PCa. This miRNA was up-regulated in high grade (P=0.041), the TMPRSS2-ERG fusion-positive tumors (P=0.026), and in patients with subsequently developed biochemical recurrence (BCR; P=0.054) after radical prostatectomy. MiRNAs highly expressed in PCa tissues were also detectable in urine from PCa patients. Moreover, the urinary levels of miR-21 had significant discriminatory power (P=0.010) to separate PCa patients from BPH, while the combined analysis of urinary miR-19a and miR-19b was prognostic for BCR. In PCa, the diagnostic potential of urinary miRNA panel (miR-21, miR-19a, and miR-19b) was higher than that of the PSA test (AUC=0.738 vs. AUC=0.514). CONCLUSIONS Measurement of urinary levels of PCa-specific miRNAs could assist in more specific detection of PCa and prediction of BCR.

Combined Analysis of TMPRSS2-ERG and TERT for Improved Prognosis of Biochemical Recurrence in Prostate Cancer

Prostate cancer (PCa) is a heterogeneous disease with diverse clinical outcomes. TMPRSS2–ERG is the most common gene fusion in PCa, whereas activation of telomerase is a common feature of various malignancies. The aim of our study was to explore the combined utility of these and some other biomarkers in predicting biochemical recurrence after radical prostatectomy. Prostate specimens and urine sediments from 179 previously untreated patients with pT2‐pT3 stage PCa were analyzed for expression of telomerase (TERT and TR) and the TMPRSS2–ERG fusion gene by means of reverse transcription PCR. Real‐time PCR was used for quantification of ERG and SPINK1 expression. In total, 74% (117/158) of the prostate adenocarcinomas were positive for the TMPRSS2–ERG and/or TERT expression. Noninvasively, these transcripts were identified in 31% (19/61) of catheterized urine specimens. Significantly higher expression of ERG was detected in TMPRSS2–ERG‐positive tumors (P < 0.0001), whereas more intense expression of SPINK1 was characteristic for the TMPRSS2–ERG‐negative tumors (P = 0.003). TERT‐positive cases also had elevated levels of ERG (P = 0.016), suggesting a possible link between aberrant expression of ERG and reactivation of TERT in prostate tumors. The cases negative for both transcripts, TMPRSS2–ERG and TERT, rarely recurred (P = 0.014) and showed significantly longer biochemical recurrence‐free period (P = 0.022) as compared to the TMPRSS2–ERG and/or TERT‐positive cases. The results of our study suggest that combined analysis of TMPRSS2–ERG and TERT expression can be a valuable tool for early prediction of biochemical recurrence of PCa after radical prostatectomy.

Frequent down-regulation of ABC transporter genes in prostate cancer.

BackgroundATP-binding cassette (ABC) transporters are transmembrane proteins responsible for the efflux of a wide variety of substrates, including steroid metabolites, through the cellular membranes. For better characterization of the role of ABC transporters in prostate cancer (PCa) development, the profile of ABC transporter gene expression was analyzed in PCa and noncancerous prostate tissues (NPT).MethodsTaqMan Low Density Array (TLDA) human ABC transporter plates were used for the gene expression profiling in 10 PCa and 6 NPT specimens. ABCB1 transcript level was evaluated in a larger set of PCa cases (N = 78) and NPT (N = 15) by real-time PCR, the same PCa cases were assessed for the gene promoter hypermethylation by methylation-specific PCR.ResultsExpression of eight ABC transporter genes (ABCA8, ABCB1, ABCC6, ABCC9, ABCC10, ABCD2, ABCG2, and ABCG4) was significantly down-regulated in PCa as compared to NPT, and only two genes (ABCC4 and ABCG1) were up-regulated. Down-regulation of ABC transporter genes was prevalent in the TMPRSS2-ERG-negative cases.A detailed analysis of ABCB1 expression confirmed TLDA results: a reduced level of the transcript was identified in PCa in comparison to NPT (p = 0.048). Moreover, the TMPRSS2-ERG-negative PCa cases showed significantly lower expression of ABCB1 in comparison to NPT (p = 0.003) or the fusion-positive tumors (p = 0.002). Promoter methylation of ABCB1 predominantly occurred in PCa and was rarely detected in NPT (p < 0.001).ConclusionsThe study suggests frequent down-regulation of the ABC transporter genes in PCa, especially in the TMPRSS2-ERG-negative tumors.

Frequent Methylation of RASSF1 and RARB in Urine Sediments From Patients with Early Stage Prostate Cancer

BACKGROUND. Prostate cancer (PCa) is the second most prevalent malignancy among males, characterized by high mortality rates. Aberrant DNA methylation in promoters of tumor suppressor genes is an early and frequent event during prostate carcinogenesis. Modern techniques allow a sensitive detection of DNA methylation biomarkers in bodily fluids from cancer patients offering a noninvasive tool for PCa monitoring. Our study aimed at the analysis of DNA methylation in urine sediments from PCa patients for the selection of most informative noninvasive biomarkers. MATERIAL AND METHODS. Real-time methylation-specific polymerase chain reaction was used for the detection of methylated RASSF1, RARB, and GSTP1 genes in catheterized urine specimens from 34 patients with biopsy-proven early or medium stage PCa. RESULTS. At least one gene was methylated in urine sediments from 28 cases with PCa, with a sensitivity of the test reaching 82%. RASSF1 was methylated in 71% (24 of 34), RARB in 44% (15 of 34), and GSTP1 in 3% (1 of 34) of the specimens. High level of methylation (≥50%) in RARB and RASSF1 genes was detected in 40% and 20% of cases, respectively. A significant association was observed between high level of RARB methylation and Gleason score (P=0.01), while methylation of at least one gene occurred more frequently in urine DNA of older patients (P=0.02). CONCLUSIONS. Results of our study show a high sensitivity of DNA methylation biomarkers, especially RASSF1 and RARB, for the early and noninvasive detection of PCa.

Dual-Parameter Immunoflow Cytometry in Diagnosis and Follow-Up of Patients with Bladder Cancer

Objectives: The aim of this study was to evaluate the clinical significance of a dual-parameter immunoflow cytometry (DPI-FCM) assay in the detection of tumor cells in barbotage specimens of bladder cancer patients. Methods: DPI-FCM is an automated method, based on the utilization of two monoclonal antibodies (mAbs) either used for a preselection of urothelial cells (mAb Due AUT 2) or the analysis of the expression of a differentiation antigen within the preselected urothelial cells (mAb Due ABC 3). The ratio of ABC 3-positive urothelial cells was used to discriminate between the normal and malignant state of the urothelium. At the time of examination 40 patients had endoscopically overt bladder tumors. Another 30 patients without endoscopically visible tumors were examined before routine rebiopsy. Thirty barbotage specimens from patients with diseases not related to bladder cancer were examined as controls. Results: Overall, the sensitivity of DPI-FCM in patients with endoscopically overt and invisible residual tumors was 95 and 83%, respectively, regardless of concomitant urinary tract infection. The sensitivity of DPI-FCM for both patient groups was 86, 95 and 94% for tumor grades 1, 2 and 3, respectively. The specificity of the method in 30 patients with no history of bladder cancer was 93%. Conclusions: DPI-FCM appears to be a highly reliable method of recognizing tumor cells in bladder barbotage specimens even in patients with concomitant urinary tract infection. The procedure may be of value in monitoring bladder cancer patients for tumor recurrence.