Prognostic Value of Liquid-Biopsy-Based Biomarkers in Upper Tract Urothelial Carcinoma.

Currently, there are no reliable prognostic factors to determine which upper tract urothelial carcinoma (UTUC) patients will progress after radical nephroureterectomy (RNU). We aim to evaluate whether liquid-biopsy-based biomarkers (circulating tumor cells (CTCs), cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA)) were able to predict clinical outcomes in localized UTUC patients undergoing RNU. Twenty patients were prospectively enrolled between 2021 and 2023. Two blood samples were collected before RNU and three months later. CTCs and cfDNA were isolated and evaluated using the IsoFlux system and Quant-iT PicoGreen dsDNA kit, respectively. Droplet digital PCR was performed to determine ctDNA status. Cox regression analysis was performed on CTCs, cfDNA, and ctDNA at two different follow-up time points to examine their influence on tumor progression and cancer-specific survival (CSS). During a median follow-up of 18 months, seven (35%) patients progressed and three (15%) died. Multivariate analysis demonstrated that cfDNA levels three months after RNU are a significant predictor of tumor progression (HR = 1.085; p = 0.006) and CSS (HR = 1.168; p = 0.029). No associations were found between CTC enumeration and ctDNA status with any of the clinical outcomes evaluated. The evaluation of cfDNA levels in clinical practice could improve the disease management of UTUC patients.

Tumor-Agnostic Circulating Tumor DNA Testing for Monitoring Muscle-Invasive Bladder Cancer.

Circulating tumor DNA (ctDNA) has recently emerged as a real-time prognostic and predictive biomarker for monitoring cancer patients. Here, we aimed to ascertain whether tumor-agnostic ctDNA testing would be a feasible strategy to monitor disease progression and therapeutic response in muscle-invasive bladder cancer (MIBC) patients after radical cystectomy (RC). Forty-two MIBC patients who underwent RC were prospectively included. Blood samples from these patients were collected at different follow-up time points. Two specific mutations (TERT c.1-124C>T and ATM c.1236-2A>T) were analyzed in the patients' plasma samples by droplet digital PCR to determine their ctDNA status. During a median follow-up of 21 months, 24% of patients progressed in a median of six months. ctDNA status was identified as a prognostic biomarker of tumor progression before RC and 4 and 12 months later (HR 6.774, HR 3.673, and HR 30.865, respectively; p < 0.05). Lastly, dynamic changes in ctDNA status between baseline and four months later were significantly associated with patient outcomes (p = 0.045). In conclusion, longitudinal ctDNA analysis using a tumor-agnostic approach is a potential tool for monitoring MIBC patients after RC. The implementation of this testing in a clinical setting could improve disease management and patients' outcomes.

Gene variation impact on prostate cancer progression: Lymphocyte modulator, activation, and cell adhesion gene variant contribution.

BACKGROUND: The view of prostate cancer (PCa) progression as a result of the interaction of epithelial cancer cells with the host's immune system is supported by the presence of tumor infiltrating lymphocytes (TILs). TILs fate and interaction with the tumor microenvironment is mediated by accessory molecules such as CD5 and CD6, two signal-transducing coreceptors involved in fine-tuning of T cell responses. While the nature of the CD5 ligand is still controversial, CD6 binds CD166/ALCAM, a cell adhesion molecule involved in progression and dissemination of epithelial cancers, including PCa. The purpose of the present study was to determine the role of CD5, CD6, and CD166/ALCAM gene variants in PCa. METHODS: Functionally relevant CD5 (rs2241002 and rs2229177), CD6 (rs17824933, rs11230563, and rs12360861) and CD166/ALCAM (rs6437585, rs579565, rs1044243, and rs35271455) single nucleotide polymorphisms (SNPs) were genotyped in germline DNA samples from 376 PCa patients. Their association with PCa prognostic factors, namely biochemical recurrence (BCR) and International Society of Urological Pathology (ISUP) grade was analyzed by generalized linear models and survival analyses. RESULT: Proportional hazards regression showed that the minor CD6 rs12360861AA and CD166/ALCAM rs579565AA genotypes were associated with earlier BCR, with hazard ratios of 2.65 (95% CI: 1.39-5.05, p = 0.003) and 1.86, (95% CI: 1.02-3.39, p = 0.043), respectively. Individually, none of the analyzed SNPs was significantly associated with ISUP grade, but haplotype analyses revealed association of the CD5 rs2241002C -rs2229177T haplotype with ISUP grade ≥2, with odds ratio of 1.52 (95% CI: 1.05-2.21, p = 0.026). CONCLUSION: The results show the impact on PCa aggressiveness and recurrence brought about by gene variants involved in modulation of lymphocyte activation (CD5, CD6) and immune-epithelial cell adhesion (CD166/ALCAM) in PCa aggressiveness and recurrence, thus supporting a role for host immune response in PCa pathophysiology.

Multicentric validation of diagnostic tests based on BC-116 and BC-106 urine peptide biomarkers for bladder cancer in two prospective cohorts of patients.

BACKGROUND: Non-invasive urine-based biomarkers can potentially improve current diagnostic and monitoring protocols for bladder cancer (BC). Here we assess the performance of earlier published biomarker panels for BC detection (BC-116) and monitoring of recurrence (BC-106) in combination with cytology, in two prospectively collected patient cohorts. METHODS: Of the 602 patients screened for BC, 551 were found eligible. For the primary setting, 73 patients diagnosed with primary BC (n = 27) and benign urological disorders, including patients with macroscopic haematuria, cystitis and/or nephrolithiasis (n = 46) were included. In total, 478 patients under surveillance were additionally considered (83 BC recurrences; 395 negative for recurrence). Urine samples were analysed with capillary electrophoresis-mass spectrometry. The biomarker score was estimated via support vector machine-based software. RESULTS: Validation of BC-116 biomarker panel resulted in 89% sensitivity and 67% specificity (AUCBC-116 = 0.82). A diagnostic score based on cytology and BC-116 resulted in good (AUCNom116 = 0.85) but not significantly better performance (P = 0.5672). A diagnostic score including BC-106 and cytology was evaluated (AUCNom106 = 0.82), significantly outperforming both cytology (AUCcyt = 0.72; P = 0.0022) and BC-106 (AUCBC-106 = 0.67; P = 0.0012). CONCLUSIONS: BC-116 biomarker panel is a useful test for detecting primary BC. BC-106 classifier integrated with cytology showing >95% negative predictive value, might be useful for decreasing the number of cystoscopies during surveillance.

Prognostic implication of TERT promoter mutation and circulating tumor cells in muscle-invasive bladder cancer.

PURPOSE: Current clinical prognostic factors are not accurate enough to identify and monitor those muscle-invasive bladder cancer (MIBC) patients at high risk of progression after radical cystectomy (RC). Here, we determined genetic alterations in the tumor and circulating tumor cell (CTC) enumeration to find biomarkers useful for the management of MIBC after RC. METHODS: Thirty-nine MIBC patients undergoing RC were included. Tumoral tissue DNA was analyzed by next generation sequencing. CTCs were isolated from blood collected before RC and one, four and 12 months later. RESULTS: Sixteen (41%) patients progressed in a median time of 8.5 months and 11 (69%) of these patients harbored the TERT c.-124C > T mutation. All progressive patients harboring the TERT c.-124C > T mutation presented a significant increase in CTC number 12 months after RC compared to those without the mutation. Additionally, CTC number at 12 months was identified as an independent prognostic biomarker for tumor progression and cancer specific survival (CSS). Ten (63%) progressive patients showed an increment of CTC number with a median anticipation period of four months compared with imaging techniques. CONCLUSIONS: The TERT c.-124C > T mutation could be considered a biomarker of aggressivity. CTC enumeration is a useful tool for identifying MIBC patients at high risk of progression and CSS after RC and for detecting tumor progression earlier than imaging techniques.

Cell-Free DNA as a Prognostic Biomarker for Monitoring Muscle-Invasive Bladder Cancer.

Cell-free DNA (cfDNA) has recently emerged as a real-time biomarker for diagnosis, monitoring and prediction of therapy response in tumoral disease. Here, we evaluated cfDNA as a prognostic biomarker for monitoring muscle-invasive bladder cancer (MIBC) patients at different follow-up time points. Blood samples from 37 MIBC patients who underwent radical cystectomy (RC) were collected at cystectomy and 1, 4, 12 and 24 months later. Plasma cfDNA amount and fragmentation patterns were determined. Four mutations were analyzed in cfDNA to detect circulating tumor DNA (ctDNA) during patient follow-up. During a median follow-up of 36 months, 46% of patients progressed; median time to progression was 10 months. cfDNA levels and ctDNA status four months after RC were identified as independent prognostic biomarkers of tumor progression (HR 5.290; p = 0.033) and cancer-specific survival (HR 4.199; p = 0.038), respectively. Furthermore, ctDNA clearance four months after RC was significantly associated with patients' clinical outcomes. In conclusion, cfDNA levels and ctDNA status four months after RC have prognostic implications in MIBC patients. In addition, cfDNA monitoring is useful to predict patient outcomes after RC. cfDNA analysis in the clinical setting could greatly improve MIBC patient management.

The Role of Fluorescence In Situ Hybridization for Predicting Recurrence after Adjuvant bacillus Calmette-Guérin in Patients with Intermediate and High Risk Nonmuscle Invasive Bladder Cancer: A Systematic Review and Meta-Analysis of Individual Patient Data.

PURPOSE: The objective of this study was to assess the value of fluorescence in situ hybridization to predict early recurrence in patients with nonmuscle invasive bladder cancer at intermediate and high risk treated with bacillus Calmette-Guérin. MATERIALS AND METHODS: We performed a systematic review using MEDLINE®, Embase® and the Cochrane Library. Individual patient data from prospective observational studies of fluorescence in situ hybridization in patients treated with bacillus Calmette-Guérin were included. A 2-stage individual patient data meta-analysis was done to assess the value of fluorescence in situ hybridization to predict tumor recurrence after bacillus Calmette-Guérin induction therapy. RESULTS: From a total of 4 studies we obtained individual data on 422 patients, of whom 408 with a median followup of 18.8 months were included in the final analysis. When fluorescence in situ hybridization was positive, the recurrence HR was 1.20 (95% CI 0.81-1.79) before bacillus Calmette-Guérin (time 0), 2.23 (95% CI 1.31-3.62) at 6 weeks (time 1), 3.70 (95% CI 2.34-5.83) at 3 months (time 2) and 23.44 (95% CI 5.26-104.49) at 6 months (time 3). CONCLUSIONS: A positive fluorescence in situ hybridization test after bacillus Calmette-Guérin correlated with higher risk of recurrent tumor. Fluorescence in situ hybridization could aid urologists in risk stratifying and counseling patients. Based on the HR and the narrowest CI the preferred timing of fluorescence in situ hybridization is 3 months after transurethral resection of bladder tumor. This is also in time for patients in whom induction therapy fails to enter clinical trials or change the treatment strategy.

Mediterranean Dietary Pattern is Associated with Low Risk of Aggressive Prostate Cancer: MCC-Spain Study.

PURPOSE: We explored the association of the previously described Western, prudent and Mediterranean dietary patterns with prostate cancer risk by tumor aggressiveness and extension. MATERIALS AND METHODS: MCC-Spain (Multicase-Control Study on Common Tumors in Spain) is a population based, multicase-control study that was done in 7 Spanish provinces between September 2008 and December 2013. It collected anthropometric, epidemiological and dietary information on 754 histologically confirmed incident cases of prostate cancer and 1,277 controls 38 to 85 years old. Three previously identified dietary patterns, including Western, prudent and Mediterranean, were reconstructed using MCC-Spain data. The association of each pattern with prostate cancer risk was assessed by logistic regression models with random, province specific intercepts. Risk according to tumor aggressiveness (Gleason score 6 vs greater than 6) and extension (cT1-cT2a vs cT2b-cT4) was evaluated by multinomial regression models. RESULTS: High adherence to a Mediterranean dietary pattern rich not only in fruits and vegetables but also in fish, legumes and olive oil was specifically associated with a lower risk of Gleason score greater than 6 prostate cancer (quartile 3 vs 1 relative RR 0.66, 95% CI 0.46-0.96 and quartile 4 vs 1 relative RR 0.68, 95% CI 0.46-1.01, p-trend = 0.023) or with higher clinical stage (cT2b-T4 quartile 4 vs 1 relative RR 0.49, 95% CI 0.25-0.96, p-trend = 0.024). This association was not observed with the prudent pattern, which combines vegetables and fruits with low fat dairy products, whole grains and juices. The Western pattern did not show any association with prostate cancer risk. CONCLUSIONS: Nutritional recommendations for prostate cancer prevention should consider whole dietary patterns instead of individual foods. We found important differences between the Mediterranean dietary pattern, which was associated with a lower risk of aggressive prostate cancer, and Western and prudent dietary patterns, which had no relationship with prostate cancer risk.

Using gene expression from urine sediment to diagnose prostate cancer: development of a new multiplex mRNA urine test and validation of current biomarkers.

BACKGROUND: Additional accurate non-invasive biomarkers are needed in the clinical setting to improve prostate cancer (PCa) diagnosis. Here we have developed a new and improved multiplex mRNA urine test to detect prostate cancer (PCa). Furthermore, we have validated the PCA3 urinary transcript and some panels of urinary transcripts previously reported as useful diagnostic biomarkers for PCa in our cohort. METHODS: Post-prostatic massage urine samples were prospectively collected from PCa patients and controls. Expression levels of 42 target genes selected from our previous studies and from the literature were studied in 224 post-prostatic massage urine sediments by quantitative PCR. Univariate logistic regression was used to identify individual PCa predictors. A variable selection method was used to develop a multiplex biomarker model. Discrimination was measured by ROC curve AUC for both, our model and the previously published biomarkers. RESULTS: Seven of the 42 genes evaluated (PCA3, ELF3, HIST1H2BG, MYO6, GALNT3, PHF12 and GDF15) were found to be independent predictors for discriminating patients with PCa from controls. We developed a four-gene expression signature (HIST1H2BG, SPP1, ELF3 and PCA3) with a sensitivity of 77% and a specificity of 67% (AUC = 0.763) for discriminating between tumor and control urines. The accuracy of PCA3 and previously reported panels of biomarkers is roughly maintained in our cohort. CONCLUSIONS: Our four-gene expression signature outperforms PCA3 as well as previously reported panels of biomarkers to predict PCa risk. This study suggests that a urinary biomarker panel could improve PCa detection. However, the accuracy of the panels of urinary transcripts developed to date, including our signature, is not high enough to warrant using them routinely in a clinical setting.

SPARC mediates metastatic cooperation between CSC and non-CSC prostate cancer cell subpopulations.

BACKGROUND: Tumor cell subpopulations can either compete with each other for nutrients and physical space within the tumor niche, or co-operate for enhanced survival, or replicative or metastatic capacities. Recently, we have described co-operative interactions between two clonal subpopulations derived from the PC-3 prostate cancer cell line, in which the invasiveness of a cancer stem cell (CSC)-enriched subpopulation (PC-3M, or M) is enhanced by a non-CSC subpopulation (PC-3S, or S), resulting in their accelerated metastatic dissemination. METHODS: M and S secretomes were compared by SILAC (Stable Isotope Labeling by Aminoacids in Cell Culture). Invasive potential in vitro of M cells was analyzed by Transwell-Matrigel assays. M cells were co-injected with S cells in the dorsal prostate of immunodeficient mice and monitored by bioluminescence for tumor growth and metastatic dissemination. SPARC levels were determined by immunohistochemistry and real-time RT-PCR in tumors and by ELISA in plasma from patients with metastatic or non-metastatic prostate cancer. RESULTS: Comparative secretome analysis yielded 213 proteins differentially secreted between M and S cells. Of these, the protein most abundantly secreted in S relative to M cells was SPARC. Immunodepletion of SPARC inhibited the enhanced invasiveness of M induced by S conditioned medium. Knock down of SPARC in S cells abrogated the capacity of its conditioned medium to enhance the in vitro invasiveness of M cells and compromised their potential to boost the metastatic behavior of M cells in vivo. In most primary human prostate cancer samples, SPARC was expressed in the epithelial tumoral compartment of metastatic cases. CONCLUSIONS: The matricellular protein SPARC, secreted by a prostate cancer clonal tumor cell subpopulation displaying non-CSC properties, is a critical mediator of paracrine effects exerted on a distinct tumor cell subpopulation enriched in CSC. This paracrine interaction results in an enhanced metastatic behavior of the CSC-enriched tumor subpopulation. SPARC is expressed in the neoplastic cells of primary prostate cancer samples from metastatic cases, and could thus constitute a tumor progression biomarker and a therapeutic target in advanced prostate cancer.

Identification of blood serum micro-RNAs associated with idiopathic and LRRK2 Parkinson's disease.

Blood-cell-free circulating micro-RNAs (miRNAs) have been proposed as potential accessible biomarkers for neurodegenerative diseases such as Parkinson's disease (PD). Here we analyzed the serum levels of 377 miRNAs in a discovery set of 10 idiopathic Parkinson's disease (IPD) patients, 10 PD patients carriers of the LRRK2 G2019S mutation (LRRK2 PD), and 10 controls by using real-time quantitative PCR-based TaqMan MicroRNA arrays. We detected candidate differentially expressed miRNAs, which were further tested in a first validation set consisting of 20 IPD, 20 LRRK2 PD, and 20 control samples. We found four statistically significant miRNAs that were downregulated in either LRRK2 or IPD (miR-29a, miR-29c, miR-19a, and miR-19b). Subsequently, we validated these findings in a third set of samples consisting of 65 IPD and 65 controls and confirmed the association of downregulated levels of miR-29c, miR-29a, and miR-19b in IPD. Differentially expressed miRNAs are predicted to target genes belonging to pathways related to ECM-receptor interaction, focal adhesion, MAPK, Wnt, mTOR, adipocytokine, and neuron projection. Results from our exploratory study indicate that downregulated levels of specific circulating serum miRNAs are associated with PD and suggest their potential use as noninvasive biomarkers for PD. Future studies should further confirm the association of these miRNAs with PD.

Validation study of a noninvasive urine test for diagnosis and prognosis assessment of bladder cancer: evidence for improved models.

PURPOSE: We validated the performance of our previously reported test for bladder cancer based on urine gene expression patterns using an independent cohort. We also ascertained whether alternative models could achieve better accuracy. MATERIALS AND METHODS: Gene expression patterns of the previously reported 48 genes, including the 12 + 2 genes of the signature, were analyzed by TaqMan® arrays in an independent set of 207 urine samples. We pooled all samples analyzed to date to obtain a larger training set of 404 and used it to search for putative improved new models. RESULTS: Our 12 + 2 gene expression signature had overall 80% sensitivity with 86% specificity (AUC 0.914) to discriminate between bladder cancer and control samples. It had 75% sensitivity and 75% specificity (AUC 0.83) to predict tumor aggressiveness in the validation set of urine samples. After grouping all samples 3 new signatures for diagnosis containing 2, 5 and 10 genes, respectively, and 1 containing 6 genes for prognosis were designed. Diagnostic performance of the 2, 5, 10 and 12-gene signatures was maintained or improved in the enlarged sample set (AUC 0.913, 0.941, 0.949 and 0.944, respectively). Performance to predict aggressiveness was also improved in the 14 and 6-gene signatures (AUC 0.855 and 0.906, respectively). CONCLUSIONS: This validation study confirms the accuracy of the 12 + 2 gene signature as a noninvasive tool for assessing bladder cancer. We present improved models with fewer genes that must be validated in future studies.

Clinical implications in the shift of syndecan-1 expression from the cell membrane to the cytoplasm in bladder cancer.

BACKGROUND: To determine the diagnostic and prognostic capability of urinary and tumoral syndecan-1 (SDC-1) levels in patients with cancer of the urinary bladder. METHODS: SDC-1 levels were quantitated by enzyme-linked immunosorbent assay (ELISA) in 308 subjects (102 cancer subjects and 206 non-cancer subjects) to assess its diagnostic capabilities in voided urine. The performance of SDC-1 was evaluated using the area under the curve of a receiver operating characteristic curve. In addition, immunohistochemical (IHC) staining assessed SDC-1 protein expression in 193 bladder specimens (185 cancer subjects and 8 non-cancer subjects). Outcomes were correlated to SDC-1 levels. RESULTS: Mean urinary levels of SDC-1 did not differ between the cancer subjects and the non-cancer subjects, however, the mean urinary levels of SDC-1 were reduced in high-grade compared to low-grade disease (p < 0.0001), and in muscle invasive bladder cancer (MIBC) compared to non-muscle invasive bladder cancer (NMIBC) (p = 0.005). Correspondingly, preliminary data note a shift from a membranous cellular localization of SDC-1 in normal tissue, low-grade tumors and NMIBC, to a distinctly cytoplasmic localization in high-grade tumors and MIBC was observed in tissue specimens. CONCLUSION: Alone urinary SDC-1 may not be a diagnostic biomarker for bladder cancer, but its urinary levels and cellular localization were associated with the differentiation status of patients with bladder tumors. Further studies are warranted to define the potential role for SDC-1 in bladder cancer progression.

Prognostic value of microRNA expression pattern in upper tract urothelial carcinoma.

OBJECTIVE: To examine the microRNA (miRNA) expression pattern in tumour samples from patients with progressing and non-progressing upper tract urothelial carcinoma (UTUC) in order to identify putative miRNAs that may be used as prognostic markers. PATIENTS AND METHODS: We conducted a multicentre, retrospective study of formalin-fixed paraffin-embedded tissue samples from 150 patients with UTUC who had undergone radical nephroureterectomy. Global miRNA expression patterns were analysed in 18 selected samples from patients with UTUC using TaqMan arrays. The differential expression of five key miRNAs was validated by quantitative polymerase chain reaction in an independent cohort of 132 samples from patients with UTUC. Models to predict tumour progression and cancer-specific survival that included miRNA expression patterns were developed by Cox regression analysis. RESULTS: Twenty-six miRNAs were found to be aberrantly expressed between samples from patients with progressing and non-progressing UTUC and five of these were selected for subsequent studies. The regression analysis identified tumour stage and miR-31 and miR-149 expression as independently associated with tumour progression and tumour stage and miR-149 expression as independently associated with cancer-specific survival. The risk scores derived from these miRNA models were able to discriminate two groups with a highly significantly different probability of tumour progression (hazard ratio [HR] 4.78; P < 0.001) and death (HR 276; P = 0.004). CONCLUSIONS: There is a differential miRNA expression pattern between patients with progressing and non-progressing UTUC. The identification of new miRNAs associated with a high probability of tumour recurrence and cancer-specific survival in patients with UTUC and their combination in a robust, easy-to-use and reliable algorithm may help tailor treatment and surveillance strategies in these patients.

Using microRNA profiling in urine samples to develop a non-invasive test for bladder cancer.

Current standard methods used to detect and monitor bladder urothelial cell carcinoma (UCC) are invasive or have low sensitivity. The incorporation into clinical practice of a non-invasive tool for UCC assessment would enormously improve patients' quality of life and outcome. This study aimed to examine the microRNA (miRNA) expression profiles in urines of UCC patients in order to develop a non-invasive accurate and reliable tool to diagnose and provide information on the aggressiveness of the tumor. We performed a global miRNA expression profiling analysis of the urinary cells from 40 UCC patients and controls using TaqMan Human MicroRNA Array followed by validation of 22 selected potentially diagnostic and prognostic miRNAs in a separate cohort of 277 samples using a miRCURY LNA qPCR system. miRNA-based signatures were developed by multivariate logistic regression analysis and internally cross-validated. In the initial cohort of patients, we identified 40 and 30 aberrantly expressed miRNA in UCC compared with control urines and in high compared with low grade tumors, respectively. Quantification of 22 key miRNAs in an independent cohort resulted in the identification of a six miRNA diagnostic signature with a sensitivity of 84.8% and specificity of 86.5% (AUC = 0.92) and a two miRNA prognostic model with a sensitivity of 84.95% and a specificity of 74.14% (AUC = 0.83). Internal cross-validation analysis confirmed the accuracy rates of both models, reinforcing the strength of our findings. Although the data needs to be externally validated, miRNA analysis in urine appears to be a valuable tool for the non-invasive assessment of UCC.

Multiplex protein signature for the detection of bladder cancer in voided urine samples.

PURPOSE: Accurate urine assays for bladder cancer detection would benefit patients and health care systems. Through extensive genomic and proteomic profiling of urine components we previously identified a panel of 8 biomarkers that can facilitate the detection of bladder cancer in voided urine samples. In this study we confirmed this diagnostic molecular signature in a diverse multicenter cohort. MATERIALS AND METHODS: We performed a case-control, phase II study in which we analyzed voided urine from 102 subjects with bladder cancer and 206 with varying urological disorders. The urinary concentration of 8 biomarkers (IL-8, MMP-9 and 10, PAI-1, VEGF, ANG, CA9 and APOE) was assessed by enzyme-linked immunosorbent assay. Diagnostic performance of the panel of tested biomarkers was evaluated using ROCs and descriptive statistical values, eg sensitivity and specificity. RESULTS: Seven of the 8 urine biomarkers were increased in subjects with bladder cancer relative to those without bladder cancer. The 7 biomarkers were assessed in a new model, which had an AUROC of 0.88 (95% CI 0.84-0.93), and 74% sensitivity and 90% specificity. In contrast, the sensitivity of voided urine cytology and the UroVysion® cytogenetic test in this cohort was 39% and 54%, respectively. Study limitations include analysis performed on banked urine samples and the lack of voided urine cytology and cytogenetic test data on controls. CONCLUSIONS: The study provides further evidence that the reported panel of diagnostic biomarkers can reliably achieve the noninvasive detection of bladder cancer with higher sensitivity than currently available urine based assays.

Investigation of CCL18 and A1AT as potential urinary biomarkers for bladder cancer detection.

BACKGROUND: In this study, we further investigated the association of two biomarkers, CCL18 and A1AT, with bladder cancer (BCa) and evaluated the influence of potentially confounding factors in an experimental model. METHODS: In a cohort of 308 subjects (102 with BCa), urinary concentrations of CCL18 and A1AT were assessed by enzyme-linked immunosorbent assay (ELISA). In an experimental model, benign or cancerous cells, in addition to blood, were added to urines from healthy controls and analyzed by ELISA. Lastly, immunohistochemical staining for CCL18 and A1AT in human bladder tumors was performed. RESULTS: Median urinary protein concentrations of CCL18 (52.84 pg/ml vs. 11.13 pg/ml, p < 0.0001) and A1AT (606.4 ng/ml vs. 120.0 ng/ml, p < 0.0001) were significantly elevated in BCa subjects compared to controls. Furthermore, the addition of whole blood to pooled normal urine resulted in a significant increase in both CCL18 and A1AT. IHC staining of bladder tumors revealed CCL18 immunoreactivity in inflammatory cells only, and there was no significant increase in these immunoreactive cells within benign and cancerous tissue and no association with BCa grade nor stage was noted. A1AT immunoreactivity was observed in the cytoplasm of epithelia cells and intensity of immunostaining increased with tumor grade, but not tumor stage. CONCLUSIONS: Further development of A1AT as a diagnostic biomarker for BCa is warranted.

[New concepts in molecular biology applied to traslational research]. / Nuevos conceptos en biología molecular aplicada a la investigación traslacional.

This chapter intends to introduce the new concepts that have been established in molecular biology over the last years and are being applied in translational research. The chapter is divided in four big blocks, which treat the molecular biology concepts and techniques in relation to DNA, RNA, proteins and metabolites, respectively. Moreover, we give examples of translational application of these new methodologies described.

[Non-invasive diagnosis bladder cancer: new molecular markers and future perspectives]. / Diagnóstico no invasivo en cáncer de vejiga: nuevos marcadores moleculares y futuras perspectivas.

Bladder cancer represents a very important entity in the field of urologic oncology. At the time of diagnosis 70% of them are non muscle-invasive tumors with a very variable risk of recurrence and progression. These patients will require a periodic follow up, at least for 5 years, performing multiple cystoscopies and urine cytologies throughout their lifes. These diagnostic tests are either invasive or have a low sensitivity, a fact that has stimulated the search of non-invasive markers with high sensitivity for the diagnosis of bladder cancer. The great advantage in this neoplasia is the availability of a biologic sample such as urine, which being in close contact with the urothelium collects exfoliated cells, representing a valuable non invasive tool for the diagnosis and follow up of this neoplasias. In this article we try to perform a review of those markers for bladder cancer already marketed and under development, as well as future diagnostic perspectives.

Assessment of aggressive bladder cancer mutations in plasma cell-free DNA.

Background and aims: The spatial and temporal genetic heterogeneity of bladder cancer (BC) makes challenging to find specific drivers of metastatic disease, thus preventing to determine those BC patients at high risk of tumor progression. Our aim was to identify DNA mutations providing aggressive behavior to bladder tumors and analyze them in patients' cell-free DNA (cfDNA) during their follow-up after radical cystectomy (RC) in order to monitor tumor evolution. Methods: Six BC patients who underwent RC and presented disease progression during their follow-up were included. Next-generation sequencing was used to determine somatic mutations in several primary tumor and metastatic specimens from each patient. Shared DNA mutations between primary bladder tumor and metastatic sites were identified in cfDNA samples through droplet digital PCR. Results: Besides BC genetic heterogeneity, specific mutations in at least one of these genes -TERT, ATM, RB1, and FGFR3- were found in primary tumors and their metastases in all patients. These mutations were also identified in the patients' cfDNA at different follow-up time points. Additionally, the dynamic changes of these mutations in cfDNA allowed us to determine tumor evolution in response to treatment. Conclusion: The analysis of BC mutations associated with poor prognosis in plasma cfDNA could be a valuable tool to monitor tumor evolution, thus improving the clinical management of BC patients.