Redefining prostate cancer risk stratification: a pioneering strategy to estimate outcome based on Ki67 immunoscoring.

Accurate prostate cancer (PCa) patient diagnosis and risk assessment are key to ensure the best outcome. Currently, low- and favorable intermediate-risk PCa patients may be offered AS due to the indolent nature of the disease. Nonetheless, deciding between active surveillance and curative-intent treatment remains an intricate task, as a subset of these patients may eventually progress, enduring poorer prognosis. Herein, we sought to construct risk calculators based on cancer biomarkers, enabling more accurate discrimination among patients which may benefit from active interventions.Ki67 immunoscore, GSTP1 and KLF8 promoter methylation levels (me) were assessed in PCa tissues. Study endpoints included overall and biochemical recurrence-free (BCR) survival. Combination with relevant clinicopathological parameters allowed for construction of graphical calculating tools (nomograms).Higher Ki67 index correlated with worse BCR-free survival, whereas higher KLF8me levels were associated with improved overall survival, especially in patients with lower-grade tumors. GSTP1me levels had no prognostic value. Among prognostic models tested, a BCR-risk calculator - ProstARK (including Ki67 and clinicopathologic parameters) - disclosed 79.17% specificity, 66.67% sensitivity, 55% positive predictive value, 86% negative predictive value, and 75.76% accuracy. Similar results were found using an independent PCa biopsy cohort, validating its prognostication ability.Combining clinicopathologic features and Ki67 index into a risk calculator enables easy and accurate implementation of a novel PCa prognostication tool. This nomogram may be useful for a more accurate selection of patients for active surveillance protocols. Nonetheless, validation in a larger, multicentric, set of diagnostic PCa biopsies is mandatory for further confirmation of these results.

Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation.

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.

Shifting the Cancer Screening Paradigm: The Rising Potential of Blood-Based Multi-Cancer Early Detection Tests.

Cancer remains a leading cause of death worldwide, partly owing to late detection which entails limited and often ineffective therapeutic options. Most cancers lack validated screening procedures, and the ones available disclose several drawbacks, leading to low patient compliance and unnecessary workups, adding up the costs to healthcare systems. Hence, there is a great need for innovative, accurate, and minimally invasive tools for early cancer detection. In recent years, multi-cancer early detection (MCED) tests emerged as a promising screening tool, combining molecular analysis of tumor-related markers present in body fluids with artificial intelligence to simultaneously detect a variety of cancers and further discriminate the underlying cancer type. Herein, we aim to provide a highlight of the variety of strategies currently under development concerning MCED, as well as the major factors which are preventing clinical implementation. Although MCED tests depict great potential for clinical application, large-scale clinical validation studies are still lacking.

DNA methylation biomarkers accurately detect esophageal cancer prior and post neoadjuvant chemoradiation.

BACKGROUND: Esophageal cancer (ECa) is associated with high mortality, mostly due to late diagnosis, precluding curativeintent surgery. Hence, neoadjuvant chemoradiation (ChRT) is recommended in most patients regardless of histological subtype. A proportion of these patients, however, achieve complete disease remission and might be spared of radical surgery. The lack of reliable, minimally invasive biomarkers able to detect post-ChRT disease persistence is, nonetheless, a major drawback. We have previously shown that miRNA promotor methylation enables accurate cancer detection in tissues and liquid biopsies but has been seldom explored in ECa patients. AIMS: Herein, we sought to unveil and validate novel candidate biomarkers able to detect ECa prior and post ChRT. MATERIALS AND METHODS: Promoter methylation of miR129-2, miR124-3 and ZNF569 was assessed, using quantitative methylation-specific PCR (qMSP), in tissue samples from normal esophagus, treatment-naïve and post-ChRT ECa, as well as in liquid biopsies from ECa patients. RESULTS: All genes disclosed significantly different promoter methylation levels between ECa and normal esophagus, accurately detecting post-ChRT disease, especially for adenocarcinoma. Remarkably, miR129-2me /ZNF569me methylation panel identified ECa in liquid samples with 53% sensitivity and 87% specificity. DISCUSSION: MiR129-2me , miR124-3me and ZNF569me accurately discriminate ECa, either pre- or post-ChRT, from normal tissue, enabling ECa detection. Furthermore, circulalting methylation-based biomarkers are promising minimally invasive tools to detect post-ChRT residual ECa. CONCLUSION: Overall, our results encourage the use of miRNA methylation biomarkers as accurate ECa detection tools as a novel approach for ChRT response monitoring.

MiRNA biomarkers in cancers of the male reproductive system: Are we approaching clinical application?

BACKGROUND: Specific cancer types face specific clinical management challenges. Owing to their stability, robustness and fast, easy and cost-effective detection, microRNAs (miRNAs) are attractive candidate biomarkers to the clinic. OBJECTIVES: Based on a comprehensive review of the relevant literature in the field, we explore the potential of miRNAs as biomarkers to answer relevant clinical dilemmas inherent to cancers of the male reproductive tract (prostate [PCa], testis [TGCTs] and penis [PeCa]) and identify some of the challenges/limitations hampering their widely application. RESULTS AND DISCUSSION: We conclude that the use of miRNAs as biomarkers is at different stages for these distinct cancer types. While for TGCTs, miRNA-371a-3p is universally accepted to fill in important clinicals gaps and is moving fast towards clinical implementation, for PCa almost no overlap of miRNAs exists between studies, denoting the absence of a consistent miRNA biomarker, and for PeCa the field of miRNAs has just recently started, with only a few studies attempting to explore their clinical usefulness. CONCLUSION: Technological advances influencing miRNA detection and quantification will be instrumental to continue to move forward with implementation of miRNAs in the clinic as biomarkers for non-invasive diagnosis, risk stratification, treatment monitoring and follow-up.

DigiMir Test: Establishing a Novel Pipeline for MiR-371a Quantification Using Droplet Digital PCR in Liquid Biopsies From Testicular Germ Cell Tumor Patients.

Testicular germ cell tumors (TGCTs) are the most common cancers in young-adult male patients aged between 15 and 39 years. Hsa-miR-371a-3p is currently the most reliable biomarker for diagnosis and monitoring of these patients non-invasively in liquid biopsies, and it is destined to be introduced in the clinic due to improved performance compared to the classical serum tumor markers available. Current studies have focused on real-time quantitative PCR (RT-qPCR) protocols for its determination; still, some challenges remain, since these protocols often require preamplification steps (costly and time-consuming), and report relative levels normalized to a housekeeping microRNA, not always performed the same way. Droplet digital PCR (ddPCR) shows the promise to overcome these challenges, skipping normalization and preamplifications, but has hardly been explored in the field of TGCTs. In this work, we provide a report of a ddPCR-based pipeline for the quantification of hsa-miR-371a-3p (the DigiMir pipeline) and compare it with two RT-qPCR protocols. A total of 107 plasma samples were investigated in the validation setting. The DigiMir pipeline detected TGCTs in a manner representative of tumor burden, with a sensitivity and specificity of 94% and 100%, respectively, outperforming the combined sensitivity of all three classical serum tumor markers (61.5%). Therefore, in this proof-of-concept investigation, we have shown that the DigiMir pipeline constitutes a new promising methodology to accurately report hsa-miR-371a-3p in the clinical setting.

One sample fits all: a microfluidic-assisted methodology for label-free isolation of CTCs with downstream methylation analysis of cfDNA in lung cancer.

Lung cancer (LC) is a major cause of mortality. Late diagnosis, associated with limitations in tissue biopsies for adequate tumor characterization contribute to limited survival of lung cancer patients. Liquid biopsies have been introduced to improve tumor characetrization through the analysis of biomarkers, including circulating tumour cells (CTCs) and cell-free DNA (cfDNA). Considering their availability in blood, several enrichment strategies have been developed to augment circulating biomarkers for improving diagnostic, prognostic and treament efficacy assessment; often, however, only one biomarker is tested. In this work we developed and implemented a microfluidic chip for label-free enrichment of CTCs with a methodology for subsequent cfDNA analysis from the same cryopreserved sample. CTCs were successfully isolated in 38 of 42 LC patients with the microfluidic chip. CTCs frequency was significantly higher in LC patients with advanced disease. A cut-off of 1 CTC per mL was established for diagnosis (sensitivity = 76.19%, specificity = 100%) and in patients with late stage lung cancer, the presence of ≥5 CTCs per mL was significantly associated with shorter overall survival. MIR129-2me and ADCY4me panel of cfDNA methylation performed well for LC detection, whereas MIR129-2me combined with HOXA11me allowed for patient risk stratification. Analysis of combinations of biomarkers enabled the definition of panels for LC diagnosis and prognosis. Overall, this study demonstrates that multimodal analysis of tumour biomarkers via microfluidic devices may significantly improve LC characterization in cryopreserved samples, constituting a reliable source for continuous disease monitoring.

LiKidMiRs: A ddPCR-Based Panel of 4 Circulating miRNAs for Detection of Renal Cell Carcinoma.

BACKGROUND: Decreased renal cell cancer-related mortality is an important societal goal, embodied by efforts to develop effective biomarkers enabling early detection and increasing the likelihood of curative treatment. Herein, we sought to develop a new biomarker for early and minimally invasive detection of renal cell carcinoma (RCC) based on a microRNA panel assessed by ddPCR. METHODS: Plasma samples from patients with RCC (n = 124) or oncocytomas (n = 15), and 64 healthy donors, were selected. Hsa-miR-21-5p, hsa-miR-126-3p, hsa-miR-155-5p and hsa-miR-200b-3p levels were evaluated using a ddPCR protocol. RESULTS: RCC patients disclosed significantly higher circulating levels of hsa-miR-155-5p compared to healthy donors, whereas the opposite was observed for hsa-miR-21-5p levels. Furthermore, hsa-miR-21-5p and hsa-miR-155-5p panels detected RCC with high sensitivity (82.66%) and accuracy (71.89%). The hsa-miR-126-3p/hsa-miR-200b-3p panel identified the most common RCC subtype (clear cell, ccRCC) with 74.78% sensitivity. CONCLUSION: Variable combinations of plasma miR levels assessed by ddPCR enable accurate detection of RCC in general, and of ccRCC. These findings, if confirmed in larger studies, provide evidence for a novel ancillary tool which might aid in early detection of RCC.

Cadherin switches during epithelial-mesenchymal transition: CDH4/RCAD downregulation reduces bladder cancer progression.

PURPOSE: Non-muscle invasive bladder cancer (NMIBC) is a highly recurrent disease that progresses to muscle-invasive bladder cancer (MIBC) in 5-25% of the cases. Epithelial-mesenchymal transition (EMT) has been associated with features of disease progression. Thus, we aimed to characterize the cadherin switch (CS), an EMT hallmark, and its regulatory mechanisms in bladder cancer (BlCa) progression, as well as the biological role of RCAD, a lesser-known cadherin, in bladder carcinogenesis. METHODS: Cadherin mRNA and promoter methylation levels were retrieved from The Cancer Genome Atlas (TCGA). Validation was performed in an independent set of 121 primary BlCa (NMIBC and MIBC) and 40 normal bladder samples from IPO Porto, using RT-qPCR and qMSP. Immunohistochemistry was performed in these samples and in 14 additional sarcomatoid BlCa. CRISPR-Cas9 was performed to explore the potential in vitro impact of RCAD on BlCa cell migration and invasion. RESULTS: In both the TCGA and IPO Porto BlCa cohorts, cadherin gene deregulation was observed compared to normal tissue samples, independent of promoter methylation. At the protein level, decreased E-cadherin and increased P- and R-cadherin expression was noted in BlCa tissues. In sarcomatoid BlCa the same trend was observed, with a more intense staining compared to that in conventional MIBCs. RCAD knockout considerably reduced the malignant properties of BlCa cells. CONCLUSIONS: Our data indicate that E-, P- and R-cadherin switches occur in BlCa, being associated with tumor progression. Promoter methylation is not the likely mechanism underlying cadherin expression deregulation. Our findings suggest an oncogenic role of RCAD in BlCa progression.

Unveiling the World of Circulating and Exosomal microRNAs in Renal Cell Carcinoma.

Renal cell carcinoma is the third most common urological cancer. Despite recent advances, late diagnosis and poor prognosis of advanced-stage disease remain a major problem, entailing the need for novel early diagnosis tools. Liquid biopsies represent a promising minimally invasive clinical tool, providing real-time feedback of tumor behavior and biological potential, addressing its clonal evolution and representing its heterogeneity. In particular, the study of circulating microRNAs and exosomal microRNAs in liquid biopsies experienced an exponential increase in recent years, considering the potential clinical utility and available technology that facilitates implementation. Herein, we provide a systematic review on the applicability of these biomarkers in the context of renal cell carcinoma. Issues such as additional benefit from extracting microRNAs transported in extracellular vesicles, use for subtyping and representation of different histological types, correlation with tumor burden, and prediction of patient outcome are also addressed. Despite the need for more conclusive research, available data indicate that exosomal microRNAs represent a robust minimally invasive biomarker for renal cell carcinoma. Thus, innovative research on microRNAs and novel detection techniques are likely to provide clinically relevant biomarkers, overcome current clinical challenges, and improve patient management.