Inflammatory infiltration is associated with AR expression and poor prognosis in hormone naïve prostate cancer.

BACKGROUND: Tumor microenvironment inflammatory infiltration is proposed as a protumorigenic mechanism for prostate cancer with proinflammatory cytokines stimulating androgen receptor (AR) activity. However, association with patient prognosis remains unclear. This study derives an inflammatory gene signature associated with AR expression and investigates CD3+ and CD8+ T-lymphocyte infiltration association with AR and prognosis. METHODS: Gene profiling of inflammatory related genes was performed on 71 prostate biopsies. Immunohistochemistry on 243 hormone-naïve prostate cancers was performed for CD3, CD8, AR, and phosphorylated AR tumor expression. RESULTS: Multiple proinflammatory genes were differentially expressed in association with high AR expression compared with low AR expression including PI3KCA and MAKP8 (adjusted P < .05). High CD3+ and high CD8+ infiltration associated with reduced cancer-specific survival (P = .018 and P = .020, respectively). High CD3+ infiltration correlated with high tumor cytoplasmic AR expression and if assessed together, they associated with reduced cancer-specific and 5-year survival from 90% to 56% (P = .000179). High CD8+ cytotoxic infiltration associated with high androgen-independent tumor nuclear AR serine 213 phosphorylation (correlation coefficient = 0.227; P = .003) and when assessed together associated with poor clinico-pathological features including perineural invasion (P = .001). Multiple genes involved in proinflammatory signaling pathways are upregulated in high AR expressing prostate samples. CONCLUSION: T-lymphocyte infiltration in hormone-naïve disease associates with androgen-independent driven disease and provides possible therapeutic targets to reduce transformation from hormone-naïve to castrate-resistant disease.

Circulating miR-99a-5p Expression in Plasma: A Potential Biomarker for Early Diagnosis of Breast Cancer.

MicroRNAs have emerged as new diagnostic and therapeutic biomarkers for breast cancer. Herein, we analysed miR-99a-5p expression levels in primary tumours and plasma of breast cancer patients to evaluate its usefulness as a minimally invasive diagnostic biomarker. MiR-99a-5p expression levels were determined by quantitative real-time PCR in three independent cohorts of patients: (I) Discovery cohort: breast cancer tissues (n = 103) and healthy breast tissues (n = 26); (II) Testing cohort: plasma samples from 105 patients and 98 healthy donors; (III) Validation cohort: plasma samples from 89 patients and 85 healthy donors. Our results demonstrated that miR-99a-5p was significantly downregulated in breast cancer tissues compared to healthy breast tissues. Conversely, miR-99a-5p levels were significantly higher in breast cancer patients than in healthy controls in plasma samples from both testing and validation cohorts, and ROC curve analysis revealed that miR-99a-5p has good diagnostic potential even to detect early breast cancer. In conclusion, miR-99a-5p's deregulated expression distinguished healthy patients from breast cancer patients in two different types of samples (tissues and plasma). Interestingly, expression levels in plasma were significantly lower in healthy controls than in early-stage breast cancer patients. Our findings suggest circulating miR-99a-5p as a novel promising non-invasive biomarker for breast cancer detection.

PROSTAGLANDIN E2 stimulates cancer-related phenotypes in prostate cancer PC3 cells through cyclooxygenase-2.

Cyclooxygenase (COX)-derived prostaglandin E2 (PGE2 ) affects many mechanisms that have been shown to play roles in carcinogenesis. Recently, we found that, in androgen-independent prostate cancer PC3 cells, PGE2 acts through an intracrine mechanism by which its uptake by the prostaglandin transporter (PGT) results in increased intracellular PGE2 (iPGE2 ), leading to enhanced cell proliferation, migration, invasion, angiogenesis, and loss of cell adhesion to collagen I. These iPGE2 -mediated effects were dependent on hypoxia-inducible factor 1-α (HIF-1α), whose expression increased upon epidermal growth factor receptor (EGFR) transactivation by a subset of intracellular PGE2 receptors. Here, we aimed to study the role of COX in PGE2 protumoral effects in PC3 cells and found that the effects were prevented by inhibition of COX-2, which highlights its crucial role amplifying the levels of iPGE2 . Treatment with exogenous PGE2 determined a transcriptional increase in COX-2 expression, which was abolished by genetic or pharmacologic inhibition of PGT. PGE2 -induced increase in COX-2 expression and, thereby, in transcriptional increase in HIF-1α expression was due to EGFR activation, leading to the activation of Phosphoinositide 3-kinase/Akt, Extracellular signal -regulated kinases 1/2, p38 and Mitogen- and stress-activated protein kinase-1 (PI3K/Akt, Erk1/2, p38 and MSK-1). Collectively, the data suggest that EGFR-dependent COX-2 upregulation by a novel positive feedback loop triggered by iPGE2 underlies the intracrine pro-tumoral effects of PGE2 in PC3 cells. Therefore, this feedback loop may be relevant in prostate cancer for the maintenance of PGE2 -dependent cancer cell growth through amplifying the activity of the COX-2 pathway.

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.

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.

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.

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.

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.

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.

Urinary Extracellular Vesicles as Potential Biomarkers for Urologic Cancers: An Overview of Current Methods and Advances.

Urologic cancers are a heterogeneous group of tumors, some of which have poor prognosis. This is partly due to the unavailability of specific and sensitive diagnostic techniques and monitoring tests, ideally non- or minimally invasive. Hence, liquid biopsies are promising tools that have been gaining significant attention over the last decade. Among the different classes of biomarkers that can be isolated from biofluids, urinary extracellular vesicles (uEVs) are a promising low-invasive source of biomarkers, with the potential to improve cancer diagnosis and disease management. Different techniques have been developed to isolate and characterize the cargo of these vesicles; however, no consensus has been reached, challenging the comparison among studies. This results in a vast number of studies portraying an extensive list of uEV-derived candidate biomarkers for urologic cancers, with the potential to improve clinical outcome; however, without significant validation. Herein, we review the current published research on miRNA and protein-derived uEV for prostate, bladder and kidney cancers, focusing on different uEV isolation methods, and its implications for biomarker studies.

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.

Deregulation of N6-Methyladenosine RNA Modification and Its Erasers FTO/ALKBH5 among the Main Renal Cell Tumor Subtypes.

(1) Background: Methylation of N6-adenosine (m6A) is the most abundant messenger RNA (mRNA) modification in eukaryotes. We assessed the expression profiles of m6A regulatory proteins in renal cell carcinoma (RCC) and their clinical relevance, namely, as potential biomarkers. (2) Methods: In silico analysis of The Cancer Genome Atlas (TCGA) dataset was use for evaluating the expression of the m6A regulatory proteins among RCC subtypes and select the most promising candidates for further validation. ALKBH5 and FTO transcript and protein expression were evaluated in a series of primary RCC (n = 120) and 40 oncocytomas selected at IPO Porto. (3) Results: In silico analysis of TCGA dataset disclosed altered expression of the major m6A demethylases among RCC subtypes, particularly FTO and ALKBH5. Furthermore, decreased FTO mRNA levels associated with poor prognosis in ccRCC and pRCC. In IPO Porto's cohort, FTO and ALKBH5 transcript levels discriminated ccRCC from oncocytomas. Furthermore, FTO and ALKBH5 immunoexpression differed among RCC subtypes, with higher expression levels found in ccRCC comparatively to the other RCC subtypes and oncocytomas. (4) Conclusion: We conclude that altered expression of m6A RNA demethylases is common in RCC and seems to be subtype specific. Specifically, FTO and ALKBH5 might constitute new candidate biomarkers for RCC patient management, aiding in differential diagnosis of renal masses and prognostication.

Promoter methylation of DNA homologous recombination genes is predictive of the responsiveness to PARP inhibitor treatment in testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) are the most common cancers in men aged 15-39 years and are divided into two major groups, seminomas and nonseminomas. Novel treatment options are required for these patients, to limit side effects of chemotherapy. We hypothesized that promoter methylation of relevant homologous recombination (HR) genes might be predictive of response to poly-ADP ribose polymerase inhibitors (PARPis) in TGCTs. We report a study pipeline combining in silico, in vitro, and clinical steps. By using several databases and in silico tools, we identified BRCA1, RAD51C, PALB2, RAD54B, and SYCP3 as the most relevant genes for further investigation and pinpointed specific CpG sites with pronounced negative correlation to gene expression. Nonseminomas displayed significantly higher methylation levels for all target genes, where increased methylation was observed in patients with more differentiated subtypes and higher disease burden. We independently performed second-line targeted validation in tissue series from TGCT patients. A moderate and/or strong anti-correlation between gene expression (assessed by RNA-sequencing) and promoter methylation (assessed by 450k array) was found, for all of the targets. As a proof of concept, we demonstrated the sensitivity of TGCT cell lines to Olaparib, which associated with differential methylation levels of a subset of targets, namely BRCA1 and RAD51C. Our findings support the use of HR genes promoter methylation as a predictor of the therapeutic response to PARPis in patients with TGCT.

Differential methylation EPIC analysis discloses cisplatin-resistance related hypermethylation and tumor-specific heterogeneity within matched primary and metastatic testicular germ cell tumor patient tissue samples.

Testicular germ cell tumors (TGCTs) are among the most common solid malignancies in young-adult men, and currently most mortality is due to metastatic disease and emergence of resistance to cisplatin. There is some evidence that increased methylation is one mechanism behind this resistance, stemming from individual studies, but approaches based on matched primary and metastatic patient samples are lacking. Herein, we provide an EPIC array-based study of matched primary and metastatic TGCT samples. Histology was the major determinant of overall methylation pattern, but some clustering of samples related to response to cisplatin was observed. Further differential analysis of patients with the same histological subtype (embryonal carcinoma) disclosed a remarkable increase in net methylation levels (at both promoter and CpG site level) in the patient with cisplatin-resistant disease and poor outcome compared to the patient with complete response to chemotherapy. This further confirms the recent results of another study performed on isogenic clones of sensitive and resistant TGCT cell lines. Differentially methylated promoters among groups of samples were mostly not shared, disclosing heterogeneity in patient tissue samples. Finally, gene ontology analysis of cisplatin-resistant samples indicated enrichment of differentially hypermethylated promoters on pathways related to regulation of immune microenvironment, and enrichment of differentially hypomethylated promoters on pathways related to DNA/chromatin binding and regulation. This data supports not only the use of hypomethylating agents for targeting cisplatin-resistant disease, but also their use in combination with immunotherapies and chromatin remodelers.

Identification of a Two-MicroRNA Signature in Plasma as a Novel Biomarker for Very Early Diagnosis of Breast Cancer.

The early diagnosis of breast cancer is essential to improve patients' survival rate. In this context, microRNAs have been described as potential diagnostic biomarkers for breast cancer. Particularly, circulating microRNAs have a strong value as non-invasive biomarkers. Herein, we assessed the potential of a microRNA signature based on miR-30b-5p and miR-99a-5p levels in plasma as a diagnostic biomarker for breast cancer. This two-microRNA signature was constructed by Principal Component Analysis and its prognostic value was assessed in a discovery cohort and blindly validated in a second cohort from an independent institution. ROC curve analysis and biomarker performance parameter evaluation demonstrated that our proposed signature presents a high value as a non-invasive biomarker for very early detection of breast cancer. In addition, pathway enrichment analysis identified three of the well-known pathways involved in cancer as targets of the two microRNAs.

Circulating extracellular vesicles release oncogenic miR-424 in experimental models and patients with aggressive prostate cancer.

Extracellular vesicles (EVs) are relevant means for transferring signals across cells and facilitate propagation of oncogenic stimuli promoting disease evolution and metastatic spread in cancer patients. Here, we investigated the release of miR-424 in circulating small EVs or exosomes from prostate cancer patients and assessed the functional implications in multiple experimental models. We found higher frequency of circulating miR-424 positive EVs in patients with metastatic prostate cancer compared to patients with primary tumors and BPH. Release of miR-424 in small EVs was enhanced in cell lines (LNCaPabl), transgenic mice (Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG) and patient-derived xenograft (PDX) models of aggressive disease. EVs containing miR-424 promoted stem-like traits and tumor-initiating properties in normal prostate epithelial cells while enhanced tumorigenesis in transformed prostate epithelial cells. Intravenous administration of miR-424 positive EVs to mice, mimicking blood circulation, promoted miR-424 transfer and tumor growth in xenograft models. Circulating miR-424 positive EVs from patients with aggressive primary and metastatic tumors induced stem-like features when supplemented to prostate epithelial cells. This study establishes that EVs-mediated transfer of miR-424 across heterogeneous cell populations is an important mechanism of tumor self-sustenance, disease recurrence and progression. These findings might indicate novel approaches for the management and therapy of prostate cancer.

DNA Methylation-Based Testing in Liquid Biopsies as Detection and Prognostic Biomarkers for the Four Major Cancer Types.

Lung, breast, colorectal, and prostate cancers are the most incident worldwide. Optimal population-based cancer screening methods remain an unmet need, since cancer detection at early stages increases the prospects of successful and curative treatment, leading to a lower incidence of recurrences. Moreover, the current parameters for cancer patients' stratification have been associated with divergent outcomes. Therefore, new biomarkers that could aid in cancer detection and prognosis, preferably detected by minimally invasive methods are of major importance. Aberrant DNA methylation is an early event in cancer development and may be detected in circulating cell-free DNA (ccfDNA), constituting a valuable cancer biomarker. Furthermore, DNA methylation is a stable alteration that can be easily and rapidly quantified by methylation-specific PCR methods. Thus, the main goal of this review is to provide an overview of the most important studies that report methylation biomarkers for the detection and prognosis of the four major cancers after a critical analysis of the available literature. DNA methylation-based biomarkers show promise for cancer detection and management, with some studies describing a "PanCancer" detection approach for the simultaneous detection of several cancer types. Nonetheless, DNA methylation biomarkers still lack large-scale validation, precluding implementation in clinical practice.

Clinical Significance of ARID1A and ANXA1 in HER-2 Positive Breast Cancer.

BACKGROUND: trastuzumab is considered the standard of care for human epidermal growth factor receptor-2 (HER-2+) breast cancer patients. Regardless of the benefits of its use, many early-stage patients eventually recur, and usually, the disease progresses within a year. Since about half of the HER-2+ patients do not respond to trastuzumab, new biomarkers of prognosis and prediction are warranted to allow a better patient stratification. Annexin A1 (ANXA1) was previously reported to contribute to trastuzumab resistance through AKT activation. An association between adenine thymine-rich interactive domain 1A (ARID1A) loss and ANXA1 upregulation was also previously suggested by others. METHODS: in this study, we examined tissue samples from 215 HER-2+ breast cancer patients to investigate the value of ARID1A and ANXA1 protein levels in trastuzumab response prediction and patient outcome. Expression of ARID1A and ANXA1 were assessed by immunohistochemistry. RESULTS: contrary to what was expected, no inverse association was found between ARID1A and ANXA1 expression. HER-2+ (non-luminal) tumours displayed higher ANXA1 expression than luminal B-like (HER-2+) tumours. Concerning trastuzumab resistance, ARID1A and ANXA1 proteins did not demonstrate predictive value as biomarkers. Nevertheless, an association was depicted between ANXA1 expression and breast cancer mortality and relapse. CONCLUSIONS: overall, our results suggest that ANXA1 may be a useful prognostic marker in HER-2+ patients. Additionally, its ability to discriminate between HER-2+ (non-luminal) and luminal B-like (HER-2+) patients might assist in patient stratification regarding treatment strategy.