Leukocyte telomere length and hTERT genetic polymorphism rs2735940 influence the renal cell carcinoma clinical outcome.

Aim: Analysis of the genetic hTERT-1327 C>T (rs2735940) influence on leukocyte telomere length (LTL) and tumor development, progression and overall survival in renal cell carcinoma (RCC) patients. Materials & methods: Using leukocyte DNA of RCC patients and healthy individuals, LTL measurement and allelic discrimination of rs2735940 was performed by real-time PCR. Results: RCC patients showed shorter LTL than healthy individuals and LTL increased with clinical stage. CC+TC genotypes healthy carriers' presented shorter LTL. However, no statistical association between the different genotypes and RCC risk. Nevertheless, CC homozygous presented a reduced time to disease progression and a lower overall survival. The use of hTERT-1327 single nucleotide polymorphism information increased the capacity to predict risk for RCC progression. Conclusion: In fact, in healthy individuals, hTERT-1327 CC+TC genotypes were associated with shorter LTL, and this single nucleotide polymorphism was associated with time to disease progression, being a promising potential prognosis biomarker to be used in the future.

Plasma Extracellular Vesicle-Derived TIMP-1 mRNA as a Prognostic Biomarker in Clear Cell Renal Cell Carcinoma: A Pilot Study.

The tumor microenvironment has gained a lot of attention from the scientific community since it has a proven impact in the development of tumor progression and metastasis. Extracellular vesicles (EVs) are now considered one of the key players of tumor microenvironment modulation. Clear cell renal cell carcinoma (ccRCC) is the most lethal urological neoplasia and presents a high metastatic potential, which reinforces the need for the development of more effective predictive biomarkers. Our goal was to evaluate the applicability of EV-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) as prognostic biomarkers for ccRCC. To do so, we studied the plasma EV content of 32 patients with localized ccRCC and 29 patients with metastatic ccRCC. We observed that patients with localized disease and tumors larger than 7 cm presented higher levels of plasma EV-derived TIMP-1 mRNA when compared with patients presenting smaller tumors (p = 0.020). Moreover, patients with metastatic disease presented higher levels of EV-derived TIMP-1 mRNA when compared with patients with localized disease (p = 0.002) and when we stratified those patients in high and low levels of TIMP-1 EV-derived mRNA, the ones presenting higher levels had a lower overall survival (p = 0.030). EV-derived TIMP-1 mRNA may be a good prognostic biomarker candidate for ccRCC.

Influence of peripheral whole-blood microRNA-7 and microRNA-221 high expression levels on the acquisition of castration-resistant prostate cancer: evidences from in vitro and in vivo studies.

Prostate cancer (PC) is the more frequently diagnosed neoplasia in men in developed countries. The evolution of PC to castration-resistant prostate cancer (CRPC) represents real problems of clinical management, in consequence to the limited therapeutic options. MicroRNAs (miRNAs) are small noncoding RNAs that play an important role in gene expression and function regulation. The increased evidence that miRNAs are involved in cancer development and progression has made them potential biomarkers for cancer diagnosis, prognosis, and aggressiveness. Our purpose was to identify a miRNA expression profile associated with the development of CRPC. We firstly observed a miRNA expression profile differentially expressed between the castration-resistant (CR) PC3 cell line and the hormone-sensitive LnCaP cell line, where miR-7, miR-221, and miR-222 were upregulated in PC3 (11.3-fold increase, P = 0.012; 11.3-fold increase, P = 0.002; 8.6-fold increase, P = 0.002, respectively). We also observed that the trend of miR-1233 expression levels was higher in PC3 (3.7-fold increase, P = 0.057). These miRNAs differentially expressed in vitro were studied in a peripheral whole-blood samples from PC patients. We observed that patients presenting an early CR acquisition (≤ 20 months) had higher expression levels of miR-7 and miR-221 (P = 0.034 and P = 0.036, respectively). Furthermore, we found that patients diagnosed with high-Gleason score tumors and presenting simultaneous higher miR-7 expression levels have a significant reduce time to CR compared with patients who present lower miR-7 expression levels (11 vs. 51 months, log-rank test P = 0.004). We also found that patients diagnosed with high-Gleason score tumors and higher expression levels of miR-221 have an early CRPC compared to patients with lower miR-221 expression levels (10 vs. 46 months, log-rank test P = 0.012). We observed a significantly lower overall survival in patients with higher peripheral whole-blood expression levels of miR-7 (28 vs. 116 months, log-rank test P = 0.001). Our results suggest that miR-7 and miR-221 peripheral whole-blood expression levels can be potential predictive biomarkers of CRPC development.

Higher circulating expression levels of miR-221 associated with poor overall survival in renal cell carcinoma patients.

The mechanisms involved in renal cell carcinoma (RCC) development and progression remain unclear, and new biomarkers for early detection, follow-up of the disease and prognosis are needed in routine practice to improve the diagnostic and/or prognostic accuracy. There is increasing evidence that microRNAs (miRNAs) are involved in cancer development and progression. The up-regulation of miR-221/222 has been described in several human cancers, and during RCC development, this up-regulation can modulate the metastatic process. Our purpose was to investigate the circulating expression levels of miR-221/222 as potential biomarkers for RCC detection and their influence in patients' overall survival. The circulating miR-221/222 was studied by relative quantification in 77 plasma samples. A follow-up study was undertaken to evaluate the overall survival. We observed that RCC patients presented higher circulating expression levels of miR-221 and miR-222 than healthy individuals (2(-ΔΔCt) = 2.8, P = 0.028; 2(-ΔΔCt) = 2.2, P = 0.044, respectively). The RCC patients with metastasis at diagnosis also presented higher circulating expression levels of miR-221 than patients with no metastasis (2(-ΔΔCt) = 10.9, P = 0.001). We also observed a significantly lower overall survival in patients with higher expression levels of miR-221 (48 vs 116 months, respectively; P = 0.024). Furthermore, multivariate Cox regression analysis using the tumour, nodes and metastasis stage (TNM stage); Fuhrman nuclear grade and age (≥60 years) as covariants demonstrated a higher risk of specific death by cancer in patients who presented higher expression levels of miR-221 (hazard ratio (HR) = 10.7, 95% confidence interval 1.33-85.65, P = 0.026). The concordance (c) index showed that the definition of profiles that contain information regarding tumour characteristics associated with circulating miR-221 expression information presents an increased capacity to predict the risk of death by RCC (c index model 1, 0.800 vs model 2, 0.961). Our results, which identified the plasma miR-221/222 at variable levels during RCC development, suggest that these miRNAs may have a potential as noninvasive biomarkers of RCC development.

Antiandrogen Withdrawal Syndrome After Discontinuation of Enzalutamide in Patients With Metastatic Castration-Resistant Prostate Cancer: A Report of Two Clinical Cases and a Literature Review.

Metastatic prostate cancer treatment is based on androgen deprivation, with pharmacological or surgical castration. This treatment may be complemented with the addition of antiandrogenic drugs. In the setting of prostate-specific antigen (PSA) progression and subsequent suspension of the antiandrogenic drug, there might occur a phenomenon of antiandrogen withdrawal, leading to a decrease in PSA and/or improvement in imaging or clinical outcomes after discontinuation of the antiandrogenic agent. Although there are some descriptions of withdrawal after the cessation of enzalutamide, the physiological mechanism behind it, as well as its frequency and impact on patient survival, remain unknown. We present two clinical cases of antiandrogenic withdrawal after enzalutamide discontinuation and discuss potential contributing factors to this phenomenon.

EHMT2/G9a and EZH2: Epimarkers in testicular germ cell tumors.

BACKGROUND: Testicular germ cell tumors remain the most frequent solid malignancies in young males. Despite excellent prognosis, the fact that only 60% of patients at diagnosis have elevated serum tumor markers (dependent on stage and histology) and the poor quality of life of patients who develop resistance to chemotherapy cannot be neglected. Consequently, it is mandatory to bring out novel biomarkers. OBJECTIVES: The main goal was to evaluate EZH2 and EHMT2/G9a immunoexpression in a well-characterized patients' cohort of primary and metastatic testicular germ cell tumors, seeking associations with clinicopathological features and discovering differential immunoexpression patterns among specific subtypes. MATERIALS AND METHODS: First, an in silico analysis of the Cancer Genome Atlas database was performed regarding EZH2 and EHMT2/G9a. Then, immunohistochemistry for EZH2 and EHMT2/G9a was carried out in a cohort of testicular germ cell tumor patients, comprising 155 chemo-naïve primary tumors and 11 chemo-treated metastases. Immunoexpression was evaluated using a digital pathology analysis software. RESULTS: Higher EZH2 and EHMT2/G9a expression levels were found in non-seminoma in the in silico analysis, particularly in embryonal carcinoma. Through digital pathology analysis, non-seminomas showed significantly higher EZH2 and EHMT2/G9a immunoexpression, with embryonal carcinoma showing higher expression. Moreover, mixed tumors with 50% or more of embryonal carcinoma component revealed the highest nuclei positivity for both biomarkers. Cisplatin-exposed metastases demonstrated a higher EZH2-positive nuclei and H-score, as well as higher EHMT2/G9a-positive nuclei. DISCUSSION AND CONCLUSION: Overall, our data suggest that EZH2 and EHMT2/G9a might be associated with greater aggressiveness and, eventually, involved in the metastatic setting, paving the way for testing targeted therapies.

Influence of survivin (BIRC5) and caspase-9 (CASP9) functional polymorphisms in renal cell carcinoma development: a study in a southern European population.

Renal cell carcinoma (RCC) is the most common cancer of the adult kidney and its incidence and mortality has increase in the last 20 years. The disruption of cellular death is one the mechanism involved in cancer development. This process is precise regulated by apoptotic and anti-apoptotic molecules. Survivin (BIRC5) is a member of the inhibitor of apoptosis protein family and has the ability to inhibit the activation of the pro-apoptotic caspase-9 (CASP9). Thus BIRC5 and CASP9 functional polymorphisms might modulate the apoptosis and consequently RCC development. Our purpose was to investigate the potential role of BIRC5-31G/C and CASP9+83C/T functional polymorphisms in the risk for the development of RCC and metastatic disease. We studied the BIRC5-31G/C and CASP9+83C/T functional polymorphisms by PCR-RFLP and allelic discrimination using the 7300 real-time polymerase chain reaction system, respectively, in 178 RCC patients and in 305 healthy individuals. Regarding the BIRC5-31G/C polymorphism, there is a trend to an overrepresentation of CC genotype in RCC group compared with normal controls (aOR, 1.94; P=0.053). We observed, after gender stratification and age-adjustment, that BIRC5-31CC and CASP9+83CT/TT genotypes were associated with an increased risk for RCC development in the female group of our southern European study population (aOR=3.85; P=0.019; aOR=2.98; P=0.028; respectively). Concerning the waiting time for onset of metastatic disease, we observed that BIRC5-31CC homozygous developed metastasis 8 years earlier than the G carriers using a Cox proportional hazard model with gender as covariate (HR=4.9, P=0.038, P bootstrap=0.009). The Cox regression proportional hazard model was validated using bootstrap statistic with 1,000 samples of the same number of patients as the original dataset. Our results suggest that individual differences influence the susceptibility to RCC and tumor behavior. This genetic profile may help to define higher risk groups that would benefit from individualized chemoprevention strategies and therapies.

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.

Ki67 and LSD1 Expression in Testicular Germ Cell Tumors Is Not Associated with Patient Outcome: Investigation Using a Digital Pathology Algorithm.

TGCTs represent a model of curable disease afflicting especially young men. Defining tumor biological characteristics is crucial to increase current knowledge and tailor the best clinical management. Ki67, a potential prognostic marker, still exhibits heterogenous associations with patient outcomes, thus bringing the need of corroboration with larger cohorts in clinical practice. LSD1, an epigenetic enzyme, represents a future target for epigenetic drugs that may lower treatment-associated morbidity. This study aimed to assess Ki67/LSD1 immunoexpression across all TGCT histological subtypes and correlate it with clinicopathological features. Results were compared with an in silico analysis of the TCGA database. Immunohistochemistry for Ki67 and LSD1 was carried out in a cohort of 157 TGCT tumor samples and assessed using a digital pathology algorithm. LSD1 protein expression was explored in TGCT cell lines, including ATRA-differentiated clones. There was a significant positive correlation between Ki67 and LSD1 H-scores (rs = 0.182, p = 0.037). Ki67 positivity percentage and H-score were significantly higher in non-seminomas (p = 0.0316 and 0.0113, respectively). Expression was not significantly different according to clinicopathological features, including stage, IGCCCG prognosis-based system, or relapse/progression-free survival, which was corroborated by in silico analysis. Our study, making use of digital image analysis, does not confirm the utility of these biomarkers in a daily practice cohort. Although not affecting patient outcome in our cohort, LSD1 is expressed overall in TGCTs, suggesting sensitivity to LSD1 inhibitors.

The component of the m6A writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors.

BACKGROUND: Germ cell tumors (GCTs) are developmental cancers, tightly linked to embryogenesis and germ cell development. The recent and expanding field of RNA modifications is being increasingly implicated in such molecular events, as well as in tumor progression and resistance to therapy, but still rarely explored in GCTs. In this work, and as a follow-up of our recent study on this topic in TGCT tissue samples, we aim to investigate the role of N6-methyladenosine (m6A), the most abundant of such modifications in mRNA, in in vitro and in vivo models representative of such tumors. METHODS: Four cell lines representative of GCTs (three testicular and one mediastinal), including an isogenic cisplatin resistant subline, were used. CRISPR/Cas9-mediated knockdown of VIRMA was established and the chorioallantoic membrane assay was used to study its phenotypic effect in vivo. RESULTS: We demonstrated the differential expression of the various m6A writers, readers and erasers in GCT cell lines representative of the major classes of these tumors, seminomas and non-seminomas, and we evidenced changes occurring upon differentiation with all-trans retinoic acid treatment. We showed differential expression also among cells sensitive and resistant to cisplatin treatment, implicating these players in acquisition of cisplatin resistant phenotype. Knockdown of VIRMA led to disruption of the remaining methyltransferase complex and decrease in m6A abundance, as well as overall reduced tumor aggressiveness (with decreased cell viability, tumor cell proliferation, migration, and invasion) and increased sensitivity to cisplatin treatment, both in vitro and confirmed in vivo. Enhanced response to cisplatin after VIRMA knockdown was related to significant increase in DNA damage (with higher γH2AX and GADD45B levels) and downregulation of XLF and MRE11. CONCLUSIONS: VIRMA has an oncogenic role in GCTs confirming our previous tissue-based study and is further involved in response to cisplatin by interfering with DNA repair. These data contribute to our better understanding of the emergence of cisplatin resistance in GCTs and support recent attempts to therapeutically target elements of the m6A writer complex.

AGO2 expression levels and related genetic polymorphisms: influence in renal cell progression and aggressive phenotypes.

Aim: Renal cell carcinoma (RCC) is the most lethal urological cancer and up to 40% of patients submitted to surgery will relapse. Thus, the study aim was to analyze the associations of AGO2 SNPs with RCC patients' prognosis, and evaluate their effect on AGO2 mRNA levels. Materials & methods: The AGO2 rs4961280, rs3928672 and rs11996715 polymorphisms and the relative quantification of AGO2 mRNA levels were analyzed by real-time PCR. Results: We observed that AGO2 rs4961280 AC + AA genotypes carriers presented a higher cancer progression risk (odds ratio= 3.13, p < 0.001), a reduced progression-free survival (log rank test, p = 0.003) and an increased risk of an early relapse (hazard ratio= 2.26, p = 0.008). In fact, these patients also presented higher circulating levels of AGO2 mRNA (p = 0.043), with the high levels being associated with more aggressive tumors. Conclusion: The AGO2 rs4961280 AA/AC genotypes are unfavorable RCC prognostic biomarkers, with the AGO2 levels being a useful RCC aggressive phenotype biomarker.

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.

p53 and MDM2 expression in primary and metastatic testicular germ cell tumors: Association with clinical outcome.

BACKGROUND: Testicular germ cell tumors (TGCTs) are highly sensitive to platinum-based chemotherapy, and wild-type p53 seems to play a pivotal role in this susceptibility. On the other hand, overexpression of MDM2 seems to entail treatment resistance and unfavorable prognosis. OBJECTIVES: We aimed to describe p53 and MDM2 immunoexpression in a well-characterized cohort of primary and metastatic TGCTs and evaluate associations with clinicopathological and prognostic variables, including survival. MATERIALS AND METHODS: 237 primary tumor samples and 12 metastases were evaluated for p53 and MDM2 immunoexpression using digital image analysis. Clinical records of all patients were reviewed for baseline clinical/pathologic characteristics and follow-up. RESULTS: A significant positive correlation between p53 and MDM2 H-scores was found (rs  = 0.590, P < .0001). Non-seminomas showed significantly higher expression levels of both p53 and MDM2 (P = .0002, P < .0001), which peaked in embryonal carcinomas and choriocarcinomas. Percentage of immunoexpressing cells and H-score were significantly higher in chemo-treated metastases compared with chemo-naïve primary tumors for MDM2 (P ≤ .0001 for both), but not for p53 (P = .919 and P = .703, respectively). Cases with higher MDM2 immunoexpression showed a statistically significant trend for association with poorer prognosis (P = .043). Relapse/progression-free survival at 12 months post-diagnosis was lower in the "MDM2-high" (≥P50) vs. the "MDM2-low" (

Differential expression of DNA methyltransferases and demethylases among the various testicular germ cell tumor subtypes.

Aim: Characterize DNA methyltransferases/demethylases expression in testicular germ cell tumors (TGCTs). Methods:In silico analysis of TCGA database, assessment of transcript levels of most relevant enzymes in four TGCT cell lines and validation in patient cohort (real-time quantitative polymerase chain reaction; immunohistochemistry). Results:DNMT3A, DNMT3B and TET2 were the most differentially expressed between seminomas (SEs) and nonseminomas (NSs). DNMT3B was significantly overexpressed in NS-related cell lines, and the opposite was found for TET2. Significantly higher DNMT3A/B mRNA expression was observed in NS, indicating a role for de novo methylation in reprogramming. Significantly higher TET2 protein expression was observed in SEs, suggesting active demethylation contributes for SE hypomethylated state. More differentiated histologies disclosed distinct expression patterns. Conclusion: DNA-modifying enzymes are differentially expressed between TGCT subtypes, influencing reprogramming and differentiation.

Efficacy of HDAC Inhibitors Belinostat and Panobinostat against Cisplatin-Sensitive and Cisplatin-Resistant Testicular Germ Cell Tumors.

Novel treatment options are needed for testicular germ cell tumor (TGCT) patients, particularly important for those showing or developing cisplatin resistance, the major cause of cancer-related deaths. As TGCTs pathobiology is highly related to epigenetic (de)regulation, epidrugs are potentially effective therapies. Hence, we sought to explore, for the first time, the effect of the two most recently FDA-approved HDAC inhibitors (HDACis), belinostat and panobinostat, in (T)GCT cell lines including those resistant to cisplatin. In silico results were validated in 261 patient samples and differential expression of HDACs was also observed across cell lines. Belinostat and panobinostat reduced cell viability in both cisplatin-sensitive cells (NCCIT-P, 2102Ep-P, and NT2-P) and, importantly, also in matched cisplatin-resistant subclones (NCCIT-R, 2102Ep-R, and NT2-R), with IC50s in the low nanomolar range for all cell lines. Treatment of NCCIT-R with both drugs increased acetylation, induced cell cycle arrest, reduced proliferation, decreased Ki67 index, and increased p21, while increasing cell death by apoptosis, with upregulation of cleaved caspase 3. These findings support the effectiveness of HDACis for treating TGCT patients in general, including those developing cisplatin resistance. Future studies should explore them as single or combination agents.

Extracellular Vesicles Enriched in hsa-miR-301a-3p and hsa-miR-1293 Dynamics in Clear Cell Renal Cell Carcinoma Patients: Potential Biomarkers of Metastatic Disease.

Clear cell renal cell carcinoma (ccRCC) is the most aggressive subtype of kidney cancer and up to 40% of patients submitted to surgery with a curative intent will relapse. Thus, the aim of this study was to analyze the applicability of an Extracellular vesicle (EV) derived miRNA profile as potential prognosis biomarkers in ccRCC patients. We analyzed a nine-miRNA profile in plasma EVs from 32 ccRCC patients with localized disease (before and after surgery) and in 37 patients with metastatic disease. We observed that the levels of EV-derived hsa-miR-25-3p, hsa-miR-126-5p, hsa-miR-200c-3p, and hsa-miR-301a-3p decreased after surgery, whereas hsa-miR-1293 EV-levels increased. Furthermore, metastatic patients presented higher levels of hsa-miR-301a-3p and lower levels of hsa-miR-1293 when compared to patients with localized disease after surgery. Functional enrichment analysis of the targets of the four miRNAs that decreased after surgery resulted in an enrichment of terms related to cell cycle, proliferation, and metabolism, suggesting that EV-miRNA enrichment in the presence of the tumor could represent an epigenetic mechanism to sustain tumor development. Taken together, these results suggest that EVs content varies depending on the presence or absence of the disease and that an increase of EV-derived hsa-miR-301a-3p, and decrease of EV-derived hsa-miR-1293, may be potential biomarkers of metastatic ccRCC.

Detailed Characterization of Immune Cell Infiltrate and Expression of Immune Checkpoint Molecules PD-L1/CTLA-4 and MMR Proteins in Testicular Germ Cell Tumors Disclose Novel Disease Biomarkers.

Background: The immune infiltrate plays an important part in testicular germ cell tumors, but it remains scarcely studied. We aimed at thoroughly characterizing the immune infiltrate and expression of immune checkpoints PD-L1/CTLA-4 and mismatch repair (MMR) proteins in these neoplasms, seeking for associations with patient outcome. Methods: A total of 162 consecutively diagnosed patients (2005-2018) were included. Immunostaining for PD-L1, CTLA-4 and MMR proteins was independently assessed both in immune cells (ICs) and tumor cells (TCs) of primary tumors and metastases, and characterization of IC populations was pursued. Results: PD-L1 and CTLA-4 positivity in ICs was frequent (85.5% and 96.3%). Patients with absent PD-L1 positive ICs exhibited significantly worse relapse-free survival (hazard ratio = 4.481, 95% CI 1.366-14.697, p = 0.013), both in univariable and multivariable analysis. Lower CD20 and CD3 IC infiltration in seminomas associated with higher disease stage (p = 0.0216, p = 0.0291). CTLA-4 TC intensity was significantly higher in yolk sac tumor, choriocarcinoma and teratoma, while PD-L1 TC positivity was significantly more frequent in choriocarcinoma. Both PD-L1 and CTLA-4 immunoexpression in ICs of metastatic samples was frequent (100% and 88.2%). MMR proteins were differentially expressed among the different tumor subtypes. Conclusions: Immune infiltrate/checkpoints associate with patients' outcome, constituting novel (potentially targetable) disease biomarkers.

Circulating MicroRNAs as Biomarkers for Prostate Cancer Detection and Metastasis Development Prediction.

Prostate Cancer (PCa) overdiagnosis and overtreatment, as a consequence of the limited specificity of current detection and prognostication methods, remains a major challenge in clinical practice. Therefore, development and validation of new molecular biomarkers amenable of detecting clinically significant disease is crucial. MicroRNAs (miRNA) deregulation is common in cancer, constituting potential non-invasive biomarkers for PCa detection and prognostication. Herein, we evaluated the screening and prognostic biomarker potential of two onco-microRNAs (miR-182-5p and miR-375-3p) in liquid biopsies (plasma) of PCa patients with clinically localized disease undergoing curative-intent treatment. A first cohort of 98 PCa and 15 normal prostates were used to assess PCa-specificity of miR-182-5p in tissues. A cohort composed of PCa 252 patients and 52 asymptomatic controls allowed for assessment of diagnostic and prognostic value in plasmas. After RNA extraction from tissue and plasma samples, cDNA synthesis specific for miRNAs was performed followed by measurement of miR-182-5p and miR-375-3p relative expression by RT-qPCR, using U6 snRNA gene as reference. MiR-182-5p was significantly overexpressed in PCa tissues (p < 0.0001) and in plasma of PCa patients (p = 0.0020), compared to respective controls. Moreover, miR-182-5p expression identified PCa with AUC = 0.81 (95% CI: 0.725-0.892, p = 0.0001) in tissue and with 77% specificity and 99% NPV (AUC = 0.64, 95% CI: 0.561-0.709, p = 0.0021) in plasma. Both circulating miR-182-5p and miR-375-3p levels associated with more advanced pathologic stage and the former was significantly higher in patients that developed metastasis (p = 0.0145). Indeed, at the time of diagnosis, circulating miR-375-3p levels predicted which patients would develop metastasis, with almost 50% sensitivity, 76% specificity, and a NPV of 89% (AUC = 0.62, 95% CI: 0.529-0.713, p = 0.0149). We conclude that these two circulating miRNAs might be clinical useful as non-invasive biomarkers for detection and prediction of metastasis development at the diagnosis together with clinical variables used in routine practice.

XPO5 genetic polymorphisms in cancer risk and prognosis.

miRNAs are small noncoding RNA molecules that have a very important role in gene expression regulation and, therefore, in cell homeostasis. SNPs in certain miRNA-related genes have been shown to influence cancer risk and prognosis. miRNA cellular processing is complex and involves multiple proteins. XPO5 is a key factor in this process as it is responsible for the nuclear export of the precursor pre-miRNA to the cytoplasm, where it will be further processed to its final miRNA conformation in order to be loaded to RNA inducing silencing complex to exert its regulatory effect. SNPs in miRNA machinery related genes have previously been shown to influence carcinogenesis, but the role of XPO5 SNPs in its expression and function is not yet fully understood. In our review, we elaborate comprehensively on the role of XPO5 and how polymorphisms have been shown to influence cancer risk and prognosis to date.