The role of miRNAs as biomarkers in prostate cancer.

There is an urged need of non-invasive biomarkers for the implementation of precision medicine. These biomarkers are required to these days for improving prostate cancer (PCa) screening, treatment or stratification in current clinical strategies. There are several commercial kits (Oncotype DX genomic prostate score®, Prolaris®, among others) that use genomic changes, rearrangement or even non-coding RNA events. However, none of them are currently used in the routine clinical practice. Many recent studies indicate that miRNAs are relevant molecules (small single-stranded non-coding RNAs that regulate gene expression of more than 30% of human genes) to be implement non-invasive biomarkers. However, contrasting to others tumors, such as breast cancer where miR-21 seems to be consistently upregulated; PCa data are controversial. Here we reported an extended revision about the role of miRNAs in PCa including data of AR signaling, cell cycle, EMT process, CSCs regulation and even the role of miRNAs as PCa diagnostic, prognostic and predictive tool. It is known that current biomedical research uses big-data analysis like Next Generation Sequencing (NGS) analysis. We also conducted an extensive online search, including the main platforms and kits for miRNAs massive analysis (like MiSeq, Nextseq 550, or Ion S5™ systems) indicating their pros, cons and including pre-analytical and analytical issues of miRNA studies.

Methodology for Y Chromosome Capture: A complete genome sequence of Y chromosome using flow cytometry, laser microdissection and magnetic streptavidin-beads.

This study is a comparison of the efficiency of three technologies used for Y chromosome capture and the next-generation sequencing (NGS) technologies applied for determining its whole sequence. Our main findings disclose that streptavidin-biotin magnetic particle-based capture methodology offers better and a deeper sequence coverage for Y chromosome capture, compared to chromosome sorting and microdissection procedures. Moreover, this methodology is less time consuming and the most selective for capturing only Y chromosomal material, in contrast with other methodologies that result in considerable background material from other, non-targeted chromosomes. NGS results compared between two platforms, NextSeq 500 and SOLID 5500xl, produce the same coverage results. This is the first study to explore a methodological comparison of Y chromosome capture and genetic analysis. Our results indicate an improved strategy for Y chromosome research with applications in several scientific fields where this chromosome plays an important role, such as forensics, medical sciences, molecular anthropology and cancer sciences.

Genetic markers a landscape in prostate cancer.

Prostate cancer (PC) is one of the most common cancers worldwide. The observed variability in progression and responses to the same treatment between patients underlie the genetic heterogeneity of the disease. Nowadays, screening and follow-up biomarkers in PC are still having a deep lack of information, which makes difficult the cancer diagnosis, prognosis and the selection of the most suitable therapies. This is making that currently unnecessary biopsies, over-treatments and hormonoresistances have high rates of prevalence among patients. New biomarkers are urgently needed and in this sense genomic biomarkers could be the most suitable tools. These genetic markers will be helpful for improving the precision of prognostic and the predictive current tools which are employed in the clinical practice. A recent literature search up was conducted, including clinical trials and pre-clinical basic research studies. Keywords included germline variants, prostate cancer, biomarkers, androgen deprivation therapy, screening and liquid biopsy; among others. We have reviewed how germline variants, CNVs and repetitive regions are relevant to prostate carcinogenesis, treatment and progression. Moreover, we have also considered novel biomarkers for PC prognosis based on differentially expressed genes. Finally, we have included new strategies in recent markers of liquid biopsy or updated technologies for minimal samples analysis. The improvement of genetic markers use and their application to the clinical practice, will enhance the variability of simple, non-invasive, tools such as liquid biopsy and germline variants, these will reduce the number of PC needle biopsies and current over-treatments that are usual in the management of this cancer.

Circulating Tumor Cells: Markers and Methodologies for Enrichment and Detection.

Cancer is a leading cause of disease worldwide; however, nowadays many points of its initiation processes are unknown. In this chapter, we are focusing on the role of liquid biopsies in cancer detection and progression. CTCs are one of the main components of liquid biopsies, they represent a subset of tumor cells that have acquired the ability to disseminate from the primary tumor and intravasate to the circulatory system. The greatest challenge in the detection of CTCs is their rarity in the blood. Human blood consists of white blood cells (5-10 × 106/mL), red blood cells (5-9 × 109/mL), and platelets (2.5-4 × 108/mL); very few CTCs will be present even in patients with known metastatic disease, with often less than one CTC per mL of blood. CTCs are found in frequencies on the order of 1-10 CTCs per mL of whole blood in patients with metastatic disease, and it is reduced in half for non-metastatic stages. Therefore, accurate methodologies for their capture and analysis are really important. The main aim of the present chapter is to describe different methodologies for CTCs capturing and analysis.

Somatic Mutations in Prostate Cancer: Closer to Personalized Medicine.

The molecular cause of prostate cancer (PCa) is still unclear; however, its progression involves androgen, PI3K/Akt, and PTEN signaling, as cycle and apoptotic pathways. Alterations in oncogenes and tumor suppressor genes as PIK3CA, BRAF, KRAS and TP53 are not very common. Recently, somatic mutations have been discovered in relation to cancer progression mainly in genes such as PIK3CA; however, little data has been described in PCa. Nowadays genetic tools allow us to investigate multiple details about the biological heterogeneity of PCa, to better understand the mechanisms of disease progression and treatment resistance. Therefore, if the most relevant somatic mutations were included during screening, we could identify the best treatment for the right patient, bringing us closer to personalized medicine. The main objective of this article is to provide a review of the principal somatic mutations that appear to have a relevant role in hormonal cancers, like prostate cancer.

Combined analysis of copy number alterations by single-nucleotide polymorphism array and MYC status in non-metastatic breast cancer patients: comparison according to the circulating tumor cell status.

Recent technological advances have made it possible to detect circulating tumor cells (CTCs) as a prognostic marker in operable breast cancer patients. Whether the presence of CTCs in cancer patients correlates with molecular alterations in the primary tumor has not been widely explored. We identified 14 primary breast cancer specimens with known CTC status, in order to evaluate the presence of differential genetic aberrations by using SNP array assay. There was a global increase of altered genome, CNA, and copy-neutral loss of heterozygosity (cn-LOH) observed in the CTC-positive (CTC(+)) versus CTC-negative (CTC(-)) cases. As the preliminary results showed a higher proportion of copy number alteration (CNA) at 8q24 (MYC loci) and the available evidence supporting the role of MYC in the processes cancer metastases is conflicting, MYC status was determined in tissue microarray sections in a larger series of patients (n = 49) with known CTC status using FISH. MYC was altered in 62% (16/26) CTC(+) patients and in 43% (6/14) CTC(-) patients (p = 0.25). Based on the observation in our study, future studies involving a larger number of patients should be performed in order to definitively define if this correlation exists.

[Elongation of the right renal vein in 120 consecutive transplant patients. A comparative analysis]. / Alargamiento de la vena renal derecha en 120 trasplantes consecutivos. Análisis comparativo.

OBJECTIVES: Shorter length of the right renal vein (RRV) may represent an additional difficulty for transplant. This paper has aimed to present our experience with RRV elongation in the kidney from a cadaveric donor and to compare the results with the rest of kidneys transplanted in the same period of time. MATERIAL AND METHODS: We performed 377 kidneys transplants within the last 11 years. Of these, in 120, the RRV was elongated with the vena cava. The surgical technique is described and the results compared with the remaining transplants of the series. Renal function, graft survival and complications were assessed at 1, 3 and 12 months. Data were obtained retrospectively from the database of the Urology Department and Hospital medical records. RESULTS: In the 377 transplants, there were 4 (1%) venous thrombosis, 2 (1.6%) of which concerned kidneys with elongation of the RRV and 2 (0.7%) in the rest of transplanted kidneys. There was no difference in postoperative bleeding, 11 (9.1%) occurred in kidneys with elongated RRV and 22 (8.5%) in the remaining kidney. In no case was the bleeding related to the elongated segment. Graft survival and renal function were similar for both groups. CONCLUSIONS: Elongation of the RRV with the vena cava is a feasible, fast, and effective procedure that does not increase morbidity or affect renal function or graft viability. It facilitates vascular anastomosis and places the kidney in a less forced position, shortens the warm ischemia time and avoids the risk of kinking of the renal artery because it is equal to the length of the vein artery.

[RNASEL study of genetics of prostate cancer and its relation to clinical staging]. / Estudio genético de RNASEL en cáncer de próstata y su relación con la estadificación clínica.

OBJECTIVES: This study has aimed to find a possible genetic relationship between sporadic prostate cancers. An attempt is made to establish population subgroups in patients based on the genotype found and the aggressiveness of the cancer. MATERIAL AND METHODS: A total of 231 patients with sporadic prostate cancer and 68 controls were selected. The subjects were selected by an urologist using clinical parameters such as PSA level and Gleason score. Both groups (patients and controls) were genotyped in RNASEL gene by sequencing the exons 1 and 3. RESULTS: Statistically significant differences were found between controls and patients in some of the genotyped regions of the RNASEL gene (I97L, D541E and R462Q). CONCLUSIONS: Thanks to the genetic profile in some regions of the genoma, such as the RNASEL gene, together with the combination of the clinical and environmental parameters, we can suggest a care and more personalized follow-up of each patient.

[Cystitis and ketamine associated bladder dysfunction]. / Cistitis y disfunción vesical asociada al consumo de ketamina.

OBJECTIVES: To analyze the prevalence of lower urinary tract symptoms (LUTS) in recreational ketamine users and evaluate its relationship with the consumption pattern. MATERIAL AND METHODS: Evaluation of 13 ketamine users. The presence of LUTS, gross hematuria and lumbar spine pain was analyzed. The ketamine usage pattern was recorded: initiation, administration route, dose in the last month and frequency of usage. RESULTS: Six patients (46%) reported LUTS, with daily mean micturations every 42 minutes and nighttime of 3 episodes, with dysuria (100%), urgency (100%), incontinence (20%), decreased flow (80%), hypogastric or perineal pain (80%), gross hematuria (80%) and bilateral lumbar spine pain (40%). Symptomatic patients described a mean intake of inhaled ketamine of 3g/day (SD 2), 80% with a daily frequency and the asymptomatic ones of 1.03 g/day (SD 0.92) limited to weekends. The mean consumption time to the appearance of the symptoms was 31 months (SD 16.29). Intensity of the symptoms was related with the ketamine dose and improved on increasing water intake. CONCLUSIONS: There seems to be a relationship between the picture with the dose and frequency of consumption, there being factors that reinforce the hypothesis that this action of the drug is due to the harmful effect on the urothelium. The process to identify it on time should be known, since the only known effective measure is to stop the consumption in the initial phases.

Predictive value in the analysis of RNASEL genotypes in relation to prostate cancer.

BACKGROUND: We would like to compare the different RNASEL genotypes with the stage of the cancer using parameters such as PSA levels, Gleason score and T-stage, and to develop a clinical protocol for the monitoring of the disease for trying a better evolution of the patient. METHODS: A total of 231 patients with sporadic prostate cancer and 100 of controls were genotyped in RNASEL gene by sequencing the exons 1 and 3. A survey of clinical information was collected by a specialist following the Helsinki protocol. All patients and controls were interviewed by a researcher and signed their informed consent to participation in the study, which was approved by Ethics Committee of the hospital. The genetic information was processed and collected with an ABI PRISM Genetic Analyser 3130 using SeqScape software v.2.6. All the patients were analysed by comparing the genetic and clinical data. χ(2)-tests, Monte Carlo, Fisher tests and contigency tables were performed using SPSS v.15.0 and ARLEQUIN v.3.5 software on patient population. RESULTS: Significant differences were found only between patients and controls in D541E, R461Q and I97L genotypes, the remainder of the variants did not seem relevant to our population in contrast to other populations, such as north-Caucasians, Afro Americans and Ashkenazi Jews. The genotypes associated with the worst prognoses are G/G in D541E, A/A in R462Q and A/G in I97L. The controls were included in our study to determine an approximation of the genotype in our population compared with the patients, but they did not account for the statistical process. CONCLUSIONS: The genetic profile of patients with this cancer combined with other parameters could be used as a prognosis factor in deciding to give more radical and frequent treatments, depending on personal genotype.

Lung histopathological findings in fatal pandemic influenza A (H1N1).

OBJECTIVE: To describe the lung pathological changes in influenza A (H1N1) viral pneumonia. We studied morphological changes, nitro-oxidative stress and the presence of viral proteins in lung tissue. METHODS AND PATIENTS: Light microscopy was used to examine lung tissue from 6 fatal cases of pandemic influenza A (H1N1) viral pneumonia. Fluorescence for oxidized dihydroethydium, nitrotyrosine, inducible NO synthase (NOS2) and human influenza A nucleoprotein (NP) (for analysis under confocal microscopy) was also studied in lung tissue specimens. RESULTS: Age ranged from 15 to 50 years. Three patients were women, and 5 had preexisting medical conditions. Diffuse alveolar damage (DAD) was present in 5 cases (as evidenced by hyaline membrane formation, alveolo-capillary wall thickening and PMN infiltrates), and interstitial fibrosis in one case. In the fluorescence studies there were signs of oxygen radical generation, increased NOS2 protein and protein nitration in lung tissue samples, regardless of the duration of ICU admission. Viral NP was found in lung tissue samples from three patients. Type I pneumocytes and macrophages harbored viral NP, as evidenced by confocal immunofluorescence microscopy. CONCLUSIONS: Lung tissue from patients with pandemic influenza A (H1N1) viral pneumonia shows histological findings consistent with DAD. Prolonged nitro-oxidative stress is present despite antiviral treatment. Viral proteins may remain in lung tissue for prolonged periods of time, lodged in macrophages and type I pneumocytes.

Characterization of cytoskeletal proteins in follicular structures of cows with cystic ovarian disease.

The distribution of intermediate filaments (vimentin, cytokeratins, desmin) and microfilaments (alpha-smooth muscle actin and muscle specific actin) was studied immunohistochemically in bovine ovaries, with and without cystic ovarian disease. The immunohistochemically stained area (IHCSA), was quantified by image analysis, to evaluate the expression of these cytoskeletal proteins in the follicular wall of healthy antral, atretic, and cystic follicles. The granulosa cell layer of cystic follicles and atretic follicles had a significantly larger IHCSA for vimentin than did healthy antral follicles. Cytokeratins reacted lightly in the granulosa cells of antral follicles of normal ovaries, whereas granulosa cells of atretic and cystic follicles showed significantly higher IHCSA values. Immunohistochemical localization of desmin, muscle specific actin, and alpha-smooth muscle actin was restricted to the theca externa. This study supports earlier suggestions that strongly positive reactions with vimentin and cytokeratin antibodies observed in the granulosa cells of cystic follicles are due to the reorganization that occurs in the follicle during the process of cystic development, and are associated with changes in the expression of cytoskeletal proteins that are essential to proper cellular functioning.

The role of ACTH in the pathogenesis of polycystic ovarian syndrome in rats: hormonal profiles and ovarian morphology.

Numerous hypotheses have been proposed about the pathogenesis of the polycystic ovarian syndrome (PCOS). However, hormonal control of persistent follicles has not been established. The objective of the present study was to compare the follicular structure and hormonal profiles of rats treated with the adrenocorticotrophic hormone (ACTH) with two experimental models of PCOS. ACTH-treated animals were compared with those exposed to continuous light, those treated with estradiol valerate, and with control (in proestrous and diestrous). Serum hormone levels, histomorphometrical changes, and immunoexpression of vimentin, cytokeratins, cadherins, and proliferating cell nuclear antigen (PCNA) were examined. Treatment with ACTH resulted in an elevation of corticosterone secretion with LH reduction but without changes in ovarian morphology. Although stress (or ACTH) stimulation may be only one of pathophysiological mechanisms involved in follicular cyst pathogenesis in other species, we do not have important evidence to suppose that this would happen in rats.

Oestradiol induced inhibition of neuroendocrine marker expression in Leydig cells of adult rats.

The objectives of this work were to determine the changes in the expression of neuroendocrine markers in Leydig cell by oestradiol treatment, and to determine whether testosterone is able to recover partially the effects of hormonal suppression induced by oestradiol. Adult male rats were injected daily with either 50 microg of oestradiol or oestradiol plus testosterone propionate (25 mg every 3 days) for 15 days. The animals were sacrificed and testicles were dissected and processed by routine histological protocols. FSH and LH serum levels were determined by radioimmunoassay. The visualization of antigens was achieved by the streptavidin-peroxidase immunohistochemical method. Antibodies against chromogranin A (CrA), S-100 protein (S-100), P substance (PS), synaptofisin (SYN), neurofilament protein (NF), gliofibrillary acidic protein (GFAP) and neuron specific enolase (NSE) were used. The mean LH and FSH serum concentrations were consistently suppressed with hormonal treatments. Intermediate filaments (NF and GFAP) showed no difference in their expression. The expression of S-100, NSE and SYN was significantly lower in both hormone-treated groups. In oestradiol-treated rats, the immunoreactivity of CrA and SP decreased significantly but was restored after testosterone supplementation. Although the nature and functions of many of these substances in Leydig cells remain unknown, these results are consistent with the hypothesis that the expression of some neuroendocrine markers is hormonally controlled.

[Comparative study of the effect of complete prolonged neoadjuvant hormonal blockade versus standard hormonal blockade]. / Estudio comparativo del efecto del bloqueo hormonal completo neoadyuvante prolongado frente al estándar.

INTRODUCTION: Radical prostatectomy is considered as a curative treatment option in clinically localised prostate cancer patients. Therapy failure is related to positive surgical margins and/or extracapsular extension. The use of neoadjuvant combined androgen blockade (CAB) withdrawal therapy, mainly in cT2 disease, has been shown to decrease positive margin rates. However, CAB therapy remains controversial since there is no proof that this approach confers any benefit in relation to biochemical and clinical disease-free survival. Increasing negatives surgical margins and lower tumour volume (TV) with prolonged CAB therapy has been recently reported. AIM: To analyse the effect of 6 months neoadjuvant CAB therapy in front of 3 months in clinically localised prostate cancer patients submitted to radical prostatectomy. PATIENTS AND METHODS: The pathological stage and TV in forty-two patients treated by 6 months in front of thirty-four patients treated by 3 months were studied. The relationship of clinical stage and initial PSA concentration were analysed. RESULTS: TV was significantly lower in 6 months treated patients (0.97 cc vs. 0.48 cc, p = 0.05). The lowest TV was observed in cT1 patients, but significant differences only were observed in cT2 (1.5 cc vs. 0.86 cc, p = 0.04). No relationship between TV and PSA was obtained. No differences in the incidence of organ-confined disease were seen depending of the CAB length (47% vs. 43%, p = NS). However, increasing incidence of specimen-confined disease was observed in 6 months treated patients (56% vs. 74%, p = 0.05). CONCLUSION: The duration of neoadjuvant CAB can affect both TV and surgical margin status. Lower TV and increasing incidence of specimen-confined disease with 6 months CAB treatment were observed. Patients with palpable disease may be more benefited by this treatment option.

A longer duration of neo-adjuvant combined androgen blockade prior to radical prostatectomy may lead to lower tumour volume of localised prostate cancer.

OBJECTIVES: To analyse tumour volume (TV) in clinically localised prostate cancer patients treated with neo-adjuvant combined androgen blockade (CAB) therapy prior to radical prostatectomy. PATIENTS AND METHODS: Two hundred consecutive patients treated between 1996 and 2000 were retrospectively analysed. Fifty patients underwent radical prostatectomy alone and 45 were treated with CAB for 1-3 months, 83 for 4-6 months and 22 for more than 6 months before surgery. Logistic regression analysis was performed to identify the strongest independent prognosticator of organ-confined disease. RESULTS: No evidence of residual cancer was found in 11 specimens (5.6%). Regarding TV, 20 specimens showed less than 0.1cc, 33 between 0.1 and 0.49cc and 86 more than 0.5cc. Smaller TV was found in CAB-treated patients. Significant correlation was observed between treatment duration and TV. In logistic regression analysis, only CAB duration and TV were significantly correlated with organ-confined disease. CONCLUSIONS: Prominent regressive features and lower TV were found after neo-adjuvant CAB. It seems that more prolonged treatment may lead to greater tumoural regression. Only tumour burden and length of CAB therapy were independent variables significantly correlated with pathologically localised prostate cancer.

Effect of antibiotic treatment on serum PSA and percent free PSA levels in patients with biochemical criteria for prostate biopsy and previous lower urinary tract infections.

BACKGROUND: Controversy exists as to the influence of inflammatory foci on total and free prostate-specific antigen (PSA) concentrations. The objective was to analyze the biological variations of PSA and percent free PSA (%f-PSA) in patients with biochemical criteria for prostate biopsy (PSA higher than 4 ng/mL and normal rectal examination) and compare them with the variation induced by antibiotic treatment in a cohort of patients with a history of lower urinary tract infections and no clinical evidence of prostatitis. METHODS: Ninety patients with a history of lower urinary tract infections, non-suspicious digital rectal examination and PSA between 4 and 20 ng/mL were analyzed. PSA concentration and %f-PSA were determined. Forty-five patients were treated with three weeks of ofloxacin, following which marker determination was repeated. All patients underwent ultrasound-controlled transrectal six-core prostate biopsy. RESULTS: Sixty-seven patients presented benign prostatic hyperplasia (BPH) (30 with prostatitic foci) and 23 cancer. Significant variations in PSA (6.97 ng/mL vs. 5.82 ng/mL, p=0.001) and %f-PSA (14.84% vs. 17.53%, p=0.01) were found only in the treated patients. These differences were significant for patients with BPH-associated prostatitic foci and not for patients with BPH or cancer. The tendency was for PSA to decrease (15 treated patients with PSA <4 ng/mL vs. six non-treated patients) and for %f-PSA to increase. The median variation of %f-PSA was greater than that of PSA. When the cutoff for %f-PSA was set at 25%, 18.9% of unnecessary biopsies after the first determination and 20% after the second could be avoided. By associating the reduction in PSA, up to 46% could be avoided in treated patients. CONCLUSION: Biochemical criteria for prostate biopsy may be modified in patients with a history of lower urinary tract infections due to variations greater than those explained by intraindividual biological variations, and may be influenced by the antibiotic treatment. These results suggest that subclinical inflammatory foci may influence PSA and %f-PSA.

Intraindividual variations of total and percent free serum prostatic-specific antigen levels in patients with normal digital rectal examination.

PURPOSE: To analyze intraindividual variations of total and percent free serum prostate-specific antigen (PSA) in patients with normal digital rectal examination. MATERIAL AND METHODS: Total and free serum PSA were determined in two blood samples corresponding to 107 nonconsecutive patients. The period between both determinations ranged from 23 to 60 days. Prostatic biopsy was done after both determinations in all except 17 patients because the two serum PSA concentrations were <4 ng/ml. Total and free PSA were determined using double monoclonal antibody immunoassay Tandem and Tandem free (Hybritech Inc.). The diagnosis was benign prostatic hyperplasia (BPH) in 63 patients and prostate cancer (PCA) in 44. RESULTS: The variations of PSA ranged between -6.8 and +3.2 ng/ml in BPH patients and between -2.8 and +9.0 in patients with PCA. The median coefficients of variation were 15.4 and 15.7% respectively. The variations in the percent free PSA were between -30.7 and +40.9 in the BPH group and between -17.9 and +15.8 in the PCA group. The median coefficients of variation were 32.2 and 32.3% respectively. If prostatic biopsy had been indicated when percent free PSA had been