Resistance to 2-Hydroxy-Flutamide in Prostate Cancer Cells Is Associated with the Downregulation of Phosphatidylcholine Biosynthesis and Epigenetic Modifications.

In this study, we examined the metabolic adaptations of a chemoresistant prostate cancer cell line in comparison to a sensitive cell line. We utilized prostate cancer LNCaP cells and subjected them to a stepwise increase in the antiandrogen 2-hydroxy-flutamide (FLU) concentration to generate a FLU-resistant cell line (LN-FLU). These LN-FLU cells displayed characteristics of cancer stem cells, exhibited drug resistance, and showed a significantly reduced expression of Cyclin D1, along with the overexpression of p16, pointing to a proliferation arrest. In comparing the cancer stem-like LN-FLU cells to the LNCaP cells, we observed a decrease in the expression of CTP-choline cytidylyl transferase α (CCTα), as well as a decline in choline kinase, suggesting altogether a downregulation of the phosphatidylcholine biosynthetic pathway. In addition, we found decreased levels of the protein methyl transferase PRMT2 and the upregulation of the histone deacetylase Sirtuin1 (Sirt1). Analysis of the human prostate cancer samples revealed similar results in a population with high expressions of the stem cell markers Oct4 and ABCB1A1. Our findings suggest that the adaptation of prostate cancer cells to antiandrogens could induce reprogramming into stem cells that survive in a low phosphocholine metabolism and cell cycle arrest and display drug resistance.

A Highly Sensitive Immunoassay for Determination of Immune Response to SARS-CoV-2 in Capillary Blood Samples.

Throughout the pandemic, serological assays have been revealed as crucial for detecting previous exposures to the virus and determining the timing of antibody maintenance after vaccination or natural infection. This study aimed to develop an optimized enzyme-linked immunosorbent assay (ELISA)-based serology, which could be used in case of reagent shortages, such as that occurred in the beginning of this health emergency. As a result, we present a high-sensitive immunoassay for the determination of IgG levels in venous serum samples, using 2 µg/mL antigen (receptor-binding domain of the spike protein S1) for coating the plate and utilizing human samples at a dilution 1:1000. This method showed non-inferiority features versus a commercial kit, is less expensive, and has a higher spectrophotometric range that allows for a better quantification of the antibody titers. The optical density values before and after heating venous serum samples at 56 °C during 30 min was quite similar, showing that heat inactivation can be used to reduce the biohazardous risks while handling samples. Furthermore, we show that finger-stick capillary blood samples can also serve as a suitable source for IgG detection, bypassing the need for serum isolation and being suitable for point-of-care application (Pearson's coefficient correlation with capillary serum was 0.95, being statistically significant).

Increase in Ischemia-Modified Albumin and Pregnancy-Associated Plasma Protein-A in COVID-19 Patients.

This study was undertaken due to the urgent need to explore reliable biomarkers for early SARS-CoV-2 infection. We performed a retrospective study analyzing the serum levels of the cardiovascular biomarkers IL-6, TNF-α, N-terminal pro-B natriuretic peptide, cardiac troponin T (cTnT), ischemia-modified albumin (IMA) and pregnancy-associated plasma protein-A (PAPP-A) in 84 patients with COVID-19.Patients were divided into three groups according to their RT-qPCR and IgG values: acute infection (n = 35), early infection (n = 25) or control subjects (n = 24). Levels of biomarkers were analyzed in patient serum samples using commercially available ELISA kits. Results showed a significant increase in IMA and PAPP-A levels in the early infected patients. Moreover, multivariate analysis and receiver operating characteristic (ROC) curve showed that IMA and PAPP-A had excellent discrimination value for the early stage of COVID-19. For IMA, the area under the ROC curve (AUC) had a value of 0.94 (95% confidence interval (CI): 0.881-0.999). Likewise, the serum level of PAPP-A was significantly higher in patients with early infection than in the control subjects (AUC = 0.801 (95% CI: 0.673-0.929)). The combined use of IMA and PAPP-A enhanced the sensitivity for total SARS-CoV-2-infected patients to 93%. These results suggest that the increased levels of PAPP-A and IMA shed light on underlying mechanisms of COVID-19 physiopathology and might be used as efficient biomarkers with high sensitivity and specificity for the early stage of COVID-19. Importantly, when monitoring pregnancy and cardiovascular diseases using PAPP-A or IMA levels, a SARS-CoV-2 infection should be discarded for proper interpretation of the results.

Homeostasis: apoptosis and cell cycle in normal and pathological prostate.

Prostatic diseases such as hyperplasia and cancer are a consequence of glandular aging due to the loss of homeostasis. Glandular homeostasis is guaranteed by the delicate balance between production and cell death. Both cell renewal and apoptosis are part of this delicate balance. We will explore the predictive capacity for biochemical progression, following prostatectomy, of some members of the Bcl-2 family and of proteins involved in cell cycle inhibition in conjunction with established classical markers. The expression of Bcl-2, Bcl-xL, Mcl-1, Bax, Bim, Bad, PUMA, Noxa, p21, p27, Rb and p53 were analyzed by immunochemistry in 86 samples of radical prostatectomy and correlated with each of the markers established clinicopathological tests using statistical tests such as Sperman, Kaplan-Meier curves, unifactorial Cox, and multifactorial. The most relevant results are: (1) Positive correlation between: p27 with clinical T stage; and PUMA with pathological T stage; (2) Negative correlation between: Bcl-2 with clinical T stage, Bcl-xL with survival, Noxa and pRb with Gleason score.Our results suggest that the expression of Bcl-2, Bcl-xL, PUMA, Noxa, p27, and Rb were related to some of the classic markers established to predict biochemical progression after prostatectomy.

PI3K pathway and Bcl-2 family. Clinicopathological features in prostate cancer.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathways and Bcl-2 family play a central role in prostate cancer (PC). The aim was to determine influence in the biochemical progression in PC. To evaluate the association between clinic pathological and immunohistochemical variables, Spearman's test was performed. Log-rank test and Kaplan-Meier curves were used for survival comparisons. To explore the correlation of the studied immunohistochemical parameters and the established prognostic variables with biochemical progression, univariate and multivariate Cox proportional Hazard regression analyses were performed. Spearman analysis showed correlation between stroma expression and tumor expression of PI3K with biochemical progression (p = .009, p = .004), respectively, and tumor immunohistochemical score with biochemical progression (p = .051). In the multivariate Cox regression model, only PI3K was retained as independent predictors of biochemical progression. In stroma expression, PI3K is (HR 0.172, 95% CI 0.065-0.452, p = .000); tumor expression, PI3K is (HR 0.087, 95% CI 0.026-0.293, p = .000), and tumor immunohistochemical score (HR 0.382, 95% CI 0.209-0.697 p = .002). Our results suggest a role for prostatic expression of PI3K was prognostic markers for PC. PI3K/AKT/mTOR and Bcl-2 family are becoming an important therapeutic target and predictive biomarkers of onset and progression of PC.

Does [-2]Pro-Prostate Specific Antigen Meet the Criteria to Justify Its Inclusion in the Clinical Decision-Making Process?

INTRODUCTION: To assess whether [-2]pro-prostate-specific antigen (p2PSA) meets the criteria to justify its inclusion in a predictive model of prostate cancer (PCa) diagnosis and in the clinical decision-making process. MATERIALS AND METHODS: A total 172 men with total prostate-specific antigen of 2-10 ng/mL underwent measurement of free PSA and p2PSA before prostate biopsy in an observational and prospective study. From these measurements, the Prostate Health Index (PHI) was calculated. Clinical and analytical predictive models were created incorporating PHI. RESULTS: Of 172 men, 72 (42%) were diagnosed with PCa, 33 (46%) of whom were found to be with high-grade disease. PHI score was the most predictive of biopsy outcomes in terms of discriminative ability (area under the curve = 0.79), with an added gain in predictive accuracy of 17%. All the models that incorporated PHI worked better in terms of calibration close to 45° on the slope. In the decision curve analysis, at a threshold probability of 40% we could prevent 82 biopsies, missing only 16 tumors and 5 high-grade tumors. CONCLUSIONS: PHI score is a more discriminant biomarker, has superior calibration and superior net benefit, and provides a higher rate of avoided biopsies; thus, it can be useful for aiding in making a more informed decision for each patient.

A DNA hypermethylation profile reveals new potential biomarkers for the evaluation of prognosis in urothelial bladder cancer.

DNA hypermethylation has emerged as a molecular biomarker for the evaluation of cancer diagnosis and prognosis. We define a methylation signature of bladder cancer and evaluate whether this profile assesses prognosis of patients. Genome-wide methylation analysis was performed on 70 tumor and 10 normal bladder samples. Hypermethylation status of 1505 CpGs present in the promoter region of 807 genes was studied. Thirty-three genes were significantly hypermethylated in ≥10% of the tumors. Three clusters of patients were characterized by their DNA methylation profile, one at higher risk of dead of disease (p = 0.0012). Association between cluster distribution and stage (p = 0.02) or grade (p = 0.02) was demonstrated. Hypermethylation of JAK3 and absence of hypermethylation of EYA4, GAT6, and SOX1 were associated with low-grade non-invasive disease. On the other hand, in high-grade invasive disease hypermethylation of CSPG2, HOXA11, HOXA9, HS3ST2, SOX1, and TWIST1 was associated with muscle invasiveness. A panel of hypermethylated genes including APC, CSPG2, EPHA5, EYA4, HOXA9, IPF1, ISL1, JAK3, PITX2, SOX1, and TWIST1 predicted cancer-specific survival and SOX1 (HR = 3.46), PITX2 (HR = 4.17), CSPG2 (HR = 5.35), and JAK3 hypermethylation (HR = 0.19) did so independently. Silencing of genes by hypermethylation is a common event in bladder cancer and could be used to develop diagnostic and prognostic markers. Combined hypermethylation of SOX1, PITX2, or CSPG2 signals patients at higher risk of death from bladder cancer.

Does obesity modify prostate cancer detection in a European cohort?

INTRODUCTION: To investigate prostate-specific antigen (PSA) accuracy and digital rectal examination (DRE) accuracy in detecting prostate cancer according to body mass index (BMI) in Spanish men with an indication of the first prostate biopsy. MATERIAL AND METHODS: We reviewed the clinical and histopathological data of 1,319 patients who underwent transrectal ultrasound-guided prostate needle biopsy. The patients were categorised according to the BMI as follows: <25 kg/m2 (normal weight); 25-29.9 kg/m2 (overweight); and ≥30 kg/m2 (obese). Receiver operator characteristic curves were used to assess PSA accuracy and DRE accuracy by calculating the area under the curve. RESULTS: The obesity rate of the cohort was 14%. PSA accuracy for predicting prostate cancer in each BMI category was 0.52, 0.58 and 0.62, respectively (p = 0.01). After stratification by DRE findings, there was no difference in the performance accuracy of PSA in predicting the presence of cancer across BMI groups in abnormal DRE (p = 0.90). Serum PSA, DRE and BMI were strong predictors of prostate cancer diagnosis (odds ratio 1.07, 2.02 and 1.4, respectively; p <0.001). When the DRE was abnormal, a BMI ≥30 increased the risk of prostate cancer twice. With the addition of BMI to the model, the area under the curve of the combined PSA and DRE for diagnosing prostate cancer improved from 0.60 to 0.63. CONCLUSIONS: The predictive value of PSA in predicting prostate cancer is not poorer in the obese population and the predictive value of an abnormal DRE in cancer detection is significantly modified by the patient's BMI.

Survival predictors in patients with prostate adenocarcinoma with hormonal blockade.

Ki-67 index and clinical-pathological factors such as the Gleason score and the presence of neuroendocrine differentiation have been used for predicting survival in patients with prostate cancer. We examined prostate tissue from 45 patients with advanced prostate cancer who were treated with maximal androgen blockade and analysed their cancer-specific survival (CSS). We assessed the Gleason index, performed an immunohistochemical analysis of Ki-67 (MIB-1) and determined the presence of neuroendocrine differentiation (chromogranin A). A survival study was conducted using Kaplan-Meier curves (log-rank test) and a Cox regression analysis. Twenty-four patients (53.3%) died from the disease, with a mean follow-up of 68.7±7.7 months (56.6% CSS at 5 years and 31.8% at 10 years). In the univariate analysis, survival was associated with an interquartile distribution of Ki-67 (0-5, 6-12%, 13-25%, >25%; log-rank, p=0.01), Gleason 5 (total index 9-10; log-rank, p=0.002) and the presence of metastases during the diagnosis (M1; log-rank, p=0.004) but not to cT category (T3-T4; log-rank, p=0.26) or neuroendocrine differentiation (immunohistochemically positive tumour cell nests; log-rank, p=0.46). The multivariate analysis revealed that a Ki-67 index ≤12% (HR, 0.22; p=0.0009) and the absence of metastases (M0) during diagnosis (HR, 0.17; p=0.0002) were protective factors in this population. In conclusion, Ki-67 proliferation index and the lack of metastases at diagnosis predict CSS in patients with advanced prostate cancer who undergo hormonal blockade. Neuroendocrine differentiation in tumour tissue had no prognostic value in this study.

Anti-proliferative and pro-apoptotic effects of GHRH antagonists in prostate cancer.

Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in the progression of various tumors. In vitro and in vivo studies have demonstrated that GHRH antagonists inhibit the growth of several cancers. GHRH antagonists, JMR-132 and JV-1-38 inhibit the growth of androgen-independent prostate tumors. Here we investigated the involvement of GHRH antagonists in proliferative and apoptotic processes. We used non-tumoral RWPE-1 and tumoral LNCaP and PC3 human prostatic epithelial cells, as well as an experimental model of human tumor PC3 cells. We evaluated the effects of JMR-132 and JV-1-38 antagonists on cell viability and proliferation in the three cell lines by means of MTT and BrdU assays, respectively, as well as on cell cycle and apoptotic process in PC3 cells. The expression levels of PCNA, p53, p21, CD44, Cyclin D1, c-myc, Bax and Bcl2 were determined in both in vivo and in vitro models by means of Western-blot and RT-PCR. GHRH antagonists suppressed cell proliferation and decreased the levels of the proliferation marker, PCNA, in the three cell lines and in PC3 tumor. GHRH antagonists led to an increase of cells in S-phase and a decrease in G1 and G2/M phases, and induced S-phase arrest and increase of apoptotic cells. The effects of GHRH-antagonists on cell cycle could be due to the changes observed in the expression of p21, p53, Bax, Bcl2, CD44, Cyclin D1, c-myc and caspase 3. Present results confirm and extend the role of GHRH antagonists as anti-proliferative and pro-apoptotic molecules in prostate cancer.

A DNA Hypermethylation Profile Independently Predicts Biochemical Recurrence Following Radical Prostatectomy.

PURPOSE: Detection of DNA hypermethylation is emerging as a novel molecular biomarker for different malignancies. We intend to define whether a hypermethylation profile of patients with prostate cancer (PCa) predicts biochemical recurrence (BCR) after radical prostatectomy (RP). MATERIAL AND METHODS: Genome-wide methylation analysis was performed using the GoldenGate Methylation Cancer Panel-I (Illumina, Inc.) on 10 normal prostate tissues and 58 tumor samples from patients treated by RP followed for prostate-specific antigen (PSA) failure (>0.4 ng/ml) and disease progression. Patients were classified on the basis of D'Amico criteria according to clinical staging, PSA at diagnosis and Gleason score after pathologist review. Hypermethylation status of 1505 CpGs present in the promoter region of 807 genes was studied. Hierarchical clustering analysis was performed and relationships with outcome were investigated using log-rank analysis and Cox regression model. RESULTS: We found 28 genes significantly hypermethylated in >20% of the tumors analyzed. Four clusters of patients were characterized by their DNA methylation profile, one at higher risk to develop BCR (p = 0.005). Multivariate analysis revealed patients in this cluster (HR 2.56), and high-risk patients (HR 4.34) according to D'Amico classification were independent predictors of BCR after prostatectomy. From the selected genes MT1A, ALOX12, GSTM2, APC, MYCL2 and RARB hypermethylation predicted BCR and GSTM2 (HR 3.78) and MYCL2 hypermethylation (HR 2.71) did so independently. CONCLUSION: Epigenetic silencing of GSTM2 and MYCL2 comprise novel molecular markers to predict BCR after surgery for medium- and high-risk localized PCa undergoing surgical treatment and hypermethylation of these genes could be incorporated to the clinical and pathological factors defining the patient at higher risk of PSA failure after prostatectomy. The limitation of the study is that no independent validation cohort is analysed.

Development of Castration Resistant Prostate Cancer can be Predicted by a DNA Hypermethylation Profile.

PURPOSE: Detection of DNA hypermethylation has emerged as a novel molecular biomarker for prostate cancer diagnosis and evaluation of prognosis. We sought to define whether a hypermethylation profile of patients with prostate cancer on androgen deprivation would predict castrate resistant prostate cancer. MATERIALS AND METHODS: Genome-wide methylation analysis was performed using a methylation cancer panel in 10 normal prostates and 45 tumor samples from patients placed on androgen deprivation who were followed until castrate resistant disease developed. Castrate resistant disease was defined according to EAU (European Association of Urology) guideline criteria. Two pathologists reviewed the Gleason score, Ki-67 index and neuroendocrine differentiation. Hierarchical clustering analysis was performed and relationships with outcome were investigated by Cox regression and log rank analysis. RESULTS: We found 61 genes that were significantly hypermethylated in greater than 20% of tumors analyzed. Three clusters of patients were characterized by a DNA methylation profile, including 1 at risk for earlier castrate resistant disease (log rank p = 0.019) and specific mortality (log rank p = 0.002). Hypermethylation of ETV1 (HR 3.75) and ZNF215 (HR 2.89) predicted disease progression despite androgen deprivation. Hypermethylation of IRAK3 (HR 13.72), ZNF215 (HR 4.81) and SEPT9 (HR 7.64) were independent markers of prognosis. Prostate specific antigen greater than 25 ng/ml, Gleason pattern 5, Ki-67 index greater than 12% and metastasis at diagnosis also predicted a negative response to androgen deprivation. Study limitations included the retrospective design and limited number of cases. CONCLUSIONS: Epigenetic silencing of the mentioned genes could be novel molecular markers for the prognosis of advanced prostate cancer. It might predict castrate resistance during hormone deprivation and, thus, disease specific mortality. Gene hypermethylation is associated with disease progression in patients who receive hormone therapy. It could serve as a marker of the treatment response.

Evaluation of the prognostic significance of perirenal fat invasion and tumor size in patients with pT1-pT3a localized renal cell carcinoma in a comprehensive multicenter study of the CORONA project. Can we improve prognostic discrimination for patients with stage pT3a tumors?

BACKGROUND: The current TNM system for renal cell carcinoma (RCC) merges perirenal fat invasion (PFI) and renal vein invasion (RVI) as stage pT3a despite limited evidence concerning their prognostic equivalence. In addition, the prognostic value of PFI compared to pT1-pT2 tumors remains controversial. OBJECTIVE: To analyze the prognostic significance of PFI, RVI, and tumor size in pT1-pT3a RCC. DESIGN, SETTING, AND PARTICIPANTS: Data for 7384 pT1a-pT3a RCC patients were pooled from 12 centers. Patients were grouped according to stages and PFI/RVI presence as follows: pT1-2N0M0 (n=6137; 83.1%), pT3aN0M0 + PFI (n=1036; 14%), and pT3aN0M0 (RVI ± PFI; n=211; 2.9%). INTERVENTION: Radical nephrectomy or nephron-sparing surgery (NSS) (1992-2010). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Cancer-specific survival was estimated using the Kaplan-Meier method. Univariate and multivariate Cox proportional-hazards regression models, as well as sensitivity and discrimination analyses, were used to evaluate the impact of clinicopathologic parameters on cancer-specific mortality (CSM). RESULTS AND LIMITATIONS: Compared to stage pT1-2, patients with stage pT3a RCC were significantly more often male (59.4% vs 53.1%) and older (64.9 vs 62.1 yr), more often had clear cell RCC (85.2% vs 77.7%), Fuhrman grade 3-4 (29.4% vs 13.4%), and tumor size >7 cm (39.1% vs 13%), and underwent NSS less often (7.5% vs 36.6%; all p<0.001). According to multivariate analysis, CSM was significantly higher for the PFI and RVI ± PFI groups compared to pT1-2 patients (hazard ratio [HR] 1.94 and 2.12, respectively; p<0.001), whereas patients with PFI only and RVI ± PFI did not differ (HR 1.17; p=0.316). Tumor size instead enhanced CSM by 7% per cm in stage pT3a (HR 1.07; p<0.001) with a 7 cm cutoff yielding the highest prediction accuracy. CONCLUSIONS: Since the prognostic impact of PFI and RVI on CSM seems to be comparable, merging both as stage pT3a RCC might be justified. Enhanced prognostic discrimination of stage pT3a RCC appears to be possible by applying a tumor size cutoff of 7 cm within an alternative staging system. PATIENT SUMMARY: Prognosis prediction for patients with localized renal cell carcinoma up to stage pT3a can be enhanced by including tumor size with a cutoff of 7 cm as an additional parameter in the TNM classification system.

VIP induces NF-κB1-nuclear localisation through different signalling pathways in human tumour and non-tumour prostate cells.

The nuclear factor κB (NF-κB) is a powerful activator of angiogenesis, invasion and metastasis. Transactivation and nuclear localisation of NF-κB is an index of recurrence in prostate cancer. Vasoactive intestinal peptide (VIP) exerts similar effects in prostate cancer models involving increased expression of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) which are related to NF-κB transactivation. Here we studied differential mechanisms of VIP-induced NF-κB transactivation in non-tumour RWPE-1 and tumour LNCaP and PC3 human prostate epithelial cells. Immunofluorescence studies showed that VIP increases translocation of the p50 subunit of NF-κB1 to the nucleus, an effect that was inhibited by curcumin. The signalling transduction pathways involved are different depending on cell transformation degree. In control cells (RWPE1), the effect is mediated by protein kinase A (PKA) activation and does not implicate extracellular signal-regulated kinase (ERK) or phosphoinositide 3-kinase (PI3-K) pathways whereas the opposite is true in tumour LNCaP and PC3 cells. Exchange protein directly activated by cAMP (EPAC) pathway is involved in transformed cells but not in control cells. Curcumin blocks the activating effect of VIP on COX-2 promoter/prostaglandin E2 (PGE2) production and VEGF expression and secretion. The study incorporates direct observation on COX-2 promoter and suggests that VIP effect on VEGF may be indirectly mediated by PGE2 after being synthesised by COX-2, thus amplifying the initial signal. We show that the signalling involved in VIP effects on VEGF is cAMP/PKA in non-tumour cells and cAMP/EPAC/ERK/PI3K in tumour cells which coincides with pathways mediating p50 nuclear translocation. Thus, VIP appears to use different pathways for NF-κB1 (p50) transactivation in prostate epithelial cells depending on whether they are transformed or not. Transformed cells depend on pro-survival and pro-proliferative signalling pathways involving ERK, PI3-K and cAMP/EPAC which supports the potential therapeutic value of these targets in prostate cancer.

Results of a comparative study analyzing octogenarians with renal cell carcinoma in a competing risk analysis with patients in the seventh decade of life.

OBJECTIVES: To analyze clinicopathological features and survival of surgically treated patients with renal cell carcinoma (RCC) ≥ 80 years of age in comparison with patients between the ages of 60 and 70 years. MATERIALS AND METHODS: The data for 2,516 patients with a median follow-up of 57 months were retrieved from a multinational database (Collaborative Research on Renal Neoplasms Association [CORONA]), including data for 6,234 consecutive patients with RCC after radical or partial nephrectomy. Comparative analysis of clinicopathological features of 241 octogenarians (3.9% of the database) and 2,275 reference patients between the ages of 60 and 70 years (36.5%) was performed. Multivariable regression analysis adjusted for competing risks was applied to identify the effect of advanced age on cancer-specific mortality (CSM) and other-cause mortality (OCM). Furthermore, instrumental variable analysis was employed to reduce residual confounding by unmeasured parameters. RESULTS: Significantly more women were present (50% vs. 40%, P = 0.004), and significantly less often nephron-sparing surgery was performed in octogenarians compared with the reference group (11% vs. 20%, P<0.001). Although median tumor size and stages did not significantly defer, older patients less often had advanced or metastatic disease (N+/M1) (4.6% vs. 9.6%, P = 0.009). On multivariable analysis, higher CSM (hazard ratio = 1.48, P = 0.042) and OCM rates (hazard ratio = 4.32, P<0.001) were detectable in octogenarians (c-indices = 0.85 and 0.72, respectively). Integration of the variable age group in multivariable models significantly increased the predictive accuracy regarding OCM (6%, P<0.001), but not for CSM. Limitations are based on the retrospective study design. CONCLUSIONS: Octogenarian patients with RCC significantly differ in clinical features and display significantly higher CSM and OCM rates in comparison with their younger counterparts.

Signalling pathways involved in antitumoral effects of VIP in human renal cell carcinoma A498 cells: VIP induction of p53 expression.

Vasoactive intestinal peptide (VIP) decreases cell proliferation through PI3K signalling and prevents tumour progression in clear renal cell carcinoma (RCC). Here we analyzed the signalling pathways that mediate such VIP effects by using human RCC A498 cells. The effects of treatment with 1 µM VIP and/or specific protein kinase inhibitors such as H89, Wortmannin and PD98059 were studied by cell adhesion assay, ELISA of VEGF165 and ROS production assays. Semiquantitative RT-PCR and western blot were performed to study p53 expression. VIP increased cell adhesion and ROS production, and decreased VEGF165 secretion through PI3K signalling. Moreover, VIP increased nuclear expression of tumour suppressor p53. VIP effects could be blocked by cell incubation with a specific p53 inhibitor, cyclin pifithrin-α hydrobromide (CPFT-αH). In conclusion, this study provides a p53-dependent mechanism by which VIP regulates cell proliferation in RCC development. It supports a potential usefulness of VIP in new therapies of RCC.

A DNA hypermethylation profile reveals new potential biomarkers for prostate cancer diagnosis and prognosis.

BACKGROUND: DNA hypermethylation has emerged as a novel molecular biomarker for the evaluation of prostate cancer diagnosis and prognosis. Defining the specific gene hypermethylation profile for prostate cancer could involve groups of genes that specifically discriminate patients with indolent and aggressive tumors. METHODS: Genome-wide methylation analysis was performed on 83 tumor and 10 normal prostate samples using the GoldenGate Methylation Cancer Panel I (Illumina, Inc.). All clinical stages of disease were considered. RESULTS: We found 41 genes hypermethylated in more than 20% of the tumors analyzed (P < 0.01). Of these, we newly identified GSTM2 and PENK as being genes that are hypermethylated in prostate cancer and that were simultaneously methylated in 40.9% of the tumors analyzed. We also identified panels of genes that are more frequently methylated in tumor samples with clinico-pathological indicators of poor prognosis: a high Gleason score, elevated Ki-67, and advanced disease. Of these, we found simultaneous hypermethylation of CFTR and HTR1B to be common in patients with a high Gleason score and high Ki-67 levels; this might indicate the population at higher risk of therapeutic failure. The DNA hypermethylation profile was associated with cancer-specific mortality (log-rank test, P = 0.007) and biochemical recurrence-free survival (log-rank test, P = 0.0008). CONCLUSIONS: Our findings strongly indicate that epigenetic silencing of GSTM2 and PENK is a common event in prostate cancer that could be used as a molecular marker for prostate cancer diagnosis. In addition, simultaneous HTR1B and CFTR hypermethylation could help discriminate aggressive from indolent prostate tumors.

Do young patients with renal cell carcinoma feature a distinct outcome after surgery? A comparative analysis of patient age based on the multinational CORONA database.

PURPOSE: We analyzed the distinct clinicopathological features and prognosis of patients with renal cell carcinoma age 40 years or less compared to a reference group of patients 60 to 70 years old. MATERIALS AND METHODS: Overall 2,572 patients retrieved from a multicenter international database comprised of 6,234 patients with surgically treated renal cell carcinoma were included in this retrospective study. Clinical and histopathological features of 297 patients 40 years old or younger (4.8%) were compared to those of 2,275 patients (36.5%) 60 to 70 years old, who served as the reference group. Median followup was 59 months. The impact of young age and further parameters on disease specific mortality and all cause mortality was evaluated by multivariate Cox proportional hazards regression analyses. RESULTS: Young patients more frequently underwent nephron sparing surgery (27% vs 20%, p = 0.008) and regional lymph node dissection compared to older patients (38% vs 32%, p = 0.025). Organ confined tumor stage (81% vs 70%, p <0.001), smaller tumor diameter (4.5 vs 4.7 cm, p = 0.014) and chromophobe subtype (10% vs 4%, p <0.001) were significantly more frequent in young patients. On multivariate analysis older patients had a higher disease specific (HR 2.21, p <0.001) and all cause mortality (HR 3.05, p <0.001). The c indices for the Cox models were 0.87 and 0.78, respectively. However, integration of the variable age group did not significantly increase the predictive accuracy of the disease specific and all cause mortality models. CONCLUSIONS: Young patients with renal cell carcinoma (40 years old or younger) have significantly different frequencies of clinical and histopathological features, and a significantly lower all cause and disease specific mortality compared to patients 60 to 70 years old.

Clinical significance of both tumor and stromal expression of components of the IL-1 and TNF-α signaling pathways in prostate cancer.

IL-1 and TNF-α, the two major proinflammatory cytokines, have been involved in initiation and progression of several malignancies. They could influence the biological behavior of prostatic tumors and patient outcome, and could be useful as prognostic factors. This study evaluated the prognostic capability for biochemical progression after radical prostatectomy of expression of IL-1, TNF-α and related signaling components, in the tumor and surrounding stroma, as well as its correlation with other clinicopathological features. Expression of IL-1α, IL-1ß, IL-1Ra, IL-1RI, IL-1RII, IRAK-1, TRAF6, TNF-α, TNFRI and TRAF2 was analyzed by immunohistochemistry in radical prostatectomy samples from 93 prostate cancer patients. Spearman's test, Kaplan-Meier curves, and univariate and multivariate Cox proportional hazard regression analyses were performed. Expression of TNF-α, TNFRI, TRAF2, ILRI, IRAK-1 and TRAF6 correlated with at least one clinicopathological feature (clinical T stage, pathological T stage, preoperative serum PSA or Gleason score). Increased tumor expression of TNF-α, TNFRI and IL-1RI, and reduced tumor expression of IRAK-1 were significantly correlated with a poor prognosis in univariate analysis. Reduced stromal expression of IL-1ß and IL-1RII, and increased stromal expression of IRAK-1 were also adverse prognostic factors in univariate analysis. Remarkably, tumor IL-1ß and stromal IL-1RII and IRAK-1 remained as independent prognostic factors after adjustment for preoperative serum PSA, pathological T stage and Gleason score in multivariate Cox models. Our results suggest that prostatic expression of TNF-α, IL-1ß and related signaling proteins (TNFRI, IL-1RI, IL-1RII and IRAK-1) predicts clinical outcome in prostate cancer, and support the involvement of TNF-α and IL-1ß signaling in prostate cancer progression.

Antitumoral effects of vasoactive intestinal peptide in human renal cell carcinoma xenografts in athymic nude mice.

We studied antitumor effect of VIP in human renal cell carcinoma (RCC) (A498 cells xenografted in immunosuppressed mice). VIP-treated cells gave resulted in p53 upregulation and decreased nuclear ß-catenin translocation and NFκB expression, MMP-2 and MMP-9 activities, VEGF levels and CD-34 expression. VIP led to a more differentiated tubular organization in tumours and less metastatic areas. Thus, VIP inhibits growth of A498-cell tumours acting on the major issues involved in RCC progression such as cell proliferation, microenvironment remodelling, tumour invasion, angiogenesis and metastatic ability. These antitumoral effects of VIP offer new therapeutical possibilities in RCC treatment.