Analysis of AR-FL and AR-V1 in Whole Blood of Patients with Castration Resistant Prostate Cancer as a Tool for Predicting Response to Abiraterone Acetate.

PURPOSE: Reliable molecular diagnostic tools are still unavailable for making informed treatment decisions and monitoring the response in patients with castration resistant prostate cancer. We evaluated the significance of whole blood circulating androgen receptor transcripts of full length (AR-FL) and splice variants (AR-V1, AR-V3 and AR-V7) as biomarkers of abiraterone acetate treatment resistance in patients with castration resistant prostate cancer. MATERIALS AND METHODS: After retrospective analysis in 112 prostate specimens AR-FL, AR-V1, AR-V3 and AR-V7 were evaluated in 185 serial blood samples, prospectively collected from 102 patients with castration resistant prostate cancer before and during abiraterone acetate therapy via reverse transcription quantitative polymerase chain reaction. RESULTS: AR-FL was present in all samples while AR-V1, AR-V3, AR-V7 and at least 1 of them was detected in 17%, 55%, 65% and 81% of castration resistant prostate cancer blood samples, respectively. The highest amount of AR-V1 was found in blood of patients whose response time was short and medium in comparison to extended. Patients with a higher level of AR-FL and/or AR-V1 had the shortest progression-free survival and overall survival (p <0.0001). CONCLUSIONS: Blood circulating AR-FL or AR-V1 can serve as blood based biomarkers for identification of the primary resistance to abiraterone acetate and the tool to monitor de novo resistance development during abiraterone acetate treatment.

Frequent DNA methylation changes in cancerous and noncancerous lung tissues from smokers with non-small cell lung cancer.

Cancer deaths account for nearly 10 million deaths worldwide each year, with lung cancer (LCa) as the leading cause of cancer-related death. Smoking is one of the major LCa risk factors, and tobacco-related carcinogens are potent mutagens and epi-mutagens. In the present study, we aimed to analyse smoking-related epigenetic changes in lung tissues from LCa cases. The study cohort consisted of paired LCa and noncancerous lung tissues (NLT) from 104 patients, 90 of whom were smokers or ex-smokers (i.e. ever smokers) at the time of diagnosis. DNA methylation status of tumour suppressor genes DAPK1, MGMT, p16, RASSF1 and RARB was screened by means of methylation-specific PCR (MSP) and further analysed quantitatively by pyrosequencing. Methylation of at least one gene was detected in 59% (61 of 104) of LCa samples and in 39% (41 of 104) of NLT. DAPK1 and RASSF1 were more frequently methylated in LCa than in NLT (P = 0.022 and P = 0.041, respectively). The levels of DNA methylation were higher in LCa than NLT at most of the analysed CpG positions. More frequent methylation of at least one gene was observed in LCa samples of ever smokers (63%, 57 of 90) as compared with never smokers (36%, 5 of 14; P = 0.019). In the ever smokers group, methylation of the genes also occurred in NLT, but was rare or absent in the samples of never smokers. Among the current smokers, RASSF1 methylation in LCa showed association with the number of cigarettes smoked per day (P = 0.017), whereas in NLT it was positively associated with the duration of smoking (P = 0.039). Similarly, p16 methylation in LCa of current smokers correlated with the larger number of cigarettes smoked per day (P = 0.047). Overall, DNA methylation changes were present in both cancerous and noncancerous tissues of LCa patients and showed associations with smoking-related parameters.

Asbestos-associated genome-wide DNA methylation changes in lung cancer.

Previous studies have revealed a robust association between exposure to asbestos and human lung cancer. Accumulating evidence has highlighted the role of epigenome deregulation in the mechanism of carcinogen-induced malignancies. We examined the impact of asbestos on DNA methylation. Our genome-wide studies (using Illumina HumanMethylation450K BeadChip) of lung cancer tissue and paired normal lung from 28 asbestos-exposed or non-exposed patients, mostly smokers, revealed distinctive DNA methylation changes. We identified a number of differentially methylated regions (DMR) and differentially variable, differentially methylated CpGs (DVMC), with individual CpGs further validated by pyrosequencing in an independent series of 91 non-small cell lung cancer and paired normal lung. We discovered and validated BEND4, ZSCAN31 and GPR135 as significantly hypermethylated in lung cancer. DMRs in genes such as RARB (FDR 1.1 × 10-19 , mean change in beta [Δ] -0.09), GPR135 (FDR 1.87 × 10-8 , mean Δ -0.09) and TPO (FDR 8.58 × 10-5 , mean Δ -0.11), and DVMCs in NPTN, NRG2, GLT25D2 and TRPC3 (all with p <0.05, t-test) were significantly associated with asbestos exposure status in exposed versus non-exposed lung tumors. Hypomethylation was characteristic to DVMCs in lung cancer tissue from asbestos-exposed subjects. When DVMCs related to asbestos or smoking were analyzed, 96% of the elements were unique to either of the exposures, consistent with the concept that the methylation changes in tumors may be specific for risk factors. In conclusion, we identified novel DNA methylation changes associated with lung tumors and asbestos exposure, suggesting that changes may be present in causal pathway from asbestos exposure to lung cancer.

The utility of urine-circulating miRNAs for detection of prostate cancer.

BACKGROUND: In this paper, the utility of urine-circulating microRNAs (miRNAs) as the potential biomarker of prostate cancer (PCa), the second most prevalent male cancer worldwide, was evaluated. METHODS: Cancerous (N=56) and non-cancerous (N=16) prostate tissues were analysed on TaqMan Low Density Array, with the initial screening of 754 miRNAs in a subset of the samples. The abundance of selected miRNAs was analysed in urine specimens from two independent cohorts of patients with PCa (N=215 overall), benign prostatic hyperplasia (BPH; N=23), and asymptomatic controls (ASC; N=62) by means of quantitative reverse transcription PCR. RESULTS: Over 100 miRNAs were found deregulated in PCa as compared with non-cancerous prostate tissue. After thorough validation, four miRNAs were selected for the analysis in urine specimens. The abundance of miR-148a and miR-375 in urine was identified as specific biomarkers of PCa in both cohorts. Combined analysis of urine-circulating miR-148a and miR-375 was highly sensitive and specific for PCa in both cohorts (AUC=0.79 and 0.84) and strongly improved the diagnostic power of the PSA test (AUC=0.85, cohort PCa1), including the grey diagnostic zone (AUC=0.90). CONCLUSIONS: Quantitative measurement of urine-circulating miR-148a and miR-375 can serve as the non-invasive tool for sensitive and specific detection of PCa.

Detection of miRNAs in urine of prostate cancer patients.

BACKGROUND AND AIM: Prostate cancer (PCa) is the second most prevalent oncologic disease among men worldwide. Expression of various transcripts, including miRNAs, is markedly deregulated in cancerous prostate tissue. This study aimed at identifying a PCa-specific expression profile of miRNAs for subsequent use in noninvasive diagnostics. MATERIALS AND METHODS: MiRNA expression was profiled in 13 PCa tissues using human miRNA microarrays. Highly expressed miRNAs were selected for the analysis in urine of patients with PCa (N=143) and benign prostate hyperplasia (BPH; N=23) by means of real time PCR, while miRNAs showing the expression differences in relation to clinical variables were further analyzed in 52 PCa and 12 noncancerous prostate tissues (NPT) on TaqMan Low Density Arrays (TLDA). RESULTS: Analysis of miRNA expression in prostate tissue linked miR-95 to aggressive form of PCa. This miRNA was up-regulated in high grade (P=0.041), the TMPRSS2-ERG fusion-positive tumors (P=0.026), and in patients with subsequently developed biochemical recurrence (BCR; P=0.054) after radical prostatectomy. MiRNAs highly expressed in PCa tissues were also detectable in urine from PCa patients. Moreover, the urinary levels of miR-21 had significant discriminatory power (P=0.010) to separate PCa patients from BPH, while the combined analysis of urinary miR-19a and miR-19b was prognostic for BCR. In PCa, the diagnostic potential of urinary miRNA panel (miR-21, miR-19a, and miR-19b) was higher than that of the PSA test (AUC=0.738 vs. AUC=0.514). CONCLUSIONS: Measurement of urinary levels of PCa-specific miRNAs could assist in more specific detection of PCa and prediction of BCR.

High-resolution melting-based quantitative analysis of RASSF1 methylation in breast cancer.

BACKGROUND AND OBJECTIVE: Breast cancer is the leading cause of death from cancer among women worldwide. The aberrant promoter methylation of tumor suppressor genes is a typical epigenetic alteration for breast cancer and can be detected in early carcinogenesis. High-throughput and cost-effective methods are needed for the early and sensitive detection of epigenetic changes in clinical material. The main purpose of our study was to optimize a high-resolution melting (HRM) assay for the reliable and quantitative assessment of RASSF1 gene methylation, which is considered one of the earliest epigenetic alterations in breast cancer. MATERIAL AND METHODS: A total of 76 breast carcinomas and 10 noncancerous breast tissues were studied by means of HRM and compared with the results obtained by means of quantitative methylation-specific polymerase chain reaction (QMSP) and methylation-specific polymerase chain reaction (MSP). RESULTS: Both quantitative methods, HRM and QMSP, showed a similar specificity and sensitivity for the detection of RASSF1 methylation in breast cancer (about 80% and 70%, respectively). In breast cancer, the mean methylation intensity of RASSF1 was 42.5% and 48.6% according to HRM and QMSP, respectively. Both methods detected low levels of methylation (less than 5%) in noncancerous breast tissues. In comparison with quantitative methods, MSP showed a lower sensitivity (70%), but a higher specificity (80%) for the detection of RASSF1 methylation in breast cancer. CONCLUSIONS: HRM is as a simple, cost-effective method for the reliable high-throughput quantification of DNA methylation in clinical material.

Investigating the relationship between the severity of periodontitis and rheumatoid arthritis: a cross-sectional study.

OBJECTIVES: This paper evaluates the prevalence and severity of periodontitis (PD) in patients with rheumatoid arthritis (RA), focusing on the link between the severity of PD with RA disease activity/disability scores, the influence of RA treatment on PD, and levels of vitamin D. METHODS: A total of 93 RA patients were enrolled in the cross-sectional study and analyzed accordingly as RA-PD (N = 63, 67.8%) and RA-only (N = 30, 32.2%) groups. A number of associations between rheumatological clinical data, i.e., Disease Activity Score (DAS28 CRP), health assessment questionnaires, and PD severity (measured by periodontal outcome parameters) with regard to serum levels of vitamin D were assessed. The outcome variables were compared by parametric and non-parametric tests. RESULTS: A total of 29% of RA patients were diagnosed with severe PD. The RA-PD group presented a higher mean DAS28 CRP score in moderate-severe PD compared to periodontally healthy-initial stage PD subjects (4.49 ± 1.22 vs. 3.86 ± 1.58, p = 0.033). RA patients treated with biologic disease-modifying antirheumatic drugs (bDMARDs) were less likely to be diagnosed with PD (p = 0.022) and revealed significantly lower PD outcome parameters, i.e., bleeding on probing (%) and bone loss (%) (p < 0.05). Vitamin D concentration was significantly lower in RA-PD group with diagnosed advanced severe PD (IV stage) compared to moderate PD (II stage) (39.61 ± 17.12 vs. 52.07 ± 18.23 nmol/l, p = 0.031). CONCLUSIONS: The study revealed a high prevalence of severe PD in RA patients, being significantly associated with higher RA disease activity and lower vitamin D level in RA-PD group, while bDMARD treatment was related to lower PD outcome parameters. Key Points • Severe PD is prevalent amongst RA patients and is associated with RA disease activity. The higher RA DAS28 CRP score is associated with moderate-severe PD compared to periodontally healthy-initial stage PD in RA patients. • Biologic DMARDs treatment used for RA is linked to lower PD rates and PD outcome parameters. • Significantly lower vitamin D level is found in advanced severe PD compared to moderate PD stage in RA-PD subjects.

Associations of periodontal status in periodontitis and rheumatoid arthritis patients.

PURPOSE: The aim of this study was to assess the association between the clinical status of rheumatoid arthritis (RA) and periodontitis (PD) in patients diagnosed with PD and to evaluate the impact of RA treatment on the severity of PD. METHODS: The study included 148 participants with PD, of whom 64 were also diagnosed with RA (PD+RA group), while 84 age-matched participants were rheumatologically healthy (PD-only group). PD severity was assessed by the following periodontal parameters: clinical attachment loss, probing pocket depth (PPD), bleeding on probing (BOP), alveolar bone loss, and number of missing teeth. RA disease characteristics and impact of disease were evaluated by the Disease Activity Score 28 using C-reactive protein, disease duration, RA treatment, the RA Impact of Disease tool, and the Health Assessment Questionnaire. Outcome variables were compared using parametric and non-parametric tests and associations were evaluated using regression analysis with the calculation of odds ratios (ORs). RESULTS: Participants in the PD+RA group had higher mean PPD values (2.81 ± 0.59 mm vs. 2.58 ± 0.49 mm, P=0.009) and number of missing teeth (6.27±4.79 vs. 3.93±4.08, P=0.001) than those in the PD-only group. A significant association was found between mean PPD and RA (OR, 2.22; 95% CI, 1.16-4.31; P=0.016). Within the PD+RA group, moderate to severe periodontal disease was significantly more prevalent among participants with higher RA disease activity (P=0.042). The use of biologic disease-modifying antirheumatic drugs (bDMARDs) was associated with a lower BOP percentage (P=0.016). CONCLUSIONS: In patients with PD, RA was associated with a higher mean PPD and number of missing teeth. The severity of PD was affected by the RA disease clinical activity and by treatment with bDMARDs, which were associated with a significantly lower mean BOP percentage.

MiRNA profiling of gastrointestinal stromal tumors by next-generation sequencing.

Deregulation of miRNAs has been observed virtually in all major types of cancer, whereas the miRNA signature in GIST is not well characterized yet. In this study the first high-throughput miRNA profiling of 15 paired GIST and adjacent normal tissue samples was performed using small RNA-seq approach and differentially expressed miRNAs as well as isomiRNAs were defined. Highly significantly deregulated miRNAs were selected for validation by Taq-Man low-density array in replication group of 40 paired samples. Validated miRNAs were further subjected to enrichment analysis, which revealed significantly enriched KEGG pathways in the main GIST associated pathways. Further, we used an integrated analysis of miRNA-mRNA correlations for KIT and PDGFRA target genes and found a significant correlation between all of the enriched miRNAs and their target gene KIT. Results of the phenotype analysis showed miR-509-3p to be up-regulated in epithelioid and mixed cell types compared to spindle type, whereas miR-215-5p showed negative correlation with risk grade of GIST. These data reveal a detailed miRNA profile of GIST and highlight new candidates that may be important in the development of malignant disease.