A sensitive assay to measure biomarker glycosylation demonstrates increased fucosylation of prostate specific antigen (PSA) in patients with prostate cancer compared with benign prostatic hyperplasia

BackgroundProstate specific antigen (PSA) measurement is used for the diagnosis of prostate cancer (PCa) but the test lacks specificity due to the number of false positive readings. The glycosylation of PSA is altered in PCa but studies in this area have been limited to few clinical samples and/or require advanced laboratory facilities. An assay to assess PSA glycosylation was established using equipment available in most routine biomedical testing laboratories.MethodsSerum samples from patients with PCa or benign prostatic hyperplasia (BPH) were used. PSA (range 4–10 ng/ml) was affinity purified, separated and probed with the lectin Ulex europaeus (UEA-1; specific for α1,2 linked fucose). An enzyme-linked immunosorbent lectin assay (ELLA) with colorimetric detection was devised and PSA fucosylation assessed in a further independent set of 26 samples.ResultsFree PSA (fPSA) from PCa patients showed a significant increase in fucosylation compared with fPSA from patients with BPH. The ELLA was 92% specific and 69% sensitive for PCa over BPH. In comparison, fPSA measurement was 70% specific and 56% sensitive (threshold set to 25% tPSA) for PCa over BPH.ConclusionsChanges in glycosylation of PSA were identified using 50 μl of serum with PSA in the range of 4–10 ng/ml, this represents a more specific and sensitive test for PCa based on fucosylation changes of fPSA.     

Prognostic value of miR-26a and HMGA1 in urothelial bladder cancer

BackgroundMicroRNA-26a (miR-26a) functions as a tumor suppressor by regulating its direct target gene high mobility group AT-hook 1 (HMGA1). This study was aimed to investigate the associations of differential expression of miR-26a and HMGA1 with tumor progression and prognosis in urothelial bladder cancer (UBC) patients.Materials and methodsOne hundred and twenty-six UBC patients were selected and quantitative real-time PCR was performed to detect the expression of miR-26a and HMGA1 mRNA in the respective tumors.ResultsOur data showed the decreased expression of miR-26a and the increased expression of HMGA1 mRNA in UBC tissues compared with corresponding non-cancerous tissues (both P < 0.001). Then, the expression levels of miR-26a in UBC tissues were negatively correlated with those of HMGA1 mRNA significantly (r = –0.72, P < 0.001). In addition, UBC patients with combined miR-26a downregulation and HMGA1 upregulation (miR-26a-low/HMGA1-high) more frequently had advanced pathological stage (P < 0.001) and high tumor grade (P < 0.001). Moreover, miR-26a-low/HMGA1-high expression was associated with a significantly shortest disease-free survival (P < 0.001) and overall survival (P < 0.001) of all miR-26a/HMGA1 combined expression groups. Furthermore, multivariate analysis indicated that miR-26a/HMGA1 expression was an independent prognostic factor for both disease-free survival and overall survival (both P = 0.001) in UBC patients.ConclusionInteraction between miR-26a and its target gene HMGA1 may contribute to the malignant progression of human UBC. Tumors with miR-26a downregulation in combination with high expression of HMGA1 showed a worse prognosis than the other tumors. Combined detection of their expression might be particularly helpful for surveillance of disease progression and treatment stratification.     

Individual and cumulative association of prostate cancer susceptibility variants with clinicopathologic characteristics of the disease

BackgroundRecent genome-wide association studies have identified several independent single nucleotide polymorphisms (SNPs) strongly associated with prostate cancer (PCa) risk. However, their individual and cumulative associations with clinicopathologic characteristics of the disease remain inconclusive.MethodsWe systematically evaluated 20 PCa risk SNPs in a cohort of 320 localized PCa patients receiving radical prostatectomy, and the associations of these variants with age at diagnosis, preoperative prostate-specific antigen concentration, Gleason score, pathologic stage, surgical margin, and lymph node metastasis were evaluated.ResultsEight SNPs, rs10486567 at 7p15, rs6465657 at 7q21, rs6983267, rs1447295, and rs4242382 at 8q24, 10993994 at 10q11, rs4430796 at 17q12, and rs266849 at 19q13, were significantly (P ≤ 0.048) associated with some specific clinicopathologic features. In combination of these 8 SNPs, men who carried 4 or 5, or more than 5 unfavorable alleles had an increasing likelihood of adverse clinicopathologic features, as compared with men who carried fewer than 4 unfavorable alleles (P for trend, 0.031, < 0.001, 0.006, and 0.003, for Gleason scores 8–10, advanced pathologic stage, positive surgical margin, and lymph node metastasis, respectively).ConclusionsOur results suggested that loci associated with PCa risk might also have a cumulative and significant association with disease aggressiveness.     

Serum testosterone improves the accuracy of Prostate Health Index for the detection of prostate cancer

ObjectiveProstate cancer (PCa) detection suffers from low specificity when using the prostate-specific antigen (PSA) alone. The aim of this study was to investigate the applicability of total testosterone (tT), free testosterone (fT), the fraction (%) of fT to tT (%fT), and bioavailable testosterone (bioT) in serum to improve the diagnostic validity of the serum (− 2)pro-PSA-based Prostate Health Index (PHI).Design and methodsTotal and free PSA (tPSA, fPSA), (− 2)pro-PSA, testosterone, and sex-hormone-binding globulin were measured by automated immunoassays from serum of 193 men scheduled for prostate biopsy (99 PCa, 94 without PCa). fT and bioT were calculated using an online calculator. Statistical analyses were performed by non-parametric tests (Wilcoxon signed rank, Mann–Whitney, Kruskal–Wallis), binary logistic regression, and receiver operating characteristic (ROC) analyses.ResultsCompared with the non-malignant controls, PCa patients had significantly higher tPSA concentrations and PHI values, but lower %fPSA values and lower concentrations of tT, fT, and bioT. PCa could be differentiated from controls by PHI, tT, fT, bioT, and %fPSA. PHI showed the largest area under the ROC curve (AUC = 0.73) that was increased further by the inclusion of bioT or tT in a binary logistic regression model. The AUC of PHI in patients with tT concentrations of < 8 nmol/L (indicating biochemical hypogonadism) was significantly larger than that in patients with higher tT values (0.86 vs. 0.70; P = 0.024).ConclusionsThe PHI-based discrimination between PCa patients and non-malignant controls could be improved by the simultaneous determination of testosterone. Patients with testosterone concentrations of < 8 nmol/L have the greatest benefit.     

Post-treatment circulating plasma BMP6 mRNA and H3K27 methylation levels discriminate metastatic prostate cancer from localized disease

BackgroundWe evaluated the utility of post-treatment plasma levels of the circulating bone-morphogenetic protein-6-specific mRNA (cBMP6 mRNA), cell-free DNA (cf-DNA), apoptotic nucleosomes and Histone H3 lysine 27 trimethylation (H3K27me3), in discriminating metastatic prostate cancer (PCa) from organ confined, locally controlled disease.MethodsPeripheral blood was taken from the patients at the end of therapy, and quantitative PCR was performed to amplify cBMP6 mRNA or cf-DNA from plasma while apoptotic nucleosomes and H3K27me3 were determined by ELISA-based approaches. Following blinded measurements, the markers were compared between the patients with local (n = 22), local advanced (n = 11) or metastatic disease (n = 28).ResultsOf the four markers investigated, the cBMP6 mRNA and H3K27me3 levels revealed significant differences between the three subgroups. We found higher levels of cBMP6 mRNA in the patients with metastases than in those with localized (p = 0.001) or local advanced disease (p = 0.05). When compared to cBMP6, H3K27me3 displayed an inverse distribution and was significantly lower in the patients with metastatic disease than in those with localized (p = 0.05) or local advanced disease (p = 0.024). There was no correlation between the different markers and total PSA levels or Gleason score at diagnosis.ConclusionOur study provides evidence that post-treatment analysis of cBMP6 mRNA and H3K27me3 may be used to distinguish metastatic PCa from organ confined, locally controlled disease.     

A prospective study investigating the impact of multiparametric MRI in biopsy-naïve patients with clinically suspected prostate cancer: The PROKOMB study

BackgroundIn patients with suspected prostate cancer (PCa) according to current guidelines systematic transrectal ultrasound (TRUS)-guided biopsy of the prostate is performed to verify or rule out PCa. However, TRUS-guided biopsy can result in underdetection of clinically significant cancers as well as diagnosis of clinically insignificant cancers. Multiparametric MRI (mpMRI) might improve the diagnostic pathway and help to avoid unnecessary biopsies.Design and methodsThe PROKOMB (Prostata – Kooperatives MRT-Projekt Berlin) study is a prospective two-arm multicentre study designed to evaluate the potential role of mpMRI as a triage test before biopsy. Up to 600 biopsy-naïve men with suspicion for PCa undergo mpMRI at two dedicated imaging centers. Only patients with equivocal or suspicious lesions on mpMRI undergo prostate biopsy including systematic as well as MRI-guided targeted biopsies at several different community-based urologists or hospitals. The PROKOMB study is designed to evaluate how many biopsies can be avoided, how many clinically insignificant cancers are diagnosed on prostate biopsy in patients with positive findings on mpMRI, and how many clinically significant cancers are missed using this alternative diagnostic pathway. For the purpose of this study clinically significant PCa is defined as Gleason ≥ 3 + 4 cancer. In addition, the detection rates of different techniques for MRI-guided biopsy are evaluated as well as psychological distress before mpMRI and after the diagnosis of PCa.ConclusionThe PROKOMB study might help in defining the role of mpMRI in biopsy-naïve patients with suspected PCa in an ambulatory care setting.     

Expression of STK15 mRNA in hepatocellular carcinoma and its prognostic significance

ObjectivesTo investigate whether the STK15 mRNA expression correlates with clinicopathologic features and the prognosis of HCC patients.Design and methodsThree hepatoma cell lines, two normal liver epithelial cell lines, hepatoma tissues, adjacent tumor tissues and normal liver tissues were obtained from 46 HCC patients. Semi-quantitative RT-PCR assays were performed to detect the expression of STK15 mRNA in above cell lines and tissues. Moreover, the expression of STK15 protein in hepatoma tissues, adjacent tumor tissues and normal liver tissues was also examined by immunohistochemical staining. Finally, correlations between STK15 mRNA expression and the clinicopathological features and prognosis of HCC patients were evaluated.ResultsSTK15 mRNA showed higher levels in hepatoma cell lines than in normal liver epithelial cell lines. Moreover, the mean levels of STK15 mRNA and protein expression showed statistical difference between tumor tissues, tumor adjacent tissues and normal liver tissues (P < 0.01). By immunohistochemical analysis, we found that paraffin-embedded archival HCC tissues showed higher expression of STK15 than adjacent tumors and normal liver tissues. Furthermore, HCC patients with higher STK15 mRNA expression showed poorer prognosis than those with lower STK15 mRNA expression. The high level of STK15 mRNA expression was significantly correlated with tumor stage (P = 0.0081), more frequent lymph node (P = 0.0380) or hematogenous metastasis (P = 0.0066), and a higher incidence of cancer-related death (P = 0.0083). Furthermore, the disease-free survival (DFS) and overall survival (OS) rates of HCC patients with higher STK15 mRNA expression group (47.6% and 52.7%) were significantly lower than those of patients with low STK15 mRNA expression group (56.9% and 68.8%, P = 0.0018 and 0.0047).ConclusionsSTK15 mRNA might be a good marker for predicting the prognosis of HCC patients.     

Prostate cancer biomarker annexin A3 detected in urines obtained following digital rectal examination presents antigenic variability

ObjectivesAnnexin A3 (ANXA3) is a potential marker for prostate cancer (PCa). We aimed to develop robust immunoassays suitable for quantifying ANXA3 in urine samples obtained following digital rectal examination (DRE) in order to facilitate the diagnostic performance evaluation of this marker.Design and methodsAnti-ANXA3 monoclonal antibodies were generated and their epitopes mapped. Two different ANXA3 assay prototypes were established on the VIDAS® automated immunoanalyser and analytical validation was carried out using post-DRE urine samples obtained from patients with PCa (n = 23) or benign prostate hyperplasia (n = 31).ResultsThe assays had the same capture antibody (TGC44) but different detection antibodies (13A12 or 5C5), recognizing novel distinct epitopes. Both had a lower limit of quantification < 1 ng/mL and were highly specific for ANXA3, not cross-reacting with other annexins. Interassay imprecision was ≤ 11% and ≤ 15% for 13A12 and 5C5 assays, respectively. Surprisingly, a total lack of correlation was observed between ANXA3 levels measured by these two assays in post-DRE urines, indicating detection of distinct antigenic variants. Two freeze–thaw cycles did not affect analyte stability in either assay, whereas a lack of stability of antigenic variants was observed when samples were stored at − 80 °C for 1 month.ConclusionsTwo different antigenic variants of ANXA3 are present in post-DRE urines and their clinical significance for diagnosis of prostate cancer should be further investigated. These variants are not stable over time in samples preserved at − 80 °C. Until this issue is resolved, ANXA3 should only be measured in freshly collected samples.     

Up-regulated microRNA-155 expression is associated with poor prognosis in cervical cancer patients

BackgroundMicroRNAs (miRNAs) play important roles in tumor development and progression. The purposes of the study was to investigate the role of miR-155 in cervical cancer.MethodsQuantitative real-time RT-PCR (qRT-PCR) was performed to examine miR-155 expression in cervical cancer tissues and adjacent non-cancerous tissues. The association with overall survival of patients was analyzed by Kaplan-Meier survival analysis. Small interfering RNA (siRNA) was used to suppress miR-155 expression in cervical cancer cells. In vitro assays were performed to further explore the biological functions of miR-155 in cervical cancer.ResultsWe found that miR-155 expression was markedly up-regulated in cervical cancer tissues and correlated with FIGO stage, lymph nodes metastasis, vascular invasion and HPV. Patients with high miR-155 expression level had poorer overall survival than those with low miR-155 expression. Furthermore, multivariate Cox regression analysis suggested that increased miR-155 was an independent prognostic indicator for cervical cancer (P = 0.007; HR = 2.320; 95%CI: 1.259–4.276). Moreover, knockdown of miR-155 was demonstrated to inhibit cell proliferation, migration, and invasion in vitro.ConclusionOur study presents that miR-155 is a novel molecule involved in cervical cancer progression, which provide a potential prognostic biomarker and therapeutic target.     

BCL2L12: A promising molecular prognostic biomarker in breast cancer

ObjectivesBCL2-like 12 (BCL2L12) is a new member of the BCL2 gene family that was discovered and cloned by members of our group and found to be expressed in the mammary gland. Many genes of the BCL2 family were found to be implicated in breast carcinogenesis and to serve as possible prognostic markers. The aim of the present study was the quantification of BCL2L12 mRNA expression in order to assess its value as a prognostic tissue biomarker in breast cancer (BC).Design and methodsBCL2L12 mRNA levels were determined in a statistically significant sample size of cancerous (N = 108) and adjacent non-cancerous (N = 71) breast tissues using a highly sensitive quantitative real-time polymerase chain reaction (qRT-PCR) method. Relative quantification analysis was conducted using the comparative C_T (2^− ΔΔC_T) method, whereas the association between BCL2L12 expression and clinopathological data, disease-free survival (DFS) and overall survival (OS) were estimated by statistical analysis.ResultsBCL2L12 mRNA expression was decreased in malignant samples compared to the histologically normal counterparts (p = 0.012). Significant relationships between BCL2L12 expression and TNM stages (p = 0.009), metastatic potential (p = 0.012), tumor size (p = 0.04) and age (p = 0.024) were observed. Moreover, Kaplan–Meier and Cox univariate analyses indicated that BCL2L12 expression is associated with longer DFS, whereas multivariate analysis pointed out the independent favorable prognostic value of BCL2L12.ConclusionsAccording to our results, BCL2L12 mRNA expression is a favorable prognostic marker of DFS for BC patients, suggesting its possible application as a novel prognostic indicator of this malignancy.     

Association of sociodemographic factors and prostate-specific antigen (PSA) testing

ObjectivesThere are conflicting recommendations regarding the use of prostate specific antigen (PSA) as a screening test. Integral to this debate is an understanding of who is currently being tested. The purpose of this study was to provide a detailed account of PSA testing practices in a major Canadian city (Calgary, Alberta) and to identify variables that may affect access to the PSA test.Design and methodsPSA test counts were retrieved from Calgary Laboratory Services' Laboratory Information System from January 1, 2011 to December 31, 2011. A total of 75,914 individual PSA tests were included in our analysis. The frequency of PSA testing was plotted onto a dissemination area map of Calgary using ArcGIS software. Associations with sociodemographic variables were tested using Poisson regression.ResultsThe median PSA value was 0.93 μg/L and the median age at collection was 58 years. Forty-three percent of men aged 60–69 received a PSA test. Visible minority status ‘Black’ (P = 0.0002) and Métis status (P = 0.0075) were associated with lower PSA testing frequencies, while median household income (P = < 0.0001) and university education (P = < 0.0001) were associated with higher PSA testing frequencies.ConclusionThere are areas in Calgary which are significantly over or under tested relative to the mean. The amount of PSA testing in men < 50 years of age is increasing, which is contrary to PSA testing guidelines.     

Inverse correlation of ribosomal protein S27A and multifunctional protein YB-1 in hepatocellular carcinoma

ObjectiveThe detection of possible correlation between ribosomal protein RPS27A and multifunctional YB-1 expression in hepatocellular carcinoma (HCC).Design and methodsTissue microarray slides containing totally 80 cores with 19 tissues of HCC, 1 tissue of hepatocholangiocarcinoma, 10 tissues of liver cirrhosis and 10 normal liver tissues in duplicates were analyzed for expression of RPS27A and YB-1 by immunohistochemistry.ResultsAmong each of 10 LC and normal liver tissues all (100%) showed RPS27A positive expression but only 11 out of 19 HCC tissues (57.89%) showed RPS27A positive expression with significant difference compared (P < 0.05) with both LC and normal tissues. We found positive expression of YB-1 in 17 tissues out of 19 HCC tissues (89.47%) but only 4 tissues out of each 10 LC as well as normal liver tissues showed positive expression with significant (P < 0.01) difference compared to HCC tissues. A statistically significant inverse weak correlation (rho = − 0.293) between YB-1 expression and RPS27A expression was found.ConclusionThe present investigation concludes that the ribosomal protein RPS27A was down-regulated in viral induced HCC patients. RPS27A expression was found to have a weak inverse correlation with overexpression of multifunctional protein YB-1 in HCC tissues. This study opens up a new window for YB-1–RPS27A axis in HCC.     

Fyn-related kinase expression predicts favorable prognosis in patients with cervical cancer and suppresses malignant progression by regulating migration and invasion

AimTo investigate expression pattern, clinical significance and potential roles of fyn related kinase (FRK) in cervical cancer.MethodsExpression of FRK protein and mRNA in 100 pairs of cervical cancer and matched non-cancerous tissue samples were detected by Western blot, immunohistochemistry and quantitative PCR, respectively. Statistical analyses were performed to evaluate associations of FRK protein expression with various clinicopathologic features and patients' prognosis. The effects of FRK on cell migration and invasion were examined using in vitro migration and invasion assays, respectively.ResultsWeak/negative immunostaining of FRK protein was observed in 86 (86.00%) of 100 cervical cancer tissues. Low FRK expression was significantly associated with several aggressive clinicopathologic features of cervical cancer, such as higher International Federation of Gynecology and Obstetric stage (P = 0.01), the presence of lymph node metastasis (P = 0.01) and recurrence (P = 0.02). In addition, the survival analysis showed that cervical cancer patients with low FRK expression often had shorter overall survival than those with high FRK expression. The multivariate analysis also identified FRK expression as an independent prognostic factor of cervical cancer. Functionally, the enforced expression of FRK could efficiently inhibit cell migration and invasion of cervical cancer cells, but the knockdown of FRK dramatically enhanced cell migration and invasion.ConclusionOur findings suggest that loss of FRK protein may be implicated into the tumorigenesis and cell motility of human cervical cancer. More importantly, FRK expression may function as a promising prognostic marker of this malignancy, highlighting its potentials as a candidate target for gene therapy.     

ADD-ASPIRIN: A phase III,double-blind,placebo controlled,randomised trial assessing the effects of aspirin on disease recurrence and survival after primary therapy in common non-metastatic solid tumours

BackgroundThere is a considerable body of pre-clinical, epidemiological and randomised data to support the hypothesis that aspirin has the potential to be an effective adjuvant cancer therapy.MethodsAdd-Aspirin is a phase III, multi-centre, double-blind, placebo-controlled randomised trial with four parallel cohorts. Patients who have undergone potentially curative treatment for breast (n = 3100), colorectal (n = 2600), gastro-oesophageal (n = 2100) or prostate cancer (n = 2120) are registered into four tumour specific cohorts. All cohorts recruit in the United Kingdom, with the breast and gastro-oesophageal cohort also recruiting in India. Eligible participants first undertake an active run-in period where 100 mg aspirin is taken daily for approximately eight weeks. Participants who are able to adhere and tolerate aspirin then undergo a double-blind randomisation and are allocated in a 1:1:1 ratio to either 100 mg aspirin, 300 mg aspirin or a matched placebo to be taken daily for at least five years. Those participants ≥ 75 years old are only randomised to 100 mg aspirin or placebo due to increased toxicity risk.ResultsThe primary outcome measures are invasive disease-free survival for the breast cohort, disease-free survival for the colorectal cohort, overall survival for the gastro-oesophageal cohort, and biochemical recurrence-free survival for the prostate cohort, with a co-primary outcome of overall survival across all cohorts. Secondary outcomes include adherence, toxicity including serious haemorrhage, cardiovascular events and some cohort specific measures.ConclusionsThe Add-Aspirin trial investigates whether regular aspirin use after standard therapy prevents recurrence and prolongs survival in participants with four non-metastatic common solid tumours.     

The association of Phosphatase and tensin homolog (PTEN) deletion and prostate cancer risk: A meta-analysis

ObjectivePhosphatase and tensin homolog (PTEN) deleted on chromosome 10, a tumor suppressor that negatively regulates the phosphoinositide-3-kinase(PI3 K) which has been implicated in a number of human malignancies including prostate cancer. However the prognostic value of PTEN deletion in prostate cancer patient’s diagnosis and the mechanism of PTEN deletion in prostate cancer development still remain unclear.MethodA meta-analysis of 26 published studies including 8097 prostate cancer patients was performed.ResultsCompared to PTEN normal patients, PTEN deletion patients showed a higher aggressive Gleason score(OR: 1.284, 95%CI = 1.145–1.439) and pathological stage(OR: 1.628, 95%CI = 1.270–2.087) which generally had a higher risk in prostate replace(HR: 1.738, 95%CI = 1.264–2.390). Significant association between PTEN deletion and ERG rearrangements in prostate cancer development was also proved that compared to PTEN normal patients, patients with PTEN deletion showed a higher risk in ERG rearrangements(OR: 1.345, 95%CI = 1.102–1.788).ConclusionThis study indicated that patients with PTEN deletion were associated with higher pathological stage or Gleason score and a higher risk in prostate cancer replace potentially represent a novel clinically relevant event to identify individuals at increased risk for the occurrence, progression and prognosis of prostate cancer. Prostate cancer patients with PTEN deletion usually had a higher risk in ERG rearrangements than other patients may be a potential new area for identifying poor prognosis patients and selecting patients for targeted therapies which required confirmation through adequately designed prospective studies.     

Prognostic significance of RASSF1A promoter methylation in operable breast cancer

ObjectivesThe aim of our study was to evaluate the prognostic significance of RASSF1A promoter methylation status in operable breast cancer.Design and methodsBy using Methylation Specific PCR, we evaluated the specificity of RASSF1A promoter methylation in 10 breast tumors and matching normal tissues, 10 breast fibroadenomas and 11 normal breast tissues. The prognostic significance of RASSF1A methylation was validated in 93 formalin fixed paraffin-embedded (FFPE) tissues obtained from patients with operable breast cancer.ResultsMethylation of RASSF1A promoter was observed in 1/31 (3.2%) non-cancerous breast tissues and 53/93 (57.0%) early stage breast tumors. The only positive sample in the non-cancerous breast tissues group was found in a histological normal tissue surrounding the tumor. During the follow-up period, 24/93 (25.8%) patients relapsed and 19/93 (20.4%) died. Disease-Free-Interval (DFI) was significantly associated with RASSF1A methylation (p = 0.028).ConclusionsRASSF1A promoter methylation provides important prognostic information in early stage breast cancer patients.