Differential expression of microRNAs in plasma of patients with colorectal cancer: A potential marker for colorectal cancer screening

Objective: MicroRNAs (miRNAs) have been shown to offer great potential in the diagnosis of cancer. We investigated whether plasma miRNAs could discriminate between patients with and without colorectal cancer (CRC). Methods: This study was divided into three phases: (1) marker discovery using real-time PCR-based miRNA profiling on plasma, corresponding cancerous and adjacent non-cancerous colonic tissues of five patients with CRC, along with plasma from five healthy individuals as controls; (2) marker selection and validation by real-time quantitative RT-PCR on a small set of plasma; and (3) independent validation on a large set of plasma from 90 patients with CRC, 20 patients with gastric cancer, 20 patients with inflammatory bowel disease (IBD) and 50 healthy controls. Results: Of the panel of 95 miRNAs analysed, five were upregulated both in plasma and tissue samples. All the five miRNAs were validated on the plasma of 25 patients with CRC and 20 healthy controls. Both miR-17-3p and miR-92 were significantly elevated in the patients with CRC (p<0.0005). The plasma levels of these markers were significantly reduced after surgery in 10 patients with CRC (p<0.05). Further validation with an independent set of plasma samples (n=180) indicated that miR-92 differentiates CRC from gastric cancer, IBD and normal subjects. This marker yielded a receiver operating characteristic curve area of 88.5%. At a cut-off of 240 (relative expression in comparison to RNU6B snRNA), the sensitivity was 89% and the specificity was 70% in discriminating CRC from control subjects. Conclusion: MiR-92 is significantly elevated in plasma of patients with CRC and can be a potential non-invasive molecular marker for CRC screening.published_or_final_versio

Circulating microRNAs as Specific Biomarkers for Breast Cancer Detection

Background: We previously showed microRNAs (miRNAs) in plasma are potential biomarkers for colorectal cancer detection. Here, we aimed to develop specific blood-based miRNA assay for breast cancer detection. Methodology/Principal Findings: TaqMan-based miRNA profiling was performed in tumor, adjacent non-tumor, corresponding plasma from breast cancer patients, and plasma from matched healthy controls. All putative markers identified were verified in a training set of breast cancer patients. Selected markers were validated in a case-control cohort of 170 breast cancer patients, 100 controls, and 95 other types of cancers and then blindly validated in an independent set of 70 breast cancer patients and 50 healthy controls. Profiling results showed 8 miRNAs were concordantly up-regulated and 1 miRNA was concordantly down-regulated in both plasma and tumor tissue of breast cancer patients. Of the 8 up-regulated miRNAs, only 3 were significantly elevated (p<0.0001) before surgery and reduced after surgery in the training set. Results from the validation cohort showed that a combination of miR-145 and miR-451 was the best biomarker (p<0.0001) in discriminating breast cancer from healthy controls and all other types of cancers. In the blind validation, these plasma markers yielded Receiver Operating Characteristic (ROC) curve area of 0.931. The positive predictive value was 88% and the negative predictive value was 92%. Altered levels of these miRNAs in plasma have been detected not only in advanced stages but also early stages of tumors. The positive predictive value for ductal carcinoma in situ (DCIS) cases was 96%. Conclusions: These results suggested that these circulating miRNAs could be a potential specific biomarker for breast cancer screening. © 2013 Ng et al.published_or_final_versio

A Toolkit and Robust Pipeline for the Generation of Fosmid-Based Reporter Genes in C. elegans

Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene locus contained within genomic clones by homologous recombination (“recombineering”) represents the most straightforward method to generate these reporters. In this methodology paper, we describe a simple and robust pipeline for recombineering of fosmids, which we apply to generate reporter constructs in the nematode C. elegans, whose genome is almost entirely covered in an available fosmid library. We have generated a toolkit that allows for insertion of fluorescent proteins (GFP, YFP, CFP, VENUS, mCherry) and affinity tags at specific target sites within fosmid clones in a virtually seamless manner. Our new pipeline is less complex and, in our hands, works more robustly than previously described recombineering strategies to generate reporter fusions for C. elegans expression studies. Furthermore, our toolkit provides a novel recombineering cassette which inserts a SL2-spliced intercistronic region between the gene of interest and the fluorescent protein, thus creating a reporter controlled by all 5′ and 3′ cis-acting regulatory elements of the examined gene without the direct translational fusion between the two. With this configuration, the onset of expression and tissue specificity of secreted, sub-cellular compartmentalized or short-lived gene products can be easily detected. We describe other applications of fosmid recombineering as well. The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans

Differential expression of microRNAs in plasma of colorectal cancer patients: a potential marker for colorectal cancer screening

Topic Forum: Oral Sessions - Scientific Sessions: Microrna and digestive cancers, Oral Presentation no. 1070OBJECTIVE: MicroRNAs (miRNA), a class of small non-coding RNAs, play important roles in cancers and have been shown to have great cancer diagnostic potential. Since cell-free miRNAs are recently detected in the plasma and serum, we investigated whether plasma miRNAs enable to discriminate patients with and without colorectal cancer (CRC). METHODS: This study was divided into three phases: (i) Marker discovery using real-time PCR-based miRNA profiling on plasma, corresponding cancerous and adjacent non-cancerous colonic tissues of 5 CRC patients, along with plasma from 5 healthy controls. (ii) miRNA marker selection and validation by real-time quantitative RT-PCR on plasma from 25 CRC and 20 healthy controls. (iii) Validation on an independent set of plasma from 90 CRC patients, 20 gastric cancers, 20 inflammatory bowel disease and 50 healthy controls. RESULTS: Of the panel of 95 miRNAs analyzed, 5 miRNAs were up-regulated both in plasma and tissue samples. All 5 miRNAs were validated on the plasma of 25 CRC patients and 20 healthy controls. Both miR-17-3p and miR-92 were significantly elevated in CRC patients (p<0.0005). The plasma levels of these markers were significantly reduced after surgery in 10 CRC patients (p<0.05). Further validation with an independent set of plasma samples (n=180) indicated that miR-92 differentiates CRC from gastric cancer, IBD and normal subjects. This marker yielded a receiver operating characteristic curve area of 88.5%. At a cutoff of 240, the sensitivity was 89% and the specificity was 70% in discriminating CRC from control subjects. Conclusions: Our findings demonstrated that plasma miRNAs have great potential for CRC screening. This opens up a new class of molecular markers for CRC diagnosis

Prospective study on the performance of the prostate-specific antigen isoform P2PSA and its derivative prostate health index in prediction of prostate cancer in local Chinese men with total prostate-specific antigen levels of 4-10 ng/mL

This free journal suppl. entitled: Special Issue: 14th Urological Association of Asia Congress 2016 SingaporeConference Theme: Urological Advancement in AsiaAbstract and Poster PresentationOBJECTIVES To evaluate the accuracy of the prostate-specific antigen isoform P2PSA and its derivatives including Prostate Health Index (PHI) in determining the presence of prostate cancer in prostate biopsies with serum total prostate-specific antigen (tPSA) at 4–10 ng/mL. METHODS This was a prospective cohort study involving two urology centers. Patients with tPSA at 4–10 ng/mL with a negative digital rectal examination who consented to prostate biopsy to detect underlying prostate cancer were recruited from March 2013 to March 2016. Their serum tPSA and P2PSA were checked before the biopsy and evaluated with respect to their prostate biopsy results. RESULTS Two hundred and forty-seven patients were recruited with cancer detected in 43 (17.4%) patients. %P2PSA and PHI were significantly higher in patients with cancer (P < 0.0001) whilst %fPSA and tPSA were similar between the two groups. ROC analysis showed the AUC for tPSA, %fPSA, %PSPSA and PHI to be 0.50, 0.58, 0.77 and 0.76 respectively. %P2PSA and PHI were the best predictor of underlying prostate cancer and were significantly better than tPSA (P < 0.0001 and 0.0005 respectively). At a sensitivity of 90%, PHI has the highest specificity (43.1%) amongst all the markers (tPSA: 7.4%, % fPSA: 27.0%, %P2PSA: 37.7%). When compared with tPSA, the use of the PHI would avoid 73(39%) of patients from unnecessary biopsies. CONCLUSIONS PHI is a superior marker to tPSA in detection of prostate cancer in our cohort of local Chinese men. Its use in addition to tPSA can improve its accuracy and aids in patient counseling regarding whether to proceed with prostate biopsies.link_to_OA_fulltex