Rapid and Sensitive Detection of Breast Cancer Cells in Patient Blood with Nuclease-Activated Probe Technology.

A challenge for circulating tumor cell (CTC)-based diagnostics is the development of simple and inexpensive methods that reliably detect the diverse cells that make up CTCs. CTC-derived nucleases are one category of proteins that could be exploited to meet this challenge. Advantages of nucleases as CTC biomarkers include: (1) their elevated expression in many cancer cells, including cells implicated in metastasis that have undergone epithelial-to-mesenchymal transition; and (2) their enzymatic activity, which can be exploited for signal amplification in detection methods. Here, we describe a diagnostic assay based on quenched fluorescent nucleic acid probes that detect breast cancer CTCs via their nuclease activity. This assay exhibited robust performance in distinguishing breast cancer patients from healthy controls, and it is rapid, inexpensive, and easy to implement in most clinical labs. Given its broad applicability, this technology has the potential to have a substantive impact on the diagnosis and treatment of many cancers.

A web-based registry for patients with sarcoidosis.

BACKGROUND/OBJECTIVE: The objective is to present the development of a novel web-based patient registry for sarcoidosis. We describe recruitment efforts and assess efficacy of internet-based advertising on recruitment. METHODS: "Worldwide Sarcoidosis Research Study (WISE)" started in 2011 under the domain www.sarcoidstudy.org. The registry includes thirteen patient-reported surveys about patient characteristics, diagnosis, and treatment. Effects of two internet-based advertising methods (geographically-broad versus geographically-targeted to high sarcoidosis search areas) on recruitment were analyzed with time series regression. RESULTS: Since 2011, over 1500 participants have registered (82% whites, 9% African Americans, 5% mixed, 4% other), with 23% of participants providing saliva samples for DNA. Median age is 43 years (range 21-80). African Americans were more frequently recruited via support groups, while whites had a higher frequency of finding the registry via internet. Generalized internet-based advertising significantly improved recruitment in all demographic groups (p<0.001). However, a higher response rate to internet-based advertising was seen in whites compared to African Americans(p<0.001), females versus males(p=0.043), higher income categories(p=0.048), and increased education level(p<0.001). Targeting advertising campaigns to geographical areas with high internet-search patterns for sarcoidosis, with different demographics, was not effective in raising registry recruitment above baseline or increasing diversity. CONCLUSIONS: A web-based registry is an effective method for establishing a cohort of patients with sarcoidosis invested in clinical research with DNA specimens. Despite limitations, opportunities for research in patient-oriented outcomes and broad internet-based research methodology are possible. Our results demonstrate that web-based approaches to recruit study subjects need to be focused to match different target populations.

Downregulation of dystroglycan glycosyltransferases LARGE2 and ISPD associate with increased mortality in clear cell renal cell carcinoma.

BACKGROUND: Dystroglycan (DG) is a cell-surface laminin receptor that links the cytoskeleton to the extracellular matrix in a variety of epithelial tissues. Its function as a matrix receptor requires extensive glycosylation of its extracellular subunit αDG, which involves at least 13 distinct genes. Prior work has shown loss of αDG glycosylation in an assortment of carcinomas, including clear cell renal cell carcinoma (ccRCC) though the cause (s) and functional consequences of this loss are still unclear. METHODS: Using The Cancer Genome Atlas (TCGA) database, we analyzed the DG glycosylation pathway to identify changes in mRNA expression and correlation with clinical outcomes. We validated our findings with a cohort of 65 patients treated with radical nephrectomy by analyzing DG glycosylation via immunohistochemistry and gene expression via qRT-PCR. RESULTS: Analysis of TCGA database revealed frequent dysregulation of a subset of DG glycosyltransferases. Most notably, there was a frequent, significant downregulation of GYLTL1B (LARGE2) and ISPD. DG glycosylation is frequently impaired in ccRCC patient samples and most strongly associates with downregulation of GYLTL1B. CONCLUSIONS: Reduced levels of GYLTL1B and ISPD mRNA associated with increased patient mortality and are the likely cause of αDG hypoglycosylation in ccRCC.

The glycosyltransferase LARGE2 is repressed by Snail and ZEB1 in prostate cancer.

Reductions in both expression of the dystroglycan core protein and functional glycosylation of the α-dystroglycan (αDG) subunit have been reported in a number of cancers and may contribute to disease progression. In the case of prostate cancer, one mechanism that contributes to αDG hypoglycosylation is transcriptional down-regulation of LARGE2 (GYLTY1B), a glycosyltransferase that produces the functional (laminin-binding) glycan on αDG, but the mechanism(s) underlying reduction of LARGE2 mRNA remain unclear. Here, we show that αDG hypoglycosylation is associated with epithelial-to-mesenchymal transition (EMT)-like status. We examined immunoreactivity for both functionally-glycosylated αDG and E-cadherin by flow cytometry and the relative expression of ZEB1 mRNA and the αDG glycosyltransferase LARGE2 mRNA in prostate and other cancer cell lines by quantitative RT-PCR. To study the role of ZEB1 and other transcription factors in the regulation of LARGE2, we employed overexpression and knockdown approaches. Snail- or ZEB1-driven EMT caused αDG hypoglycosylation by repressing expression of the LARGE2 mRNA, with both ZEB1-dependent and -independent mechanisms contributing to Snail-mediated LARGE2 repression. To examine the direct regulation of LARGE2 by Snail and ZEB1 we employed luciferase reporter and chromatin immunoprecipitation assays. Snail and ZEB1 were found to bind directly to the LARGE2 promoter, specifically to E/Z-box clusters. Furthermore, analysis of gene expression profiles of clinical samples in The Cancer Genome Atlas reveals negative correlation of LARGE2 and ZEB1 expression in various cancers. Collectively, our results suggest that LARGE2 is negatively regulated by Snail and/or ZEB1, revealing a mechanistic basis for αDG hypoglycosylation during prostate cancer progression and metastasis.