Researchers Find RNA Molecules in Urine and Tissue That Detect Prostate Cancer


Potential Biomarkers may pave way to a more sensitive, specific, and noninvasiveprostate cancer screening assay, according to Report in The Journal ofMolecular Diagnostics

Philadelphia, PA, October 9, 2014

Researchers at Sanford-Burnham Medical Research Institutehave identified a set of RNA molecules that are detectable in tissue samplesand urine of prostate cancer patients but not in normal healthy individuals.The study sets the stage for the development of more sensitive and specificnoninvasive tests for prostate cancer than those currently available, whichcould result in fewer unnecessary prostate biopsies with less treatment-relatedmorbidity, according to a new study in TheJournal of Molecular Diagnostics.

According to the American Cancer Society, prostate canceris the second most common type of cancer in American men (after skin cancer),and the second-leading cause of cancer-related death in men (after lungcancer). In 2014, more than 230,000 new cases of prostate cancer will bediagnosed. One in seven American men will get prostate cancer during hislifetime, and one in 36 will die from it. Since most men with prostate cancerhave indolent (nonaggressive) disease for which conservative therapy orsurveillance is appropriate, the clinical challenge is not only how to identifythose with prostate cancer, but also how to distinguish between those who wouldbenefit from surgical or other aggressive treatment from those who would not.

Prostate cancer is primarily detected and monitored bytesting for high concentrations of prostate-specific antigen (PSA) in bloodsamples. High PSA levels are often followed by a biopsy to confirm the presenceof cancer and whether it is slow growing or aggressive.

"While elevated PSA can be an alert to a lethal cancer,it can also detect less aggressive cancers that may never do any harm," saidVipul Patel, MD, medical director of the Global Robotics Institute at FloridaHospital in Orlando. "Moreover, only 25 percent of men with raised PSA levelsthat have a biopsy actually have prostate cancer. Prostate cancer needs to bescreened for; we just need to find a better marker."

The researchers believe that they have identified a groupof RNA molecules – known as long noncoding RNAs (lncRNAs) – that hold thepotential for serving as better prognostic markers for prostate cancer. lncRNAswere dismissed until recently by scientists as non-functional noise in thegenome. However, they are now thought to regulate normal cellular developmentand are increasingly reported as contributing to a range of diseases, includingcancer.

"We have identified a set of lncRNAs that appear to havean important role in prostate cancer diagnostics," commented Ranjan J. Perera,PhD, associate professor and scientific director of Analytical Genomics andBioinformatics at Sanford-Burnham's Lake Nona campus in Orlando. "The findingsadvance our understanding of the role of lncRNAs in cancer biology and,importantly, broaden the opportunity to use lncRNAs as biomarkers to detectprostate cancer."

The study profiled the lncRNAs in three distinct groups:(1) human prostate cancer cell lines and normal prostate epithelial cells; (2)prostate adenocarcinoma tissue samples and matched normal tissue samples; and(3) urine samples from patients with prostate cancer or benign prostate hyperplasia,and normal healthy individuals. In each case, the lncRNAs were elevated inprostate cancer patient samples, but not in patients with benign prostate hyperplasiaor normal healthy individuals.

One advantage of lncRNAs is that the molecules can bedetected in urine samples, which are more easily available than blood tests. OnelncRNA, PCA3, was recently commercialized in a urine test to identify which mensuspected of having prostate cancer should undergo repeat prostate biopsy.However, discrepancies exist between PCA3 levels and clinicopathologicfeatures, noted Dr. Perera. In the current study, PCA3 was detected in some,but not all of the study samples, suggesting that reliance on a singlebiomarker may be insufficient for prostate cancer detection, whereas combiningadditional markers may increase the specificity and sensitivity of the test.

"There is a tremendous unmet clinical need for betternon-invasive screening tools for early detection of prostate cancer to reducethe overtreatment and morbidity of this disease," added Dr. Patel. "Ourfindings represent a promising approach to meet this demand."

Technical detailsof the study
The goal of the first experiment was to see whetherlncRNAs are differentially expressed in prostate cancer by measuring total RNAfrom prostate cancer cell lines and normal epithelial prostatic cells usingNCode human ncRNA array and SurePrint G3 human lncRNA microarrays. Hierarchicalclustering revealed distinguishable lncRNA expression profiles. Thirty lncRNAswere up-regulated and the expression levels of three top-ranking candidates [XLOC_007697,LOC100287482, and AK024556 (also known as SPRY4-IT1)]were confirmed in prostate cancer cell lines by quantitative real-timepolymerase chain reaction (qPCR) analysis. The SPRY4-IT1 was found to be up-regulated more than 100-fold in PC3cells compared with prostatic epithelial cells.

In a second experiment, lncRNA expression was compared inpooled prostate cancer tissue samples and matched normal tissues from 10 frozenbiopsy specimens. Hierarchical clustering of the differentially expressedlncRNAs was observed and 10 up-regulated lncRNAs were detected usingmicroarrays. An additional set of 18 prostate cancer tissue samples wasanalyzed by qPCR and five lncRNAs were found to be significantly higher inprostate tumor tissues compared with matched normal tissues.

Researchers used qPCR to analyze total RNA isolated fromurine in another experiment. Urine was collected from 13 prostate cancerpatients and 14 healthy controls. All six lncRNAs were found to besignificantly up-regulated in the urine samples from the prostate cancerpatients compared with normal patient controls, whereas there were nodifferences between normal and benign prostatic hyperplasia patient samples.

In other studies focused particularly on SPRY4-IT1, using both qPCR and highlysensitive droplet digital PCR, expression of SPRY-IT1 was found to be increased in 16 of 18 (89 percent) tissuesamples from patients with prostatic adenocarcinoma, compared to normal tissuesamples. The researchers developed chromogenic in situ hybridization (CISH) techniques to visualize SPRY4-IT1 expression in cancerous andmatched normal tissue. Intense staining was seen in all adenocarcinoma samples,but not in normal prostatic tissue. Finally, the investigators showed that reductionof SPRY4-IT1 in prostate cancer cellsthrough the use of small interfering RNA (siRNA) leads to decreased cellviability and cellular invasion as well as increased apoptosis, similar to whatis seen in melanoma cells.

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Notes for editors
"Long Noncoding RNAs as PutativeBiomarkers for Prostate Cancer Detection," byBongyong Lee, Joseph Mazar,Muhammed Nauman Aftab, Feng Qi, John Shelley, Jian-Liang Li, SubramaniamGovindarajan, Felipe Valerio, Inoel Rivera, Tadzia Thurn, Tien Anh Tran, DarianKameh, Vipul Patel, and Ranjan J. Perera, DOI: http://dx.doi.org/10.1016/j.jmoldx.2014.06.009. Published online ahead of The Journal of Molecular Diagnostics, Volume 16, Issue 6 (November 2014) published by Elsevier.

Full text of the article is available to credentialedjournalists upon request; contact Eileen Leahy at + 1 732 238 3628 or jmdmedia@elsevier.com.Journalists wishing to interview the authors should contact Susan Gammon at +1 858 795 5012or sgammon@sanfordburnham.org. Accompanying video at http://www.youtube.com/watch?v=2QrZdTWljmA&list=UUuMr-NyvQParnW4vXHPGC_Q.

This research was supported by NIH/National CancerInstitute Grant 5P30CA030199 and the International Prostate Cancer Foundation.

About The Journal of Molecular Diagnostics
The Journal of Molecular Diagnostics, (http://jmd.amjpathol.org), the official publication of the Associationfor Molecular Pathology, co-owned by the American Society for InvestigativePathology, and published by Elsevier, Inc., seeks to publish high qualityoriginal papers on scientific advances in the translation and validation ofmolecular discoveries in medicine into the clinical diagnostic setting, and thedescription and application of technological advances in the field of moleculardiagnostic medicine. The editors welcome for review articles that contain: noveldiscoveries or clinicopathologic correlations including studies in oncology,infectious diseases, inherited diseases, predisposition to disease, or thedescription of polymorphisms linked to disease states or normal variations; theapplication of diagnostic methodologies in clinical trials; or the developmentof new or improved molecular methods for diagnosis or monitoring of disease ordisease predisposition.

The Journal of Molecular Diagnostics, withan Impact Factor of 3.955, ranks 13th among 76 journals inPathology, according to 2013 JournalCitation Reports® Thomson Reuters, 2014

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