oa A Circulating MicroRNA Signature for the Diagnosis of Clinically Significant Prostate Cancer

Gold nanoparticle cores functionalized with highly oriented shells of oligonucleotides, referred to as spherical nucleic acids (SNAs), are novel three-dimensional oligonucleotide structures with unique properties that are distinct from their linear counterparts. As a result, SNAs exhibit novel biochemical activities that enable them to address many fundamental challenges associated with basic biological processes. They have been utilized to develop a number of powerful platforms for biomarker detection due to intrinsic unusual properties such as their amplifiable light scattering properties to achieve high assay sensitivity. In addition, these nanoconjugates show high binding affinities for complementary targets (reflected in elevated melting temperatures) and narrow subsequent melting transitions relative to oligonucleotide duplexes formed from conventional DNA probes of the same sequence. This behavior can be translated into significantly higher assay specificity and sensitivity to study the role of critical biomarkers in many forms of cancer, including prostate cancer (PCa).

PCa is the most common noncutaneous malignancy among men in the United States and the second most common cause of cancer mortality. Despite its prevalence, there are no specific accurate diagnostic or prognostic biomarkers. Indeed, although serum prostate specific antigen (PSA) concentration is used as a routine screening tool for prostate cancer, up to 11% of men with a PSA <  2.0 ng/ml may still have prostate cancer, and based on the serum level alone, it is not possible to distinguish between high and low risk prostate cancers. Due to the lack of specificity with PSA-based screening and harm associated with overtreatment and overdiagnosis, the United States Preventive Services Task Force has recommended that physicians do not routinely perform PSA-based prostate cancer screening. In an effort to separate diagnosis from treatment, active surveillance for men with low and very low risk prostate cancer, which combines PSA screening with rigorous scheduled prostate biopsies, has been implemented to decrease rates of overtreatment. However, active surveillance is a potential option only in a very select group of men with low grade and low volume PCa. From studies of men that meet strict pathologic criteria to begin active surveillance, nearly 70% can avoid treatment over five years. Yet, many urologists and patients are reluctant to monitor their cancer on active surveillance due to concerns for delaying treatment or potentially missing treatment during a window of cure. For aggressive PCa, some have concluded that it is undergraded at the time of diagnosis in up to 40% of prostate biopsies due to the limited accuracy of the technique. Thus, significant discrepancies between prostatic needle biopsy and radical prostatectomy (RP) specimens may be attributed to diagnostic pitfalls. Resolving such screening paradigms can be achieved by identifying novel molecular signatures capable of discriminating aggressive forms of PCa, which could lead to avoiding unnecessary biopsies, patient anxiety, or biopsy-related complications.

Detection of molecular signatures that are indicative of molecular changes related to cancer progression would provide a means for early diagnosis of PCa. MicroRNAs (miRNA, miR) are critical gene regulatory elements that are present in stable forms in serum and have emerged as potential non-invasive biomarkers for cancer diagnosis. However, advancement in analytical chemistry is required to detect low abundance miRNAs with high specificity. The research described here report the development of a novel scanometric-based miRNA profiling array, called the Scano-miR assay. This platform is highly sensitive and able to detect 1 femtomolar concentrations of miRNAs from serum and highly selective with the capability of identifying single nucleotide polymorphisms (i.e. SNPs). Indeed, it provides increased sensitivity for miRNA targets compared to molecular fluorophore-based detection systems, where 88% of the low abundance miRNA targets could not be detected under identical conditions. The application of the Scano-miR platform to high density array formats demonstrates its utility for high throughput and multiplexed miRNA profiling from various biological samples.

To assess the accuracy of the Scano-miR system, we studied the miRNA profiles of samples from men with PCa, and identified a novel molecular signature based on the differential expressions of circulating miRNA in serum samples specific to patients with clinically significant PCa. We analyzed the circulating miRNA profiles from patients with aggressive forms of PCa and compared them with those from healthy individuals and ones with indolent forms of the disease. From this study, a panel of five miRNAs PCa biomarkers has been identified as potentially useful for tracking both indolent and aggressive forms of the disease. Importantly, all patients with highly aggressive PCa in the study were the only cohort that exhibited elevated levels of an exclusively expressed miRNA in their serum samples. In addition, another miRNA biomarker was present in all samples from those with either aggressive or indolent forms of the disease but not detectable by qRT-PCR methods in samples from healthy volunteers. In addition, it is differentially expressed in patients with aggressive versus indolent forms of the disease. The other three identified miRNAs show different expression patterns, depending on the state of the disease. The signature is determined using a scoring method that calculates the aggregated levels of all five miRNAs by substracting the expression levels of down-regulated miRNAs from up-regulated ones and could be use to differentiate patients with aggressive forms of the diseases from those with indolent forms as well as healthy individuals with at least 94% and potentially 100% accuracy. This approach is important since it can be used to identify novel and low abundant miRNA targets for a wide variety of cancer diagnostic applications.