Samuil R. Umansky

Circulating cell-free microRNA as biomarkers for screening, diagnosis and monitoring of neurodegenerative diseases and other neurologic pathologies

Many neurodegenerative diseases, such as Alzheimers disease, Parkinson disease, vascular and frontotemporal dementias, as well as other chronic neurological pathologies, are characterized by slow development with a long asymptomatic period followed by a stage with mild clinical symptoms. As a consequence, these serious pathologies are diagnosed late in the course of a disease, when massive death of neurons has already occurred and effective therapeutic intervention is problematic. Thus, the development of screening tests capable of detecting neurodegenerative diseases during early, preferably asymptomatic, stages is a high unmet need. Since such tests are to be used for screening of large populations, they should be non-invasive and relatively inexpensive. Further, while subjects identified by screening tests can be further tested with more invasive and expensive methods, e.g., analysis of cerebrospinal fluid or imaging techniques, to be of practical utility screening tests should have high sensitivity and specificity. In this review, we discuss advantages and disadvantages of various approaches to developing screening tests based on analysis of circulating cell-free microRNA (miRNA). Applications of circulating miRNA-based tests for diagnosis of acute and chronic brain pathologies, for research of normal brain aging, and for disease and treatment monitoring are also discussed.

Early detection of neurodegenerative diseases: circulating brain-enriched microRNA.

As has been demonstrated in numerous studies, the development of neurodegenerative diseases, such as Alzheimer (AD), Parkinson (PD), Huntington diseases, vascular and frontotemporal (FTD) dementias, begins 10–20 y prior to the clinical manifestation. Although molecular mechanisms behind various neurodegenerative diseases are different, many processes, e.g., neurite retraction, dysfunction and destruction of synapses and ultimately neuronal death,1 are characteristic of neurodegeneration in general. AD is the most common and, thus, actively investigated neurodegenerative disease, for which diagnostic tools and therapeutic treatments are being developed. Recent failures of anti-AD therapies in late-stage clinical trials (including dimebon of Medivation and Pfizer, solanezumab of Eli Lilly and bapineuzumab of Pfizer and Johnson & Johnson) highlighted two important points associated with the development of effective treatment for AD: first, therapy in late stages of AD is ineffective, most likely due to massive neuronal death, which precedes symptoms of dementia; and second, detailed stratified analysis of clinical data reveals promising results for treatment of AD patients with earlier, mild stages of the disease. Two approaches were demonstrated to be effective for early detection of AD: PET scan for in vivo detection of β-amyloid depositions, and analysis of levels of β-amyloid protein 1–42, total tau protein and phosphorylated tau181P protein in the cerebrospinal fluid.2,3 It needs to be mentioned, however, that invasiveness and high cost of these approaches hinder their use for primary screening. Our paper4 describes a new approach for early detection of neurodegenerative diseases based on quantitative analysis of circulating cell-free miRNA in the bloodstream. Two approaches are commonly used for analysis of miRNA as potential biomarkers: (1) miRNA arrays to analyze huge numbers of circulating miRNA and (2) analysis of tissue miRNA to identify miRNA, whose expression is changed in this tissue as a result of a particular pathology. Many potential biomarkers of various diseases have been found;5,6 however, these approaches have significant limitations: concentrations of many circulating miRNA are too low to be detected by microarray analysis, and, as a consequence, only about 30% of miRNA are detected in plasma or serum; in many cases there is no correlation between miRNA concentrations in a particular organ and plasma;7,8 and finally, since many miRNA are associated with a particular pathology type, e.g., cancer or inflammation, changes in concentrations of such miRNA decrease test specificity.5,6 To address these challenges, we propose a RT-PCR measurement of circulating miRNA that are significantly enriched in a particular organ, tissue or even cell type. Further, in addition to normalization of miRNA concentrations per spiked or ubiquitous miRNA, we used “miRNA pairs” approach,9,10 i.e., analysis of ratios of all measured miRNA concentrations to select most promising miRNA biomarker pairs. The innovations reported in our paper can be applied to pathologies of various organs; however, their use for the detection of neurodegeneration appears especially productive for the following reasons: (1) certain miRNA are enriched in neurons of distinct brain compartments and can be used for differentiating diseases characterized by pathological processes in hippocampus (AD), midbrain (PD) or frontal lobe (FTD); (2) disease progression gradually affects new brain areas, and changes in the levels of circulating miRNA enriched in these brain areas can be used for disease monitoring; (3) there are miRNA enriched in neurites and synapses that may appear in the extracellular space and ultimately in the blood stream as a result of axon, dendrite and spine pruning and synaptic loss; and (4) neurodegenerative diseases as well as normal aging are often accompanied by changes in the blood-brain barrier permeability; the use of brain-enriched miRNA pairs may compensate for this factor. In the first proof-of-principle study, patients with mild cognitive impairment (MCI), a syndrome characteristic of early stages of many neurodegenerative diseases, were compared with age matched controls. Two sets of miRNA biomarker pairs capable of differentiating MCI from controls with 82–92% accuracy were identified.4 These results are especially encouraging since 10–20% of MCI cases are reversible, and further, some of the normal age-matched subjects could be in a pre-symptomatic stage of MCI. A small longitudinal study demonstrated that in 70% of cases, MCI was detectable by the selected miRNA biomarker pairs at a pre-symptomatic stage, 1–5 y prior to the clinical diagnosis. MCI patients and age-matched control subjects had various non-neurological conditions. The ability of the reported miRNA biomarker pairs to differentiate MCI from age-matched control in the presence of other pathologies supports the proposed selection of biomarkers among the brain-enriched miRNA. It is estimated that approximately 50% of MCI patients progress to AD, i.e., the identified miRNA biomarker pairs detect early stages of AD, although they cannot distinguish MCI cases preceding AD dementia from other MCI cases. Interestingly, the same miRNA biomarker pairs differentiated age-matched controls (76–86-y-old) from younger subjects (21–59-y-old). The data suggest that the identified miRNA pairs detect processes common for normal aging and neurodegenerative diseases, e.g. synapse destruction, although these processes are less prominent during normal aging. Other miRNA should be tested for prognosis of MCI development, for example, progression to AD dementia. Additional studies focused on promising miRNA candidates are currently underway at DiamiR.

Analysis of organ-enriched microRNAs in plasma as an approach to development of Universal Screening Test: feasibility study

BackgroundEarly disease detection with a minimally invasive screening test will significantly increase effectiveness and decrease the cost of treatment. Here we propose a framework of a novel approach – Universal Screening Test (UST) for the detection of pathological processes in a particular organ system, organ, or tissue by RT-qPCR analysis of circulating cell-free miRNAs in plasma. As the first step towards assessing the feasibility of this concept, the present study was designed to analyze whether the same microRNAs (miRNAs) can detect various diseases of a particular organ system.MethodsRNA was extracted from plasma using Trizol treatment and silica binding. Levels of miRNAs were measured by single target RT-qPCR. The following innovations have been tested and proven effective: (i) the use of organ system/organ/tissue-enriched miRNAs; (ii) the use of miRNAs associated with broad disease categories, such as cancer and inflammation, in combination with the organ-enriched miRNAs; and (iii) the use of “miRNA pairs” for selecting miRNA combinations with the highest sensitivity and specificity.ResultsHere we report biomarker miRNA pairs effectively differentiating (i) patients with pulmonary system diseases (asthma, pneumonia and non-small cell lung cancer) and gastrointestinal (GI) system diseases (Crohn’s disease, stages I/II esophageal, gastric and colon cancers) from controls, each with 95% accuracy; (ii) patients with a pathology of the pulmonary system from patients with a pathology of the GI system with 94% accuracy; and (iii) cancer patients (stages I/II esophageal, gastric, colon cancers, or non-small cell lung cancer) from patients with inflammatory diseases (asthma, pneumonia, or Crohn’s disease) with 93%-95% accuracy.ConclusionsThe results obtained in the present study, along with the data reported by us and others previously, are encouraging and lay the ground for further investigation of the described approach for UST development.

Circulating brain-enriched microRNAs as novel biomarkers for detection and differentiation of neurodegenerative diseases

BackgroundMinimally invasive specific biomarkers of neurodegenerative diseases (NDs) would facilitate patient selection and disease progression monitoring. We describe the assessment of circulating brain-enriched microRNAs as potential biomarkers for Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS).MethodsIn this case-control study, the plasma samples were collected from 250 research participants with a clinical diagnosis of AD, FTD, PD, and ALS, as well as from age- and sex-matched control subjects (n = 50 for each group), recruited from 2003 to 2015 at the University of Pennsylvania Health System, including the Alzheimer’s Disease Center, the Parkinson’s Disease and Movement Disorders Center, the Frontotemporal Degeneration Center, and the Amyotrophic Lateral Sclerosis Clinic. Each group was randomly divided into training and confirmation sets of equal size. To evaluate the potential of circulating microRNAs enriched in specific brain regions affected by NDs and present in synapses as biomarkers of NDs, the levels of 37 brain-enriched and inflammation-associated microRNAs in the plasma of all participants were measured using individual qRT-PCR. A “microRNA pair” approach was used for data normalization.ResultsMicroRNA pairs and their combinations (classifiers) capable of differentiating NDs from control and from each other were defined using independently and jointly analyzed training and confirmation datasets. AD, PD, FTD, and ALS are differentiated from control with accuracy of 0.89, 0.90, 0.88, and 0.83 (AUCs, 0.96, 0.96, 0.94, and 0.93), respectively; NDs are differentiated from each other with accuracy ranging from 0.77 (AUC, 0.87) for AD vs. FTD to 0.93 (AUC, 0.98) for AD vs. ALS. The data further indicate sex dependence of some microRNA markers. The average increase in accuracy in distinguishing ND from control for all and male/female groups is 0.06; the largest increase is for ALS, from 0.83 for all participants to 0.92/0.98 for male/female participants.ConclusionsThe work presented here suggests the possibility of developing microRNA-based diagnostics for detection and differentiation of NDs. Larger multicenter clinical studies are needed to further evaluate circulating brain-enriched microRNAs as biomarkers for NDs and to investigate their association with other ND biomarkers in clinical trial settings.

DETECTION AND DIFFERENTIATION OF MILD COGNITIVE IMPAIRMENT, ALZHEIMER'S AND PARKINSON’S DISEASES BY ANALYSIS OF BRAIN-ENRICHED MICRORNAS IN PLASMA

Background: Recent genome-wide association studies (GWAS) have identified around 20 variants as late-onset Alzheimer’s disease (LOAD) susceptibility loci in whites. In addition to these single loci tests, it is important to detect and understand combined effects of multiple associated genes on LOAD. We performed a preliminary network analysis incorporating human protein-protein interaction database mined from 12 different sites including BIND, BioGRID, IntAct etc. to the HapMap2-imputed combined ADGC data set. Post-GWAS, this helps researchers to prioritize functionally related genes and networks that are of the highest biological relevance underlying the pathogenesis of LOAD. Methods: We combined HapMap2-imputed data sets from 15 studies after performing strict quality control. We performed a case-control association for LOAD adjusting for population sub-structure and study sites on a set of 19,692 unrelated individuals using PLINK and those results were used to perform a gene-wide analysis using VEGAS. The gene-wide association results were then integrated into the human protein-protein interaction network using a dense module searching (DMS) method to identify candidate genes or sub-networks for LOAD. We then attempted to functionally validate candidate genes from this network in vivo using a transgenic C. elegans model of Ab 1-42 toxicity. Results: The network analysis identified several of the known LOAD risk loci as well as other genes such as ALB, BAG1 and UBC to be strongly associated with LOAD. RNAi knockdown of the C. elegans orthologs of UBC (ubq-1 or ubq-2) significantly accelerated the age-associated onset of Ab 1-42 toxicity. Conclusions:We were able to identify a set of significant modules and candidate genes, including some well-studied genes not detected in the single-marker analysis of GWA studies for LOAD, and to demonstrate a role for two of these genes as modifiers of Ab toxicity in C. elegans. This approach provides complementary data to a GWAS of a complex disease phenotype by incorporating biological knowledge derived from protein-protein interactions and allows for initial functional validation in vivo. Further functional enrichment analysis is needed to determine whether these novel loci may provide targets for interventions to ameliorate LOAD. THURSDAY, JULY 17, 2014 ORAL SESSIONS O5-05 BIOMARKERS: NOVEL MOLECULAR FLUID MARKERS

Universal screening test based on analysis of circulating organ-enriched microRNAs: a novel approach to diagnostic screening.

Early disease detection leads to more effective and cost-efficient treatment. It is especially important for cancer and neurodegenerative diseases, because progression of these pathologies leads to significant and frequently irreversible changes in underlying pathophysiological processes. At the same time, the development of specific screening tests for detection of each of the hundreds of human pathologies in asymptomatic stage may be impractical. Here, we discuss a recently proposed concept: the development of minimally invasive Universal Screening Test (UST) based on analysis of organ-enriched microRNAs in plasma and other bodily fluids. The UST is designed to detect the presence of a pathology in particular organ systems, organs, tissues or cell types without diagnosing a specific disease. Once the pathology is detected, more specific, and if necessary invasive and expensive, tests can be administered to precisely define the nature of the disease. Here, we discuss recent studies and analyze the data supporting the UST approach.

Early detection of Alzheimer's disease by analysis of brain-enriched miRNA biomarkers in plasma

IL7was significantly negatively associated with depression (OR1⁄40.01, 95% CI .00-0.47). Serum IL7 was significantly positively associated with depression (OR1⁄43.01, 95% CI 1.02-9.31). Preliminary analyses suggest that diagnosis (AD versus control) also impacted the relation between GDS scores and IL7. Plasma IL7 levels were significantly negatively associated with GDS scores among women diagnosed with AD whereas serum IL7 was significantly negatively associated with GDS scores among female normal controls. Conclusions: Our results confirm prior work that IL7 is significantly associated with depression scores and shows that the IL7 depression link varies by gender and blood fraction. IL7 levels are weakly correlated across blood fractions. Future work examining blood-based markers of depression must consider gender as well as blood fraction in analyses. If IL-7 is considered in as a therapeutic agent for depression or AD, the current findings suggest that gender should be considered in trial design.