Yaron Goren

Serum MicroRNAs Are Promising Novel Biomarkers

Background Circulating nucleic acids (CNAs) offer unique opportunities for early diagnosis of clinical conditions. Here we show that microRNAs, a family of small non-coding regulatory RNAs involved in human development and pathology, are present in bodily fluids and represent new effective biomarkers. Methods and Results After developing protocols for extracting and quantifying microRNAs in serum and other body fluids, the serum microRNA profiles of several healthy individuals were determined and found to be similar, validating the robustness of our methods. To address the possibility that the abundance of specific microRNAs might change during physiological or pathological conditions, serum microRNA levels in pregnant and non pregnant women were compared. In sera from pregnant women, microRNAs associated with human placenta were significantly elevated and their levels correlated with pregnancy stage. Conclusions and Significance Considering the central role of microRNAs in development and disease, our results highlight the medically relevant potential of determining microRNA levels in serum and other body fluids. Thus, microRNAs are a new class of CNAs that promise to serve as useful clinical biomarkers.

MicroRNAs accurately identify cancer tissue origin

MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that is involved in oncogenesis and shows remarkable tissue specificity. Their potential for tumor classification suggests they may be used in identifying the tissue in which cancers of unknown primary origin arose, a major clinical problem. We measured miRNA expression levels in 400 paraffin-embedded and fresh-frozen samples from 22 different tumor tissues and metastases. We used miRNA microarray data of 253 samples to construct a transparent classifier based on 48 miRNAs. Two-thirds of samples were classified with high confidence, with accuracy >90%. In an independent blinded test-set of 83 samples, overall high-confidence accuracy reached 89%. Classification accuracy reached 100% for most tissue classes, including 131 metastatic samples. We further validated the utility of the miRNA biomarkers by quantitative RT-PCR using 65 additional blinded test samples. Our findings demonstrate the effectiveness of miRNAs as biomarkers for tracing the tissue of origin of cancers of unknown primary origin.

Serum levels of microRNAs in patients with heart failure

Diagnosis and risk stratification of patients with heart failure remain a challenge. The small non‐coding RNAs known as microRNAs regulate gene expression and seem to play an important role in the pathogenesis of heart failure. In the current study, we aim to characterize the levels of microRNAs in the sera of chronic systolic heart failure patients vs. controls and assess the possible correlation between elevation in the levels of specific microRNAs and clinical prognostic parameters in heart failure patients.

A Diagnostic Assay Based on MicroRNA Expression Accurately Identifies Malignant Pleural Mesothelioma

The definitive identification of malignant pleural mesothelioma (MPM) has significant clinical implications, yet other malignancies often involve the lung pleura, confounding the diagnosis of MPM. In the absence of accurate markers, MPM can be difficult to distinguish from peripheral lung adenocarcinoma and metastatic epithelial cancers. MicroRNA expression is tissue-specific and highly informative for identifying tumor origin. We identified microRNA biomarkers for the differential diagnosis of MPM and developed a standardized microRNA-based assay. Formalin-fixed, paraffin-embedded samples of 33 MPM and 210 carcinomas were used for assay development. Using microarrays, we identified microRNAs differentially expressed between MPM and various carcinomas. Hsa-miR-193-3p was overexpressed in MPM, while hsa-miR-200c and hsa-miR-192 were overexpressed in peripheral lung adenocarcinoma and carcinomas that frequently metastasize to lung pleura. We developed a standardized diagnostic assay based on the expression of these microRNAs. The assay reached a sensitivity of 100% and a specificity of 94% in a blinded validation set of 68 samples from the lung and pleura. This diagnostic assay can provide a useful tool in the differential diagnosis of MPM from other malignancies in the pleura.

Predicting progression of bladder urothelial carcinoma using microRNA expression

Recurrence and progression prediction in urothelial cancer is currently based on clinical and pathological factors: tumour grade, tumour stage, number of lesions, tumour size, previous recurrence rate, and presence of concomitant carcinoma in situ. These factors are not specific enough to predict progression and ∼50% of patients diagnosed as high risk in fact do not progress within 3 years. Patient follow‐up is both expensive and unpleasant (frequent invasive cystoscopies). Molecular biomarkers, including microRNAs have been studied to provide additional prognostic information for these patients, but to date no molecular biomarker has become the ‘gold standard’ for patient diagnosis and follow‐up. We used Rosetta Genomics’ highly specific microRNA expression profiling platforms to study the prognostic role of microRNAs in bladder cancer. Using microdissection we chose specific tumour microRNAs to study in order to avoid background contamination. Tumour progression was associated with altered levels of microRNAs. In particular, high expression levels of miR‐29c* were associated with a good prognosis. The study found that the use of microRNAs for determining progression and invasiveness for patients with urothelial cancer could potentially have a substantial impact on the treatment and follow‐up individual patients.

MicroRNA-486-5p is an erythroid oncomiR of the myeloid leukemias of Down syndrome.

Children with Down syndrome (DS) are at increased risk for acute myeloid leukemias (ML-DS) characterized by mixed megakaryocytic and erythroid phenotype and by acquired mutations in the GATA1 gene resulting in a short GATA1s isoform. The chromosome 21 microRNA (miR)-125b cluster has been previously shown to cooperate with GATA1s in transformation of fetal hematopoietic progenitors. In this study, we report that the expression of miR-486-5p is increased in ML-DS compared with non-DS acute megakaryocytic leukemias (AMKLs). miR-486-5p is regulated by GATA1 and GATA1s that bind to the promoter of its host gene ANK1. miR-486-5p is highly expressed in mouse erythroid precursors and knockdown (KD) in ML-DS cells reduced their erythroid phenotype. Ectopic expression and KD of miR-486-5p in primary fetal liver hematopoietic progenitors demonstrated that miR-486-5p cooperates with Gata1s to enhance their self renewal. Consistent with its activation of AKT, overexpression and KD experiments showed its importance for growth and survival of human leukemic cells. Thus, miR-486-5p cooperates with GATA1s in supporting the growth and survival, and the aberrant erythroid phenotype of the megakaryocytic leukemias of DS.

Multicentre validation of a microRNA-based assay for diagnosing indeterminate thyroid nodules utilising fine needle aspirate smears

Aims The distinction between benign and malignant thyroid nodules has important therapeutic implications. Our objective was to develop an assay that could classify indeterminate thyroid nodules as benign or suspicious, using routinely prepared fine needle aspirate (FNA) cytology smears. Methods A training set of 375 FNA smears was used to develop the microRNA-based assay, which was validated using a blinded, multicentre, retrospective cohort of 201 smears. Final diagnosis of the validation samples was determined based on corresponding surgical specimens, reviewed by the contributing institute pathologist and two independent pathologists. Validation samples were from adult patients (≥18 years) with nodule size >0.5 cm, and a final diagnosis confirmed by at least one of the two blinded, independent pathologists. The developed assay, RosettaGX Reveal, differentiates benign from malignant thyroid nodules, using quantitative RT-PCR. Results Test performance on the 189 samples that passed quality control: negative predictive value: 91% (95% CI 84% to 96%); sensitivity: 85% (CI 74% to 93%); specificity: 72% (CI 63% to 79%). Performance for cases in which all three reviewing pathologists were in agreement regarding the final diagnosis (n=150): negative predictive value: 99% (CI 94% to 100%); sensitivity: 98% (CI 87% to 100%); specificity: 78% (CI 69% to 85%). Conclusions A novel assay utilising microRNA expression in cytology smears was developed. The assay distinguishes benign from malignant thyroid nodules using a single FNA stained smear, and does not require fresh tissue or special collection and shipment conditions. This assay offers a valuable tool for the preoperative classification of thyroid samples with indeterminate cytology.

The role of microRNA profiling in prognosticating progression in Ta and T1 urinary bladder cancer

OBJECTIVE To analyze microRNA profile in Ta and T1 urinary bladder cancers in combination and separately and to relate this to the risk of later developing higher-stage disease. MATERIALS AND METHODS Formalin-fixed, paraffin-embedded samples of 44 Ta and 42 T1 bladder cancers representing cases with and without stage progression during follow-up were collected and microRNA expression levels were measured by microarray analysis. RESULTS In a comparison between the progressors and controls, in the Ta/T1 group, miR-10a-5p and miR-31-5p were differentially expressed. miR-10a-5p was also correlated to time to progression (P = 0.00012). In the subgroup analysis, 3 microRNAs, miR-10a-5p, miR-31-5p, and miR-130a-3p, were differentially expressed among Ta tumors and had a fold change of more than 1.5 (P<0.038). The comparison concerning microRNA expression between the progressors and controls in category T1 cancers revealed no significant differences. CONCLUSIONS Profiling revealed that certain microRNAs predicted the risk of developing higher-stage disease among patients with Ta cancers. Lower miR-10a-5p expression in Ta progressing tumors indicates that this microRNA could be important for later malignant potential among this group of patients.

Risk Score Model for Predicting Mortality in Advanced Heart Failure Patients Followed in a Heart Failure Clinic

The prevalence of heart failure (HF) in the population is increasing, concomitant with high incidence of rehospitalizations and mortality. The aim of this study was to characterize a prognostic risk score model for patients with chronic HF. A total of 500 patients followed at the HF clinic were evaluated by clinical, functional, laboratory, imaging, and therapeutic variables that were correlated to mortality during a follow-up period of 25 months. Risk stratification was carried out by applying a risk score model based on multivariate analysis. Predictors correlated with mortality during follow-up were systolic blood pressure <110 mm Hg, male sex, age older than 70 years, 6-minute walk distance <300 m, lack of β-blocker therapy, hyperuricemia (>7.5 mg/dL), hyponatremia, and prolonged QTc interval (>450 ms). Based on these variables, a risk score model (score 0-55) was established and included low risk, score <21 (9% mortality during 2-year follow-up); moderate risk, 21 to 29 (22%); high risk, 30 to 35 (35%), and very high risk: ≥36 points (62% 2-year mortality). The risk model had good discrimination ability (concordance index 0.75), which was better than the performance of the Seattle Heart Failure Model on our cohort (0.69). Simple noninvasive characteristics examined during the initial admission to the HF clinic can serve as prognostic markers for mortality and may help in the process of therapeutic decision-making in patients with HF.

Abstract 3017: MicroRNAs as prognostic indicators in gastric cancer

Background: Our ability to determine the prognosis of an individual patient with gastric cancer and thereby to select patients for adjuvant therapy is still limited. Preliminary data suggest that microRNAs may have a prognostic role in this disease. We hence decided to compare the microRNA profiles in the primary tumor of patients with recurrent and non-recurrent gastric cancer and to evaluate the prognostic impact of these molecules. Patients and Methods: Eligible patients, who underwent curative gastrectomies between 1995 and 2005 and did not receive any pre- or postoperative adjuvant therapy, were identified from the database of the participating institutions. Total RNA was extracted from tumor formalin-fixed paraffin embedded (FFPE) samples using proprietary protocols that preserve the small RNA fraction. Initial profiling using microRNA microarrays identified potential microRNA biomarkers that can be used to predict recurrence of gastric cancer after resection. The expression of differential microRNAs, was verified by qRT-PCR. Results: Forty-five eligible patients had adequate tumor content and microRNA expression data and were included in the analysis. Of these, 14 (31%) experienced recurrence of disease within three years from surgery (“bad prognosis” group) and 31 (69%) did not (“good prognosis” group). Advanced pathological stage and extensive surgical procedure correlated with bad prognosis. Signal levels of three microRNAs were found to be significantly differentially expressed in tumors from patients with good prognosis vs. patients with bad prognosis. High expression of each of these microRNAs was associated with significantly poorer prognosis for both recurrence and survival. Proprietary microRNA qRT-PCR showed a high correlation to microarrays and similar separation into prognosis groups based on microRNA expression. Conclusion: We identified a set of microRNAs whose high expression was predictive of tumor recurrence and poor prognosis in patients with gastric cancer. This finding provides a basis for a novel tool for risk stratification in this disease and allows further insight into its pathogenesis. Based on these results, a validation study, on a larger and independent cohort of patients, is planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3017.