Noga Yerushalmi

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.

hsa-miR-191 Is a Candidate Oncogene Target for Hepatocellular Carcinoma Therapy

Hepatocellular carcinoma (HCC) is generally a fatal disease due to a paucity of effective treatment options. The identification of oncogenic microRNAs that exert pleiotropic effects in HCC cells may offer new therapeutic targets. In this study, we have identified the human microRNA miR-191 as a potential target for HCC therapy. Inhibition of miR-191 decreased cell proliferation and induced apoptosis in vitro and significantly reduced tumor masses in vivo in an orthotopic xenograft mouse model of HCC. Additionally, miR-191 was found to be upregulated by a dioxin, a known liver carcinogen, and was found to be a regulator of a variety of cancer-related pathways. Our findings offer a preclinical proof of concept for miR-191 targeting as a rational strategy to pursue for improving HCC treatment.

Discovery of microRNAs and other small RNAs in solid tumors

MicroRNAs (miRNAs) are ∼22-nt long, non-coding RNAs that regulate gene silencing. It is known that many human miRNAs are deregulated in numerous types of tumors. Here we report the sequencing of small RNAs (17–25 nt) from 23 breast, bladder, colon and lung tumor samples using high throughput sequencing. We identified 49 novel miRNA and miR-sized small RNAs. We further validated the expression of 10 novel small RNAs in 31 different types of blood, normal and tumor tissue samples using two independent platforms, namely microarray and RT–PCR. Some of the novel sequences show a large difference in expression between tumor and tumor-adjacent tissues, between different tumor stages, or between different tumor types. We also report the identification of novel small RNA classes in human: highly expressed small RNA derived from Y-RNA and endogenous siRNA. Finally, we identified dozens of new miRNA sequence variants that demonstrate the existence of miRNA-related SNP or post-transcriptional modifications. Our work extends the current knowledge of the tumor small RNA transcriptome and provides novel candidates for molecular biomarkers and drug targets.

Novel microRNA-based assay demonstrates 92% agreement with diagnosis based on clinicopathologic and management data in a cohort of patients with carcinoma of unknown primary

BackgroundCancer of unknown or uncertain primary is a major diagnostic and clinical challenge, since identifying the tissue-of-origin of metastases is crucial for selecting optimal treatment. MicroRNAs are a family of non-coding, regulatory RNA molecules that are tissue-specific, with a great potential to be excellent biomarkers.MethodsIn this study we tested the performance of a microRNA-based assay in formalin-fixed paraffin-embedded samples from 84 CUP patients.ResultsThe microRNA based assay agreed with the clinical diagnosis at presentation in 70% of patients; it agreed with the clinical diagnosis obtained after patient management, taking into account response and outcome data, in 89% of patients; it agreed with the final clinical diagnosis reached with supplemental immunohistochemical stains in 92% of patients, indicating a 22% improvement in agreement from diagnosis at presentation to the final clinical diagnosis. In 18 patients the assay disagreed with the presentation diagnosis and was in agreement with the final clinical diagnosis, which may have resulted in the administration of more effective chemotherapy. In three out of four discordant cases in which supplemental IHC was performed, the IHC results validated the assay’s molecular diagnosis.ConclusionsThis novel microRNA-based assay shows high accuracy in identifying the final clinical diagnosis in a real life CUP patient cohort and could be a useful tool to facilitate administration of optimal therapy.

Restoring the oncosuppressor activity of microRNA-34a in glioblastoma using a polyglycerol-based polyplex

Glioblastoma multiforme (GBM) is the most common and aggressive primary neoplasm of the brain. Poor prognosis is mainly attributed to tumor heterogeneity, invasiveness, and drug resistance. microRNA-based therapeutics represent a promising approach due to their ability to inhibit multiple targets. In this work, we aim to restore the oncosuppressor activity of microRNA-34a (miR-34a) in GBM. We developed a cationic carrier system, dendritic polyglycerolamine (dPG-NH2), which remarkably improves miRNA stability, intracellular trafficking, and activity. dPG-NH2 carrying mature miR-34a targets C-MET, CDK6, Notch1 and BCL-2, consequently inhibiting cell cycle progression, proliferation and migration of GBM cells. Following complexation with dPG-NH2, miRNA is stable in plasma and able to cross the blood–brain barrier. We further show inhibition of tumor growth following treatment with dPG-NH2–miR-34a in a human glioblastoma mouse model. We hereby present a promising technology using dPG-NH2–miR-34a polyplex for brain-tumor treatment, with enhanced efficacy and no apparent signs of toxicity.

Abstract B05: Patient-derived GBM tumors versus patient-derived primary cells and GBM cell lines: lessons from microRNA profiling

Glioblastoma multiforme is a very heterogeneous tumor, highly infiltrative, angiogenic and resistant to standard therapeutic intervention. Experimental models currently used in research are mostly based on cancer cell lines, which are grown in culture for many generations and have lost many essential biological characteristics. In consequence, the xenograft tumor models derived from those cells do not maintain genomic and phenotypic characteristics present in the original tumor. It is questionable whether these artificial preclinical models can serve as reliable platforms to select the lead candidate for a novel therapeutic approach. We utilized miRNA expression patterns to evaluate the clinical relevance of some of the currently available experimental models of GBM, searching for similarities and differences in miRNA expression levels between freshly isolated tumors, patient-derived primary cells and GBM cell lines grown in culture. The study included 22 formalin-fixed paraffin-embedded (FFPE) tumors from glioblastoma resections. Those were divided into two groups of Short Term Survivors (STS, survived up to 3 months from initial diagnosis; n=12) and Long Term Survivors (LTS, survived more than 3 years from initial diagnosis; n=10).We further tested 6 patient-derived primary cells, and 5 ATCC human GBM cell lines widely used in preclinical research. Two of the cell lines included dormant and fast-growing variants previously developed in our laboratory to resemble LTS and STS phenotypes, respectively. Samples were loaded on custom microRNA (miR) arrays, including 2172 known miRNAs (miRBase 19). We found several miRNAs that were upregulated in the STS samples while others exhibited higher expression levels in the LTS samples. Both GBM cell lines and patient-derived primary cells exhibited a miRNA profile which was very different from patient samples. Brain miRNAs used as brain markers, such as hsa-miR-124-3p), show relatively low expression levels in both patient-derived primary cells and commercial cell lines but are highly expressed in tumors. We concluded that GBM experimental models must be reevaluated and pre-clinical findings derived from long-established and commonly used in vitro and in vivo models should be further validated in patient-derived models that are more suitable to evaluate the potential clinical relevance of new therapeutics. Citation Format: Paula Ofek, Nir Dromi, Noga Yerushalmi, Sharon Kredo-Russo, Rachel Grossman, Zvi Ram, Ronit Satchi-Fainaro. Patient-derived GBM tumors versus patient-derived primary cells and GBM cell lines: lessons from microRNA profiling. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B05.

Abstract 802: Development and validation of a microRNA-based diagnostic assay for the classification of renal cell tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Renal cancers account for more than 3% of adult malignancies and result in more than 13,000 deaths per year in the US alone. The four most common types of kidney tumors include the malignant renal cell carcinomas: clear cell, papillary and chromophobe, as well as the benign oncocytoma. These histological subtypes vary in their clinical course and their prognosis, and different clinical strategies have been developed for their management. The differential diagnosis between the subtypes of kidney tumors based on morphology alone can be challenging, and is subjected to inter- and intra-observer variability. Even when utilizing immunohistochemistry (IHC) markers, the ability to differentiate between sub-types can be difficult, especially in the setting of uncommon morphology and biopsy sample with small amounts of tumor tissue. We present the development and validation of a microRNA-based test for classifying primary kidney tumors. Methods: 181 Formalin Fixed Paraffin Embedded (FFPE) samples from primary kidney tumors were collected and reviewed by pathologists from different institutes according to morphology and available IHC labeling data. High-quality total RNA, including the well-preserved microRNA fraction, was extracted from the FFPE samples using a proprietary protocol. Expression levels of hundreds of microRNAs were profiled using a custom microarray platform. Technical validation of the array results was performed using qRT-PCR. A diagnostic assay was developed using a K-nearest neighbor algorithm that searches for the 5 samples in the training database (181 samples used for assay development) that are most similar to the tested sample. The result for the tested sample is defined by a majority vote of the pre-determined subtypes of these 5 closest neighbors. A validation set of 201 independent samples was classified using the assay and analyzed blindly. Results: A set of 24 differentially expressed microRNAs were found to separate the four kidney tumor subtypes and were chosen as classifiers in the KNN algorithm. Clinical validation was performed using an independent, blinded sample set. The test was able to produce results for 92% of the validation set of 201 samples with accuracy of 95%. Conclusions: Expression levels of 24 microRNAs measured on a microarray platform were found to accurately differentiate the four main types of primary kidney tumors. These findings were the basis for the development and validation of a standardized diagnostic assay for the classification of renal cell tumors in FFPE samples from resections or biopsies. This assay can serve as a reliable diagnostic tool to aid physicians with the growing unmet need for kidney tumor classification. Citation Format: Robert Wassman, Brianna St. Cyr, Eddie Fridman, Iris Barshack, Yajue Huang, Sofia Zilber, Mats Sanden, Hila Benjamin, Noga Yerushalmi, Yael Spector. Development and validation of a microRNA-based diagnostic assay for the classification of renal cell tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 802. doi:10.1158/1538-7445.AM2013-802