Abstract 3626: Quantitative analysis of microRNAs by in situ hybridization reveals prognostic value of miRNAs in melanoma and breast cancer

Abstract

Abstract Introduction: Most miRNA measurement methods require total RNA extraction which destroys spatial information and makes standardization a challenge. In situ hybridization (ISH) however, allows the direct assessment of miRNA expression levels in tissue and importantly allows for the evaluation of expression in malignant cells as well as stromal cells. We have developed and validated a novel method for the quantitative analysis of miRNA expression by ISH on formalin fixed paraffin embedded tissue microarrays (TMAs) using miR-21, miR-92a, miR-34a, miR-221, miR-205, and Let-7a. Methods: In order to quantitatively measure miRNA expression by ISH, DIG labeled LNA modified miRNA probes were employed and multiplexed with DAPI and cytokeratin or S100/GP100 immunofluorescence to apply the AQUA technology. This co-localization based, quantitative approach allows for the measurement of miRNA expression in subcellular compartments. The assay allows standardization of miRNA measurement and potential for rapid large cohort assessment. We also assessed protein targets of each miRNA by AQUA to investigate the hypothesized inverse relationship between miRNAs and target proteins. Results: In hundreds of tissue samples a broad dynamic range and a variable expression pattern specific to each miRNA was observed. Specificity of the assay was validated with anti-miR and miRNA mimic transfected cell lines, miR-21 knock out mouse tissue, and blocking oligo experiments. The assays were performed on near serial sections and were found to have high reproducibility with R*2>0.8. miR-221 and miR-205 were found to be prognostic in breast cancer and melanoma respectively (p<0.05). Finally, the inverse relationship between miRNAs and putative targets were not seen in the large population cohorts. Conclusions: This specific and reproducible method for the quantitative analysis of miRNA expression provides proof of concept for the use of miRNAs as tissue biomarkers using quantitative ISH. Future studies will further determine the prognostic value of miR-221 and miR-205, and the relationship between miRNAs and target protein expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3626. doi:1538-7445.AM2012-3626