Abstract 5564: Molecular profiling of RTOG 9813: Optimization of a pilot study to identify novel molecular biomarkers for anaplastic astrocytomas
Authors: McNulty Maureen , Bell Erica , Chakravarti Arnab 
Published: August 2012

Abstract
Anaplastic gliomas (WHO grade III) are a histologically defined and occurring subtype of glioma with a median survival of less than 3 years. In general, the histological classification and grading of tumors, combined with clinical prognostic features, guides treatment decisions. However, the optimum treatment for anaplastic gliomas remains controversial. Treatment identification is made even more difficult as histological classification of gliomas is troublesome and subject to a large (20-30%) degree of interobserver-variation. Because anaplastic gliomas are histologically and molecularly diverse, there is a clinical need to objectively identify prognostically relevant subtypes and subtypes that will respond to treatment. Therefore, we have sought to use clinical trial tissue, specifically from the Radiation Therapy Oncology Group (RTOG), to identify much needed prognostic and predictive markers for anaplastic gliomas. In particular, we have focused on RTOG 9813, a closed trial that assessed for the benefit of TMZ compared to nitrosourea in combination with radiation. We are currently performing high-throughput screening technologies, such as SNP/CN genotyping, miRNA profiling and methylation profiling. Unfortunately, clinical samples are formalin-fixed and embedded in paraffin (FFPE), resulting in partially degraded RNA and DNA. Due to the lack of literature currently about digestion of FFPE cores, which are preferred to scrolls from CNS tumors due to necrosis and other non-tumor heterogeneity, we have successfully optimized nucleic acid isolation protocols for FFPE cores to determine which procedure maximizes recovery while retaining the quality required for our downstream assays. Specifically, we determined that for DNA isolation using a combination of the Ambion RecoverAll kit with the Epicentre MasterPure protocol increases our yield 2-4X more from a 1mm core than using the Ambion RecoverAll or Epicentre MasterPure kits alone as well as other non-kit methods. Additionally, the 260/280 and 260/230 ratios were high and most consistent using the combined protocol. For RNA isolation, a traditional Trizol method gave us the best yield, but a combination of the Ambion's RecoverAll digestion buffer and the Qiagen RNeasy FFPE Kit gave us the highest yield while maintaining the 260/280 and 260/230 ratios needed for our downstream assay for miRNA (Nanostring). We will present the optimized method for both the DNA and RNA isolations from 1mm FFPE cores as well as preliminary correlative data from anaplastic astrocytoma tissue using these isolation techniques in combination with the Affymetrix SNP Array 6.0 and Nanostring miRNA expression analysis. Our ultimate goal is to develop a diagnostic tool that will help clinical decision making by further characterizing subpopulations of anaplastic gliomas that may be used for therapeutic purposes.
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 5564. doi:1538-7445.AM2012-5564