Gregory Riggins - US grants

serial analysis of gene expression; glioma; neoplasm /cancer classification /staging; molecular oncology; neoplasm /cancer genetics; nucleic acid sequence; prognosis; cooperative study; gene expression; high throughput technology; microarray technology; polymerase chain reaction; human subject; clinical research;

DESCRIPTION (provided by applicant): The main goal of this work is to locate and evaluate molecular targets for brain cancers, starting with glioblastoma. This project serves as a pilot for larger cancer re-sequencing projects. We have teamed with the Venter Institute and its associated Joint Technology Center to perform state-of-the-art and low cost sequencing using their SNP detection pipeline. For our first specific aim, we will re-sequence all known kinase domains in the cancer genomes of 30 glioblastomas. Once a kinase domain mutation is detected we expand our analysis to the entire gene in 50 additional glioblastomas and a panel of other brain cancers, including pediatric glioblastomas and medulloblastomas. In our preliminary analysis of the first 40 kinase genes, novel tyrosine receptor kinase mutations were found in FGFR1 and PDGFRA. We have also found additional mutations in PIK3CA in adult and pediatric glioblastoma. In specific aim 2, we propose to create a high-resolution copy number map of our 30 glioblastomas using Digital Karyotyping, so we can evaluate in the same samples point mutations, amplifications and deletions. We have completed Digital Karyotyping in 8 glioblastomas, and previously published on using this powerful technique to find a developmental gene genomic amplification in medulloblastoma. In specific aim 3 we will functionally evaluate the mutations we find with frequency greater than 10%, starting with two kinase mutations we have already found in glioblastoma. We will create a model system of the mutation in cell lines and determine the target of altered phosphorylation and if the cell-cycle, apoptosis and/or invasion are altered. To ensure rapid reporting and integration of our work into larger efforts we propose an online database and reporting for our fourth and final specific aim. Glioblastomas have poor survival and new treatments are needed. Our long-term goal is to choose the best molecular targets from this systematic analysis and determine if inhibition of these new mutations will be a successful therapeutic strategy.

[unreadable] DESCRIPTION (provided by applicant): Our long term goal is to develop a more accurate test that can preoperatively distinguish a benign from a malignant thyroid nodule. Currently, fine-needle aspiration (FNA) cytology is the best non-surgical diagnostic tool for evaluating a thyroid nodule. However, approximately 30% of all nodules are classified as suspicious. Because the current method is based on morphological features, and fails to improve the sensitivity and specificity, it remains a frequent clinical problem with challenges in particular to the pathologist. To address this problem, we previously performed gene expression profiling both a follicular thyroid adenoma (FTA), and a follicular thyroid carcinoma (FTC). This profiling and subsequent analysis revealed that four novel markers (DDIT3, ARG2, C1orf24 and ITM1) differed between the two tumors and a linear combination of expression levels distinguished FTC from FTA with an estimated predictive accuracy of 0.83. Commercially available antibodies for DDIT3 and ARG2 were used for further validation in an independent set of FTA and FTC paraffin-embedded sections. Sensitivity and specificity were 85% and 91% respectively for each antibody. Using the two antibodies in combination did not improve the estimates. We custom produced antibodies for C1or24 and ITM1 and tested them in the aforementioned set of FTA and FTC sections. We achieved a sensitivity of 100%. Furthermore, these novel markers can reliably classify other thyroid lesions into benign or malignant classes. Our hypothesis is that gene expression profiling will locate novel diagnostic markers that can improve the specificity of the test. We will use Serial Analysis of Gene Expression (SAGE), to profile Hurthle cell adenoma which was originally misclassified as malignant and, therefore, predicted to improve the specificity of preoperative diagnosis test. Our next goal is to develop a test that can be routinely used as an adjuvant with FNA cytology. To achieve this goal, we propose in R33 phase of this application to evaluate the effectiveness of both antibody and quantitative RT-PCR tests to diagnose thyroid nodules. To assess the accuracy and feasibility of both tests, the results from these analyses will be compared with final histology. The development of an innovative and more accurate preoperative diagnostic test for evaluating thyroid nodules will not only result in progress in cancer diagnosis but will also improve treatment decisions while reducing long-term health costs. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Driver mutations in Isocitrate Dehydrogenase 1 (IDH1) are present in 70-80% of grade II and III gliomas, which the majority eventually progress to glioblastoma multiforme (GBM). In this molecularly distinct class of malignant gliomas, mutant IDH1 enzyme produces 2-hydroxyglutarate (2-HG), an oncometabolite that inhibits a-ketoglutarate dependent histone and DNA demethylases resulting in characteristic hypermethylation of genomic DNA and suppression of cellular differentiation. We have demonstrated the preclinical efficacy and mechanism of action of the FDA approved DNA demethylating drug 5-azacytidine. In molecularly accurate models, systemic 5-azacytidine administration reduces tumor burden, extends survival and induce differentiation in vivo. For this work we have created our own patient derived model of IDH1 mutant anaplastic astrocytoma, and have additionally obtained models of IDH1 mutant grade III oligodendroglioma and oligoastrocytoma from our collaborators. The focus of this grant is to optimize and understand the mechanism of DNA demethylation therapy for the treatment of IDH1 mutant glioma. In Aim 1 we propose to demonstrate the mechanism and efficacy of 5-azacytidine induced tumor regression in orthotopic IDH1 mutant glioma systems beyond our preliminary findings. In preclinical modeling, we will optimize the mode of administration, confirm intracranial delivery, and survival benefit. To confirm the in vivo mechanism we will show target inactivation, differentiation, and key alterations in transcription and methylation. In Aim 2 we will confirm the synergistic benefit of combining 5-azacytidine with the current standard of care for high grade gliomas. Additionally, we will assess the mechanism of tumor regression in these tumors including alterations in apoptosis and differentiation. In Aim 3 we will perform a preclinical evaluation of synergistic combination of 5-azacytidine with IDH1 mutant protein inhibitors which are currently in clinical trials. Here we will determine if we can achieve a faster and more favorable therapeutic response by halting pathogenic 2-HG production while reversing pathogenic hypermethylation. Additionally, we will assess in depth the mechanism of successful therapy for inhibition of mutant IDH1 protein plus demethylation by 5- azacitidine. Overall, our goal is to elucidate the mechanism of 5-azacytidine induced tumor regression in IDH1 mutant tumors and to ascertain the most effective therapeutic strategy in vivo. Our preliminary results are promising, but optimization of drug administration, synergistic combinations as well as a more in-depth and mechanistic approach is needed to increase the chances that the work will translate successfully to the clinic. The results from this work will allow us to better design and possibly support a new trial for patients with IDH1 mutant glioma, which account for a substantial fraction of brain cancer mortality.

? DESCRIPTION (provided by applicant): Driver mutations in Isocitrate Dehydrogenase 1 (IDH1) are present in 70-80% of grade II and III gliomas, which the majority eventually progress to glioblastoma multiforme (GBM). In this molecularly distinct class of malignant gliomas, mutant IDH1 enzyme produces 2-hydroxyglutarate (2-HG), an oncometabolite that inhibits a-ketoglutarate dependent histone and DNA demethylases resulting in characteristic hypermethylation of genomic DNA and suppression of cellular differentiation. We have demonstrated the preclinical efficacy and mechanism of action of the FDA approved DNA demethylating drug 5-azacytidine. In molecularly accurate models, systemic 5-azacytidine administration reduces tumor burden, extends survival and induce differentiation in vivo. For this work we have created our own patient derived model of IDH1 mutant anaplastic astrocytoma, and have additionally obtained models of IDH1 mutant grade III oligodendroglioma and oligoastrocytoma from our collaborators. The focus of this grant is to optimize and understand the mechanism of DNA demethylation therapy for the treatment of IDH1 mutant glioma. In Aim 1 we propose to demonstrate the mechanism and efficacy of 5-azacytidine induced tumor regression in orthotopic IDH1 mutant glioma systems beyond our preliminary findings. In preclinical modeling, we will optimize the mode of administration, confirm intracranial delivery, and survival benefit. To confirm the in vivo mechanism we will show target inactivation, differentiation, and key alterations in transcription and methylation. In Aim 2 we will confirm the synergistic benefit of combining 5-azacytidine with the current standard of care for high grade gliomas. Additionally, we will assess the mechanism of tumor regression in these tumors including alterations in apoptosis and differentiation. In Aim 3 we will perform a preclinical evaluation of synergistic combination of 5-azacytidine with IDH1 mutant protein inhibitors which are currently in clinical trials. Here we will determine if we can achieve a faster and more favorable therapeutic response by halting pathogenic 2-HG production while reversing pathogenic hypermethylation. Additionally, we will assess in depth the mechanism of successful therapy for inhibition of mutant IDH1 protein plus demethylation by 5- azacitidine. Overall, our goal is to elucidate the mechanism of 5-azacytidine induced tumor regression in IDH1 mutant tumors and to ascertain the most effective therapeutic strategy in vivo. Our preliminary results are promising, but optimization of drug administration, synergistic combinations as well as a more in-depth and mechanistic approach is needed to increase the chances that the work will translate successfully to the clinic. The results from this work will allow us to better design and possibly support a new trial for patients with IDH1 mutant glioma, which account for a substantial fraction of brain cancer mortality.