Research Project
7293253
[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Aside for a vast minority of asymptomatic patients diagnosed incidentally, virtually all lung cancer patients are symptomatic at presentation. As a result only 30-35% of patients have sufficiently localized tumors to allow for surgical resection of the tumor. Cancers over expressing the epidermal growth factor receptor (EGFR) have been shown to increase resistance to chemotherapy, thus increasing the risk of metastases. Biologics targeting the tyrosine kinase (TKI) domain of EGFR are being developed, and are currently at various stages of clinical testing. Therapeutic inhibition of EGFR has resulted in significant tumor regressions in only 10% to 20% of patients. Sensitivity to EGFR inhibitors is largely dependent on the presence of somatic mutations in EGFR, thus indicating the need for a simple diagnostic method to identify patients susceptible to these drugs. Virtually all patients with EGFR activating mutations who clinically respond to the TKI, gefitinib or erlotinib, develop resistance to these agents. The emergence of a secondary mutation in EGFR (T790M), accounts 50% of the cases of resistance. Given that other EGFR inhibitors may be effective treatments in such patients, there is a critical need to accurately and efficiently identify these patients for these treatments. Since repeated tumor biopsies from such patients are often difficult (due to thoracic location of tumor which increases the chance of lung collapse with a needle biopsy), the development of a non-invasive method to assay for the emergence of resistance would be a significant advance. We believe the best approach to solve this problem is to assay circulating epithelial cells (CEC) for these mutations. [unreadable] [unreadable] We propose to develop assays for the key EGFR mutations influencing response to TKI. The assays we propose to develop will be able to identify the emergence of secondary EGFR mutations in patients. These assays would use total nucleic acids isolated from blood, and a novel allele specific amplification technology with a reported specificity of 10-7. This extraordinary specificity is possible because oligonucleotides with a 3' dideoxy terminated nucleotide that is not matched to its template DNA target are not efficiency activated by pyrophosphorolysis while oligonucleotides whose 3' ends are perfectly matched to their template can be activated. Pyrophosphorolysis, the reverse of the DNA polymerization reaction, therefore activates the polymerization of DNA only if the 3'end is matched to its target; hence the name pyrophosphorolysis-activated polymerization (PAP) for this technology developed by the City of Hope hospital. Not all DNA polymerases efficiently perform PAP. For, example a modified Taq polymerase bearing a F667Y mutation that greatly enhances the polymerase's affinity for dideoxy terminated oligonucelotides (ddNTP) (US patent 5,614,365) is more efficient at performing PAP than the wild-type polymerase. We have performed preliminary tests with a Bacillus stearothermophilus (Bst) polymerase mutant (F712Y) with similar properties. In addition, some native enzymes (like the bacteriophage T7 DNA polymerase) have reduced specificity for deoxynucleotide triphosphates (dNTP) and may thus be ideal for PAP. In Phase I, we will investigate these alternative polymerases in pyrophosphorolysis-activated polymerization, helicase dependent amplification (papHDA). Both Bst polymerase and T7 polymerase have been reported to function in HDA. In Phase I we propose to focus on developing assays targeting a missense mutation in EGFR, L858R, is the second most common EGFR mutation. A secondary mutation in EGFR (T790M) can drastically reduce this sensitivity even in patients with the responsive allele. Another EGFR inhibitor, CL-387,785, which binds to Cys-773, is still able to inhibit EGFR phosphorylation in the presence of the T790M secondary mutation, therefore detecting patients with gefitinib or erlotinib sensitive alleles and the T790M will be clinically relevant. In Phase II, we will also develop assays targeting the most common mutations conferring sensitivity to TKI; i.e., small deletions in Exon 19 of EGFR (e.g., Del747-749) and the G719C, L861Q point mutations. Patients with the deletions are particularly responsive to TKI. It is expected this innovation would facilitate the implementation of mutation screening in treating lung cancer patients. [unreadable] [unreadable] [unreadable]
