Pyrophosphorolysis-activatable helicase dependent amplification assay for lung ca

肺癌的焦磷酸解激活解旋酶依赖性扩增测定

• 批准号: 7293253
• 负责人: BERTRAND LEMIEUX
• 金额: $ 14.13万
• 依托单位: BIOHELIX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-05 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7293253
• 关键词: Accounting; Affinity; Alleles; Bacillus stearothermophilus; Bacteriophage T7; Binding; Biological Assay; Biopsy; Blood; CL 387785; Cancer Patient; Cell Line; Cells; Chest; Cities; Clinical; Clinical Research; Clinical Trials; Collaborations; Condition; DNA; DNA amplification; DNA-Directed DNA Polymerase; Dana-Farber Cancer Institute; Detection; Development; Diagnosis; Diagnostic Procedure; Disease regression; Enzymes; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Epithelial Cells; Erlotinib; Excision; Exons; Gefitinib; Generations; HKI272; Hospitals; Invasive; Legal patent; Leukocytes; Licensing; Localized; Location; Lung; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Methods; Minority; Missense Mutation; Mutation; Names; Neoplasm Metastasis; Non-Small-Cell Lung Carcinoma; Nucleic Acids; Nucleotides; Oligonucleotides; Operative Surgical Procedures; Patients; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phosphorylation; Point Mutation; Polymerase; Polymerase Chain Reaction; Problem Solving; Property; Protein Tyrosine Kinase; Puncture biopsy; Rate; Reaction; Relative (related person); Reporting; Resistance; Resistance development; Risk; Sampling; Screening procedure; Somatic Mutation; Specificity; Staging; TP53 gene; Taq Polymerase; Techniques; Technology; Temperature; Testing; Tetradecanoylphorbol Acetate; Therapeutic; Tyrosine Kinase Domain; United States Food and Drug Administration; c-erbB-1 Proto-Oncogenes; chemotherapy; clinically relevant; cold temperature; helicase; innovation; mutant; novel; polymerization; research clinical testing; resistance mechanism; response; tripolyphosphate; tumor

DESCRIPTION (provided by applicant): 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. 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.

描述（由申请人提供）： 除了偶然被诊断出的无症状患者外，几乎所有肺癌患者在表现上都是有症状的。结果，只有30-35％的患者患有足够的局部肿瘤以允许对肿瘤进行手术切除。癌症超过表达表皮生长因子受体（EGFR）的癌症已被证明会增加对化学疗法的抗性，从而增加转移的风险。针对EGFR的酪氨酸激酶（TKI）结构域的生物制剂正在开发，目前处于临床测试的各个阶段。 EGFR的治疗抑制作用导致仅10％至20％的患者导致了显着的肿瘤回归。对EGFR抑制剂的敏感性在很大程度上取决于EGFR中体细胞突变的存在，因此表明需要一种简单的诊断方法来鉴定患者敏感这些药物。几乎所有患有EGFR激活突变的患者对TKI，Gefitinib或Erlotinib临床反应，都会对这些药物产生抗性。 EGFR（T790M）中继发突变的出现，占抵抗病例的50％。鉴于其他EGFR抑制剂在此类患者中可能是有效的治疗方法，因此需要准确有效地识别这些患者的这些治疗方法。由于这种患者的重复肿瘤活检通常很困难（由于肿瘤的胸腔位置，通过针头活检增加了肺塌陷的机会），因此开发了一种非侵入性方法来测定抗性出现的方法将是一个重大进展。我们认为解决此问题的最佳方法是分析这些突变循环上皮细胞（CEC）。 我们建议开发有关影响对TKI反应的关键EGFR突变的测定。我们建议开发的测定方法将能够确定患者次级EGFR突变的出现。这些测定法将使用从血液中分离出来的总核酸，以及一种新型的等位基因特异性扩增技术，其特异性为10-7。这种非凡的特异性是可能的，因为具有3'二氧基终止核苷酸的寡核苷酸与其模板DNA靶标不匹配的寡核苷酸并非被焦磷酸化的效率激活，而3'端的寡核苷酸则与模板完美匹配。 DNA聚合反应的逆磷酸解析，因此，仅当3'End与其靶标匹配时，DNA的聚合才能激活DNA的聚合；因此，这项由Hope Hospital开发的这项技术的焦磷酸解激活聚合（PAP）的名称。并非所有DNA聚合酶都有效地执行PAP。例如，例如，具有F667Y突变的修饰的TAQ聚合酶，可极大地增强聚合酶对二维氧基终止寡核苷酸（DDNTP）（DDNTP）（US PATENT 5,614,365）的亲和力比野生型聚合酶在表演PAP上更有效。我们已经使用具有相似特性的芽孢杆菌（BST）聚合酶突变体（F712Y）进行了初步测试。此外，某些天然酶（例如噬菌体T7 DNA聚合酶）对脱氧核苷酸三磷酸盐（DNTP）的特异性降低，因此可能是PAP的理想选择。在第一阶段，我们将研究这些替代聚合酶在焦磷酸分解激活的聚合，解旋酶依赖性扩增（PAPHDA）中。据报道，BST聚合酶和T7聚合酶在HDA中起作用。在第一阶段，我们建议专注于开发针对EGFR中的错义突变的测定法，L858R是第二个最常见的EGFR突变。 EGFR（T790M）中的继发突变即使在患有反应性等位基因的患者中，也可以大大降低这种敏感性。与Cys-773结合的另一种EGFR抑制剂Cl-387,785在存在T790M二次突变的情况下仍能够抑制EGFR磷酸化，因此检测Gefitinib或erlotinib敏感的等位基因且T790M的患者在临床上相关。在第二阶段，我们还将开发针对最常见的突变赋予TKI敏感性的试验。即EGFR（例如DEL747-749）和G719C，L861Q点突变中的外显子19中的小删除。缺失的患者对TKI特别有反应。预计这种创新将有助于在治疗肺癌患者中实施突变筛查。