Pyrophosphorolysis-activatable helicase dependent amplification assay for lung ca

描述(由申请人提供)： 除了少数偶然诊断的无症状患者外，几乎所有的肺癌患者在出现症状时都是有症状的。因此，只有30%-35%的患者有足够的肿瘤部位，可以进行手术切除。研究表明，过度表达表皮生长因子受体(EGFR)的癌症会增加对化疗的耐药性，从而增加转移的风险。针对EGFR酪氨酸激酶(TKI)结构域的生物制剂正在开发中，目前正处于不同的临床测试阶段。EGFR的治疗抑制只在10%到20%的患者中导致了显著的肿瘤消退。对EGFR抑制剂的敏感性在很大程度上取决于EGFR中是否存在体细胞突变，因此需要一种简单的诊断方法来识别对这些药物敏感的患者。几乎所有对TKI、吉非替尼或厄洛替尼有临床反应的具有EGFR激活突变的患者都会对这些药物产生耐药性。EGFR(T790M)出现继发性突变，占耐药病例的50%。鉴于其他EGFR抑制剂对这类患者可能是有效的治疗方法，因此迫切需要准确和有效地识别这些患者进行这些治疗。由于这类患者的重复肿瘤活检通常很困难(由于肿瘤位于胸部，这增加了针刺活检导致肺部塌陷的机会)，开发一种非侵入性方法来检测出现耐药性将是一个重大的进步。我们认为解决这个问题的最好方法是检测循环上皮细胞(CEC)中的这些突变。 我们建议开发影响TKI反应的关键EGFR突变的分析方法。我们计划开发的检测方法将能够识别患者中继发性EGFR突变的出现。这些分析将使用从血液中提取的总核酸，以及一种新的等位基因特异性扩增技术，据报道，该技术的特异性为10-7。这种特殊的特异性是可能的，因为具有与其模板DNA靶标不匹配的3‘端双脱氧核苷酸的寡核苷酸不能被焦磷酸分解有效地激活，而其3’端与其模板完全匹配的寡核苷酸可以被激活。因此，只有当DNA聚合反应的3‘端与其目标匹配时，焦磷分解才能激活DNA聚合；因此，希望之城医院为这项技术开发了焦磷分解激活聚合(PAP)技术。并不是所有的DNA聚合酶都能有效地执行PAP。例如，携带F667Y突变的改良Taq聚合酶大大增强了聚合酶对双脱氧终止寡核苷酸(DdNTP)的亲和力(美国专利5,614,365)，在进行PAP时比野生型聚合酶更有效。我们已经对具有类似特性的脂肪嗜热芽孢杆菌(BST)聚合酶突变体(F712Y)进行了初步测试。此外，一些天然酶(如噬菌体T7DNA聚合酶)降低了对脱氧核苷酸三磷酸(DNTP)的特异性，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分解-激活聚合，解旋酶依赖扩增(PapHDA)中的作用。BST聚合酶和T7聚合酶都被报道在HDA中发挥作用。在第一阶段，我们建议专注于开发针对EGFR错义突变的检测方法，L858R是第二种最常见的EGFR突变。EGFR(T790M)的第二次突变可以极大地降低这种敏感性，即使在具有敏感等位基因的患者中也是如此。另一种与Cys-773结合的EGFR抑制剂CL-387,785在存在T790M继发突变的情况下仍然能够抑制EGFR的磷酸化，因此检测吉非替尼或厄洛替尼敏感等位基因和T790M将具有临床意义。在第二阶段，我们还将开发针对最常见的导致TKI敏感性的突变的检测方法；即EGFR外显子19的小片段缺失(例如，Del747-749)和G719C、L861Q点突变。缺失的患者对TKI特别敏感。预计这一创新将促进突变筛查在肺癌患者治疗中的实施。

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.