Genetic determinants associated with pemetrexed response and toxicity

描述(申请人提供)：培美曲塞是一种多靶点抗叶酸，最近被批准用于非小细胞肺癌(NSCLC)的二线治疗。在临床可达到的浓度下，最重要的相互作用是与胸苷酸合酶(TYMS)，尽管已知该药物至少针对另外两种叶酸依赖酶。这项建议的目的是通过采用全基因组、全面的方法来检验基因多态显著影响与培美曲塞相关的毒性和反应的易感性这一假说，以确定影响培美曲塞相关毒性和反应的其他遗传变异。为此，将对来自人类多形参考中心大型参考家系和HapMap(高加索人、约鲁班人、亚洲人)样本的淋巴母细胞系进行培美曲塞细胞毒性和凋亡的表型鉴定。公共领域的基因类型信息使我们能够应用包括连锁分析在内的家族性遗传策略来检查基因组中包含导致培美曲塞诱导的细胞毒性和细胞凋亡易感性的遗传变异的区域。此外，还将使用来自非洲、欧洲和亚洲后裔的国际HapMap样本来比较培美曲塞引起的细胞毒性的个体间和种族间差异。我们实验室产生的外显子表达阵列将被用来识别通过它们对基线基因表达的影响而起作用的遗传变异，这些基因表达与培美曲塞的敏感性有关。通过这种全基因组方法确定的遗传变异将在一组单独的LCLS样本中进行验证，并通过定量聚合酶链式反应和siRNA进行验证。我们将评估TYMS、GARFT、DHFR中的新SNPs和已知候选变异，以确定与组间(NCCTG、CALGB、ECOG、SWOG、NCI-C)研究中的反应或毒性相关的基因类型，将培美曲塞与厄洛替尼作为NSCLC的二线治疗进行比较。我们的长期目标是确定一种“药物遗传学特征”，它将预测患者对培美曲塞的反应机会减少或不良反应风险增加。具体目的是：1.确定易感性对培美曲塞的细胞毒性和细胞凋亡效应的遗传贡献。2.检测培美曲塞治疗后的基线和时间依赖的表达模式。3.通过国际人类基因组图谱项目提供的数据，确定与培美曲塞在欧洲人(CEU)、非洲人(YRI)和亚洲人(CHB，JPT)来源的细胞系中的细胞毒性和基因表达有关的SNPs。4.在一项多中心试验中，确定TYMS、GARFT和DHFR的常见多态以及特定AIMS 1-3中确定的候选多态是否与培美曲塞治疗晚期NSCLC患者的疗效和/或毒性相关。公共卫生相关性：这项研究的目的是确定肺癌患者在接受化疗药物培美曲塞治疗后存在毒性或无反应的风险。该项目旨在利用细胞系建立一个模型，以更好地理解基因变异如何解释患者对药物反应的差异。此外，我们将在接受培美曲塞治疗的肺癌患者中测试我们的模型的结果。最终，我们希望找到培美曲塞反应和毒性的预测标记物。

DESCRIPTION (provided by applicant): Pemetrexed is a multitargeted antifolate recently approved for second line therapy of non-small cell lung cancer (NSCLC). The most significant interaction at clinically achievable concentrations is with thymidylate synthase (TYMS), although the drug is known to target at least two other folate dependent enzymes. The goal of this proposal is to identify additional genetic variants that influence pemetrexed-associated toxicity and response by employing a genome-wide, comprehensive approach to test the hypothesis that genetic polymorphisms significantly influence susceptibility to toxicities and response associated with pemetrexed. To this end, lymphoblastoid cell lines, derived from large reference Centre d' Etude du Polymorphisme Humain pedigrees and HapMap (Caucasian, Yoruban, Asian) samples, will be phenotyped for pemetrexed cytotoxicity and apoptosis. Genotypic information is available in the public domain enabling us to apply familial genetic strategies, including linkage analysis, to examine regions in the genome harboring the genetic variants responsible for susceptibility to pemetrexed-induced cytotoxicity and apoptosis. In addition, inter-individual and inter-ethnic variation in pemetrexed-induced cytotoxicity will be compared using the International HapMap samples from populations of African, European and Asian descent. Exon expression array generated in our laboratory will be incorporated to identify genetic variants acting through their effect on baseline gene expression that associate with sensitivity to pemetrexed. Genetic variants identified through this whole genome approach will be validated in a separate sample set of LCLs and by quantitative PCR and siRNA. We will evaluate both novel SNPs and known candidate variants in TYMS, GARFT, DHFR to determine genotypes associated with response or toxicity in an intergroup (NCCTG, CALGB, ECOG, SWOG, NCI-C) study of pemetrexed compared to erlotinib as second line therapy for NSCLC. Our long-term goal is to identify a "pharmacogenetic signature" that will predict patients with a decreased chance for response or an increased risk for adverse reactions to pemetrexed. Specific aims are: 1. To determine the genetic contribution of susceptibility to the cytotoxic and apoptotic effects of pemetrexed. 2. To examine baseline and time dependent patterns of expression following pemetrexed treatment. 3. Identify SNPs, through data available from the International HapMap project, associated with both pemetrexed cytotoxicity and gene expression in cell lines derived from Europeans (CEU), Africans (YRI) and Asians (CHB, JPT). 4. To determine if a common polymorphism in TYMS, GARFT, and DHFR, as well as candidate polymorphisms identified in specific aims 1- 3 correlate with response and/or toxicity in advanced NSCLC patients treated with pemetrexed in a multicenter trial. PUBLIC HEALTH RELEVANCE: The objective of this research is to identify lung cancer patients at risk for toxicities or lack of response following treatment with pemetrexed, a chemotherapeutic agent. The project aims to build a model using cell lines to better understand how genetic variation explains differences in patient response to drug. In addition, we will test the results of our model in lung cancer patients treated with pemetrexed. Ultimately, we hope to find predictive markers of pemetrexed response and toxicity.