Semiparametric Methods for Efficiency-Adjusted Relative qRT-PCR Quantification

描述（由申请人提供）：定量RT-PCR广泛用于癌症的分子诊断和预后评估。虽然生物标志物的检测（存在与不存在）正在成为常规临床实践的一部分，但肿瘤负荷的实际定量（例如，淋巴结或血清中的肿瘤负荷）仍处于开发阶段。为了使这种实际定量在临床上有用，有必要具有适当的数据分析方法，该方法将在用于qRT-PCR测定的各种设备平台和化学品中提供低丰度转录物的相对qRT-PCR定量的高准确度和精确度。 本项目的总体目标是开发和验证效率调整相对qRT-PCR定量的新的通用方法，这些方法普遍适用于各种qRT-PCR设备平台和化学品。新的定量方法将基于qRT-PCR动力学数据的新型半参数模型，该模型使用平滑样条灵活地表示扩增历史，并通过适当微分方程定义的罚分纳入qRT-PCR效率动力学模型。 所提出的研究将（1）调查Michaelis-Menten模型及其扩展用于描述qRT-PCR效率动态的效用;（2）使用功能数据分析中的轮廓罚估计方法开发用于qRT-PCR动力学数据的半参数模型，该半参数模型结合PCR效率的动态;（3）基于所提出的半参数模型开发新的用于效率调整的相对qRT-PCR定量的通用方法;（4）使用模拟和广泛的动力学qRT-PCR数据比较新的和已建立的方法的准确度和精确度，所述动力学qRT-PCR数据公开可用并且在来自结直肠癌患者的新鲜组织和血液中的GUYC 2C的两个大型NCI赞助的研究的过程中收集。 公共卫生相关性：定量RT-PCR现在广泛用于癌症的分子诊断和预后评估。虽然生物标志物的检测（存在与不存在）正在成为常规临床实践的一部分，但用于肿瘤负荷的实际定量（估计拷贝数）的方法仍需要改进。本项目的总体目标是开发和验证新的通用方法，用于低表达癌症生物标志物的实际相对qRT-PCR定量，这些方法普遍适用于各种qRT-PCR设备平台和化学品。

DESCRIPTION (provided by applicant): Quantitative RT-PCR is widely used for molecular diagnostics and evaluating prognosis in cancer. While detection of biomarkers (presence vs. absent) is becoming a part of routing clinical practice, the actual quantification of tumor burden, for example, in lymph nodes or serum is still in developmental phase. In order for such actual quantification to become clinically useful, it is necessary to have in place data analysis methods that would provide high accuracy and precision of relative qRT-PCR quantification of low abundance transcripts across various equipment platforms and chemistries used for qRT-PCR assay. The overall goal of this project is to develop and validate new and universal methods of efficiency-adjusted relative qRT-PCR quantification that would be universally applicable to various qRT-PCR equipment platforms and chemistries. New quantification methods will be based on novel semiparametric models for qRT-PCR kinetic data that flexibly represent amplification history using smoothing splines and incorporate the model for dynamics of qRT-PCR efficiency through the penalty defined by suitable differential equation. The proposed studies will (1) investigate the utility of Michaelis-Menten model and its extensions for describing dynamics of qRT-PCR efficiency; (2) develop semi- parametric models for qRT-PCR kinetic data incorporating the dynamics of PCR efficiency using the profiled penalty estimation approach in functional data analysis; (3) develop new universal methods for efficiency-adjusted relative qRT-PCR quantification based on the proposed semi-parametric models; (4) compare accuracy and precision of the new and established methods using simulations and wide range of kinetic qRT-PCR data publicly available and collected during the course of two large NCI sponsored studies of GUYC2C in fresh tissue and blood from colorectal cancer patients. PUBLIC HEALTH RELEVANCE: Quantitative RT-PCR is now widely used for molecular diagnostics and evaluating prognosis in cancer. While detection of biomarkers (presence vs. absent) is becoming a part of routing clinical practice, the methods for actual quantification (estimation of copy number) of tumor burden still need improvement. The overall goal of this project is to develop and validate new and universal methods of actual relative qRT-PCR quantification of low expression cancer biomarkers that would be universally applicable to various qRT-PCR equipment platforms and chemistries.