Predoctoral to Postdoctoral Research Training in ctDNA Biology for Liquid Biopsy of Cancer

项目概要/摘要 循环肿瘤DNA（ctDNA）是反映亲代肿瘤的DNA，存在于非小细胞肺癌的血浆中， 肺癌（NSCLC）患者。ctDNA的液体活检分析可以进行早期检测和遗传监测。 然而，目前的基于PCR的液体活检方法由于ctDNA的低拷贝而通常难以检测ctDNA。 流通相比之下，电场诱导释放和测量液体活检（eLB）平台检测到 突变EGFR（L 858 R，exon 19 del，T790 M）ctDNA，在<50 μ l的 NSCLC患者的未处理血浆。初步数据表明，通过eLB检测到的ctDNA可能是 超短单链ctDNA（usssctDNA），其与通常描述的160个碱基对形成对比， 核小体间双链ctDNA。这种新的亚群是无法通过PCR为基础的液体检测 由于尺寸选择偏倚，F99阶段的目标是使用usssctDNA-seq，一种新的NGS 管道，以探索NSCLC血浆是否含有大量的超短和单链ctDNA。 该流程包括usssctDNA特异性提取、文库制备、深度测序和生物信息学分析。 分析.为了验证，将对80份晚期NSCLC EGFR突变血浆样本进行usssctDNA-seq。的 usssctDNA的发现将揭示大量以前未被注意的信息，从而改善治疗 NSCLC患者的生存率。之后，在K 00阶段，受训者希望探索生物学 生物流体ctDNA的起源和usssctDNA并入现代多分析物方法， 癌症检测这将涉及新技能的培训，如类器官建模，甲基化和白细胞 测序和生物信息学机器学习在世界领先的癌症研究所。最终的目标 该拟议的F99/K 00项目将为受训者在癌症研究领域的职业生涯做好准备， 液体活检和无细胞DNA生物学。

PROJECT SUMMARY/ABSTRACT Circulating tumor DNA (ctDNA) which mirrors the parental tumor, is present in the plasma of non-small cell lung carcinoma (NSCLC) patients. Liquid biopsy analysis of ctDNA allows early detection and genetic monitoring. However, current PCR-based liquid biopsy methods often have difficulty detecting ctDNA due to low copies in circulation. In contrast, the electric field-induced release and measurement liquid biopsy (eLB) platform, detects mutant EGFR (L858R, exon19del, T790M) ctDNA with a >90% tissue-genotype concordance in <50ul of unprocessed plasma of NSCLC patients. Preliminary data demonstrate that the ctDNA detected by eLB may be ultrashort single-stranded ctDNA (usssctDNA) which contrasts the typically described 160-basepair internucleosomal double-stranded ctDNA. This new subpopulation is undetectable through PCR-based liquid biopsy methods due to size-selection bias. The goal of the F99 Phase is to use usssctDNA-seq, a novel NGS pipeline, to explore if NSCLC plasma contains high quantities of ctDNA that are ultrashort and single-stranded. This pipeline consists of a usssctDNA-specific extraction, library preparation, deep sequencing, and bioinformatic analysis. To verify, 80 late-stage NSCLC EGFR-mutated plasma samples will undergo usssctDNA-seq. The discovery of usssctDNA will open up a wealth of previously unnoticed information leading to improved treatment and survival of NSCLC patients. Afterward, during the K00 Phase, the trainee hopes to explore the biological origins of biofluid ctDNA and the incorporation of usssctDNA into modern multi-analyte approaches for pan- cancer detection. This will involve training in new skills such as organoid modeling, methylation and leukocyte sequencing, and bioinformatic machine learning at a world-leading cancer-oriented institute. Ultimately, the goal of this proposed F99/K00 project will to prepare the trainee for a career in cancer research with an expertise in liquid biopsy and cell-free DNA biology.