Cell-free DNA Analysis for Liquid Biopsy of Cancer

项目摘要/摘要 循环肿瘤dna(Ctdna)反映了亲代肿瘤，存在于非小细胞肺癌的血浆中。 癌症(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，目前基于pcr的液体活检方法通常难以检测ctdna，因为ctdna的拷贝数较低。 发行量。相比之下，电场诱导释放和测量液体活组织检查(ELB)平台检测到 突变的EGFR(L858R，外显子19del，T790M)ctDNA在&lt；50ul；50ul中具有90%的组织基因型一致性 非小细胞肺癌患者未经处理的血浆。初步数据显示ELB检测到的ctDNA可能是 超短单链ctDNA(UsssctDNA)，与通常描述的160碱基对形成对比 核小体间双链ctDNA。这个新的亚群不能通过基于聚合酶链式反应的液体检测到 活检方法由于尺寸选择的偏差。F99阶段的目标是使用一种新型的NGS-usssctDNA-seq 管道，以探索非小细胞肺癌血浆是否含有大量超短和单链的ctDNA。 这条流水线包括usssctDNA特异性提取、文库准备、深度测序和生物信息学。 分析。为了验证，80个晚期非小细胞肺癌EGFR突变的血浆样本将接受usssctDNA-seq。这个 UsssctDNA的发现将打开大量以前未被注意到的信息，从而改进治疗方法 以及非小细胞肺癌患者的存活率。随后，在K00阶段，学员希望探索生物 生物流体ctDNA的起源和将usssctDNA纳入现代多分析方法用于PAN-DNA 癌症检测。这将包括培训新的技能，如有机物建模、甲基化和白细胞 在一家世界领先的面向癌症的研究所进行测序和生物信息机器学习。最终，我们的目标是 这一拟议的F99/K00项目将使受训人员为从事癌症研究的职业生涯做好准备，并具备以下方面的专业知识 液体活组织检查和无细胞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.