Fragment Length of Circulating Tumor DNA.
Fragment Length of Circulating Tumor DNA.
复制标题
DOI：
10.1371/journal.pgen.1006162
复制
发表时间：
2016-07
期刊：
影响因子：
4.5
通讯作者：
Shendure J
中科院分区：
文献类型：
--
作者：
Underhill HR;Kitzman JO;Hellwig S;Welker NC;Daza R;Baker DN;Gligorich KM;Rostomily RC;Bronner MP;Shendure J
恶性肿瘤将 DNA 释放到循环系统中。循环肿瘤 DNA (ctDNA) 的短暂半衰期可能提供仅通过非侵入性抽血来诊断、监测复发和评估治疗反应的机会。然而，事实证明，在正常存在的源自健康细胞的无细胞 DNA 背景下检测 ctDNA 具有挑战性，特别是在非转移性实体瘤中。在这项研究中，定义了 ctDNA 和正常游离 DNA 片段长度大小的明显差异。大鼠血浆中的人类 ctDNA 源自大鼠大脑中的人类多形性胶质母细胞瘤干细胞样细胞和大鼠胁腹中的人类肝细胞癌，发现其主片段长度比背景大鼠无细胞 DNA 更短（分别为 134-144 bp 和 167 bp）。随后，与健康对照相比，黑色素瘤和肺癌患者的 ctDNA 片段长度也发生了类似的变化。对黑色素瘤患者和健康对照之间的无细胞 DNA 片段长度进行比较发现，BRAF V600E 突变等位基因的片段长度比野生型等位基因的片段长度更短（分别为 132-145 bp 和 165 bp）。此外，对较短的无细胞 DNA 片段长度进行大小选择显着增加了人类肺癌中 EGFR T790M 突变等位基因的频率。这些发现提供了令人信服的证据，表明通过实验或生物信息学方法从无细胞 DNA 中分离特定片段长度子集可能会改善 ctDNA 的检测。在细胞死亡过程中，不包含在膜内的 DNA（即无细胞 DNA）进入循环。由于源自健康细胞的正常存在的无细胞 DNA 背景相对丰富，检测源自实体瘤的无细胞 DNA（即循环肿瘤 DNA，ctDNA），特别是尚未转移的实体瘤，已被证明具有挑战性。我们的研究定义了正常游离 DNA 和来自各种实体瘤的 ctDNA 片段长度之间微妙但明显的差异。具体而言，ctDNA 总体上始终比正常无细胞 DNA 的片段长度短。随后，我们发现较短的无细胞 DNA 片段的大小选择增加了样品中 ctDNA 的比例。这些结果提供了令人信服的证据，表明开发分离与我们研究中描述的 ctDNA 片段长度一致的游离 DNA 子集的技术可能会大大改善非转移性实体瘤的检测。因此，我们的研究结果可能会对 ctDNA 在实体瘤的非侵入性检测和诊断（即“液体活检”）、监测肿瘤复发和评估肿瘤对治疗的反应方面的临床应用产生直接影响。
Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA. During cell death, DNA that is not contained within a membrane (i.e., cell-free DNA) enters the circulation. Detecting cell-free DNA originating from solid tumors (i.e., circulating tumor DNA, ctDNA), particularly solid tumors that have not metastasized, has proven challenging due to the relatively abundant background of normally occurring cell-free DNA derived from healthy cells. Our study defines the subtle but distinct differences in fragment length between normal cell-free DNA and ctDNA from a variety of solid tumors. Specifically, ctDNA was overall consistently shorter than the fragment length of normal cell-free DNA. Subsequently, we showed that a size-selection for shorter cell-free DNA fragments increased the proportion of ctDNA within a sample. These results provide compelling evidence that development of techniques to isolate a subset of cell-free DNA consistent with the ctDNA fragment lengths described in our study may substantially improve detection of non-metastatic solid tumors. As such, our findings may have a direct impact on the clinical utility of ctDNA for the non-invasive detection and diagnosis of solid tumors (i.e., the “liquid biopsy”), monitoring tumor recurrence, and evaluating tumor response to therapy.