Research ArticleCancer

Enhanced detection of circulating tumor DNA by fragment size analysis

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Science Translational Medicine  07 Nov 2018:
Vol. 10, Issue 466, eaat4921
DOI: 10.1126/scitranslmed.aat4921

Sizing up circulating tumor DNA

Unlike solid tumors, which are often hidden deep within a patient’s body, a patient’s blood is easy to access safely. As a result, liquid biopsy, the analysis of tumor DNA in the blood, is an attractive alternative to conventional biopsy. Unfortunately, tumor DNA molecules are usually vastly outnumbered by the fragments of noncancer DNA in the blood, and detecting them can be a challenge, especially in early stages of cancer. Mouliere et al. identified characteristic differences in the size distribution of tumor-derived and noncancer DNA fragments and then used these observations to design a method of tumor DNA detection with greater sensitivity.


Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC <0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC > 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA.

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