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
(0.4x). 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