Quantitative characterization of tumor cell-free DNA shortening

Juntang Guo,Kefeng Ma,Yang Xu,Jin Huang,Mei Jiang,Xue Wu,Yang W Shao,Xiangyuan Ma,Hua Bao

doi:10.1186/s12864-020-06848-9

Abstract

BackgroundPrevious studies found that cell-free DNA (cfDNA) generated from tumors was shorter than that from healthy cells, and selecting short cfDNA could enrich for tumor cfDNA and improve its usage in early cancer diagnosis and treatment monitoring; however, the underlying mechanism of shortened tumor cfDNA was still unknown, which potentially limits its further clinical application.ResultsUsing targeted sequencing of cfDNA in a large cohort of solid tumor patient, sequencing reads harboring tumor-specific somatic mutations were isolated to examine the exact size distribution of tumor cfDNA. For the majority of studied cases, 166 bp remained as the peak size of tumor cfDNA, with tumor cfDNA showing an increased proportion of short fragments (100-150 bp). Less than 1% of cfDNA samples were found to be peaked at 134/144 bp and independent of tumor cfDNA purity. Using whole-genome sequencing of cfDNA, we discovered a positive correlation between cfDNA shortening and the magnitude of chromatin inaccessibility, as measured by transcription, DNase I hypersensitivity, and histone modifications. Tumor cfDNA shortening occurred simultaneously at both 5′ and 3′ ends of the DNA wrapped around nucleosomes.ConclusionsTumor cfDNA shortening exhibited two distinctive modes. Tumor cfDNA purity and chromatin inaccessibility were contributing factors but insufficient to trigger a global transition from 166 bp dominant to 134/144 bp dominant phenotype.

Highlights

Previous studies found that cell-free Deoxyribonucleic acid (DNA) generated from tumors was shorter than that from healthy cells, and selecting short cell-free DNA (cfDNA) could enrich for tumor cfDNA and improve its usage in early cancer diagnosis and treatment monitoring; the underlying mechanism of shortened tumor cfDNA was still unknown, which potentially limits its further clinical application
Cell-free DNA is the short DNA fragment found in plasma, urine, and other body fluids, while circulating tumor DNA is a subset of cfDNA with tumor origin. cfDNA has been increasingly used for non-invasive cancer diagnosis, residual disease monitoring, and treatment efficacy evaluation [1, 2]
Presence of 134/144 bp dominant samples was not determined by tumor cfDNA purity In order to better understand the size distribution of tumor cfDNA, we extended our analysis to a larger cohort of 5608 cfDNA samples collected from patients with various types of cancers and sequenced with targeted next-generation sequencing (NGS) (422 cancer-associated genes, gene list available in Supplementary Table S1)

Summary

Introduction

Previous studies found that cell-free DNA (cfDNA) generated from tumors was shorter than that from healthy cells, and selecting short cfDNA could enrich for tumor cfDNA and improve its usage in early cancer diagnosis and treatment monitoring; the underlying mechanism of shortened tumor cfDNA was still unknown, which potentially limits its further clinical application. Jiang et al reasoned that amplified tumor chromosomal regions would be overrepresented in the cfDNA whereas the deleted tumor chromosomal regions would be underrepresented [7] Under this hypothesis, they demonstrated that short cfDNA (< 150 bp) preferentially carried tumor-associated copy number variations (CNVs) in patients with hepatocellular carcinoma (HCC) [7]. Underhill and colleagues characterized cfDNA of human glioblastoma (GBM) and human HCC in rat xenografts and they found that tumor cfDNAs were significantly shorter [8]. They reported that the shortened cfDNA fragments were observed in a limited number of patients with melanoma and lung cancer [8]. Using in vitro and in silico size selection methods, Mourliere and colleagues demonstrated enhanced detection of tumor-specific biomarkers in the short fragments of cfDNA [16]

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Conclusion