Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer

Abstract

30Analysis of cell-free DNA (cfDNA) is promising for broad applications in clinical 31 settings, but with significant bias towards late-stage cancers. Although recent studies have 32 discussed the diverse and degraded nature of cfDNA molecules, little is known about its 33 impact on the practice of cfDNA analysis. Here we reported a new targeted sequencing by 34 combining single-strand library preparation and target capture (SLHC-seq). By applying 35 the new technology in plasma cfDNA from pancreatic cancer patients, we achieved higher 36 efficiency in analysis of mutations than previously reported using other detection assays. 37 SLHC-seq rescued short or damaged cfDNA fragments along to increase the sensitivity 38 and accuracy of circulating-tumor DNA detection. Most importantly, we found that the 39 small mutant fragments are prevalent in early-stage patients, which provides strong 40 evidence for fragment size-based early detection of pancreatic cancer. Collectively, the 41 new pipeline enhanced our understanding of cfDNA biology and provide new insights for 42 liquid biopsy. 43 44 MAIN TEXT 45 65 sequencing.(Aravanis et al, 2017; Zill et al, 2015) Moreover, the noninvasive nature of cfDNA 66 analysis enables doctors to monitor disease progression.(Couraud et al, 2014; Murtaza et al, 2013; 67 Zill et al, 2015) However, cfDNA analysis using large-scale next-generation sequencing (NGS) 68 has been largely confined to advanced or metastatic cancers, in which the ctDNA ( circulating-69 tumor DNA ) fraction is higher and is more readily detectable.(Gorgannezhad et al, 2018; Haber 70 & Velculescu, 2014) Detection of ctDNA in early-stage cancers is still filled with cautionary tales 71 that highlight the technical challenges in this field. Recently, Velculescu et al reported improved 72 targeted error correction sequencing ( TEC-Seq ) as an efficient approach for the detection of 73 ctDNA in early-stage colorectal, ovarian, breast, and lung cancers, with a detection rate ranging 74 from 59% to 71%.(Phallen et al, 2017) The massive parallel sequencing approach provides an 75 applicable paradigm guiding cfDNA analysis in early-stage cancers. 76 However, the performance of existing approaches is limited in cfDNA analysis in early-77 stage pancreatic cancers. This limitation may be partially because of a lower ctDNA content in 78 the circulation of pancreatic cancers.(Aravanis et al, 2017) Moreover, the biological features of 79

Genome‐wide profiling of circulating tumor DNA depicts landscape of copy number alterations in pancreatic cancer with liver metastasis

“…This is consistent with a recent report that pancreatic cancer is among the low ctDNA cancers [41]. Emerging evidence indicated that tumor-derived cfDNA were shorter than those from nonmalignant cells, and enrichment of short fragments could improve detection of ctDNA [41,42]. Indeed, we also found that estimated TFx for samples with selection of short reads after sequencing was significantly higher than that of original samples (data not shown).…”

Detection of Circulating Tumor DNA in Solid Tumors

“…Indeed, using this approach, ctDNA could be detected in 88% of stage I/II pancreatic adenocarcinoma cases and in 95% of those with stage III/IV disease [41]. The analysis of cfDNA fragment size revealed that the DNA fragments that contained the wild-type KRAS allele were all around 160 bp in size, while a large proportion of the DNA fragments containing the mutant KRAs allele had size below 100 bp [41]. Interestingly, the short DNA fragments were more pronounced in the pre-cancerous stage and in early-stage pancreatic cancers [38].…”

“…Liu and coworkers reported developing a single-strand library preparation and hybrid-capturebased ctDNA sequencing (SLHS-seq) approach. The procedure was applied to perform cfDNA profiling in a group of 112 pancreatic cancer patients, and the results demonstrated high specificity and sensitivity in the detection of ctDNA in pancreatic cancer patients, including those at an early stage of disease [41]. Indeed, using this approach, ctDNA could be detected in 88% of stage I/II pancreatic adenocarcinoma cases and in 95% of those with stage III/IV disease [41].…”

“…The procedure was applied to perform cfDNA profiling in a group of 112 pancreatic cancer patients, and the results demonstrated high specificity and sensitivity in the detection of ctDNA in pancreatic cancer patients, including those at an early stage of disease [41]. Indeed, using this approach, ctDNA could be detected in 88% of stage I/II pancreatic adenocarcinoma cases and in 95% of those with stage III/IV disease [41]. The analysis of cfDNA fragment size revealed that the DNA fragments that contained the wild-type KRAS allele were all around 160 bp in size, while a large proportion of the DNA fragments containing the mutant KRAs allele had size below 100 bp [41].…”

Towards a more sensitive detection of somatic mutations in cell-free DNA

“…The current article describes a single-strand library preparation assay for the sequencing of cell-free DNA (cfDNA) in a clinical cohort of patients with PC or relevant pancreatic lesions and healthy controls [3]. A major pitfall of the cfDNA analysis is the contamination with high molecular weight genomic DNA, which originates from healthy blood cells when the blood is extracted and centrifuged for the retrieval of plasma.…”