New insights into structural features and optimal detection of circulating tumor DNA determined by single-strand DNA analysis

Abstract

Circulating cell-free DNA (cfDNA) has received increasing interest as an apparent breakthrough approach in diagnostics, personalized medicine, and tumor biology. However, the structural features of cfDNA are poorly characterized. Specifically, the literature has discrepancies with regards to cfDNA size profile. We performed a blinded study of the distribution of cfDNA fragment sizes in cancer patient plasma (n = 11), by various ultra-deep-sequencing approaches and quantitative PCR (Q-PCR). Whole-genome sequencing of single-stranded DNA library preparation (SSP-S) revealed that nearly half of the total cfDNA fragment number are below 120 nucleotides, which are not readily detectable by standard double-stranded DNA library preparation (DSP) protocols. Fractional size distribution of cancer patient circulating DNA was very similar using both SSP-S-based or Q-PCR-based methods also revealing that high molecular weight (over 350 bp) cfDNA is a minor component (~2%). These extra small detected cfDNA fragments may mostly result from nicks occurring in blood circulation in one or both DNA strands, which are subsequently revealed through the denaturation step of the SSP and Q-PCR procedures. Detailed analysis of the data suggested that most of the detectable cfDNA in blood has a nucleosome footprint (∼10-bp periodicity repeats). The nucleosome is thus the most stabilizing structure of DNA in the circulation. cfDNA molecules, which are initially packed in chromatin, are released from cells and are then dynamically degraded in blood both within and between nucleosomes or transcription factor-associated subcomplexes. While this study provides new insights into cfDNA size profiles harmonizing sequencing and Q-PCR findings, our data validate the use of a specific Q-PCR method and SSP-S for obtaining an optimal qualitative and quantitative analytical signal.

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“…It may also increase following intense effort. We (Sanchez et al, 2018) and others (Jiang et al, 2017;Underhill et al, 2018) revealed that cirDNA size profile shows a characteristic nucleosome fragmentation pattern. Our recent observations revealed that high molecular weight cirDNA fraction in the plasma of healthy individuals is minor, and corresponds to approximatively 12% of the total cirDNA fragment number (unpublished data).…”

Tumor microenvironment complexity and therapeutic implications at a glance

“…These methods can also eliminate the need for biopsy, additionally providing mutation-related information that allows easy monitoring of the tumor and provides therapeutic potential [209]. Furthermore, cfDNAs contain information on the mutations that affect treatment, facilitate individualized therapeutic examining, and non-invasive follow-up that may enable better cancer management [210]. However, the extraction and amplification of cfDNA can be demanding due to high DNA fragmentation and low concentration in the bloodstream [211].…”

Blood contains circulating cell‐free respiratory competent mitochondria

“…In contrast, NcfDNA had a more homogenous distribution, conventionally peaking at 167 bp, corresponding to mono-nucleosomes ( Figure 1D). 17,28 Paired-end sequencing enables high-resolution measurement of DNA size but has a practical upper limit of readout at ~1000 bp. Since no McfDNA was detected between 500 bp and 1000 bp, Whole Genome Sequencing only detected the minor fraction formerly revealed by gel electrophoresis ( Figure 1C).…”

“…In addition, the structure of McfDNA is currently unknown. In contrast, the structure of circulating DNA of nuclear origin is being characterized and mono and di‐ nucleosomes and, to a lesser extent, transcription factors are found as stabilized cell‐free DNA (cfDNA)‐associated structures in the blood‐stream . It is expected that there are important configuration differences between nuclear and mitochondrial circulating DNA, since mitochondrial DNA is a small circular genome, without protective histones, and thus is more sensitive to degradation in the circulation.…”

“…In contrast, the structure of circulating DNA of nuclear origin is being characterized 16 and mono and di-nucleosomes and, to a lesser extent, transcription factors are found as stabilized cell-free DNA (cfDNA)-associated structures in the blood-stream. 17,18 It is expected that there are important configuration differences between nuclear and mitochondrial circulating DNA, since mitochondrial DNA is a small circular genome, without protective histones, and thus is more sensitive to degradation in the circulation. However, by revealing recently that there are approximately 50 000-fold more copies of the mitochondrial genome than the nuclear genome in the plasma of healthy individuals, 19 we confirmed that McfDNA is sufficiently stable to be detected and quantified, 12,20 implying the presence of stable structures protecting these DNA molecules.…”