Analysis of the MT fraction of ccfDNA highlighted an even stronger fragmentation with very low amount of bp sequences detected compared with the WT sequence Supplementary Table 5.
Since WT sequences can be released by both normal and tumor cells while MT sequences by tumor cells only, such results suggest a higher fragmentation of ctDNA as compared with the non-mutated fraction of ccfDNA. Moreover, the concentration and fraction of ctDNA in patient samples detected by the KRAS MT allele appeared slightly higher when using 60 bp amplicon compared with the bp one Supplementary Figure 3.
Supplementary Table 6 ; Supplementary Figure 4. Such results confirmed the pertinence of preanalytic condition standardization not only for ccfDNA integrity determination but also in general for ccfDNA studies. Figure 4. Plasma from 10 healthy individuals were used in this analysis see workflow in Supplementary Figure 1.
Wilcoxon test was used for significance analysis. Figure 5. As ccfDNA from different patients' groups were prepared with different preanalytic conditions blood collection tubes and ccfDNA extraction kits , DII comparative analysis was performed for each patient group samples using healthy subject samples collected and treated with the same procedure see workflow Figure 2. Mann—Whitney U -test was used for significance analysis. Ann Oncol. Linear regression model is given in Supplementary Figure 6.
To better characterize small, fragmented DNA fraction, we characterized ccfDNA fragments between 75 and bp shorter than mono-nucleosomal DNA fragment lengths and between and bp comprising expected mono-nucleosomal DNA fragment lengths — bp.
As shown in Figure 5B1 , a higher quantity of short fragments 75— bp was observed in mCRC patients than in healthy individuals Figure 5B1 and no difference was found for — bp fragments. The ratio of the quantity of fragments between and bp and 75 and bp was also significantly different between mCRC patients and healthy individuals Figure 5B2. These results further reinforced the potentiality of using ccfDNA integrity and its size profiling as a diagnostic or follow-up tool for mCRC patients.
Up to now, most strategies of ctDNA detection are based on the targeting of tumor-specific mutations which implies the conception of a numerous assays or large NGS gene panels to cover a maximum of possible mutations. The use of epigenetic alterations such as hypermethylated sequences has been recently described in mCRC 27 but remains cancer specific.
However, contradictory results have limited researchers' interest, which drew our attention on the importance of standardization. As different clinical studies could present different preanalytical treatments, it is necessary to investigate the potential impact of preanalytical methods on the efficiency of purification of the different sizes of DNA fragments.
Several studies have recalled the impact of the collection tube on the ccfDNA integrity 32 — The collection tubes mostly differs on their content in preservative agents that prevent blood cell hemolysis We did not observe such impact in our study Supplementary Figure 4. This could be linked to the low number of healthy individuals studied. Indeed, it has been shown that when tubes are not handled adequately, the genomic DNA coming from the blood cell could be released.
The increase of multicentric clinical studies and the centralization of the analysis platforms could cause uncontrolled delays between blood collection and plasma separation.
For these reasons, several ccfDNA preservative tubes have been developed, such as the BCT Streck tubes used in this study 36 , that could become a reference collection tube dedicated to liquid biopsy Recent studies that compared extraction methods for the isolation of ccfDNA from plasma samples have concluded that they can affect both ccfDNA yield and fragmentation 32 , Our work confirmed these results showing significant differences both in terms of fragmentation and ccfDNA concentration.
Recent works however, have been developed to specifically measure integrity indexes while targeting cancer-specific mutated sequences by qPCR These studies are nevertheless limited by the sensitivity and accuracy of basic bulk PCR techniques. Moreover, innovative procedures have also been developed for fragment-based highly sensitive detection 19 , Our results demonstrated, using two independent strategies, a significant difference of ccfDNA fragmentation in plasma of mCRC patients compared with healthy subjects.
Such results confirmed the high fragmentation of ctDNA with most fragments below bp as highlighted by both strategies used , which is consistent with other studies 18 , 41 , The two methods lead to complementary information. In addition to pursuing the development of DII measurement of ccfDNA as a universal cancer marker, such results have strong implications with regard to future assay design.
Indeed, we showed that assay targeting short amplicons allowed more sensitive and precise quantification of ctDNA, which is especially relevant for the analysis of low concentrated plasma samples.
In a more recent work, they also improved the detection of ctDNA by analyzing the shorter fragments after adding an either in vitro or in silico size selection step Xiao Liu et al. In conclusion, with the combined consideration of the importance of the standardization of preanalytical and analytical procedures as well as the increase of the sensibility and accuracy of analytical methods, the measure of DNA integrity should be an interesting parameter to develop new clinical biomarker in oncology.
In particular, even if differences have been observed between sample preparation procedures, the crucial point appeared to ensure method consistency within a single study. Indeed, even if the ext. Further analyses, involving a larger number of samples are required to allow for definitive conclusions.
Moreover, the described procedures will allow conducting similar studies on newly developed ccfDNA extraction kits. The optimizations of the described ddPCR method could involve the development of multiplex assay that allows the measurement of different fragment sizes in a single pot.
Yet, the limited number of patients in our study also requires further investigations. Another limitation is that we limited our analysis to patients with mCRC. Moreover, to fully validate the DII as cancer biomarker, further investigations about the cfDNA biology, in particular about the potential influence of aging on cfDNA fragmentation could be of interest.
In order to assess its role in a more general setting, further studies should involve various cancer stages as well as other primary tumors. Moreover, investigating the processes leading to the observed differences of fragmentation profiles between tumor and healthy cells derived DNA would greatly contribute to the use of DII measurement as a universal cancer marker.
The datasets presented in this study can be found in online repositories. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. The AGEO Group sponsored the trial and investigators were responsible for study design, data collection, data analysis, and data interpretation.
Merck Serono s. France , an affiliate of Merck KGaA Darmstadt, Germany , supported this trial through grants to AGEO, but had no role in study design, data collection, data analysis, data interpretation, or writing of the report. FGa: Bio-Rad employee. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank Dr. Bardelli A, Pantel K. Liquid biopsies, what we do not know Yet. Cancer Cell. Butt AN, Swaminathan R. Annals N Y Acad Sci. Clinical applications of circulating tumor cells and circulating tumor dna as liquid biopsy. Cancer Discovery. Detection of circulating tumor DNA in early- and late-stage human malignancies.
Sci Trans Med. The diverse origins of circulating cell-free DNA in the human body: a critical re-evaluation of the literature. Biol Rev. Cell-free nucleic acids as biomarkers in cancer patients.
Nat Rev Cancer. Mouliere F, Thierry AR. The importance of examining the proportion of circulating DNA originating from tumor, microenvironment and normal cells in colorectal cancer patients. Expert Opinion Biol Therapy.
Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clini Oncol. Droplet-based digital PCR and next generation sequencing for monitoring circulating tumor DNA: a cancer diagnostic perspective. Expert Rev Mol Diagnostics. Circulating tumor cell as the functional aspect of liquid biopsy to understand the metastatic cascade in solid cancer Elsevier Enhanced Reader. Mol Aspects Med. Detection and localization of surgically resectable cancers with a multi-analyte blood test.
Liquid biopsy: general concepts. Acta Cytol. Clinical implications of monitoring circulating tumor dna in patients with colorectal cancer. Clin Cancer Res. Circulating cell-free DNA integrity as a diagnostic and prognostic marker for breast and prostate cancers. Cancer Genetics. Circulating DNA as a strong multimarker prognostic tool for metastatic colorectal cancer patient management care.
Clinical Cancer Res. Clin Chem. Assessment of DNA integrity, applications for cancer research. Adv Clin Chem. Serum DNA integrity index as a potential molecular biomarker in endometrial cancer. J Exp Clin Cancer Res. Anal Chem. Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. High fragmentation characterizes tumour-derived circulating DNA. Lee T, editor. Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts.
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