Abstract
To our knowledge, this is the first comprehensive study on the influence of several pre-analytical and demographic parameters that could be a source of variability in the quantification of nuclear and mitochondrial circulating DNA (NcirDNA and McirDNA). We report data from a total of 222 subjects, 104 healthy individuals and 118 metastatic colorectal cancer (mCRC) patients. Approximately 50,000 and 3,000-fold more mitochondrial than nuclear genome copies were found in the plasma of healthy individuals and mCRC patients, respectively. In healthy individuals, NcirDNA concentration was statistically influenced by age (p = 0.009) and gender (p = 0.048). Multivariate analysis with logistic regression specified that age over 47 years-old was predictive to have higher NcirDNA concentration (oR = 2.41; p = 0.033). McirDNA concentration was independent of age and gender in healthy individuals. In mCRC patients, NcirDNA and McirDNA levels were independent of age, gender, delay between food intake and blood collection, and plasma aspect, either with univariate or multivariate analysis. Nonetheless, ad hoc study suggested that menopause and blood collection time might have tendency to influence cirDNA quantification. In addition, high significant statistical differences were found between mCRC patients and healthy individuals for NcirDNA (p < 0.0001), McirDNA (p < 0.0001) and McirDNA/NcirDNA ratio (p < 0.0001). NcirDNA and McirDNA levels do not vary in the same way with regards to cancer vs healthy status, pre-analytical and demographic factors. Since Mandel and Metais discovered the presence of nucleic acids in serum in the 1940s 1 , different studies have reported elevated levels of circulating DNA (cirDNA) in the blood of patients suffering from various diseases 2-6 , especially cancer 7-10. Despite scant early consideration, interest in the feasibility of cirDNA analysis has increased exponentially, over the last decade, among researchers working on a large range of disorders. CirDNA was first clinically implemented in prenatal diagnosis of sex-determination and pregnancy-associated disorders by assaying fetal DNA in maternal plasma 11-13. The main sources of cirDNA are cell death, either by necrosis or apop-tosis, and active release by viable cells, including exocytosis and NETosis 14,15. Note, cirDNA may derive from either nuclear (NcirDNA) or mitochondrial DNA (McirDNA). To date, research and development of cirDNA analysis has focused on the qualitative rather than the quantitative information provided. For example, cirDNA analysis is now clinically validated for detecting specific sequences or mutations to guide the oncologist toward the most appropriate treatment. CirDNA analysis is also performed for prenatal and embryo-culture genetic testing. There have been several years of intensive studies validating cirDNA quantitation in different clinical scenarios, including sepsis, transplant recipients and immune disorders. CirDNA quantification is also now taken into consideration in oncology as, it was recently shown that, the level of mutant cirDNA is useful for following-up cancer patients to detect minimal residual disease and to monitor response to therapy and disease recurrence. Although total cirDNA levels were first examined in the early phase of cirDNA research and development , it is now not considered as a single biomarker because of its lack of specificity. Nevertheless, all cirDNA analysis relies on the optimal quantification of total amount of cirDNA. Biological biomarkers should be highly dynamic, and their diagnostic performance may vary depending on internal and external changes. The clinical efficacy of cirDNA will require the identification and the control of various patient-related confounders that may 1 iRcM, institute