Invited critical reviewCirculating cell free DNA: Preanalytical considerations☆
Introduction
The discovery of circulating cell-free DNA (ccfDNA) in the human circulatory system has led to intensive research on its use in various clinical fields. CcfDNA was discovered in 1948 by Mandel and Metais [1] although at the time, it did not attract much curiosity. Thirty to 40 years later, however, the interest of ccfDNA was demonstrated by several groups: Leon et al. [2] found that ccfDNA concentration was significantly increased in cancer patients and Stroun et al. [3], [4] described a proportion of ccfDNA that was tumor derived and carried its molecular characteristics, thus leading to the concept of a “liquid biopsy”. Additionally, ccfDNA fragmentation has grown in interest in terms of diagnosis since the revelation of significant differences between cancer patients and healthy subjects [1], [2], [3], [4], [5], [6], [7], [8]. Therefore, ccfDNA analysis could provide diagnostic, pronostic, and theranostic information. Several researchers are intensively developing techniques that allow detection and characterization of genetic and epigenetic alterations of tumor cells using ccfDNA analysis in the plasma/serum of cancer patients. Such techniques could revolutionize the management care of cancer patients through the detection of mutations leading to resistance to targeted therapies, personalized therapeutic monitoring and non-invasive follow-up of the disease. Several works and reviews have been published on this topic over the last decade [9], [10], [11], [12], [13], [14], [15]. In the field of prenatal diagnosis, ccfDNA was used to develop a risk-free, non-invasive method to analyze fetal molecular genetics in pregnant women to avoid the risks associated with certain practices, such as amniocentesis [16]. In 1997, Lo et al. [17] showed fetal DNA in the plasma of pregnant women, which stimulated vigorous research in this field and the implementation of current clinical tests, such as fetal sex assessment, fetal rhesus D genotyping, or detection of fetal chromosomal aneuploidy [18]. Other groups have demonstrated the interest of ccfDNA in other clinical fields, such as autoimmune diseases [9], [19], [20], [21], trauma, sepsis [22], or myocardial infarction [23].
Despite intensive research, few ccfDNA-based tests have been translated to clinical practice. Currently, some tests are available for specific prenatal diagnosis [18] and only one technique exists for oncology, namely BEAMing [24], a sophisticated technique allowing detection of mutations in various genes, particularly in colorectal cancer (CRC) patients. Several techniques are under development to detect and characterize ccfDNA in cancer patients including restriction fragment length polymorphism, direct sequencing, high-resolution melting analysis, digital PCR, cold PCR, and other techniques usually used for tumor-tissue analysis [14]. Nevertheless, ccfDNA concentration has not yet been validated as a cancer biomarker as the literature reveals conflicting data: plasma ccfDNA concentrations in cancer patients range from a few ng/ml to several thousand ng/ml, which overlaps with the concentration range for healthy individuals [10], [11], [15], [25]. Furthermore, the estimation of ccfDNA fragmentation in cancer patients has been found to be lower, equivalent, or higher than in control subjects [1], [2], [3], [4], [5], [6], [7], [8]. Our group's work on ccfDNA analysis in CRC cancer patients found that ccfDNA fragmentation was higher in cancer patients than in healthy subjects [26], [27]. Such differences may be due not only to bias when selecting patients, but also to variation in the technical procedures for extracting and quantifying ccfDNA used by each laboratory, since no standard operating procedure (SOP) for ccfDNA analysis is available.
Over the last decade, ccfDNA reviews have regularly highlighted the lack of a SOP. Thus, a SOP for ccfDNA analysis is necessary to translate ccfDNA analysis to clinical practice [9], [10], [11], [12], [13], [14], [15], [28], [29]. The lack of preanalytical and analytical consensus for ccfDNA analysis on variables such as type of matrix, storage conditions, or particular handling of blood sampling, affects ccfDNA concentration and fragmentation values, thus presenting major obstacles to clinical application.
This is the first review that examines the main preanalytical factors affecting ccfDNA analysis, from blood drawing to the storage of ccfDNA extracts, and provides a summary of the optimal conditions for preanalytical handling of samples for ccfDNA analysis.
Data from the literature presented in this review are supported by our own observations on the impact of different handling protocols on ccfDNA concentration. Particular attention was given to the study of ccfDNA fragmentation considering that it is an indicator of ccfDNA stability during handling and the storage of samples. Our robust and precise ccfDNA quantification method enabled us to precisely study the pre-analytical handling and portability of ccfDNA analysis.
Section snippets
Serum or plasma
The matrix of choice, i.e. serum or plasma, is the first question to ask when standardizing ccfDNA analysis. Several works comparing ccfDNA concentrations in paired plasma and serum samples [25], [30], [31], [32], [33], [34], [35], [36] have revealed significantly higher ccfDNA concentrations in serum than in plasma. Table 1 summarizes some of the results published in various clinical fields. Nevertheless, several publications have demonstrated that the increased level of ccfDNA in serum is due
Influence of blood sample storage conditions on ccfDNA concentration
Influence of time delay and temperature storage between venipuncture and blood processing are two parameters widely studied and reported in the literature [34], [35], [36], [37], [43], [44], [45], [46], [47], [48]. It is now well known that between blood drawing and processing, ccfDNA concentrations slightly increase with time. However, some authors have shown a significant increase after 2 h of storage compared to the baseline level (i.e. blood sample immediately processed after venipuncture)
Long-term storage of plasma samples and ccfDNA extracts
Table 7 summarizes the main data reported in the literature [35], [57], [58], [59], [60] on the long-term storage of plasma samples and ccfDNA extracts. Each study compares data obtained from two consecutive analyses at varying lengths of time. The results are quite conflicting and clear conclusions cannot be drawn.
Our group carried out an unpublished statistical study by analyzing data from samples we used for a blinded multicenter prospective clinical study comparing KRAS/BRAF mutational
Conclusions
The numerous discrepancies reported in the literature on ccfDNA studies are mainly due to poor reproducibility and differences in handling procedures highlighting their crucial importance. Analysis of data in the literature and our results reveals the influence of preanalytical factors on ccfDNA analysis. By evaluating all the factors potentially affecting ccfDNA concentration and fragmentation, we describe here, for the first time, the optimal pre-analytical handling conditions for ccfDNA
Acknowledgments
We wish to thank the technical assistance of Mireille Cavalier. Authors thank Profs. D. Pezet, M. Mathonnet, and M. Ychou for providing the patient blood collection and Dr. E. Crapez and Dr. P.J. Lamy for their helpful discussions. The study was granted by the GEFLUC (DCMLP10-184, France). A.R. Thierry is supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) (France).
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The study was granted from the GEFLUC (DCMLP10-184, France). A.R. Thierry is supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) (France).