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A Study of Pre-Analytical Variables and Optimization of Extraction Method for Circulating Tumor DNA Measurements by Digital Droplet PCR

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A Study of Pre-Analytical Variables and Optimization of Extraction Method for Circulating Tumor DNA Measurements by Digital Droplet PCR Published OnlineFirst March 1, 2019; DOI: 10.1158/1055-9965.EPI-18-0586 Research Article Cancer Epidemiology, Biomarkers A Study of Pre-Analytical Variables and & Prevention Optimization of Extraction Method for Circulating Tumor DNA Measurements by Digital Droplet PCR Luca Cavallone1, Mohammed Aldamry2, Josiane Lafleur1, Cathy Lan1, Pablo Gonzalez Ginestet3, Najmeh Alirezaie4, Cristiano Ferrario1, Adriana Aguilar-Mahecha1, and Mark Basik1 Abstract Background: Circulating free DNA (cfDNA) is an exciting amplification on tumor and circulating tumor DNA novel method to diagnose, monitor, and predict resistance and (ctDNA) from patients with breast cancer. response to cancer therapies, with the potential to radically Results: Making use of a novel protocol for cfDNA extraction alter the management of cancer patients. To fulfill its potential, we increased cfDNA quantities, up to double that of commer- greater knowledge about preanalytical variables is required to cial kits. We found that a second centrifugation at 3,000 g optimize and standardize the collection process, and maxi- on frozen plasma is as efficient as a high-speed (16,000 g) mize the yield and utility of the small quantities of cfDNA centrifugation on fresh plasma and does not affect cfDNA extracted. levels. Methods: To this end, we have compared the cfDNA Conclusions: These results allow for the implementation of extraction efficiency of three different protocols, including protocols more suitable to the clinical setting. Finally, we a protocol developed in house (Jewish General Hospital). found that, unlike targeted gene amplification, whole genome We evaluated the impact on cfDNA levels of preanalytical amplification resulted in altered fractional abundance of variables including speed and timing of the second centri- selected ctDNA variants. fugation and the use of k-EDTA and CTAD blood collection Impact: Our study of the preanalytical variables affecting tubes. Finally, we analyzed the impact on fractional abun- cfDNA recovery and testing will significantly enhance the dance of targeted pre-amplification and whole genome quality and application of ctDNA testing in clinical oncology. Introduction acquired resistance in cancer patients (4–7). More importantly, evidence suggests that ctDNA measurement may provide a ThepresenceofcirculatingtumorDNA(ctDNA)inblood more comprehensive picture of tumor heterogeneity than provides an accessible source of genetic material from solid tumor biopsies and allow the detection of genomic alterations tumors and holds great promise for the development of clini- in tumors present at different locations throughout the body cal tools for less invasive diagnostic testing in cancer pati- thus providing a more accurate prediction of patient's tumor ents (1–3).Owingtotherelativeeaseofaccessandthe burden (8, 9). The clinical applicability and utility of ctDNA is minimally invasive nature of blood collection, the detection highly promising, and the recent approval by the FDA of the of tumor genomic modifications in ctDNA with highly sensitive Cobas EGFR mutation test to guide therapy in patients with sequencing methods has demonstrated great potential to pro- non-small cell lung carcinoma opens a new era in clinical liquid vide sensitive biomarkers for non-invasive diagnosis, progno- biopsy testing (10, 11). Nonetheless, many challenges remain sis, prediction and monitoring of treatment response, and to be overcome for ctDNA testing to be adopted as a routine clinical tool (12, 13), and for now, the use of tissue biomarkers remains the gold standard to guide therapy for most cancer 1Department of Oncology, Lady Davis Institute, McGill University, Montreal, patients. The major limiting factor comes from the fact that Quebec, Canada. 2Department of Surgery, Jewish General Hospital, Montreal, ctDNA represents only a small fraction of the total amount of Quebec, Canada. 3Department of Epidemiology, Biostatistics and Occupational circulating cell free DNA (cfDNA; refs. 1, 5). Therefore, even 4 Health, Mcgill University, Montreal, Quebec, Canada. McGill University and though ctDNA contains genetic modifications suitable to be Genome Quebec Innovation Center, Montreal, Quebec, Canada. tested as potential cancer biomarkers its use and reliability is Note: Supplementary data for this article are available at Cancer Epidemiology, hampered by its relative scarcity. In fact, although some have Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). reported difficulties in detecting tumor-derived mutations in Corresponding Author: Mark Basik, Lady Davis Institute and Jewish General plasma(14),othershavebeenabletoisolatectDNAinboth Hospital, 3755 Cote Ste Catherine, Montreal, Quebec, Canada H3T1E2. metastatic and nonmetastatic patients (14–16). These incon- Phone: 514-340-8222 ext. 24930; Fax: 514-340-8302; E-mail: sistencies are likely due not only to the inherent variability [email protected] of the fractional abundance of tumor-derived circulating doi: 10.1158/1055-9965.EPI-18-0586 DNA (from <0.01% to >90%), but also to the different sensi- Ó2019 American Association for Cancer Research. tivity of methodologies used for mutation detection and www.aacrjournals.org 909 Downloaded from cebp.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 1, 2019; DOI: 10.1158/1055-9965.EPI-18-0586 Cavallone et al. quantification (17–20). Furthermore, it has been shown that one aliquot was either centrifuged at low (3,000 Â g) or high theamountofisolatedcfDNAdependsontheefficiency of the (16,000 Â g) centrifugal force, respectively. Samples were cen- extraction method (21–23) and can be affected by pre-analyt- trifuged for 10 minutes at RT and 1 mL of supernatant from each ical variables including type of blood collection tube, centri- condition was collected for cfDNA extraction (Fig. 1B). fugation speed, and storage temperature (23–28). These pre- analytical variables can also impact on the release of nonmutated Plasma processing in EDTA and CTAD tubes. Peripheral blood DNA from leucocytes that result in the dilution of the ctDNA from seven (n ¼ 7) healthy volunteers was collected by veni- fraction. In this work, we aimed at optimizing the yield of cfDNA puncture in 2 Â 4mLEDTAtubesand2Â 4.5 mL BD Vacutainer and studied the effect of different pre-analytical variables on CTAD Tubes (referred to as CTAD Tubes in text; BD#367599; ctDNA detection using digital droplet PCR (ddPCR). We devel- Becton Dickinson). Tubes were inverted 10 times and imme- oped an improved protocol for cfDNA extraction from plasma, diately centrifuged at 1,500 Â g for 10 minutes at RT. The which formed the basis for the investigation of other variables plasma fraction was carefully collected and a second centrifu- such as the type of anticoagulant in blood collection tubes, and gation at 16,000 Â g was carried out at RT for 10 minutes. The centrifugation parameters. Finally, to obtain a larger amount of supernatant was collected and 1 mL aliquot was used for cfDNA DNA for downstream applications, we also tested if pre-amplifi- extraction (Fig. 1C). cation of cfDNA would affect the fractional abundance of ctDNA. Our findings contribute to the pre-analytical optimization of Plasma processing Q-CROC-03 patients. As part of the Q-CROC-03 cfDNA processing which is required for precise ctDNA results, trial, patients provided blood at different time points while thus strengthening the potential biomarker value of ctDNA mea- undergoing neoadjuvant chemotherapy for breast cancer. Blood surements in cancer patients. was collected in 2 Â 4.5 mL CTAD tubes and centrifuged within 30 minutes of collection following standard operating pro- Materials and Methods cedures (SOP) submitted to the NCI-SOP database (https:// brd.nci.nih.gov/brd/sop/show/1961): samples were centrifuged Samples at 1,500 Â g for 15 minutes at RT and plasma immediately Cell lines. BT-20 and MDA-MB-436 from American Type Culture aliquoted and stored at À80C. For this study, aliquots were Collection (ATCC) were cultured according to ATCC recom- thawed and a second centrifugation at 16,000 Â g was performed mendation. Cells were authenticated using array comparative for 10 minutes at RT prior to analysis. genomic hybridization (aCGH) and mycoplasma testing con- firmed the cells were mycoplasma-free. Cells were grown to Breast cancer tumor DNA 70% to 80% confluence and harvested for DNA isolation using Patients in the Q-CROC-03 trial provided tissue samples QIAamp DNA Mini Kit (Qiagen; Catalog No. 51304). prior to or following standard neoadjuvant chemotherapy. DNA was extracted from tumor biopsies as per reported SOPs Human samples. Blood was collected from healthy control volun- (29). DNA from tumor samples and cell lines underwent whole teers, and plasma and tumor DNA were obtained from patients exome sequencing (WES) and mutation calling at the McGill with breast cancer taking part in the Q-CROC-03 (NCT01276899) University Genome Center (Supplementary methods). Data clinical trial. All participants provided informed consent and the have been deposited in the European Nucleotide Archive data- study was conducted in accordance with recognized ethical guide- base with study primary accession number PRJEB30048. lines (e.g., Declaration of Helsinki), and approved by the Jewish General Hospital Ethics Committee Review Board (JGH; proto- Generation of spiked DNA samples cols 05-006 and A04-M31-12B). We selected genes with single nucleotide variants (SNV) present in cell lines
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