Evaluation of commercial kits for purification of circulating free DNA

Cancer Genetics 228–229 (2018) 21–27

ORIGINAL ARTICLE

Evaluation of commercial kits for purification of circulating free DNA Russell J. Diefenbach a,c, Jenny H. Lee a,c, Richard F. Kefford b,c,d, Helen Rizos a,c,∗ a

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; b Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; c The Poche Centre, Melanoma Institute Australia, NSW 2065, Australia; d Department of Medical Oncology, Crown Princess Mary Cancer Centre, Westmead and Blacktown Hospitals, Sydney, NSW 2145, Australia Abstract Analysis of liquid biopsies and the identification of non-invasive biomarkers for the diagnosis and prognosis of solid tumors has grown exponentially over the last few years. This has led to an increasing number of commercial kits optimised for the purification of circulating free (cf) DNA and RNA/miRNA from biofluids such as plasma, serum and urine. To optimise and standardise current practices we sought to evaluate the performance of spin column-based and magnetic bead-based commercial kits. The following commercial cfDNA purification kits were analysed in this study: QIAamp circulating nucleic acid kit (Qiagen, Germany); Plasma/serum cell-free circulating DNA Purification midi kit (Norgen Biotek, Canada); QIAamp minElute ccfDNA mini kit (Qiagen); Maxwell RSC ccfDNA plasma kit (Promega, USA); MagMAX cell-free DNA isolation kit (Applied Biosystems, USA); and NextPrep-Mag cfDNA isolation kit (Bioo Scientific, USA). Extracted DNA from the plasma of healthy individuals, either nonspiked or spiked with DNA fragments or cfDNA, was evaluated for recovery using either a BioRad Experion or ddPCR analysis. This study represents the first to use a comprehensive size distribution of spiked-in DNA fragments to evaluate commercial cfDNA kits. The commonly used spin column-based Qiagen QIAamp circulating nucleic acid kit was found to be the most consistent performing kit across the two evaluation assays employed. The Qiagen QIAamp minElute ccfDNA mini kit represented the best performing magnetic bead-based kit and provides an alternative based on lower cost/sample with a simpler workflow than spin column-based kits. Keywords Circulating free DNA, Circulating tumor DNA, Liquid biopsy, Biomarker. © 2018 Elsevier Inc. All rights reserved.

Introduction Development of liquid biopsies for monitoring cancer progression and response to therapy is a rapidly growing field [1–3]. A liquid biopsy typically involves extraction and analysis of DNA, RNA, proteins, vesicles or cells derived from biofluids such as blood, urine, saliva and cerebrospinal fluid. Circulating free DNA (cfDNA), specifically the tumor-derived

Received May 3, 2018; received in revised form July 27, 2018; accepted August 19, 2018 ∗ Corresponding author at: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia. E-mail address: [email protected] 2210-7762/$ - see front matter © 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.cancergen.2018.08.005

circulating tumor DNA (ctDNA) fraction is a promising cancer biomarker. Circulating DNA is highly fragmented DNA with a size distribution of ∼130–170 bp [4–6], which is equivalent to the size of nuclease-cleaved nucleosomes, and may arise from multiple mechanisms including apoptosis, necrosis and active secretion [7–9]. ctDNA levels often increase with tumor volume, can predict response to targeted and immunotherapies [10–18] and can be used to monitor tumor heterogeneity and reveal expanding resistant tumor clones [19–21]. One major challenge remains the sensitivity of ctDNA; circulating DNA is not abundant and does not reliably detect patients with early stage cancer [16,22]. It is difficult to evaluate ctDNA sensitivity, however, as there is no standardised method of biofluid sampling, ctDNA extraction or analysis. Typically, a liquid biopsy workflow consists of three steps: biofluid collection; biomarker isolation and biomarker

22 analysis/detection. Each of these steps provides an opportunity for optimisation and standardisation to improve both sensitivity and consistency. For blood-derived cfDNA, a number of studies have sought to optimise the yield and stability of cfDNA by comparing a range of collection tubes and other factors during blood collection [23–29] and a range of commercial cfDNA purification kits [30–33]. These previous studies compared either spin column-based [30] or magnetic bead-based [31–33] commercial kits to assess the recovery of nonspiked cfDNA in plasma. In some cases the additional recovery of spiked-in fragmented zebrafish DNA [30] or KRAS WT and mutant DNA [33] was also assessed. In this study, we compared a wider range of commercial kits consisting of two spin column-based kits (including the Qiagen kit used in previous studies) and four magnetic bead-based kits. We evaluated the efficiency of cfDNA recovery and for the first time analysed the size distribution of recovered DNA, in order to define an optimal, standard process for purifying cfDNA from plasma.

Materials and methods Plasma preparation Written consent was obtained from all healthy individuals under an approved Human Research ethics committee protocol from Macquarie University (5201300412). Blood (10 ml) was collected in EDTA tubes (Becton Dickinson, USA) and processed immediately. Tubes were spun at 800 g for 15 min at room temperature. Plasma was then removed into new 15 ml tubes without disturbing the buffy coat and respun at 1600 g for 10 min at room temperature to remove cellular debris. Plasma was stored in 1 ml aliquots at −80 °C.

Purification of DNA from plasma For spiking of DNA into healthy human plasma we used either a commercial low molecular weight DNA ladder or cfDNA purified from a BRAFV600E -mutant melanoma cell line. A total of 3 ml of 4 ml thawed pooled plasma from four healthy individuals (1 ml each) was spiked with 500 ng/ml of low molecular weight DNA ladder (DNA size range 25–766 bp; NEB Cat#N3233S, USA) prior to DNA extraction. The 3 ml of spiked pooled healthy plasma was then divided into 1 ml replicates and along with the remaining 1 ml of nonspiked healthy pooled plasma was processed through a commercial cfDNA purification kit. Commercial cfDNA purification kits, used according to the manufacturer’s instructions, included: QIAamp circulating nucleic acid kit (Qiagen Cat#55114; Qiagen spin (QiaS)); Plasma/serum cell-free circulating DNA Purification midi kit (Norgen Biotek Cat#55600; Norgen spin (NorS)); QIAamp minElute ccfDNA mini kit (Qiagen Cat#55284; Qiagen magnetic (QiaM)); Maxwell RSC ccfDNA plasma kit (Promega Cat#AS1480; Promega magnetic (ProM)); MagMAX cell-free DNA isolation kit (Applied Biosystems Cat#A29319; Applied Biosystems magnetic (ABioM)); and NextPrep-Mag cfDNA isolation kit (Bioo Scientific Cat#3825-01; Bioo Scientific magnetic (BSciM)). The elution volume (based on recommendations for each kit), in the order given above, was 50, 40, 30, 50, 15 and 20 µl, respectively.

R.J. Diefenbach et al. For spiked-in plasma cfDNA experiments, cfDNA was first extracted from the harvested supernatant (3 ml) of melanoma cell line M229 (BRAFV600E ) [34] using the QiaS kit. Plasma (1 ml) from five healthy individuals was spiked with either a 10-fold dilution or undiluted purified M229 cfDNA. Plasma with and without spiked-in cfDNA was extracted using either the QiaS kit or the QiaM kit according to the manufacturer’s instructions. In each case final elution volume was 50 µl.

Analysis of purified spiked-in DNA ladder from plasma Purified spiked-in low molecular weight DNA ladder from plasma was analysed using an Experion automated electrophoresis system (BioRad, USA) running a DNA 1K analysis kit according to the manufacturer’s specifications. Samples included: input consisting of 500 ng of low molecular weight DNA ladder diluted in the same final volume of elution buffer used for purification; purified spiked-in DNA; and elution buffer only. In each case, 1 µl of DNA sample was loaded in duplicate with the exception of elution buffer only which was a single loading. Automatic DNA peak assignment and integration was carried out using Experion software (version 3.2). The extraction efficiency (%) for each DNA fragment was calculated using the ratio of integrated peaks (purified/input). For comparison between kits the average recovery, based on individual average recovery of DNA fragments in the size range 75– 766 bp for each kit, was compared using a one-way ANOVA analysis (paired, parametric) in Prism version 7b.

Analysis of purified cfDNA from plasma The recovery of nonspiked cfDNA or spiked-in cfDNA from plasma was analysed as previously described [12]. The QX200 droplet digital PCR (ddPCR) system was used to detect nonspiked wild-type NRAS or spiked-in mutant BRAF as previously described (Bio-Rad) [12]. The DNA copy number was determined with Quantasoft software version 1.7.4 (BioRad) using a manual threshold setting. For comparison of the kits a two-tailed t-test (paired, parametric) on four replicate ddPCR runs (for nonspiked) or one ddPCR run (for spiked-in) was undertaken using Prism version 7b.

Results Commercial cfDNA purification kits from a growing range of manufacturers typically employ one of two technologies, the more established spin column-based approach or the more recently introduced magnetic bead-based approach (Table 1). Typically spin column-based methods are more time consuming and more costly than magnetic bead-based approaches (Table 1). All of these kits have, or soon will have, the capacity to process several mls of plasma (and other biofluids), an important consideration for maximising sensitivity especially in next generation sequencing analysis of cfDNA (Table 1). The linear recovery of cfDNA using the QiaS kit has been previously demonstrated over a range (5–17.5 ml) of input plasma volumes [35]. The majority of cfDNA kits also have the ability

b

a

Applied Biosystems Bioo Scientific ABioM

BSciM

Promega ProM

Capable of isolating RNA. No manual option, price adjusted to 2 ml sample (although only currently available as 1 ml sample kit with 4 ml sample kit in development).

Chemagic 360 18.8 Plasma 1–3 60

Kingfisher 15.92 Plasma/serum/urine 0.1–10 70

Maxwell RSC 31.26 Plasma 1 70

QIAcube 18.86 Plasma/serum 1–2 70 Qiagen QiaM

Magnetic beads Magnetic beads Magnetic beads Magnetic beads

Manual 33 Plasma/serum 1–4 Norgen Biotek NorS

Spin column 80

QIAcube 36.78 Plasma/serum/urine 1–5 Spin column 90 Qiagen QiaS

QIAamp circulating nucleic acid kita Plasma/serum cell-free circulating DNA midi kit QIAamp minelute ccfDNA mini kit Maxwell RSC ccfDNA plasma kitb MagMax cell-free DNA isolation kit NextPrep-Mag cfDNA isolation kit

Kit type Manufacturer cfDNA purification kit Code for this study

Table 1 Comparison of cfDNA purification kits used in this study.

Processing time (min)

Sample volume (ml)

Sample type

Price/2 ml sample in manual mode (AUD)

Automation option

Evaluation of commercial kits for purification of circulating free DNA

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to be partially or fully automated, another important consideration if high throughput is required, especially in a diagnostic setting (Table 1). Initial evaluation of cfDNA purification kits (listed in Table 1) was based on assessing the recovery of low molecular weight DNA fragments spiked into healthy human plasma. These DNA fragments span the size range of 25–766 bp which encompasses the reported size of cfDNA (130–170 bp) [4–6]. Analysis of the recovery of each individual DNA marker was based on separation on an appropriate DNA chip in the Experion automated electrophoresis system (Supplementary Figure 1). All of the kits recovered spiked-in DNA fragments in the range 50–808 bp to varying degrees (Fig. 1(A)). Aside from the 25 bp fragment, which was not efficiently recovered by any kit (Supplementary Figure 1), the 50 bp fragment was consistently recovered, albeit at lower efficiency levels, by all kits (Fig. 1(A)). This is in agreement with the recommended size range given by the manufacturer for the QiaS kit who indicate excellent recovery for fragments 75 bp and above. No specific recovery rate based on fragment size is reported by the manufacturers of the other kits. The QiaS, NorS, QiaM, ProM and ABioM kits had consistent levels of recovery across the range 75–808 bp (Fig. 1(A)). In contrast, the BSciM kit displayed the most variable profile of recovery (Fig. 1(A)). Overall the spin column-based QiaS and NorS kits showed no significant difference in overall recovery based on mean recovery of total DNA fragments (Fig. 1(B)). Comparison of QiaS or NorS kits to the magnetic bead-based QiaM, ProM, ABioM and BSciM kits showed a significantly greater recovery rate (p < 0.001) in all cases for QiaS and NorS (Fig. 1(B)). Comparison within the magnetic bead-based kits showed a significant difference in recovery between QiaM and ProM (Fig. 1(B), p = 0.019). The QiaM was significantly better in recovery than kits ABioM (p < 0.001) and BSciM (p = 0.002) (Fig. 1(B)). Likewise, the ProM kit was significantly better in recovery than kits ABioM (p < 0.001) and BSciM (p = 0.002) (Fig. 1(B)). The lowest level of overall recovery was observed for the BSciM kit which was significantly lower in recovery than the ABioM kit (Fig. 1(B), p = 0.007). Based on the analysis of total recovery efficiency using spiked-in DNA (Fig. 1(B)), one of the best performing spin column-based kits (in this case the QiaS kit), was matched against the best performing magnetic bead-based kit (in this case the QiaM). This time analysis was based on extraction of either nonspiked cfDNA or spiked-in cfDNA carrying a somatic mutation (BRAFV600E ) from healthy plasma. Quantification of recovered nonspiked wild-type NRAS or varying amounts of spiked-in mutant BRAF was performed using ddPCR [12]. Comparison of the average DNA copy number extracted from the plasma of 5 healthy individuals showed a significant overall difference in recovery between the two kits tested, for both nonspiked cfDNA (Fig. 2(A), p = 0.049) and the highest spiked-in quantity of cfDNA (Fig. 2(B), p = 0.0025). Although the lowest spiked-in quantity of cfDNA showed no significant difference in overall recovery between the two kits there was a trend towards lower recovery for the QiaM kit compared to the QiaS kit (Fig. 2(C)). As expected no BRAFV600E was detected in nonspiked healthy individuals (data not shown). Overall, this was in agreement with the previous analysis based on spiked-in low molecular weight DNA ladder which showed a significant difference between the QiaS and QiaM kits based on overall recovery of DNA fragments (Fig. 1(B), p < 0.001).

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Fig. 1 Comparison of the recovery efficiency of various commercial cfDNA kits based on spiked-in DNA fragments. (A) Comparison based on recovery of each individual spiked-in DNA fragment. (B) Comparison based on total DNA fragment recovery. A total of 3 ml of thawed pooled plasma from healthy individuals was spiked with 500 ng/ml of low molecular weight DNA ladder. The 3 ml of spiked plasma was then divided into 1 ml replicates and processed through a number of commercial cfDNA purification kits (listed in Table 1). Elution volume for each kit, in the order given above, was 50, 40, 30, 50, 15 and 20 µl. Purified DNA was analysed using an Experion automated electrophoresis system running a DNA 1K analysis kit. Individual DNA fragment recovery was calculated using the ratio of integrated peaks (input/purified) from each Experion run. Total DNA fragment recovery was calculated based on the average of the recovery of individual DNA fragments in the size range 75–808 bp. ns, not significant.

Discussion In this study, we evaluated six commercial cfDNA purification kits for efficiency in extracting DNA from plasma samples. From our initial analysis, based on extraction of spikedin low molecular weight DNA fragments, we found that spin column-based kits (Table 1, QiaS and NorS) consistently out-performed the magnetic bead-based kits (Table 1, QiaM, ProM, ABioM and BSciM) in recovering DNA ranging in size from 50–808 bp. Previous studies, using different evaluation assays and a different subset of magnetic bead-based cfDNA kits (QiaS was common to all studies), also showed a similar trend [31–33]. Further comparison of the spin column-based QiaS kit against the best performing magnetic bead-based kit (QiaM), based on extraction of nonspiked cfDNA from healthy human plasma, also showed a significant difference in recovery efficiency. Our study is the first to use a comprehensive size distribution of spiked-in DNA fragments to evaluate commercial

cfDNA kits. We observed that regardless of the overall yield of cfDNA most kits, except the BSciM kit, are relatively consistent in recovery of a range of DNA sizes which span the oligomeric forms observed for cfDNA [8]. A similar observation was reported for the QiaS kit [30], although this study did not assess the recovery of as many DNA fragment sizes as used in the current study. cfDNA forms include mono(166 bp), di- and tri-oligonucleosomal forms of nuclear DNA bound to histones, most likely arising from apoptotic events [8]. Furthermore, ctDNA has been reported to be more fragmented than non-tumor derived cfDNA with a size range of 132–145 bp or even <100 bp in some cases [4,36,37], and circulating mitochondrial DNA derived from cancer cells is much smaller and not protected by histones [38]. Therefore, establishing the performance of commercial cfDNA kits for recovery of variable sizes of DNA is important for optimising the utility of circulating DNA as a cancer biomarker. One further consideration is the impact that blood collection tubes that stabilise cells have on the efficiency of cfDNA

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Fig. 2 Comparison of cfDNA kits based on ddPCR analysis of cfDNA. Plasma (1 ml) from five healthy individuals was used as a source of nonspiked cfDNA (A), or spiked-in with M229 (BRAFV600E ) cell line cfDNA either undiluted (B) or 10 fold-diluted (C). Plasma was processed manually through either the spin column-based QiaS kit or the magnetic bead-based QiaM kit (listed in Table 1). Elution volume for each kit was 50 µl. DNA copy number was determined using ddPCR with a primer/probe based on detection of the nonspiked wild-type NRAS gene (A) or spiked-in mutant BRAF gene (B and C). ns, not significant.

extraction. Stabilised tubes typically contain a preservative which minimises the release of genomic DNA by preventing the lysis of nucleated blood cells. The presence of this preservative required an extended proteinase digestion step for optimal cfDNA extraction when using the QiaS kit [29]. Given most cfDNA extraction kits have a proteinase digestion step, they would also require optimisation if using stabilised blood collection tubes. Overall, this study illustrates that currently available cfDNA extraction kits regardless of the technology employed (spin column vs magnetic), are currently not able to surpass the QiaS kit in enhancing the sensitivity of cfDNA detection. Our studies support the continued use of the spin column-based QiaS kit as the gold standard for cfDNA isolation for biomarker analysis. The alternative choice of a magnetic bead-based kit (such as the QiaM kit) would be based on lower cost per sample and reduced processing time. The development of a larger sample volume-based option for the ProM kit may lead to lower cost per sample and also make this kit a viable alternative, assuming it performs as well as the QiaS kit in extracting nonspiked cfDNA, with the caveat this is only available in an automated mode.

Acknowledgments RJD was supported in part by a donation to Melanoma Institute Australia from the Clearbridge Foundation. This work was also supported in part by the National Health and Medical Research Council (APP1093017 and APP1128951). HR is

supported by a National Health and Medical Research Council Research Fellowship.

Conflicts of interest RFK sits on advisory boards for BMS and Merck, makers of pembrolizumab, nivolumab and ipilimumab. All remaining authors have declared no conflicts of interest.

Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cancergen.2018. 08.005.

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