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Quantitative Measurement of Cell-Free Plasma DNA and Applications for Detecting Tumor Genetic Variation and Promoter Methylation in a Clinical Setting

2012; Elsevier BV; Volume: 14; Issue: 4 Linguagem: Inglês

10.1016/j.jmoldx.2012.03.001

1943-7811

Sunil Kadam, Mark W. Farmen, John T. Brandt,

Prenatal Screening and Diagnostics

An elevated cell-free DNA (cfDNA) level is often reported in patients with advanced cancer and is thought to represent nuclear material from a distant inaccessible tumor. cfDNA can become a valuable source to monitor tumor dynamics and evaluate genetic markers for predictive, prognostic, and diagnostic testing. DNA extraction and quantification were optimized with plasma collected from 20 patients with advanced cancer and 16 healthy controls. Plasma cfDNA from patients with advanced cancer was evaluated for TP53 genetic variation and methylation status of CpG islands in several promoters of known disease-related genes. Tumor biopsy and corresponding plasma specimens were collected from study participants to determine whether the same genetic variations were present in both samples. The cfDNA isolation method provided a lower DNA detection limit of 144 pg, equivalent to DNA from approximately 24 cells. Normal pooled human plasma cfDNA averaged 110 copies/mL of the ACTB gene. Extracted cfDNA was suitable for gene-specific variant detection, sequencing, and promoter methylation analysis. DNA extracted from tumor biopsy and corresponding plasma specimens from two patients with advanced cancer revealed an identical, nonsynonymous variant present in both samples. Immunohistochemical analysis confirmed the TP53 mutant phenotype in the tumor specimens. Quantitative measurement of cfDNA represents a useful biomarker to follow treatment outcome and is a valuable tool with which to characterize specific genetic alterations for both patient selection and personalized treatment. An elevated cell-free DNA (cfDNA) level is often reported in patients with advanced cancer and is thought to represent nuclear material from a distant inaccessible tumor. cfDNA can become a valuable source to monitor tumor dynamics and evaluate genetic markers for predictive, prognostic, and diagnostic testing. DNA extraction and quantification were optimized with plasma collected from 20 patients with advanced cancer and 16 healthy controls. Plasma cfDNA from patients with advanced cancer was evaluated for TP53 genetic variation and methylation status of CpG islands in several promoters of known disease-related genes. Tumor biopsy and corresponding plasma specimens were collected from study participants to determine whether the same genetic variations were present in both samples. The cfDNA isolation method provided a lower DNA detection limit of 144 pg, equivalent to DNA from approximately 24 cells. Normal pooled human plasma cfDNA averaged 110 copies/mL of the ACTB gene. Extracted cfDNA was suitable for gene-specific variant detection, sequencing, and promoter methylation analysis. DNA extracted from tumor biopsy and corresponding plasma specimens from two patients with advanced cancer revealed an identical, nonsynonymous variant present in both samples. Immunohistochemical analysis confirmed the TP53 mutant phenotype in the tumor specimens. Quantitative measurement of cfDNA represents a useful biomarker to follow treatment outcome and is a valuable tool with which to characterize specific genetic alterations for both patient selection and personalized treatment. The presence of cell-free DNA (cfDNA) in human plasma has been known for several decades.1Bendich A. Wilczok T. Borenfreund E. Circulating DNA as a possible factor in oncogenesis.Science. 1965; 148: 374-376Google Scholar, 2Leon S.A. Shapiro B. Sklaroff D.M. Yaros M.J. Free DNA in the serum of cancer patients and the effect of therapy.Cancer Res. 1977; 37: 646-650Google Scholar cfDNA in blood is thought to be released by apoptotic and necrotic cells of the primary tumor during advanced tumorigenesis, which causes an increase in plasma nucleic acid levels.3Kamat A.A. Bischoff F.Z. Dang D. Baldwin M.F. Han L.Y. Lin Y.G. Merritt W.M. Landen Jr., C.N. Lu C. Gershenson D.M. Simpson J.L. Sood A.K. Circulating cell-free DNA: a novel biomarker for response to therapy in ovarian carcinoma.Cancer Biol Ther. 2006; 5: 1369-1374Google Scholar Elevated nucleic acid levels, relative to normal, have been reported for a wide variety of clinical disorders.4Behera D. Gumbhir K. Malik S.K. Dani H.M. Lung cancer and circulating DNA.Indian J Chest Dis Allied Sci. 1986; 28: 54-55Google Scholar, 5Anker P. Mulcahy H. Chen X.Q. Stroun M. Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients.Cancer Metastasis Rev. 1999; 18: 65-73Google Scholar, 6Theodor L. Melzer E. Sologov M. Idelman G. Friedman E. Bar-Meir S. Detection of pancreatic carcinoma: diagnostic value of K-ras mutations in circulating DNA from serum.Dig Dis Sci. 1999; 44: 2014-2019Google Scholar Apart from cancer, elevated DNA levels are also observed in patients with benign lesions, inflammation, trauma, and acute or chronic diseases; however, the level of cfDNA in blood of these patients was generally lower.7Iizuka N. Sakaida I. Moribe T. Fujita N. Miura T. Stark M. Tamatsukuri S. Ishitsuka H. Uchida K. Terai S. Sakamoto K. Tamesa T. Oka M. Elevated levels of circulating cell-free DNA in the blood of patients with hepatitis C virus-associated hepatocellular carcinoma.Anticancer Res. 2006; 26: 4713-4719Google Scholar, 8Musacchio J.G. Carvalho Mda G. Morais J.C. Silva N.H. Scheliga A. Romano S. Spector N. Detection of free circulating Epstein-Barr virus DNA in plasma of patients with Hodgkin's disease.Sao Paulo Med J. 2006; 124: 154-157Google Scholar, 9van der Vaart M. Pretorius P.J. Circulating DNA Its origin and fluctuation.Ann N Y Acad Sci. 2008; 1137: 18-26Google Scholar The potential utility of circulating cfDNA, however, was recently realized when KRAS and EGFR mutations were detected in plasma DNA, which is a characteristic of solid tumors.10Marchese R. Muleti A. Brozzetti S. Gandini O. Brunetti E. French D. Low value of detection of KRAS2 mutations in circulating DNA to differentiate chronic pancreatitis to pancreatic cancer.Br J Cancer. 2004; 90: 2243Google Scholar, 11Haihua Y. Zhonh-Zeng Z. Yachao L. Feng L. Wenying Z. Gang H. Guanshan Z. Jiang B. A modified extraction method of circulating free DNA for epidermal growth factor receptor mutation analysis.Yonsei Med J. 2012; 53: 132-137Google Scholar Others have suggested circulating DNA as a source to monitor specific epigenetic changes, leading to personalized cancer treatment.11Haihua Y. Zhonh-Zeng Z. Yachao L. Feng L. Wenying Z. Gang H. Guanshan Z. Jiang B. A modified extraction method of circulating free DNA for epidermal growth factor receptor mutation analysis.Yonsei Med J. 2012; 53: 132-137Google Scholar Recently, Danese et al12Danese E. Montagnana M. Minicozzi A.M. De Matteis G. Scudo G. Salvagno G.L. Cordiano C. Lippi G. Guidi G.C. Real-time polymerase chain reaction quantification of free DNA in serum of patients with polyps and colorectal cancers.Clin Chem Lab Med. 2010; 48: 1665-1668Google Scholar reported that serum cfDNA is significantly increased in patients with early-stage colorectal cancer with polyps, raising the possibility of its application as a marker for identifying high-risk individuals. However, a key observation that cfDNA can induce oncogenic transformation in susceptible cultured cells might suggest that such DNA can be sufficient to trigger tumor metastasis in distant organs.13García-Olmo D.C. Domínguez C. García-Arranz M. Anker P. Stroun M. García-Verdugo J.M. García-Olmo D. Cell-free nucleic acids circulating in the plasma of colorectal cancer patients induce the oncogenic transformation of susceptible cultured cells.Cancer Res. 2010; 70: 560-567Google Scholar Tumors release genomic DNA into the systemic circulation of many cancer patients, probably through cellular necrosis, active release, cell detachment, and apoptosis.9van der Vaart M. Pretorius P.J. Circulating DNA Its origin and fluctuation.Ann N Y Acad Sci. 2008; 1137: 18-26Google Scholar This DNA is admixed with DNA released from normal cells. Tumor-specific DNA in the plasma can be detected by the presence of genetic and epigenetic alterations that are specific to a primary tumor.14Xue X. Zhu Y.M. Woll P.J. Circulating DNA and lung cancer.Ann N Y Acad Sci. 2006; 1075: 154-164Google Scholar The easy accessibility of plasma and serum DNA has made these body fluids appealing noninvasive approaches to follow up patients after treatment. Fiegl and colleagues15Fiegl H. Millinger S. Mueller-Holzner E. Marth C. Ensinger C. Berger A. Klocker H. Goebel G. Widschwendter M. Circulating tumor-specific DNA: a marker for monitoring efficacy of adjuvant therapy in cancer patients.Cancer Res. 2005; 65: 1141-1145Google Scholar have used the presence of RASSF1A DNA methylation of cfDNA in serum to monitor response of women with breast cancer. Persistence of RASSF1A methylation 1 year after primary surgery and adjuvant tamoxifen therapy was an independent predictor of poor outcome in these patients.15Fiegl H. Millinger S. Mueller-Holzner E. Marth C. Ensinger C. Berger A. Klocker H. Goebel G. Widschwendter M. Circulating tumor-specific DNA: a marker for monitoring efficacy of adjuvant therapy in cancer patients.Cancer Res. 2005; 65: 1141-1145Google Scholar Thus, cfDNA measurement provides a powerful tool for noninvasive monitoring of therapeutic response in patients with solid tumors. Collection of and access to a peripheral sample for DNA measurement are often readily available, whereas serial biopsy material is difficult and cost prohibitive. However, the sources of variability in the methods used to collect, handle, extract, and quantify cfDNA are not well documented. A significant proportion of the variation is thought to be due to the disparity in protocols for sample processing,16Fujimoto A. O'Day S.J. Taback B. Elashoff D. Hoon D.S. Allelic imbalance on 12q22-23 in serum circulating DNA of melanoma patients predicts disease outcome.Cancer Res. 2004; 64: 4085-4088Google Scholar, 17van der Vaart M. Pretorius P.J. Is the role of circulating DNA as a biomarker of cancer being prematurely overrated?.Clin Biochem. 2010; 43: 26-36Google Scholar substantial differences among the absolute DNA levels,18Gormally E. Caboux E. Vineis P. Hainaut P. Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance.Mutat Res. 2007; 635: 105-117Google Scholar and a decrease in concentration on extended storage of collected plasma.19Sozzi G. Roz L. Conte D. Mariani L. Andriani F. Verderio P. Pastorino U. Effects of prolonged storage of whole plasma or isolated plasma DNA on the results of circulating DNA quantification assays.J Natl Cancer Inst. 2005; 97: 1848-1850Google Scholar The most commonly noted variation was attributed to delay in separating plasma from whole blood, variation in centrifugation and filtration steps, storage temperature, DNA extraction conditions, and DNA quantification methods, ranging from Picogreen DNA dipstick to quantitative PCR.17van der Vaart M. Pretorius P.J. Is the role of circulating DNA as a biomarker of cancer being prematurely overrated?.Clin Biochem. 2010; 43: 26-36Google Scholar Even among the quantitative PCR methods reported, the choice of gene amplified has been a significant source of variation.17van der Vaart M. Pretorius P.J. Is the role of circulating DNA as a biomarker of cancer being prematurely overrated?.Clin Biochem. 2010; 43: 26-36Google Scholar In this report we address and evaluate some of the sources of variability in the collection, processing, storage, and measurement of cfDNA and establish a collection protocol that can be readily adopted at multiple sample collection sites. We further examine the suitability of the extracted DNA for analysis of specific genetic variations in TP53 and the promoter methylation status of selected genes of clinical relevance. The findings suggest that in cases of advanced disease, measurable quantity of cfDNA is present in cell-free plasma, cfDNA can be quantified, and cfDNA is of adequate quality to allow genetic variation analysis and determine sequence-specific DNA methylation status. We have extended the molecular and phenotypic analysis to DNA extracted from tumor biopsy and corresponding plasma cfDNA specimens to show that cfDNA in blood originated from tumor-associated cells. Blood was collected from 44 patients with advanced cancer and 16 healthy controls for the development and optimization of analytical conditions and molecular analysis. Tumor biopsy specimens were collected at surgery with appropriate consent and embedded into paraffin after overnight formalin treatment. Where specified, blood was collected in direct draw tubes with EDTA as the anticoagulant or into Streck cfDNA blood collection tubes (Streck Laboratories, Omaha, NE), containing a proprietary preservative (Streck Laboratories). Plasma was separated by two consecutive centrifugations at 1600 × g for 15 minutes followed by careful recovery of the upper plasma layer and centrifugation in a separate tube at 16,000 × g for 15 minutes. The supernatant was carefully removed and stored at −80°C for further analysis. Blood from 10 of 44 cancer patients was collected in both EDTA and Streck tubes to test the effect of tube type, DNA extraction procedures, and storage conditions. Blood from another 10 cancer patients, collected in EDTA tubes, was used to test the effect of centrifugation speed on cfDNA measurement. Plasma was tested immediately or after 24 hours at ambient temperature. To test the effect of centrifugation speed and timing, tubes were divided into two groups for processing. For group A samples, plasma was separated with two centrifugation steps at 1600 × g each at the collection site. Plasma was then frozen and shipped to the analytical laboratory, where a high-speed centrifugation step at 16,000 × g was performed just before analysis for DNA quantification. For group B samples, plasma was separated with a single centrifugation step at 1600 × g followed by high-speed centrifugation at 16,000 × g at the collection site. Plasma was then frozen and shipped to the analytical laboratory; no further centrifugation steps were performed for group B samples. The remaining 24 samples were used for molecular analysis using the optimized preanalytical condition for blood collection, plasma separation, and DNA extraction derived from this study. Plasma samples were extracted immediately where specified, and aliquots were stored at –80°C until recovered for analysis. Commercially available pooled human plasma (AllCells, Emeryville, CA) was used to determine matrix effect and for DNA spike-in experiments. All blood samples from healthy controls and advanced cancer patients were collected with study participant consent and consideration of local ethical guidelines. DNA was normally extracted from 800 μL of plasma using the QIAamp DNA blood mini kit (Qiagen Inc., Valencia, CA) using manufacturer's instructions. Where stated, DNA from whole blood collected in the EDTA tube or the Streck DNA blood collection tube was extracted using a NucleoSpin Plasma XS kit (Machery-Nagel Inc., Bethlehem, PA) according to the manufacturer's protocol (see Supplemental Table S1 at http://jmd.amjpathol.org). To accurately quantify cfDNA, plasma was spiked with known amounts of single-copy ACTB gene containing plasmid (OriGene Technologies, Rockville, MD) extracted with the Qiagen DNA extraction kit and resuspended in reduced Tris EDTA Buffer (TEKNova, Hollister, CA). A standard curve was generated for comparison with plasmid suspended in nuclease-free water to a final concentration of 106 copies per 5 μL and stored in −20°C. Fragmented DNA (fDNA) was obtained from the Apoptotic DNA-Ladder Kit positive control reagent (Roche, Basel, Switzerland) and used to simulate degraded and apoptotic cell DNA that might be present in clinical specimens from cancer patients. The CT method of absolute quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized using the ACTB gene containing plasmid and fDNA to determine copy number equivalents as previously described.20Wittwer C.T. Herrmann M.G. Moss A.A. Rasmussen R.P. Continuous fluorescence monitoring of rapid cycle DNA amplification.Biotechniques. 1997; 22 (134–138): 130-131Google Scholar To determine the assay precision, six replicate analyses for quantification of fDNA were performed on the same day and on different days. fDNA was quantified using a Nanodrop ND-1000 spectrophotometer. Briefly, the spectrophotometer was initialized with nuclease-free water then blanked with 1 μL of elution buffer from the Roche kit. The absorbance of a 1-μL sample of fDNA was determined at 260 nm in triplicate, and the mean values were calculated. Low, medium, and high control concentrations were made and stored as separate aliquots. To develop robust assay conditions, several linearity and precision experiments were performed using commercially available frozen peripheral blood plasma from healthy donors. Sequences of the primers for the human ACTB gene (targeted to Gene Bank accession number NM_001101) used were as follows: forward, 5′-CCTGGCACCCAGCACAAT-3′; reverse, 5′-GCCGATCCACACGGAGTACT-3′, with a melting temperature of 59°C. The probe was dual-labeled with the reporter 6-carboxy-fluorescein (FAM) and a quencher (Black Hole Quenchers) at the 5′ and 3′ ends, respectively. The sequence of the probe was 5′-TCATTGCTCCTCCTGAGCGC-3′, with a melting temperature of 69°C. The primers and probe were both ordered from Sigma-Aldrich (St. Louis, MO). Quantitative PCR assays were developed for both a Bio-Rad (Hercules, CA) Chromo Four-Color Real-Time PCR Detection System (p/n/359-1590G) and an ABI HT-7900 instrument (Life Technologies, Foster City, CA). Tumor tissue biopsy specimens were treated overnight with 10% neutral buffer formalin, embedded into paraffin blocks, and sectioned to 4 nm, and slides were developed with anti-p53 antibody (AB-5, MS-186-R7; Thermo Scientific, Pittsburgh, PA) according to the manufacturer's instructions. Nuclear staining positivity was determined and ×20 images were recorded. Tissue DNA was extracted from 5 × 4-μm deparaffinized tissue sections determined to contain >50 tumor content after H&E staining. To standardize the assay method for genetic variant detection in the clinical context, we evaluated the lower level of sensitivity by spiking varying amounts of DNA extracted from previously characterized TP53 mutant cell lines into normal plasma. The cell lines used were the human pancreatic adenocarcinoma cell line BxPC3 (ATCC, Manassas, VA), harboring a nonsynonymous genetic variant in exon 6 of TP53 (p.Y220C), and the TP53 wild-type (WT) human alveolar basal epithelial cell line A549 (ATCC). The previously characterized TP53 genetic variant and wild-type status were confirmed by sequence analysis. Extracted cellular DNA from the mutant cell lines was spiked into normal plasma at various amounts (50 and 100 ng). The DNA was subsequently reextracted from the plasma. To verify the presence of the mutation from the extracted DNA, Surveyor Nuclease (Transgenomic, Omaha, NE) digestion coupled to a size-based analysis platform (SpectruMedix RVL9612 Genetic Analysis System; Transgenomic) was performed, and the mutation was then verified by bidirectional sequencing. Surveyor nuclease is an endonuclease that recognizes all heterozygous variations and works by cleaving double-stranded DNA on the 3′ side of a mismatch.21Gerard G.F. Shandilya H. Qiu P. Shi Y. Lo J. Genetic variance detection using Surveyor nuclease.in: Hecker K.H. Genetic Variance Detection: Technologies for Pharmacogenomics. DNA Press, LLC, Eagleville PA2006: 95-129Google Scholar A highly sensitive size-based analysis platform, such as the SpectruMedix Genetic Analysis System or the WAVE HS Fragment Analysis System (Transgenomic), was then used to separate the digested products from the full-length, undigested WT. In addition, to determine the limit of detection, various WT to mutant ratios from the two cell lines were prepared, added to plasma, extracted, amplified, digested, and finally sequenced. This step was performed for the assessment of detection limits in a WT background, rationalizing that TP53 genetic alteration and tumor heterogeneity may be reflected in peripheral DNA from shed tumor cells. Electropherograms were used to show the presence of mutation, and a bidirectional sequence analysis was performed to confirm the genetic alteration. Plasma samples from cancer patients were analyzed for TP53 genetic alterations using PCR-based methods in which the final end product was analyzed using the highly sensitive WAVE HS system. PCR was performed using Jumpstart Polymerase (Sigma-Aldrich), and the DNA template used for PCR varied between 0.2 and 5 ng. After heteroduplex formation, the PCR products were treated with Surveyor Nuclease. All samples identified as positive for the presence of a genetic variation by Surveyor Nuclease/WAVE HS analysis were further characterized by DNA sequencing. Amplicons of approximately 50-bp length of the flanking 5′ and 3′ intronic sequences were designed for denaturing high-performance liquid chromatography analysis of each exon (2 to 11). The resulting profiles were compared with the profiles obtained from the normal reference (WT cell line) DNA samples. The analysis of the methylation status of the promoter region of each gene was conducted by a PCR-based method using DNA treated with bisulfite followed by real-time pyrosequencing, which targeted CpG islands (EpigenDx Inc., Worcester, MA). This method enabled the quantification of methylation at multiple CpG sites individually.22Colella S. Shen L. Baggerly K.A. Issa J.P. Krahe R. Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites.Biotechniques. 2003; 35: 146-150Google Scholar, 23Ronaghi M. Pyrosequencing for SNP genotyping.Methods Mol Biol. 2003; 212: 189-195Google Scholar The target sequences within each promoter are described elsewhere (Table 1).Table 1Promoter CpG Island Methylation Sites Interrogated for Five Genes That Were Analyzed from cfDNA in Advanced Cancer PatientsGene namePrimer nameSequence to analyzeDispensation orderTarget regionCyclin D2ASY611Seq5′-GATGYGTTAGAGTAYGTGTTAGGGTTYGATYGTGTTGGYGGYGA-3′5′-TGATAGTCGTAGAGTGATCGTGTATGTCGTATCGTGTAGTCTGTCG-3′5′-GGGAGGAAGGAGGTGAAGAAAYGTTATTAGATYGTATTTTTTTGTAAAGATAGTTTTGATTTAAGGATGYGTTAGAGTAYGTGTTAGGGYGYGATYGTGTTGGYGGYGATTTTATYGTAGTYGGTTTTTAGGGAGAAAGTTTTGGYGAGTGAGGYGYGAAATYGGAGGGGTYGGYGAGGATGYGGGYGAAGGTATYGAGYGTGGAGGTTTTATGTTTTYGGGGAAAGGAAGGGGTGGTGGTGT-3′Hin1ASY575Seq15′-GTTAYGAGGTTTTTTATATTYGGTTTTYGTTTTTTTAGYGTYGGTTTYGTTYGYGTTTTTGAGAAAGTTTTGTTYGTTTYGTTTAYGGTYGTGTTTTGG-3′5′-TGTGATCGAGTTATGATCTGTTCGTTATGTCAGTCTGTTCAGTCTGTCGTTGAGAGTTAGTCTGTTCGTGATCAGTCGT-3′5′-CTTGTTTTTAGAGGGTTTTAGYGTTTGTTAAGAGGAAGTTTTYGAGGTTYGGGTAGGGAAGGGGGTAYGGGTTTTTTAGGGTTYGTYGGTYGTAGTAGGAAGTTGGTTAGGGTAYGGTYGTGAGYGGGYGGGTAGGGTTTTTTTAGGAGYGYGGGYGAGGTYGGYGTTGGAGGGGYGAGGATYGGGTATAAGAAGTTTYGTGGTTTTGTTYGGGTAGTYGTAGGTTTTTYGYGYGTTTYGAGTTTTYGYGTTAT-3′p16ADS1067FS15′-GGGTGGGGYGGATYGYGTGYGTTYGGYGGTTGYGGA-3′5′-AGTGGTCGTATCAGTCGTAGTCTGTCAGTCGCTAGTCG-3′5′-GAGGAGGGGTTGGTTGGTTATTAGAGGGTGGGGYGGATYGYGTGYGTTYGGYGGTTGYGGAGAGGGGGAGAGTAGGTAGYGGGYGGYGGGGAGTAGTATGGAGTYGGYGGYGGGGAGTAGTATGGAGTTTTYGGTTGATTGGTTGGTTAYGGTYGYGGTTYGGGGTYGGGTAGAGGAGGTGYGGGYGTTGTTGGAGGYGGGGGYGTTGTTTAAYGTATYGAATAGTTAYGGTYGGAGGTYGATTTAGGTGGGTAGAGGGTTTGT-3′RASSF1ASY574FS (A1512FS)5′-YGTTYGGTTYGYGTTTGTTAGYGTTTAAAGTTAGYGAAGTAYGGGTTTAATYGGGTTATGTYGGGGGA-3′5′-GTCTGTCGTCTGTCGTGCTATGTCGTAGTATGTCGAGTGATCGTGATCGTATAGTCGG-3′5′-AGTTTGGATTTTGGGGGAGGYGTTGAAGTYGGGGTTYGTTTTGTGGTTTYGTTYGGTTYGYGTTTGTTAGYGTTTAAAGTTAGYGAAGTAYGGGTTTAATYGGGTTATGTYGGGGGAGTTTGAGTTTATTGAGTTG-3′SFRP1ADS181FS5′-YGAATTYGTTYGYGAGGGAGGYGATTGGTTTTYGYGTYGGTGA-3′5′-GTCGTATCAGTCTGTCGAGATGTCGATAGTTCTGTCAGTCGTG-3′5′-GAGTYGYGTTTGGTTTTAGTAAATYGAATTYGTTYGYGAGGGAGGYGATTGGTTTTYGYGTYGGTGAYGGAYGTGGTAAYGAGTGYGGTTYGTTTYGTYGGGAGTTGATTGGTTGYGYGGGGYGGTTTYGAGGGTTYGGTYGTAGGAGT-3′The targeted methylated cytosine residues are indicated as Y, and the first position is italicized. The analyzed sequence within the targeted region is in bold. Open table in a new tab The targeted methylated cytosine residues are indicated as Y, and the first position is italicized. The analyzed sequence within the targeted region is in bold. DNA was extracted as described herein, and 1 μg of sample DNA was treated with bisulfite using the Zymo DNA Methylation Kit (Zymo Research, Orange, CA). Bisulfite-treated DNA was eluted in a 10-μL volume. Bisulfite reaction conditions typically yielded DNA with complete conversion of guanine residues. PCR fragments were amplified in a PCR reaction mix containing 1 μL of bisulfite-converted DNA or internal controls, 10× PCR buffer, 3.0 mmol/L MgCl2, 200 μmol/L of each dNTP, 0.2 μmol/L each of forward and reverse primers, and 1.25 U of HotStart DNA polymerase (Qiagen Inc.). Products were immobilized and denatured at 80°C for 2 minutes. For the pyrosequencing reaction, the corresponding sequencing primer was added to the single-stranded DNA and nucleotides dispensed automatically by the PSQ HS 96A instrument and software version 1.0 (Biotage, Uppsala, Sweden). For each reaction, the DNA from peripheral blood mononuclear cells served as unmethylated controls, and the DNA from human colon cancer cell line SW48 (ATCC) served as the methylated control. Pyrograms were read at least twice. The collection and flow of sample for various measurements from cell-free plasma were as follows. Briefly, whole blood collected at clinical study sites was processed, and the plasma aliquots were distributed in separate tubes for cfDNA quantification, genetic variant analysis, and epigenetic testing for promoter methylation status. To develop a robust cfDNA quantification method, we initially tested the linearity and dynamic range of detection with four separate plasmids each containing the ACTB, HBB, RPL19, and PROS1 genes at half log serial dilutions starting at 105 copies based on molecular weight (data not shown). In repeated experiments, the ACTB gene measurement was consistently linear over a broad dynamic range and was sensitive to detect 10 copies. All subsequent accuracy, precision, and extraction recovery analyses were performed by absolute quantification with β-actin as the control gene, and cfDNA was expressed in ACTB copy equivalents. The accuracy and precision of the cfDNA measurement were determined using fDNA (Table 2) and reflect acceptable performance (SD <8%) at DNA levels well below the background level of 13 ng/mL for healthy controls reported earlier.24Huang Z.H. Li L.H. Hua D. Quantitative analysis of plasma circulating DNA at diagnosis and during follow-up of breast cancer patients.Cancer Lett. 2006; 243: 64-70Google ScholarTable 2Assay Performance: Accuracy and Precision of β-Actin Copy Number Equivalents in Fragmented DNAfDNA (ng)Interday (copies)⁎Interday measurements were an average of six experimental values in triplicate.SD (%)Intraday (copies)†Intraday measurements were an average of triplicate experimental values performed during 6 days.SD (%)104094842547.8138322.842817.70.14336.14922.4 Interday measurements were an average of six experimental values in triplicate.† Intraday measurements were an average of triplicate experimental values performed during 6 days. Open table in a new tab To evaluate the effect of whole blood draw tube, the efficiency of cfDNA extraction, and its stability at ambient temperature, plasma was separated from blood drawn in either the EDTA or Streck tubes. The one-way analysis of tube type and isolation kit demonstrated that in tubes where DNA was isolated using the Qiagen kit, the copy number was higher compared with the Nucleospin extraction column. In addition, there was more DNA degradation (ACTB copy number equivalents: 4951 at 0 hours versus 3703 at 24 hours) when the DNA was isolated by Nucleospin extraction column compared with Qiagen DNA extraction kit (ACTB copy number equivalents: 9721 at 0 hours versus 8760 at 24 hours), suggesting that DNA isolated by the Qiagen kit was more stable and not prone to degradation when compared with the Nucleospin extraction column as shown (Figure 1, A and B). The data reveal a similar range of values for both the EDTA and Streck tubes. However, the range of values of cfDNA measured from the Qiagen column extracted DNA compared with the Nucleospin extracted DNA was broader, indicating perhaps that the Nucleospin column might have saturated its DNA-binding capacity, whereas the Qiagen column remained functional. Despite the sensitivity of quantitative PCR in detecting DNA in an aqueous system, the efficiency and reproducibility of cell-free DNA recovery from a plasma matrix are important clinical analysis parameters. We tested the plasma matrix effect using a fDNA from apoptotic cells as a suitable alternative to peripheral DNA shed in advanced cancer. Known quantities of fDNA were spiked into normal plasma and collected and extracted under previously optimized conditions, and β-actin copy equivalents were determined. The data represented in Table 3 indicate that the addition of as low as 0.045 ng of spiked fDNA could be recovered and produced a measurable change over the normal background. Copies increase with spiked DNA amount. However, linearity is lost at higher DNA levels. ACTB copies are being lost in the matrix perhaps due to saturation of the Qiagen DNA-binding capacity.Table 3Spiked fDNA Recovery of ACTB Copies and Copy Equivalents of cfDNA from Pooled Normal PlasmafDNA (ng)β-actin copiesPlasmid DNA copiescfDNA β-actin copies026202620.045449313211.449923160229645.5215,290316,00022,998Normal range50–407Purified ACTB containing plasmid DNA and copy equivalent range of normal circulating cfDNA in plasma collected from healthy controls.