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Cancer Diagnostics |
1 Department of Molecular Oncology and 2
Division of Surgical Oncology, John Wayne Cancer Institute, Santa Monica, CA.
3 Section of Gastrointestinal Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA.
aAddress correspondence to this author at: Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404. Fax 310-449-5282; e-mail hoon{at}jwci.org.
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Abstract |
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Methods: Sera from 32 patients with CRC (3 stage I, 14 stage II, 6 stage III, and 9 stage IV patients), 19 patients with PACs (2 stage I, 9 stage II, 1 stage III, and 7 stage IV patients), and 51 healthy volunteers were assessed by quantitative real-time PCR of ALU repeats (ALU-qPCR) with 2 sets of primers (115 and 247 bp) amplifying different lengths of DNA. We used serum directly as a template for ALU-qPCR without DNA purification. DNA integrity was determined as ratio of qPCR results of 247-bp ALU over 115-bp ALU.
Results: ALU-qPCR had a detection limit of 0.01 pg of DNA. Eliminating DNA purification reduced technical artifacts and reagent/labor costs. Serum DNA integrity was significantly increased for stage I/II and III/IV CRC and stage I/II and III/IV PACs (P = 0.002, P = 0.006, P = 0.022, and P <0.0001, respectively). ROC curves for detecting CRC and PACs had areas under the curves of 0.78 and 0.80, respectively.
Conclusions: Direct ALU-qPCR is a robust, highly sensitive, and high-throughput method to measure serum DNA integrity. DNA integrity is a potential serum biomarker for detection and evaluation of CRC and PACs.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Free circulating DNA in serum or plasma is a proposed diagnostic and prognostic biomarker for malignant tumors (4)(5)(6). Increased DNA integrity in plasma, derived from threshold cycle numbers assessed by quantitative real-time PCR (qPCR) for 2 amplicons (400 bp and 100 bp) of a specific gene, was reported to indicate the presence of gynecologic and breast cancers (7). The premise is that DNA released from necrotic malignant cells varies in size, whereas DNA released from apoptotic cells is uniformly truncated into 185- to 200-bp fragments (8). Because the main source of free circulating DNA in healthy individuals is apoptotic cells, a preponderance of longer DNA fragments could be a marker for malignant tumor detection (9). However, assessment of the integrity of free circulating DNA is not yet practical for clinical use because the sensitivity and specificity of these methods have not been validated. A potential limitation may be the purification of DNA from serum or plasma, which decreases DNA yield. DNA loss may be inversely dependent on fragment size, which would affect DNA integrity values.
Recently, we developed a robust, highly sensitive, high-throughput method to measure the integrity of free circulating DNA in serum by qPCR for ALU repeats in a 0.1-µL equivalent volume of serum as a template without DNA purification. The ALU is the most abundant repeated sequence in the human genome, with a copy number of 
1.4 x 106 per genome (10)(11). ALU sequences are short interspersed elements, typically 300 nucleotides in length, that account for more than 10% of the human genome (12). ALU elements multiply within the genome in a retroposition process through RNA polymerase III–derived transcripts from evolution (13)(14); therefore, qPCR of ALU repeats with a properly designed primer set can dramatically increase the sensitivity of size-dependent DNA measurement. In this pilot study of CRC and PACs, we describe this method in detail and validate the practical utility of serum DNA integrity as a sensitive tumor biomarker.
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Materials and Methods |
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quantitative pcr of alu repeats
The target for ALU-qPCR in this study was a consensus sequence of human ALU interspersed repeats (see Fig. 1 in the Data Supplement that accompanies the online version of this article available at http://www.clinchem.org.content/vol52/issue6). We designed 2 sets of primers for ALU repeats: the primer set for the 115-bp amplicon (ALU115) amplifies both shorter (truncated by apoptosis) and longer DNA fragments, whereas the primer set for the 247-bp amplicon (ALU247) amplifies only longer DNA fragments. The sequences of the ALU115 primers were as follows: forward, 5'-CCTGAGGTCAGGAGTTCGAG-3'; reverse, 5'-CCCGAGTAGCTGGGATTACA-3'. The ALU247 primers were as follows: forward, 5'-GTGGCTCACGCCTGTAATC-3'; reverse, 5'-CAGGCTGGAGTGCAGTGG-3'.
The reaction mixture for each ALU-qPCR consisted of a template, 0.2 µM each of forward primer and reverse primer (ALU115 or ALU247), 1.0 U of iTaq DNA polymerase (Bio-Rad Laboratories), 0.02 µL of fluorescein calibration dye (Bio-Rad Laboratories), and a 1x concentration of SYBR Gold (Molecular Probe) in a total reaction volume of 20 µL with 5 mM Mg2+. Real-time PCR amplification was performed with precycling heat activation of DNA polymerase at 95 °C for 10 min, followed 35 cycles of denaturation at 95 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 30 s in an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories). The absolute equivalent amount of DNA in each sample was determined by use of a calibration curve with serial dilutions (10 ng to 0.01 pg) of gently prepared genomic DNA obtained from peripheral blood leukocytes of a healthy volunteer. A negative control (without template) was run in each reaction plate. All qPCR assays were performed in a blinded fashion without knowledge of the specimen identity, and the mean values were calculated from triplicate reactions. PCR products were electrophoresed on 2% agarose gels to confirm product size and the specificity of the PCR.
DNA integrity was calculated as the ratio of qPCR results with the 2 primer sets: Q247/Q115, where Q115 and Q247 are the ALU-qPCR results obtained with the ALU115 and ALU247 primers, respectively. Because the annealing sites of ALU115 are within the ALU247 annealing sites, the qPCR ratio (DNA integrity) would be 1.0 when template DNA is not truncated and 0.0 when all template DNA is truncated into fragments smaller than 247 bp. Because the ALU115 primers can amplify most fractions of circulating DNA, ALU-qPCR results obtained with ALU115 primers represent the absolute amount of DNA.
serum preparation and direct ALU-QPCR
We collected 1-mL blood samples into CORVAC serum separator tubes (Sherwood-Davis & Geck). Samples were processed within 6 h as follows: the blood was separated by centrifugation (1000g for 15 min) and passed through a 13-mm serum filter (Fisher Scientific) to remove potential contaminating cells. Serum was immediately stored at –80 °C. To deactivate or eliminate proteins that bind to template DNA or DNA polymerase and might invalidate qPCR results, we mixed 20 µL of each serum sample with 20 µL of a preparation buffer that contained 25 mL/L Tween 20, 50 mmol/L Tris, and 1 mmol/L EDTA. This mixture was digested with 16 µg of proteinase K solution (Qiagen) at 50 °C for 20 min, followed 5 min of heat deactivation and insolubilization at 95 °C. After subsequent centrifugation at 10 000g for 5 min, 0.2 µL of the supernatant (containing 0.1-µL equivalent volume of serum) was used as a template for each direct ALU-qPCR reaction.
evaluation of ALU-QPCR
Because the ALU-qPCR method used in this study was newly developed, we initially evaluated the performance of ALU-qPCR itself, using purified DNA or serum as a template.
We evaluated the sensitivity and linearity of ALU-qPCR with ALU115 or ALU 247 primers by use of a serially diluted, known amount of purified DNA obtained from peripheral blood leukocytes (PBLs) of a healthy volunteer. In addition, we compared the results of ALU-qPCR with ALU115 primer for serum DNA with the results obtained with the PicoGreen (Molecular Probes) reagent, which is a sensitive fluorescent nucleic acid stain for quantifying double-stranded DNA. Serum DNA from 15 healthy volunteers and 8 patients with PACs (evaluation set) was extracted and purified by a conventional technique; 500 µL of each separated and filtered serum was digested with 400 µg of proteinase K solution (Qiagen) along with 10 g/L sodium dodecyl sulfate, and DNA was purified by phenol–chloroform–isoamyl alcohol and ethanol precipitation as described previously (6).
We assessed the reproducibility of direct ALU-qPCR with ALU115 and ALU247 primers by triplicate reactions using a 0.1-µL equivalent volume of each serum of evaluation set.
We also evaluated the interfering effect of substances in serum on direct ALU-qPCR. Samples containing 10 ng of purified PBL DNA (P), 0.1-µL equivalent volume of serum (S), and a mixture of them (P+S) were prepared for each serum or evaluation set. DNA amounts in (P), (S), and (P+S) were quantified separately by ALU-qPCR with ALU115 and ALU247 primers. The interfering effect of serum on ALU-qPCR was calculated as follows: 1.0 – [Q115 (P+S) – Q115 (S)]/Q115 (P) for ALU115 primers and 1.0 – [Q247 (P+S) – Q247 (S)]/Q247 (P) for ALU247 primers, where Q115 (x) and Q247 (x) are the ALU-qPCR results for sample x with ALU115 and ALU247 primers, respectively. The interfering effect of serum on DNA integrity was calculated as follows: 1.0 – [Q247 (P+S) – Q247 (S)]/[Q115 (P+S) – Q115 (S)].
statistical analysis
We used Dunnet’s multiple comparison to compare the absolute concentration or integrity of serum DNA with clinicopathologic characteristics and used ROC curve analysis to assess the discriminating ability of assessments. The statistical package SAS JMP, Ver. 5.1 (SAS Institute Inc.) was used for statistical analyses. A P value <0.05 (two-tailed) was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The calculated relative efficiency of ALU-qPCR in relation to fragment length of template DNA is shown in Fig. 1
. The solid line shows the estimated efficiency of ALU-qPCR with ALU115 primer set: 0% for DNA fragments <115 bp and >90% for DNA fragments >1150 bp; the dotted line is for the ALU247 primer set: 0% for fragments <247 bp and 90% for fragments >2470 bp. As a result, DNA fragments between 115 and 247 bp, which covers the length of DNA cleaved by the apoptotic process, can be amplified with ALU115 primers but not with ALU247 primers.
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The threshold cycles of ALU-qPCR with the ALU115 or ALU247 primers on serially diluted genomic DNA (10 ng to 0.01 pg) obtained from PBLs of a healthy volunteer are shown in Fig. 2A
of the online Data Supplement. With both primer sets, linearity was maintained in the 106 range, and logarithmic regression lines had R = 0.998 for ALU115 and R = 0.999 for ALU247 primers; the detection limit was as low as 0.01 pg, equivalent to approximately a 1/300 copy of genome in a single cell. Agarose gel electrophoresis of PCR products obtained with the ALU115 and ALU247 primer sets on 10, 1, 0.1, and 0.01 pg of genomic DNA templates with 28 cycles of thermal cycling confirmed that the target sequence was specifically amplified without major aberrant bands (see Fig. 2B in the online Data Supplement).
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We also evaluated the sensitivity of ALU-qPCR, using clinical samples from 15 healthy volunteers and 8 patients with PACs (Fig. 2
). The amount of DNA purified from 500 µL of each serum specimen in the evaluation set was measured 2 ways: by the PicoGreen assay, which consumed one-tenth of the total extracted DNA; and by ALU-qPCR with ALU115 primers, which consumed only one-five thousandth of the total extracted DNA. Most of the serum samples from healthy volunteers contained too little DNA for accurate quantification by the PicoGreen assay. In contrast, ALU-qPCR had sufficient sensitivity and linearity for serum DNA quantification. ALU-qPCR results for specimens having relatively high serum DNA concentrations showed 1:1 linearity with the PicoGreen assay (Fig. 2
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We evaluated the reproducibility of direct ALU-qPCR by use of triplicate reactions using 0.1-µL equivalent volume of each serum specimen in the evaluation set. The median replicate CVs of the DNA concentrations quantified with for ALU115 and ALU247 primers were only 9% [interquartile range (IQR), 7%–14%] and 17% (IQR, 8%–24%), respectively. These CV values were equivalent to those for qPCR with specific primers for other genes (data not shown).
interfering effects of serum on direct ALU-QPCR
Because unpurified serum DNA directly used as a template for ALU-qPCR can inhibit the reaction efficiency, we tested its interfering effect on ALU-qPCR.
The median interfering effects of serum on direct ALU-qPCR with ALU115 and ALU247 primers were 0.09 (IQR, 0.03–0.20) and 0.24 (IQR, 0.09–0.37), respectively. The median interfering effect on DNA integrity was 0.12 (IQR, 0.08–0.18). Because the high sensitivity of ALU-qPCR lowered the requirement of serum template for ALU-qPCR, the interfering effect of serum was limited.
absolute concentration and integrity of serum dna
Healthy volunteers.
The mean (SD) age of 51 healthy volunteers (18 males and 33 females) was 48 (11) years. The mean (SE) absolute serum DNA concentration in healthy volunteers was 0.34 (0.25) ng/µL, and the mean serum DNA integrity was 0.13 (0.01). The absolute concentrations and integrity of serum DNA in healthy volunteers were independent of sex and age.
Patients with CRC.
The mean (SD) age of 32 patients with CRC (19 males and 12 females) was 66 (14) years. Mean absolute serum DNA concentrations in patients with stage I/II and stage III/IV CRC were 1.63 (0.43) and 1.73 (0.45) ng/µL, respectively, which were significantly higher than in healthy volunteers (P = 0.006 and 0.004, respectively; Fig. 3A
). The area under the ROC curve (AUC) for distinguishing patients with CRC from healthy volunteers by absolute DNA concentrations was 0.75 (Fig. 3B
). The mean (SD) serum DNA integrity in patients with stage I/II and stage III/IV CRC was 0.22 (0.02) and 0.22 (0.02), respectively, significantly higher than in healthy volunteers (P = 0.002 and 0.006, respectively; Fig. 4A
). The AUC of the ROC curve for discriminating patients with CRC from healthy volunteers by serum DNA integrity was 0.78 (Fig. 4B
). The ROC curves for serum DNA integrity and absolute serum DNA concentration were similar, indicating that serum DNA integrity may be equivalent to absolute serum DNA concentration with respect to CRC detection.
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Patients with PACs.
The mean (SD) age of 19 patients with PACs (12 males and 7 females) was 68 (9) years. Mean absolute serum DNA concentrations in patients with stage I/II and stage III/IV PACs were 0.84 (0.53) and 0.66 (0.62) ng/µL, respectively. There was no significant difference between cancer patients and healthy volunteers (P = 0.85 and 0.98, respectively; Fig. 5A
). The AUC of the ROC curve for discriminating patients with PACs from healthy volunteers by absolute DNA concentrations was only 0.59 (Fig. 5B
). The mean serum DNA integrity in patients with stage I/II and stage III/IV PACs was 0.22 (0.03) and 0.30 (0.03), respectively, significantly higher than in healthy volunteers (P = 0.022 and P <0.0001, respectively; Fig. 6A
). The AUC for the ROC curve for discriminating patients with PACs from healthy volunteers by serum DNA integrity was 0.80 (Fig. 6B
). This was greater than the AUC for absolute serum DNA concentration, indicating that serum DNA integrity was more informative than absolute serum DNA concentration with respect to the detection of PACs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA integrity may represent cancer cell death and thus could be a widely applicable biomarker for cancer existence or progression. However, difficulties in handling the very low concentrations of DNA in serum/plasma have been a technical barrier for practical applications. DNA purification steps introduce loss of DNA, which in itself is a problem in the assessment of free circulating DNA. In addition, because the recovery of serum/plasma DNA depends on DNA fragment size, it becomes a critical fluctuating factor for DNA integrity. To overcome these problems, we developed the ALU-qPCR method to directly measure the absolute amount and integrity of DNA in serum.
Because the ALU is the most abundant repeated sequence (10)(11), ALU-qPCR has sufficient sensitivity for direct assessment of serum. Elimination of the DNA purification step in direct ALU-qPCR stabilized the ratio of shorter and longer DNA fragments in serum. In addition, a calibration curve created by simultaneously performed qPCR on serially diluted genomic DNA in each reaction plate minimized the variance of ALU-qPCR results between reaction plates. Elimination of DNA purification also reduced the reagent and labor costs for the assessment, which is an important factor for implementation of screening tools. For large-scale future assessments, direct ALU-qPCR is easily adaptable to robotic automation. The extremely small volume of serum needed for this assessment is compatible with its use as a screening tool.
The concentrations of free circulating DNA are 4- to 6-fold higher in serum than in plasma (22)(23)(24). Because this difference does not reflect contaminated extraneous DNA during separation, serum is a better specimen source for circulating disease-related DNA (24). In addition, the reproducibility of direct ALU-qPCR when we used plasma as a template was inferior to the reproducibility when we used serum. However, during serum separation, cell lysis of PBLs may cause an artificial increase in DNA integrity. Increases in serum DNA were reportedly observed with overnight clotting after blood drawing (22) but not at 8 h (data not shown). We therefore processed blood within 6 h after blood drawing.
This pilot study demonstrates that serum DNA integrity is a clinically useful biomarker for detecting CRC and PACs. Serum DNA integrity was significantly increased even in localized CRC and PACs. It may therefore be useful for mass screening of malignant diseases. However, because any necrotic or mechanically ruptured cells release longer DNA fragments, patients with nonneoplastic diseases such as injury (25), acute inflammation, or infarctions may have high serum/plasma DNA integrity. In addition, pregnancy may cause a false positive because of fetal DNA in the maternal bloodstream (26). Such conditions may represent exclusion criteria for the assay as a screening tool for malignancy. Whether sera from patients with benign lesions such as colonic polyps have higher DNA integrity is unknown. A future study will help determine this.
The absolute concentration of serum DNA had a predictive value for CRC but not for PACs in this pilot study. We therefore consider serum DNA integrity a better molecular biomarker than absolute serum DNA concentration. There are some possible reasons for the wide distribution in absolute DNA concentrations seen in Fig. 3A
, such as aggressiveness of the disease, rapid turnover of the tumor, or immune response to the tumor. However, we did not find any specific conditions in the patients studied. Sera from certain cases of advanced cancers in this study showed very high ALU-qPCR values with ALU115 primers, which lowered the serum DNA integrity. In such cases, absolute serum DNA may be a better serum biomarker than DNA integrity. Therefore, a combined index of absolute concentration and integrity of serum DNA may decrease false negatives for cancer detection. For example, the {index of [mean +1.5 x SD of log(absolute DNA concentration)] or (mean + 1.5 x SD of DNA integrity)} achieves 92% specificity with 63% sensitivity for detection of stage I-IV CRC/PACs. To derive a definite index for clinical use, a large-scale clinical trial is needed.
DNA integrity has potential to help in CRC screening and surveillance, which now require colonoscopy, an invasive and relatively expensive examination (27). Although subsequent early operative intervention has improved CRC survival (28), a disproportionately high percentage of patients (15%–20%) with early-stage CRC suffer from local and distant recurrences (1). Surveillance mandates expensive follow-up procedures that are not cost-effective for the vast majority of postoperative patients (29).
PACs, specifically pancreatic cancer, are the fourth most common cause of cancer-related deaths in the United States (1). Of the estimated 32 190 new cases of pancreatic cancer in 2005, 80%–90% presented with clinically apparent metastatic disease or radiographic evidence of unresectability (1). Unfortunately, effective screening programs for pancreatic cancer have not been developed, and patients are often detected incidentally or after development of symptoms. Assessment of DNA integrity as an easy, simple, inexpensive screening method that could provide the impetus to initiate more aggressive radiographic evaluation. For those who are fortunate to have early diagnosis, surgical resection offers the only hope for cure. Despite adherence to rigorous surgical technique and histopathology-based diagnosis, up to 80% of patients will suffer early, locoregional recurrences (30)(31). Therefore, DNA integrity can also be used as a postoperative surveillance tool.
In conclusion, direct ALU-qPCR is a simple, robust, highly sensitive, and high-throughput method for measuring the integrity of free circulating DNA in serum. The elimination of DNA purification steps reduces technical artifacts and reagent and labor costs. Serum DNA integrity was significantly increased in patients with CRC and PACs and thus is a promising biomarker for detecting CRC and PACs. However, large-scale prospective studies are needed to establish the clinical utility of this index. In addition, the high sensitivity of direct ALU-qPCR suggests that it may be applicable for measurement of DNA concentration or DNA integrity in other human body fluids.
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Acknowledgments |
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Footnotes |
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References |
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