[CANCER RESEARCH 61, 1659 –1665, February 15, 2001]
`
`DNA Fragments in the Blood Plasma of Cancer Patients: Quantitations and
`Evidence for Their Origin from Apoptotic and Necrotic Cells1
`Sabine Jahr,2 Hannes Hentze, Sabine Englisch, Dieter Hardt, Frank O. Fackelmayer, Rolf-Dieter Hesch, and
`Rolf Knippers
`Department of Biology [S. J., H. H., F. O. F., R. K.] and Steinbeis-Transfer-Laboratory for Biomolecular Medicine [S. E., R-D. H.], Universita¨t Konstanz, D-78457 Konstanz, and
`Department of Oncology, Klinikum Konstanz [D. H.], D-78464, Konstanz, Germany
`
`could frequently be identified in the plasma DNA of carcinoma
`patients (10 –12). These and similar findings indicate that a certain
`percentage of circulating DNA originates from degenerating tumor
`cells. It has therefore been proposed that analyses of plasma DNA
`could be useful for prognostic purposes or for early diagnosis to
`detect, e.g., subclinical disease recurrence in disease-free patients
`(13). An analysis might also constitute a tool to follow the develop-
`ment of tumors by monitoring genetic changes in the circulating DNA
`of cancer patients. However, the amounts of DNA in plasma vary
`widely even in clinically similar situations (1, 2). Moreover, reports
`quite commonly include cases where mutated genes could be detected
`in tumor tissue but not in circulating DNA (13), suggesting that not all
`of the plasma DNA originates from tumor cells, whereas other studies
`suggest that tumor DNA is the predominant subtype in at least some
`cancer patients because tumor-specific loss of heterozygosity was
`detected in plasma DNA (7–9). In fact, the mechanism of how DNA
`is released into blood circulation is unknown, though there are many
`hypotheses, such as tumor cell apoptosis or necrosis or active release
`of DNA (13).
`Because the issue of circulating DNA is not only of clinical rele-
`vance but also of considerable biological interest, we decided to
`investigate the origin of DNA in the plasma samples of 30 cancer
`patients of different tumor types. Using highly sensitive methods of
`quantitative PCR, we determined the fraction of DNA in the circula-
`tion that originates from tumor cells and from nontumor cells. We also
`sought for evidence showing that at least some of the DNA could be
`released from apoptotic or necrotic cells, a conclusion that we support
`by studies using cultured cells and mice with in vivo-induced apo-
`ptosis and necrosis.
`
`MATERIALS AND METHODS
`
`ABSTRACT
`
`Increased levels of DNA fragments have frequently been found in the
`blood plasma of cancer patients. Published data suggest that only a
`fraction of the DNA in blood plasma is derived from cancer cells. How-
`ever, it is not known how much of the circulating DNA is from cancer or
`from noncancer cells. By quantitative methylation-specific PCR of the
`promoter region of the CDKN2A tumor suppressor gene, we were able to
`quantify the fraction of plasma DNA derived from tumor cells. In the
`plasma samples of 30 unselected cancer patients, we detected quantities of
`tumor DNA from only 3% to as much as 93% of total circulating DNA.
`We investigated possible origins of nontumor DNA in the plasma and
`demonstrate here a contribution of T-cell DNA in a few cases only. To
`investigate the possibility that plasma DNA originates from apoptotic or
`necrotic cells, we performed studies with apoptotic (staurosporine) and
`necrotic (staurosporine plus oligomycin) cells in vitro and with mice after
`induction of apoptotic (anti-CD95) or necrotic (acetaminophen) liver in-
`jury. Increasing amounts of DNA were found to be released in the
`supernatants of cells and in the blood plasma samples of treated animals.
`A clear discrimination of apoptotic and necrotic plasma DNA was possible
`by gel electrophoresis. The same characteristic patterns of DNA fragments
`could be identified in plasma derived from different cancer patients. The
`data are consistent with the possibility that apoptotic and necrotic cells are
`a major source for plasma DNA in cancer patients.
`
`INTRODUCTION
`
`It is known that double-stranded DNA fragments frequently occur
`in considerable quantities in the serum or plasma of cancer patients (1,
`2). The quantitation of this free DNA in the serum of patients with
`various types of cancer and healthy individuals showed that the DNA
`concentration in the normal controls had a mean of 13 ng/ml, whereas
`in the cancer patients the mean was 180 ng/ml. Although no correla-
`tion was found between circulating DNA levels and the size or
`location or the primary tumor, significantly higher DNA levels were
`found in the serum of patients with metastases (2). Other studies
`performed with lung cancer patients found plasma DNA levels to be
`higher in patients with advanced disease (3, 4). Investigations on the
`characteristics of the DNA found in the plasma of cancer patients
`showed that an important part of the DNA originates from the tumor
`cells (5). Furthermore, the presence of oncogene or tumor suppressor
`gene mutations that characterize DNA in tumor cells were detected in
`plasma DNA. For example, the K-ras mutations in the DNA derived
`from pancreatic tumors were also found in the circulating DNA of the
`same patients (6), just as the same microsatellite alterations, detected
`in head and neck carcinomas, small cell lung carcinomas, or renal
`carcinomas, could be determined in patients’ plasma DNA (7–9).
`Furthermore, tumor-specific epigenetic alterations such as the hyper-
`methylation of sequences in the promoters of tumor suppressor genes
`
`Plasma Sample Collection and DNA Extraction. Blood samples were
`withdrawn from a peripheral vein and placed in EDTA-containing tubes from
`a total of 30 unselected informed cancer patients diagnosed at the Department
`of Oncology at the municipal hospital. Similarly, blood was drawn from 20
`healthy donor volunteers. The study was approved by the Ethics Committee of
`the University of Konstanz. Plasma was immediately separated from blood
`cells by centrifugation at 3000 3 g for 20 min. Tumor tissue from patients was
`collected at surgery. DNA was extracted from blood plasma and tumor tissue
`using the QIAamp Blood Kit (Qiagen, Hilden, Germany) using the blood and
`body fluid protocol (11, 14) or the QIAamp Tissue Kit, respectively (11).
`Quantitation of Total Plasma DNA. The amounts of plasma DNA were
`determined by competitive PCR according to the method of Diviacco et al.
`(15), using the lamin B2 locus as a typical example for a single copy gene. The
`competitor molecule carrying a 20-bp insert was obtained directly from two
`amplification products by the overlap extension method (15). Quantitation of
`competitive templates was obtained by OD260 measurement. For quantitation,
`a fixed amount of plasma DNA was mixed with increasing amounts of the
`competitor template. For competitive PCR, two additional primers (Q-EF:
`59-TCCAATGATTTGTAATATAC-39 and Q-ER: 59-ATCTTTCTTAGA-
`CATCCGCTT-39) were designed. After PCR amplification (40 cycles: 94°C,
`1 min; 52°C, 1 min; 72°C, 1 min) and PAGE, two products of 153 and 173 bp
`were evident, corresponding to genomic and competitor templates, respec-
`tively. The ratios of the amplified products precisely reflect the initial concen-
`1659
`
`Received 6/12/00; accepted 12/07/00.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`1 Supported by Deutsche Forschungsgemeinschaft.
`2 To whom requests for reprints should be addressed, at the Department of Biology,
`Universita¨t Konstanz, Universita¨tsstr. 10, D-78457 Konstanz, Germany. E-mail: Sabine.
`Jahr@web.de.
`
`00001
`
`EX1047
`
`

`

`ON THE ORIGIN OF PLASMA DNA IN CANCER PATIENTS
`
`TTTAAGTATCGTGGATATTTTCG-39 (sense) and 59-AAAAACAACTAA-
`ACACTACTTCG-39 (antisense). The sequence-specific primers cover three
`CpG islands, two of which are positioned at the 39 end of the primers.
`Thirty-five PCR cycles were performed, with each cycle consisting of 1 min at
`94°C, 1 min at 50°C, and 1 min at 72°C. DNA of HeLa cells and of HUVECs3
`served as negative and positive controls, respectively. PCR products (151 bp)
`were visualized after electrophoresis on 6% polyacrylamide gels and ethidium
`bromide staining.
`Induction of Apoptosis and Necrosis in Vitro. Jurkat T cells in serum-free
`medium without glucose were induced to undergo necrosis (with 2.5 mM
`oligomycin plus 1.2 mM staurosporine) or apoptosis (with 1.2 mM staurospo-
`rine) as previously described (18). At different times after the addition of
`drugs, the supernatant was separated from the cells by centrifugation. The
`DNA of the supernatants was purified (QIAamp blood kit; Qiagen) and
`quantified by competitive PCR. Apoptotic and necrotic cell death were con-
`firmed by monitoring the cleavage of the nuclear SAF-A in Western blot
`analyses. Only during apoptotic, but not necrotic cell death, is SAF-A cleaved
`by caspase-3 (19, 20).
`Induction of Apoptotic and Necrotic Liver Injury in Mice. Specific
`pathogen-free male BALB/c mice (;25 g, from the in-house animal breeding
`station of the University of Konstanz) were maintained under controlled
`conditions (22°C and 55% humidity, constant 12-h day/night cycle) and fed a
`standard laboratory chow. All animals received humane care in accordance
`with the NIH guidelines as well as with the legal requirements in Germany.
`Mice were starved overnight before the onset of experiments. To induce
`hepatocyte apoptosis, activating anti-CD95 antibody (aCD95, clone Jo-2, 2 mg
`per animal; PharMingen) was injected i.v. in a volume of 300 ml endotoxin-
`free saline supplemented with 0.1% human serum albumin (21, 22). Hepato-
`cyte necrosis was induced by i.p. treatment of mice with acetaminophen (250
`mg/kg in 300 ml endotoxin-free saline; EGA, Steinheim, Germany; Ref. 20).
`At the time points indicated, mice were killed by i.v. injection of 150 mg/kg
`pentobarbital plus 1.2 mg/kg sodium citrate as an anticoagulant. Blood was
`withdrawn by cardiac puncture and centrifuged (5 min, 14,000 3 g, 4°C) to
`obtain plasma. The extent of liver damage was assessed by measuring plasma
`ALT activity with an EPOS 5060 analyzer (Netheler & Hintz, Hamburg,
`Germany) as previously described (23). DNA was extracted from murine
`plasma using a DNA extraction kit (QIAamp blood kit; Qiagen) and quantified
`by real-time PCR (LightCycler; Roche Diagnostics). The region of amplifica-
`tion was part of exon 3 of the murine a-actin gene. The following primers were
`used for amplification: 59-TGAACATGGCATCATCACC-39 and 59-CTG-
`GATAGCCACATACATG-39, resulting in a product of 199 bp. Real-time
`quantitation was carried out by the SYBR-Green reaction mix (Roche Diag-
`nostics). A fixed amount of 4 ml of eluted DNA was used in each PCR
`reaction. Forty PCR cycles were performed, with each cycle consisting of 1 s
`at 95°C, 5 s at55°C, and 10 s at 72°C.
`Analysis of Plasma DNA Fragments. DNA of human or murine plasma
`was extracted using a DNA extraction kit (QIAamp blood kit; Qiagen) and
`separated on 6% polyacrylamide gels (patients’ plasma DNA samples) or 1.5%
`agarose gels (murine plasma DNA samples). Fragments were then visualized
`after ethidium bromide staining.
`
`tration of genomic DNA versus that of the added competitor. Quantitation of
`competitor and genomic bands was obtained by densitometric scanning of the
`ethidium bromide-stained gel (15). The results obtained by means of compet-
`itive PCR were confirmed by quantitation with the Control Kit DNA in the
`LightCycler System (Roche Diagnostics). For that purpose we used the Light-
`Cycler Control Kit DNA to amplify a 110-bp fragment of the human b-globin
`gene. The amplicon was detected by fluorescence using a specific pair of
`hybridization probes (LC-Red 640). As standards we used serially diluted
`genomic DNA of the kit. After completion of PCR, the LightCycler software
`calculated the copy number of target molecules by plotting logarithm of
`fluorescence versus cycle number and setting a baseline x-axis. The concen-
`trations of the samples were extrapolated from the standard curve by the
`LightCycler software.
`T Cells. The presence of T-cell DNA in plasma samples was examined by
`PCR amplification of a region of the T-cell receptor b chain, which exhibits a
`somatic rearrangement by VDJ recombination (16). For amplification, a mix of
`sense primers (Vbz5, Vbz6) and one antisense primer (Jb1i) were used (16).
`This leads to the amplification of DNA fragments with defined sizes: 907, 767,
`and 155 bp, depending on the rearranged Jb-segment. DNA from Jurkat T
`cells, HeLa cells, and human lymphocytes was included as internal controls
`with each run. As an additional control, we amplified plasma DNA with
`primers specific for the germline configuration of the T-cell receptor using the
`primers G1F: 59-AATGATTCAACTCTACGGGA-39 (sense) and G1R: 59-
`TGAGTCCTCCACTTGTGAG-39 (antisense), resulting in a product of 250
`bp. One hundred ng of purified plasma DNA samples were used in each PCR
`assay. PCR products were analyzed by 6% PAGE and ethidium bromide
`staining.
`Hypermethylation of CDKN2A. Detection of hypermethylated CpG is-
`lands in the promoter region of the CDKN2A tumor suppressor gene was
`carried out by methylation-specific PCR as previously described (17). Bisulfite
`treatment of tumor or plasma DNA was carried out using the CpGenome DNA
`modification kit (Intergen). As a positive control, normal human dermal
`fibroblast cell DNA was methylated in vitro using the CpG methylase (New
`England BioLabs). PCR products were analyzed after electrophoresis on 6%
`polyacrylamide gels.
`Quantitation of Hypermethylated CDKN2A. The ratio between un-
`methylated and hypermethylated CDKN2A alleles in plasma of cancer patients
`was analyzed using methylation-specific quantification in the LightCycler
`system (Roche Diagnostics). For amplification of unmethylated DNA, we used
`primers according to Herman et al. (17). For the methylated CDNK2A, the
`following set of PCR primers was used: 59-GGTGGGGCGGATCGC-39
`(sense) and 59-CCGAACCGCGACCGTAA-39 (antisense). Two sets of LC-
`Red 640-labeled, methylation-specific hybridization probes were used for
`detection of real-time PCR products in the LightCycler reaction: for the
`unmethylated reaction 59-CTCCCCACCACCCACTACCTACTCT-39 (p16N
`FL) and 59-CCCCTCTCCACAACCACCAAACAC-39 (p16N LC) and for the
`methylated reaction 59-CCGCCGCCCGCTACCTACTCT-39 (p16M FL) and
`59-CCTCTCCGCAACCGCCGAAC-39 (p16M LC). PCR (40 cycles of dena-
`turation for 10 s at 95°C, annealing for 10 s at 69°C, and extension for 10 s at
`72°C) was performed with the LightCycler FastStart DNA Master Hybridiza-
`tion Probes Kit (Roche Diagnostics). A fixed volume of 4 ml deaminated
`plasma DNA was applied in each PCR assay. In vitro methylated DNA and
`DNA from normal human lymphocytes were used as standard DNA for the
`quantitation of methylated and unmethylated CDKN2A, respectively. The
`efficiency of the in vitro methylation of the standard DNA was analyzed by
`real-time PCR before quantitations: in the unmethylated CDKN2A reaction no
`product was visible using this in vitro methylated DNA as template. For all of
`the assays the same standard DNA was used. The methylation-specific meas-
`urements of plasma DNA were all performed in duplicates. The difference
`between the samples in each LightCycler assay was about 610% for both
`unmethylated and methylated DNA.
`Endothelial Cells. To investigate the presence of endothelial cell DNA in
`the plasma, a methylation-specific PCR-based method was established. After
`bisulfite treatment of purified plasma DNA (CpGenome Modification Kit;
`Intergene), a methylation-specific PCR in the promoter region of the selectin
`E (SELE) gene was carried out. Two different sets of primers were used to
`distinguish the unmethylated from the hypermethylated SELE promoter: un-
`methylated: 59-ATTTTAAGTATTGTGGATATTTTTG-39 (sense) and 59-
`3 The abbreviations used are: HUVEC, human vascular endothelial cell; SAF, scaffold
`CAAAAACAACTAAACACTACTTCA-39 (antisense); hypermethylated: 59-
`attachment factor; ALT, alanine aminotransferase.
`1660
`
`RESULTS
`
`Quantitations. We determined the concentration of DNA in
`plasma samples of 30 unselected cancer patients (Table 1). For that
`purpose, we used primers corresponding to a subtelomeric single-copy
`region of chromosome 19 in the quantitative PCR procedure of
`Diviacco et al. (15). The data obtained by quantitative PCR were
`verified by real-time quantitation in a LightCycler instrument (Roche
`Diagnostics). The difference between the results obtained by these
`two methods was 613%. All quantitations were performed in dupli-
`cate, with a reproducibility of 612.5% in the competitive assays and
`610% in the LightCycler assays. The results of quantitation revealed
`a wide spectrum of DNA concentrations in the plasma of cancer
`patients, between 10 and 1200 ng/ml, with a mean of 219 ng/ml
`
`00002
`
`

`

`Cancer
`
`DNA (ng/ml)
`
`Table 1 Plasma DNA: diagnoses; quantitations; presence of T-cell DNA; and
`hypermethylation of CDKN2A promoter
`CDKN2Ab
`T cellsa
`2
`2
`167
`Colorectal
`C1
`NDc
`1
`19
`Angiosarcoma
`C2
`2
`ND
`100
`NSCLC
`C3
`2
`2
`66
`Breast
`C4
`2
`ND
`134
`Breast
`C5
`2
`ND
`33
`SCLC
`C6
`2
`ND
`170
`Breast
`C7
`1
`2
`83
`Breast
`C8
`1
`2
`60
`Ewing’s sarcoma
`C9
`1
`ND
`20
`Breast
`C10
`2
`ND
`84
`NSCLC
`C11
`1
`ND
`10
`Head and neck
`C12
`1
`ND
`20
`Melanoma
`C13
`2
`1
`1200
`Pancreas
`C14
`2
`2
`67
`Colorectal
`C15
`ND
`ND
`25
`Colorectal
`C16
`2
`ND
`50
`Colorectal
`C17
`2
`ND
`38
`Breast
`C18
`2
`2
`90
`Breast
`C19
`1
`2
`48
`Pancreas
`C20
`2
`2
`84
`HCC
`C21
`2
`2
`168
`Urothel
`C22
`1
`2
`360
`Breast
`C23
`1
`2
`550
`NSCLC
`T1
`1
`2
`555
`Colorectal
`T2
`1
`2
`833
`Esophagus
`T3
`2
`1
`500
`Stomach
`T5
`2
`2
`980
`Colorectal
`T6
`2
`ND
`38
`Colorectal
`T7
`2
`ND
`34
`Colorectal
`T8
`a 1 or 2, presence or absence, respectively, of T-cell DNA in plasma samples.
`b 1, hypermethylation of CDKN2A gene promoter; 2, no hypermethylation detected.
`c ND, not done; NSCLC, non-mall cell lung cancer; SCLC, small cell lung cancer;
`HCC, hepatocellular carcinoma.
`
`Patient
`
`ON THE ORIGIN OF PLASMA DNA IN CANCER PATIENTS
`
`of sensitivity of the competitive assays. Thus, we conclude that
`elevated levels of circulating DNA appear to be a characteristic
`feature of most, but not all of the carcinoma diseases.
`T-Cell DNA. We considered the possibility that degenerating tu-
`mor-infiltrating T lymphocytes contribute to the DNA levels in blood
`plasma (24). We, therefore, prepared two sets of PCR primer pairs:
`one pair of primers, originally designed for the detection of different
`clonal rearrangements, identifies rearranged T-cell receptor b chain
`genes in mature T cells (16), whereas a second newly designed primer
`pair determines the germline configuration found in all of the non-T
`cells (Fig. 2A). In control experiments, the T-cell-specific primers
`amplified the clonal rearranged genomic sequence in the Jurkat T-cell
`line (Fig. 2B, Lane J) and in WBCs (Fig. 2B, Lane B) but gave
`negative results with HeLa DNA (Fig. 2B, Lane H). Conversely,
`primers specific for the germline configuration amplified the expected
`sequence in HeLa cell DNA and WBC DNA, but not in Jurkat cell
`DNA (Fig. 2C, Lanes H, J, and B). Using these primers, we detected
`mature T-cell DNA sequences in the plasma DNA samples C14
`(pancreatic adenocarcinoma, 1200 ng DNA/ml plasma; Fig. 2B) and
`T5 (stomach cancer, 500 ng/ml), whereas all of the other plasma DNA
`samples examined were devoid of detectable T-cell DNA (Table 1).
`All of the plasma samples of cancer patients examined gave positive
`results with the germline PCR (Fig. 2C). This result leads to the
`conclusion that DNA in the circulation of cancer patients does not
`very frequently contain T-lymphocyte DNA.
`Origin of Plasma DNA. The primary aim of the present study was
`to gain insight into the origin of circulating DNA in cancer patients.
`As we have just seen, lysis of T cells does usually not contribute to
`plasma DNA, and the question arises how much of the plasma DNA
`originates from tumor cells. For that purpose, we determined the
`
`Fig. 1. DNA levels in the plasma of tumor groups (24 patients) and 14 healthy
`individuals, obtained by competitive PCR. The values represent the average of duplicate
`determinations. Control, 14 healthy individuals. For patients, see Table 1.
`
`(Fig. 1). We observed no clear correlation between the concentrations
`of DNA in the plasma and the clinical situation such as diagnosis,
`tumor staging, or therapy, e.g., advanced pancreatic adenocarcinoma
`was diagnosed in patients C20 and C14, yet patient C20 had the
`relatively low concentration of 48 ng DNA/ml plasma, whereas pa-
`tient C14 had the highest plasma DNA concentration (1200 ng/ml;
`Fig. 2. Presence of T-cell DNA in plasma of cancer patients. A, position of PCR
`primers in the region of human T-cell receptor b chain in mature T-cell or germline
`Fig. 1; Table 1). In fact, some of the values in the group of cancer
`configurations. B, representative examples of mature T-cell PCR. As controls we used
`patients were not higher than the DNA concentrations determined in
`DNA of HeLa cells, Jurkat T cells, and WBCs (Lanes H, J, and B, respectively). Lanes
`C1, C3, C5, C7, and C14, T-cell PCR of plasma DNA from cancer patients (see Table 1).
`the plasma of two persons in the control group (mean, 3.7 ng DNA/ml
`The products of 907 (p) and 767 bp (pp) represent the PCR products of two different
`plasma; highest values, 10 –15 ng/ml). However, most of the control
`clonal rearrangements (J1.1 and J1.2, respectively). C, germline PCR of the same samples.
`cases had ,2 ng/ml DNA in their plasma (Fig. 1), which is at the limit
`M, molecular weight marker in bp.
`1661
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`ON THE ORIGIN OF PLASMA DNA IN CANCER PATIENTS
`
`methylation status of the human CDKN2A gene promoter. Gene
`CDKN2A encodes a cyclin-dependent kinase inhibitor, p16INK4A,
`with an important regulatory function in the cell cycle (25). It is
`known that the CDKN2A gene promoter is hypermethylated and
`thereby inactivated in a large number of diverse human cancer types
`(26, 27). The advantage of this approach is that unmethylated and
`hypermethylated DNA can be assayed side by side, allowing an
`estimation of the fraction of tumor-specific DNA in the samples
`examined. The experimental procedure is based on the deamination of
`cytosin, but not of 5-methylcytosin residues, by treatment of DNA
`with sodium bisulfite (17). Thus, an unmethylated CpG island, up-
`stream of the CDKN2A gene, acquires a number of uracil residues,
`whereas a methylated sequence retains its 5-methylcytosines after
`bisulfite treatment. Consequently, different sets of primers specifi-
`cally amplify unmethylated and methylated DNA. We used the primer
`pairs designed by Herman et al. (17) for the amplification of normal
`and hypermethylated CDKN2A promoter sequences. With these tools
`we examined a total of 25 plasma DNA samples and found evidence
`for hypermethylation of the CDKN2A-gene promoter in 11 cases
`(44%; Table 1), a percentage that is in agreement with published
`studies (26 –32). In the six cases in which both plasma and tumor
`tissue were available, the results of the methylation-specific PCR were
`identical in corresponding plasma and tissue samples. Most of the
`positive plasma DNA samples contained DNA with both methylated
`and unmethylated CDKN2A-promoter sequences. As mentioned, the
`clear differentiation between unmethylated nontumor and methylated
`tumor DNA segments in the same plasma DNA sample allows a
`real-time PCR quantitation by the LightCycler technology (Fig. 3A).
`The results of six experiments are shown in Fig. 3B and reveal that the
`fraction of DNA with hypermethylated CDKN2A-promoter sequences
`varies from .90% (sample C10) to ,10% (sample 23). Interestingly,
`samples with a high percentage of tumor-specific hypermethylated
`
`Fig. 3. Quantitation of the tumor-derived DNA in plasma by analysis of methylated and
`unmethylated CDKN2A alleles. A, position of sequence-specific primers and hybridization
`probes used in the unmethylated or the methylated LightCycler reactions. B, percentages
`of methylated (tumor DNA) and unmethylated (nontumor cell DNA) CDKN2A sequences
`in plasma of six cancer patients as analyzed by real-time quantitation. Samples C10, C8,
`and C23 are from advanced breast carcinoma cases (Table 1).
`
`Fig. 4. Methylation-specific PCR of the promoter region of SELE gene. A, scheme for
`the unmethylated and methylated PCR reactions. The sequence-specific primers cover
`three CpG islands (L). B, methylation-specific PCR of controls: HUVECs, HeLa cells,
`and HL60 cells. C, methylation-specific PCR of representative samples: tumor tissue of
`patient T2 and plasma samples of cancer patients C11 and C14. Lanes U and M,
`unmethylated and methylated reactions, respectively.
`
`DNA tend to belong to a group with lower than average concentra-
`tions of circulating DNA: C10 (from a breast cancer patient with 20
`ng DNA/ml plasma), C2 (angiosarcoma patient with only 19 ng
`DNA/ml), and C13 (melanoma patient with 20 ng DNA/ml plasma;
`Fig. 3B; Table 1). In contrast, sample C23 (breast cancer) has ,10%
`tumor-specific hypermethylated DNA in a total of 360 ng DNA/ml
`plasma.
`Where does the nontumor fraction of circulating DNA come from?
`A likely possibility is that it originates from degenerating normal cells
`in the vicinity of the expanding carcinoma tissue. This is difficult to
`investigate because DNA markers that distinguish defined cell types
`are not available. An exception may be the promoter of the endothe-
`lium-specific human gene SELE that has been described as unmeth-
`ylated in endothelial cells but hypermethylated in other cells (33). The
`human gene SELE encodes the endothelial leukocyte adhesion mol-
`ecule 1, a typical membrane component of endothelial cells (34). We
`confirmed the earlier observation studying DNA from human cell
`lines. The procedure used was methylation-specific PCR with primers
`corresponding to the sequences around the CpG islands upstream of
`the SELE gene (Fig. 4A). Following the reasoning explained above,
`we found the gene promoter unmethylated in a HUVEC line and
`hypermethylated in human HeLa cells and HL60 cells (Fig. 4B).
`Using the same PCR procedure for an investigation of surgically
`removed tumor tissue, we always detected a small fraction of un-
`methylated DNA in the presence of a much larger fraction of meth-
`ylated SELE gene promoter DNA (Fig. 4C). This was expected,
`because endothelial cells constitute a small fraction of the cells in a
`surgical preparation. In contrast to DNA from tumor tissue, we found
`no evidence for unmethylated C8, C11, C14, C23, T2, T3, T6, and T7
`(Fig. 4C; Table 1). We, therefore, conclude that very little, if any,
`DNA in the plasma DNA samples of cancer patients derives from the
`degeneration of endothelial cells.
`Does Free DNA Originate from Apoptotic or Necrotic Cells?
`We have analyzed the size distribution of purified plasma DNA from
`six of the cancer patients by PAGE. The DNA was visualized by
`1662
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`ON THE ORIGIN OF PLASMA DNA IN CANCER PATIENTS
`
`IU ALT. In contrast to the mono- and dinucleosomal DNA fragments
`appearing in the plasma of aCD95-treated mice, we here found a
`time-dependent increase of DNA fragments .10,000 bp, as expected
`for necrotic cell death (Fig. 6C, right). These results support the idea
`that the DNA in the circulation of cancer patients could originate from
`both apoptotic and necrotic cells in cancer tissue and that a discrim-
`ination of DNA originating from either type of cell death is possible
`by the determination of DNA size distribution.
`
`DISCUSSION
`
`In this study, we investigated the plasma of cancer patients and
`detected, in agreement with previous research, concentrations of DNA
`that are on the average much higher than the DNA levels in the plasma
`of healthy controls (1– 4). The range of DNA levels in the circulation
`of cancer patients varies widely, from levels like those in some of the
`controls (10 –20 ng/ml) to levels that exceed values of 1000 ng
`DNA/ml plasma. The levels of plasma DNA show no obvious corre-
`
`Fig. 5. A, size distribution of purified plasma DNA fragments; results of the six cancer
`patients. Mononucleosomal (mo), dinucleosomal (di), and trinucleosomal (tri) DNA
`fragments. B, size determination of the large-molecular-weight DNA of patient C4. M,
`molecular weight marker in bp.
`
`ethidium bromide staining when sufficient amounts of DNA could be
`recovered. The size distribution of the DNA fragments varied from
`sample to sample, but most frequently, the observed size of the DNA
`fragments was ;180 bp, sometimes accompanied by DNA fragments
`two, three, or four times this size (Fig. 5A; patients C7, T5, and T6).
`We also detected high-molecular-weight DNA fragments in some
`samples (Fig. 5, A and B; patients C4 and C9). Plasma DNA of patient
`C14 consisted of both types of fragment sizes. The spectrum of
`multiples of 180-bp fragments is reminiscent of the oligonucleosomal
`DNA ladder characteristic for apoptotic cell death when cellular
`chromatin is degraded by a caspase-activated DNase (35). On the
`other hand, DNA fragments larger than ;10,000 bp could originate
`from cells dying via necrosis.
`We, therefore, performed model studies to determine whether DNA
`can in principle be released from dying cells in form of soluble
`fragments, because this would be a necessary condition for its appear-
`ance in the circulation. In a first set of experiments, we induced
`apoptosis and necrosis in human Jurkat T cells. As shown in Fig. 6A,
`chromatin began to appear in the supernatant 3 h after induction of
`both necrosis or apoptosis.
`It is known, however, that degenerating apoptotic and necrotic cells
`in vivo are efficiently taken up by macrophages (36), and the possi-
`bility must be considered that fragmented chromatin is so rapidly
`removed that it cannot appear in the circulation. To investigate this
`point, we used established murine models for the induction of liver
`cell apoptosis or necrosis by aCD95 and acetaminophen, respectively
`(21, 22, 37). As shown in Fig. 6B (left), we could detect a dramatic
`increase of DNA in plasma of mice treated with aCD95. The appear-
`ance of plasma DNA 4 h after induction of apoptosis paralleled the
`Fig. 6. Extracellular DNA after induction of cell death in vitro and in vivo. A,
`increase in plasma ALT activity, which reached a maximum after 8 h
`supernatant DNA quantities of apoptotic or necrotic Jurkat T cells. Apoptosis was induced
`by staurosporine; necrosis was induced by staurosporine plus oligomycin (14). DNA in
`(2,790 IU ALT). Furthermore, the sizes of DNA fragments in the
`supernatants was quantitated by competitive PCR at the time points indicated. B, apoptosis
`plasma corresponded to mono- and dinucleosomal DNA (Fig. 6B,
`in vivo. Mice were treated with anti-CD95 antibody (17). Quantities of plasma DNA
`right). The results of plasma DNA quantitation of mice after treatment
`analyzed by real-time PCR quantitation (averages from three animals per time point).
`Right: Lane M, DNA fragments of known sizes (in bp); Lane P, size distribution of plasma
`with acetaminophen is shown in Fig. 6C (left). The increase of plasma
`DNA fragments at 8 h after injection of anti-CD95 antibody (mo and di, mononucleosomal
`DNA occurred with similar kinetics and was even more dramatic
`and dinucleosomal DNA fragments, respectively). C, necrosis in vivo. Mice were treated
`compared with the apoptosis model, reaching levels of .150 mg/ml
`with acetaminophen (17), and plasma DNA was analyzed by real-time PCR quantitation
`at the time points indicated (averages from three animals per time point). Right: Lane M,
`plasma 6 h after treatment of mice. At this time point, the extent of
`DNA marker (in bp); Lanes 0, 3, 4, and 6, plasma DNA at 0, 3, 4, and 6 h after injection
`liver injury was maximal, as expressed by a plasma ALT level of 2200
`of acetaminophen, respectively.
`1663
`
`00005
`
`

`

`ON THE ORIGIN OF PLASMA DNA IN CANCER PATIENTS
`
`in expanding tumor tissue. It is possible to distinguish between the
`two models of cell death: apoptosis produces DNA fragments of 180
`bp (and multiples of this), whereas necrosis results in much larger
`fragments. Both types of fragment sizes are found in the blood stream
`of cancer patients. In some of the analyzed cancer patients we found
`no evidence for apoptotic DNA. In fact, necrosis of tumor cells has
`been postulated to be the origin of plasma DNA in cancer patients in
`a number of earlier studies (2– 4, 13), including one study that showed
`an increase of plasma DNA after radiation therapy (40). However, we
`have shown here that apoptosis of cancer cells is at least as likely to
`lead to increased plasma DNA levels.
`As a conclusion, we envisage the following scenario. As the size of
`a tumor increases, vascularization becomes a problem, causing hy-
`poxia in regions remote from blood vessels. Hypoxia