Cell-free nucleic acids in plasma, serum and urine: a ...

Review Article Cell-free nucleic acids in plasma, serum and urine: a new tool in molecular diagnosis Allen KC Chan, Rossa WK Chiu and YM Dennis Lo

Abstract Address Department of Chemical Pathology The Chinese University of Hong Kong Prince of Wales Hospital 30-32 Ngan Shing Street Shatin, New Territories, Hong Kong SAR Correspondence Prof. YM Dennis Lo E-mail [email protected] This article was prepared at the invitation of the Clinical Sciences Reviews Committee of the Association of Clinical Biochemists.

There has recently been an upsurge of interest in the analysis of circulating nucleic acids (DNA and/or RNA) in blood plasma or serum as a clinical diagnostic tool. Occasional earlier reports suggested the existence of circulating nucleic acids, but the potential clinical implication was not realized until 1996, when DNA with tumourspeci c characteristics was demonstrated in the plasma/serum of cancer patients. This nding opened up possibilities for non-invasive cancer diagnosis. Potential applications have been reported in cancer diagnosis, prenatal diagnosis, transplantation and traumatology. Some of the ndings are on the verge of being translated into clinical use. DNA is also now being sought in other body uids such as urine. Ann Clin Biochem 2003; 40: 122–130

Introduction The existence of extracellular nucleic acids in the circulation was ¢rst reported by Mandel and Metais in 1948.1 They showed that DNA and RNA could be detected in the plasma of sick as well as healthy individuals. Their report was particularly remarkable as it was published only a few years after the demonstration that DNA was probably the material carrying genetic information and even before the classic paper of Watson and Crick on the double helical structure of DNA.2 Their work did not attract much attention, and further development of the ¢eld had to wait some 30 years until Leon et al. showed that cancer patients had much higher concentrations of circulating DNA than those with non-malignant diseases.3 Leon et al. also showed that, in some cases, the levels of circulating DNA decreased after successful anti-cancer therapy.3 The cellular origin of the extracellular DNA could not be determined at that time because of technological limitations. In 1989, Stroun et al.4 showed that DNA circulating in cancer patients exhibits some characteristic features of tumour DNA, such as decreased strand stability. This was con¢rmed in 1994 by Sorenson et al.5 and Vasioukhin et al.,6 who detected speci¢c oncogene mutations in the plasma /serum and tumour tissue of patients with pancreatic cancer,5 myelodys122

plastic syndrome and acute myeloid leukaemia.6 Signi¢cant progress was made in 1996, when two groups simultaneously reported that microsatellite alterations, such as loss of heterozygosity (LOH), could be detected in the DNA from the plasma /serum of some cancer patients when corresponding changes were detected in the primary tumours.7,8 These ¢ndings suggested that tumour DNA could constitute a major portion of the circulating DNA in cancer patients. Moving beyond the ¢eld of cancer detection, Lo et al. demonstrated fetal DNA in the plasma of pregnant women in 1997,9 and more recently circulating DNA has been shown to have potential in monitoring organ-transplant recipients10 and trauma patients.11 This review summarizes the development of clinical applications of the measurement of cell-free nucleic acids (see Table 1) and emphasizes the technical issues involved.

Plasma/serum nucleic acids as a tumour marker The ¢rst evidence of the existence of tumour-derived DNA circulating in cancer patients was the detection of speci¢c oncogene mutations in their plasma /serum. In 1994, Sorenson et al. described the detection of mutated KRAS sequences in the blood of pancreatic © 2003 The Association of Clinical Biochemists

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Circulating nucleic acids as a diagnostic tool

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Table 1.

Chronology of developments concerning circulating nucleic acids

1948 1953 1977 1989 1994

Discovery of the existence of extracellular nucleic acids in the circulation by Mandel and Metais Discovery of the double helical structure of DNA by Watson and Crick Demonstration of elevated circulating DNA concentrations in cancer patients by Leon and Shapiro Demonstration of decreased strand stability in DNA from cancer patients by Stroun Detection of point mutations of N-ras gene in the plasma of patients with myelodysplastic syndrome or acute myelogenous leukaemia by Vasioukhin Detection of mutated K-ras gene in the serum of pancreatic cancer patients by Sorenson Demonstration of LOH in plasma DNA of lung cancer patients by Chen Demonstration of LOH in serum DNA of head and neck cancer patients by Nawroz Demonstration of fetus-derived DNA in maternal plasma by Lo Detection of EBV DNA in the serum of NPC patients by Mutirangura Development of non-invasive prenatal diagnosis methods of fetal RhD status by Lo First clinical service utilizing plasma/serum DNA analysis (non-invasive prenatal diagnosis of fetal RhD status)

1996 1997 1998 1998 2001

LOH=loss of heterozygosity; EBV=Epstein-Barr virus; NPC=nasopharyngeal carcinoma.

cancer patients5 and Vasioukhin et al. reported the presence of point mutation of NRAS in the plasma of patients with myelodysplastic syndrome or acute myelogenous leukaemia.6 Since then, di¡erent groups have reported that matched mutations of oncogenes were present in the primary tumours and the blood of patients su¡ering from a wide variety of malignant diseases.12^15 However, the detection rates of mutated oncogene sequences in the plasma /serum of cancer patients were highly variable among di¡erent reports when di¡erent oncogenes were studied.16,17 Among these oncogenes, KRAS mutations were the most extensively studied because they could be detected in the plasma/serum of a proportion of patients su¡ering from pancreatic or gastrointestinal cancers, and the repertoire of KRAS mutations is relatively limited.12 In contrast, the mutational patterns of other oncogenes are more complex and thus the characterization of speci¢c oncogene mutations usually requires DNA sequencing. The need for this more costly and labourintensive technique is a potential hindrance in utiliz ing the detection of circulating oncogene mutations for the screening for cancers in clinical practice. Both microsatellite instability and LOH are frequently demonstrated in tumour tissues. In 1996, Chen et al.7 and Nawroz et al.8 simultaneously described the presence of microsatellite alterations in plasma DNA that were identical to those found in the DNA of primary tumours of the respective patients. Their ¢ndings have partially resolved the problem concerning the origin of circulating DNA in cancer patients, favouring the proposition that release of DNA is due to the high turnover of cells in tumour tissues. To date, LOH has been detected in the plasma/serum of patients having cancer of the lung18, breast, 20 melanoma 21 and squamous carcinoma of the head and neck.19 Although positive results can be seen in a small proportion of patients having small tumours or in situ carcinomas,19,20 patients having LOH in their plasma /serum are more likely to have invasive

tumours, regional spread and distant metastases.14,21^23 Furthermore, LOH changes in the plasma /serum of some patients disappeared after successful anti-cancer therapy14,18 and some investigators suggested that microsatellite markers might be useful for the monitoring of cancer patients under treatment. However, as the detection rate of LOH with a single microsatellite marker is rather low, a panel of microsatellite markers is usually required for the detection of LOH in tumour tissues and plasma /serum in order to achieve the necessary sensitivity. Although the development of £uorescence-based allelotyping techniques has greatly improved the robustness of microsatellite analysis, the study of microsatellite alterations is still impractical on a large scale for routine application. Viral infections have been implicated in the pathogenesis of several types of cancers.Viral DNA has been shown to be detectable in the circulation of patients su¡ering from nasopharyngeal carcinoma (NPC),24 lymphoma, 25 head and neck tumours 26 and cervical carcinoma. 27 A good example of the use of circulating viral DNA as a tumour marker is the detection of circulating Epstein- Barr virus (EBV) DNA in the clinical management of NPC patients. Lo et al. were able, by using real-time quantitative polymerase chain reaction (PCR) analysis, to detect circulating EBV DNA in 96% of NPC patients but only 7% of normal subjects.24 The EBV DNA concentrations correlated positively with the clinical staging of the NPC patients. 24 The plasma/serum EBV DNA concentration has also been shown to be an independent prognostic factor for long-term survival of NPC patients,28 and plasma /serum EBV DNA has been found to be a valuable marker for post-radiotherapy monitoring: in most patients with tumour recurrence, a rise in plasma/ serum EBV DNA concentration was noted before or at the time of clinical relapse.29 Thus, EBV DNA has been shown to be useful in the detection, monitoring and prognostication of NPC patients. A major advantage of Ann Clin Biochem 2003; 40: 122–130


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using real-time quantitative analysis for circulating EBV DNA is that it does not require any post-PCR handling of samples and therefore reduces the chance of carry-over contamination, which could be a major problem in molecular diagnosis. As the quantitative assays for EBV DNA are accurate, quick, robust and relatively cheap, EBV DNA seems to be a desirable tumour marker for routine clinical practice. Similarly, human papilloma virus (HPV) DNA has been demonstrated in the circulation of patients with cervical carcinoma 27 and some squamous head and neck carcinomas. 26 In contrast to circulating EBV DNA, which is detectable in most NPC patients, HPV DNA could only be detected in the plasma /serum of patients su¡ering from more advanced disease and is associated with the presence of metastasis. 26,27 Therefore, circulating HPV DNA is more useful as a marker for advanced disease where adjunctive therapy to conventional treatment should be considered. DNA methylation is an epigenetic characteristic associated with the silencing of gene expression. Alterations of DNA methylation patterns, including global genome hypomethylation and regional hypermethylation of tumour suppressor genes, DNA repairing genes and metastasis inhibitor genes or their promoters, are increasingly found in di¡erent types of tumours. With the development of methylation-speci¢c polymerase chain reaction (MSP), small amounts of hypermethylated sequences may be detected in a background of wild-type sequences. MSP is based on the principle that treatment of DNA with bisulphite converts unmethylated cytosine residues into uracil whereas methylated cytosine residues remain unchanged. Consequently, methylated and unmethylated DNA sequences are distinguishable by sequence-speci¢c PCR primers. Currently, several groups have detected cancer-associated epigenetic changes in the plasma /serum of patients with carcinoma of the lung,30 oesophagus,31 liver 32 and breast 33, and leukaemia.34 Real-time quantitative MSP has also been developed and could detect as few as ten genome equivalents of methylated and unmethylated p16 sequences.35 A major advantage of quantitative analysis is that it allows simultaneous quanti¢cation of methylated sequences, unmethylated sequences and any residual sequences that are not converted by bisulphite. This is particularly important as the e¤ciency of bisulphite conversion is variable and a substantial amount of DNA could be destroyed during the process.36 With real-time quantitative MSP, hypermethylated p15 or p16 DNA sequences were detected in the plasma/ serum of 92% of liver cancer patients whose tumours showed the corresponding epigenetic changes.35 In a recent study involving 99 patients with lung cancer, hypermethylated adenomatous polyposis coli gene

was detected in the plasma/serum of 47% of the patients with tumours showing the same changes and could predict poorer survival. 37 Mitochondrial DNA (mtDNA) has been found in the plasma of human subjects.38 Mutations of mtDNA have been detected in a number of cancers. Jeronimo et al. detected identical mtDNA mutations in the plasma and tumour tissues of each of three patients with prostate cancer.39 Recently, Nomoto et al., using the more sensitive oligonucleotide mismatch ligation assay, were able to detect tumour-associated mtDNA mutations in the plasma of eight out of ten patients with hepatocellular carcinoma, although the mtDNA mutant signal in DNA extracted from the plasma was much weaker than that from the tumour.40 One major concern in the study of mtDNA is the possible ampli¢cation of nuclear-encoded pseudogenes during the PCR process. For this reason fairly long segments of mtDNA are usually chosen for these studies. This might be why the detection rates of tumour-derived mtDNA mutations in the plasma /serum of cancer patients are low, as most of the circulating DNA molecules are fairly short, containing only some 180 base -pairs, which is characteristic of tissue that has undergone apoptotic cell death.41,42 The signi¢cance of this observation will be discussed below. The presence of circulating RNA was reported in 1999 by Lo et al., when they showed that EBV RNA could be detected in the plasma of NPC patients.43 This was unexpected since RNase has been shown to be present in the circulation of healthy individuals and in higher concentrations in cancer patients.44 At the same time, Kopreski et al. showed that tyrosinase mRNA could be detected in the serum of a third of patients with malignant melanoma but in none of the healthy control subjects.45 Tumour-associated RNA was later detected in the plasma /serum of patients su¡ering from carcinoma of the breast,46 colon 47 and lung.48 Silva et al. further showed that the amount of epithelial mRNA in the plasma of breast cancer patients signi¢cantly correlated with tumour size and the proliferative index.47 Because RNA molecules are labile, specimens used for RNA analysis must be processed immediately and special precautions have to be taken so as to avoid contamination by naturally occurring RNases present in the laboratory environment. RNases are present in all living cells and are responsible for the rapid degradation of mRNA and the regulation of gene expression in the cells.

Non-invasive prenatal diagnosis Prenatal diagnosis of fetal genetic diseases currently relies on the use of the invasive methods chorionic villus sampling and amniocentesis. The inherent risk of fetal loss from these procedures is a major deterrent

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to couples who are faced with the decision to opt for or against them.

Fetal DNA in maternal blood

In 1997, Lo et al.9 ¢rst reported the existence of fetusderived DNA in maternal plasma by demonstrating the positive ampli¢cation of Y-chromosome -speci¢c sequences in the plasma of women carrying a male fetus. This ¢nding challenged the traditional understanding of fetomaternal physiology. Diagnostically, the ¢nding opened up new possibilities of non-invasive prenatal diagnosis through the sampling of fetal genetic material in maternal blood. The real-time quantitative PCR approach to the quanti¢cation of fetal DNA in maternal plasma 49 has the advantage of high sensitivity and speci¢city, as it can detect the equivalent of one male cell among a background of up to 12 800 female cells, and the results are linear over ¢ve orders of magnitude. Surprisingly, in contrast to the very rare presence of intact fetal cells 50,51 in the maternal circulation, quite a high proportion of circulating DNA in maternal plasma is of fetal origin. 49 The fetal DNA concentration increases as the pregnancy advances, contributing 3Í4% and 6Í2% of the total DNA in maternal plasma in early and late gestation, respectively. The fetal-to-maternal ratios of circulating DNA in early and late gestation were 970 and 775 times the ratio of the number of fetal to maternal cells in maternal whole blood. In the same study, the Y-chromosome signal was detectable in the plasma of women carrying male fetuses from the 7th week of gestation onwards, indicating the potential of the assay of circulating fetal DNA for early prenatal diagnosis. If fetal DNA were to persist in maternal plasma after parturition this would hinder its use for prenatal diagnosis in multiparous women, but Lo et al. showed 52 that the fetus-speci¢c sequences rapidly disappeared from the maternal circulation during the post-partum period, with a mean half-life of 16Í3 min. This ¢nding raised the hope that this assay might be a dynamic marker that could be used for real-time monitoring of fetomaternal wellbeing.

Translation into clinical use

The pilot study 9 paved the way for the ¢rst potential application of circulating fetal DNA: the prenatal diagnosis of male-sex-linked diseases. Using conventional PCR, Honda et al. achieved 100% sensitivity in the detection of Y-chromosome signals from the 7th week of gestation, and the more sensitive real-time quantitative PCR achieved the same sensitivity from the 5th week of gestation.53 Sekizawa et al. assessed the fetal sex in 302 pregnancies in which serum was collected between the 7th and 16th weeks of gestation, and correctly identi¢ed 97 ¢2% of the male fetuses and

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100% of the female fetuses.54 All similar studies to date have reported 100% speci¢city in identifying a male fetus.49,55^57 In a case of suspected congenital adrenal hyperplasia, Rijnders et al.58 correctly identi¢ed the fetus as male by this technique, which obviated the need for prenatal dexamethasone therapy to prevent virilization of female fetuses. The analysis of circulating fetal DNA was soon applied to the prenatal diagnosis of autosomal dominant traits. The translation of fetal DNA analysis into prenatal diagnostic tests for autosomal dominant traits involves the exploitation of the di¡erences between the fetal-speci¢c allele from the father and the maternal DNA. The presence of the gene RHD in the plasma of an RhD-negative woman, or of an autosomal dominant mutation in an una¡ected woman’s plasma, points to the inheritance of such alleles in the current pregnancy. The most well-developed application to date is the non-invasive prenatal assessment of fetal RhD status in RhD-negative women.57,59,60 In general, from the second trimester onwards, fetal RhD genotyping by maternal plasma analysis is 100% accurate, and this technique is now o¡ered as a clinical service (http://www.blood.co.uk/ibgrl/Reference%20Services /RefSer_genotyping.htm). The analysis of circulating fetal DNA has also been reported to be feasible for the non-invasive prenatal diagnosis of genetic disorders where the father is a¡ected, including myotonic dystrophy,61 achondroplasia 62 and fetal aneuploidy.63,64 The situation is more complex for autosomal recessive conditions. Chiu et al. showed the feasibility of using maternal plasma to exclude the inheritance of an autosomal recessive disease in a couple with a previous child born with congenital adrenal hyperplasia. 65 The approach involved detecting, in maternal plasma, polymorphic markers linked to the normal (non-mutated) paternal allele. The presence of these markers indicated that the fetus had inherited the normal paternal allele and would not manifest the autosomal recessive disease.

Quantitative analysis of fetal DNA in maternal plasma Besides the detection of speci¢c fetal alleles in maternal plasma, quantitative analysis of fetal DNA concentration in maternal plasma may be useful for the prediction and monitoring of certain pregnancyassociated conditions. Several independent studies have shown that fetal DNA concentrations in maternal plasma are higher in aneuploid pregnancies,55,66 pregnancies complicated by pre-eclampsia,67^70 spontaneous preterm labour,71 fetomaternal haemorrhage 72 and invasive placentae.73 Fetal DNA is commonly elevated in pregnancies with complications, and another potential application of quantitative fetal DNA assay is in combination with other Ann Clin Biochem 2003; 40: 122–130


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existing markers commonly assessed during pregnancy, such as human chorionic gonadotrophin.74,75 The clinical utility remains to be established. As mentioned, the use of quantitative PCR o¡ers many advantages for the analysis of circulating fetal DNA, including high precision, and the robustness and throughput of the system could be further enhanced by development of an automated sample preparation system.76 However, there is high initial capital outlay and there is great potential for contamination; anti-contamination measures need to be strictly implemented.49 Besides the analytical issues, the choice of procedure for sample preparation impacts greatly on the interpretation of the data.77 van Wijk et al. reported apoptotic fetal cells in maternal plasma processed by Percoll gradient separation.78 The implications are twofold. First, the ease of isolation of fetal cells by this method may allow simple methods for non-invasive fetal cell detection to be developed; for example, analysis of these cells has been shown to be applicable for the prenatal diagnosis of fetal aneuploidy.79,80 Second, the presence of such fetal cells in maternal `plasma’ has prompted the rede¢nition of `plasma’ (normally taken to mean blood £uid free of cells). By processing maternal plasma samples using di¡erent centrifugation protocols, Chiu et al. showed that the sample processing procedure a¡ects the quantitative results for total DNA but not for fetal DNA.77 Hence, one should be cautious when comparing total and maternal plasma DNA concentrations and calculating the fetal:maternal DNA ratio. This also highlights the need for consensus to be reached on pre-analytical and analytical standards (see later).

Future developments in maternal plasma analysis The assays developed so far have relied on detecting paternally inherited traits or alleles in fetus-derived DNA from maternal plasma. Fetal DNA circulates among a background of maternal DNA in maternal plasma. It is technically di¤cult to identify the fetal origin of a maternally inherited allele in maternal plasma. This has been achieved 81 by means of methylation-speci¢c PCR of DNA obtained from maternal plasma, which distinguished a maternally inherited fetus-derived allele from the mother’s counterpart at a di¡erentially methylated locus, IGF2-H19. At this locus, a paternally inherited allele is methylated and its maternally inherited counterpart is unmethylated. This approach is applicable to pregnancies where the fetus has inherited an allele from its mother who originally inherited the allele from her father. In addition to novel developments in fetal DNA analysis, our group recently reported82 the detection of fetalspeci¢c RNA sequences in maternal plasma. Malespeci¢c RNA species were detected in the plasma of

women carrying male fetuses. This interesting ¢nding has expanded the potential applications of maternal plasma analysis for fetal assessment and opened up the possibility of non-invasive analysis of fetal gene expression. Although numerous potential applications have been reported for the analysis of fetal nucleic acids in maternal plasma, the origin, release and clearance mechanisms remain unknown. There is much to be learned about this new insight into fetomaternal physiology, and it remains to be clari¢ed whether fetal DNA and RNA molecules have any functional role in the maternal circulation.

Urinary DNA analysis DNA in urine is also a potential source of material for molecular diagnosis. In 1995, Fitzgerald et al. showed that point mutations of HRAS could be detected in urine sediments of 44% of patients with carcinoma of the bladder.83 Other groups also showed cancerderived DNA in the urine of patients su¡ering from prostate 84 and bladder cancer.85 In 1999, Zhang et al. showed that male-speci¢c DNA sequences could be detected in female transplant recipients who had received male kidneys,86 and that urinary DNA concentrations increased markedly during acute rejection, rapidly returning to normal following antirejection treatment.86 In 2000, Botezatu et al. further showed that malespeci¢c DNA sequences could be detected by nested PCR in the urine of females who had been transfused with male blood or were carrying a male fetus.87 In the same study, they reported that tumour-derived DNA could be detected in the urine of patients with carcinoma of the colon and pancreas.87 They suggested that the kidney may be involved in the clearance of circulating DNA, whereas studies involving patients with renal transplant or cancers of urological origin suggested that DNA is released directly into the urine. However, another group failed to detect fetal DNA in maternal urine.88 One possibility is that the highly sensitive nested PCR can detect male-speci¢c DNA sequences in spermatozoa contaminating the urine of tested subjects if the urine was collected shortly after sexual intercourse. Special precautions for avoiding contamination of urine, including the avoidance of sexual intercourse before the test and cleansing of the external urinary ori¢ce, should be applied when urine is used for molecular diagnostic purposes.

Source and signi cance of circulating nucleic acids Although the mechanism of release of non-cellular DNA into the circulation remains unclear, cell death has been believed to play an important role. For this



reason the levels of circulating DNA in patients after major trauma have been studied.11 A 60-fold higher median plasma DNA level was observed in patients after major trauma than in healthy subjects.11 Patients who went on to develop complications and died had signi¢cantly higher plasma DNA concentrations upon admission than those who did not develop complications.11 It has been suggested that tissue necrosis could generate circulating DNA, but the number of cells undergoing necrosis is limited in healthy subjects, and other possible mechanisms have been investigated. Apoptosis is believed to play an important role in the generation of circulating DNA in healthy subjects as well as in cancer patients. Jahr et al.41 and Giacona et al.42 have shown that most DNA molecules present in plasma/serum have lengths that are multiples of nucleosomal DNA, which is a characteristic of apoptotic cell death. Furthermore, Mutirangura et al. have demonstrated a relationship between the presence of EBV DNA in the plasma/ serum of NPC patients and the apoptosis of tumour cells.89 In an investigation of the tissue origin of circulating nucleic acids, Lo et al. studied 36 female patients who had received kidney or liver transplants.10 Malespeci¢c DNA sequences were detected in the plasma of 87% of patients who had received an organ from a male donor but in none of those receiving from a female donor.10 In 2002, Lui et al. showed by studying sex-mismatched bone marrow transplantation patients that most of the circulating DNA molecules were of haematopoietic origin.90

Preparation of samples for assay of circulating nucleic acids The pre-analytical factors a¡ecting di¡erent studies are very heterogeneous, so that highly variable results are obtained. For example, the detection rates of EBV DNA in the circulation of NPC patients in di¡erent studies ranged from 31% to 96%.24,89 The ¢ndings of the blood-processing protocol study by Chiu et al.77 may apply in other areas of plasma nucleic acid analysis. Lui et al. have shown that DNA concentrations are higher in serum than in plasma, probably because blood cells release DNA during the clotting process,90 so that for the assay of tumour- or fetus-derived nucleic acids the use of plasma rather than serum is recommended, and the more vigorous the centrifugation the better the signal-to-noise ratio. Standardization of specimen type and of sample preparation is necessary if results from di¡erent groups are to be meaningfully compared.

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Future directions Assay of circulating nucleic acids is a rapidly evolving ¢eld, encompassing cancer detection and monitoring, prenatal diagnosis and monitoring, transplantation medicine and traumatology. Urine has also been shown to be a convenient source of DNA in some clinical situations. More recently, increased awareness has been paid to the existence of plasma RNA, exempli¢ed by the ¢nding of tumour- and fetus-derived plasma RNA. Plasma RNA analysis allows for the assessment of genetic function in a minimally invasive manner and there is an increasing amount of research devoted to this area. With the rapid advancements in molecular techniques, there is no doubt that the clinical applications of circulating nucleic acids will continue to expand and will eventually be available on user-friendly platforms. However, before such techniques are applied clinically, the issues of standardization need to be addressed.

Glossary of terms Circulating nucleic acids: Cell-free nucleic acids (DNA or RNA) present in the plasma or serum of the blood. DNA methylation: Modi¢cation of the cytosine residue in a dinucleotide DNA sequence CG by attachment of a methyl group. This modi¢cation is believed to be involved in the silencing of gene expression of that particular region. Epigenetics: The study of molecular mechanisms that alter gene expression but do not involve a change in gene structure or DNA sequence. Fetal DNA: DNA molecules derived from fetal cells. Loss of heterozygosity (LOH):The loss of an allele in a heterozygous locus. Methylation-speci¢c PCR (MSP): A technique used for the discrimination of methylated and unmethylated sequence.The principle of this technique is based on the conversion of unmethylated cytosine residues to uracil by bisulphite treatment while methylated cytosine residues remain unchanged. The di¡erences between methylated and unmethylated sequences after bisulphite treatment are recognized by sequencespeci¢c primers. Microsatellite: One of many short, highly polymorphic, non-coding sequences [e.g. poly(TG)] that are found well spaced throughout the genome and which can serve as landmarks during physical mapping. Microsatellite instability: The emergence of a new allele in a speci¢c locus during microsatellite analysis. Polymerase chain reaction (PCR): A technique used for the ampli¢cation of a speci¢c sequence of DNA. Quantitative real-time PCR: A technique used to quantify the concentration of a speci¢c sequence in a Ann Clin Biochem 2003; 40: 122–130


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DNA solution. In a PCR reaction, a £uorescence signal is generated during the extension phase. A CCD camera is used to monitor the intensity of the £uorescence signal during the PCR. The number of cycles that each sample needs to generate a certain £uorescence signal is inversely proportional to the logarithm of the concentration of that sample. Therefore, the initial concentration of each sample can be calculated by comparison with a standard of known concentration. RNase: The enzyme responsible for the degradation of RNA.

Acknowledgements YMDL and RWKC are supported by the Innovation and Technology Fund (ITS/195/01).

References 1 Mandel P, Metais P. Les acides nucleiques du plasma sanguin chez l’homme. C R Acad Sci Paris 1948; 142: 241-3 2 Watson J, Crick F. A structure for deoxyribose nucleic acid. Nature 1953; 171: 737 3 Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 1977; 37: 646-50 4 Stroun M, Anker P, Maurice P, Lyautey J, Lederrey C, Beljanski M. Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology 1989; 46: 318-22 5 Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL. Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol Biomarkers Prev 1994; 3: 67-71 6 Vasioukhin V, Anker P, Maurice P, Lyautey J, Lederrey C, Stroun M. Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia. Br J Haematol 1994; 86: 774-9 7 Chen XQ, Stroun M, Magnenat JL, Nicod LP, Kurt AM, Lyautey J, et al. Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat Med 1996; 2: 1033-5 8 Nawroz H, Koch W, Anker P, Stroun M, Sidransky D. Microsatellite alterations in serum DNA of head and neck cancer patients. Nat Med 1996; 2: 1035-7 9 Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350: 485-7 10 Lo YMD, Tein MS, Pang CC, Yeung CK, Tong KL, Hjelm NM. Presence of donor-specic DNA in plasma of kidney and livertransplant recipients. Lancet 1998; 351: 1329-30 11 Lo YMD, Rainer TH, Chan LYS, Hjelm NM, Cocks RA. Plasma DNA as a prognostic marker in trauma patients. Clin Chem 2000; 46: 319-23 12 Sorenson GD. A review of studies on the detection of mutated KRAS2 sequences as tumor markers in plasma/serum of patients with gastrointestinal cancer. Ann N Y Acad Sci 2000; 906: 13-6 13 Gocke CD, Benko FA, Kopreski MS, McGarrity TJ. p53 and APC mutations are detectable in the plasma and serum of patients with colorectal cancer (CRC) or adenomas. Ann N Y Acad Sci 2000; 906: 44-50

14 Gonzalez R, Silva JM, Sanchez A, Dominguez G, Garcia JM, Chen XQ, et al. Microsatellite alterations and TP53 mutations in plasma DNA of small-cell lung cancer patients: follow-up study and prognostic signicance. Ann Oncol 2000; 11: 1097-104 15 Mayall F, Jacobson G, Wilkins R, Chang B. Mutations of p53 gene can be detected in the plasma of patients with large bowel carcinoma. J Clin Pathol 1998; 51: 611-3 16 Hibi K, Robinson CR, Booker S, Wu L, Hamilton SR, Sidransky D, et al. Molecular detection of genetic alterations in the serum of colorectal cancer patients. Cancer Res 1998; 58: 1405-7 17 Mulcahy HE, Lyautey J, Lederrey C, qi Chen X, Anker P, Alstead EM, et al. A prospective study of K-ras mutations in the plasma of pancreatic cancer patients. Clin Cancer Res 1998; 4: 271-5 18 Sozzi G, Conte D, Mariani L, Lo Vullo S, Roz L, Lombardo C, et al. Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung cancer patients. Cancer Res 2001; 61: 4675-8 19 Nunes DN, Kowalski LP, Simpson AJ. Circulating tumor-derived DNA may permit the early diagnosis of head and neck squamous cell carcinomas. Int J Cancer 2001; 92: 214-9 20 Chen X, Bonnefoi H, Diebold-Berger S, Lyautey J, Lederrey C, Faltin-Traub E, et al. Detecting tumor-related alterations in plasma or serum DNA of patients diagnosed with breast cancer. Clin Cancer Res 1999; 5: 2297-303 21 Taback B, Fujiwara Y, Wang HJ, Foshag LJ, Morton DL, Hoon DS. Prognostic signicance of circulating microsatellite markers in the plasma of melanoma patients. Cancer Res 2001; 61: 5723-6 22 Fujiwara Y, Chi DD, Wang H, Keleman P, Morton DL, Turner R, et al. Plasma DNA microsatellites as tumor-specic markers and indicators of tumor progression in melanoma patients. Cancer Res 1999; 59: 1567-71 23 Silva JM, Dominguez G, Garcia JM, Gonzalez R, Villanueva MJ, Navarro F, et al. Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations. Cancer Res 1999; 59: 3251-6 24 Lo YMD, Chan LYS, Lo KW, Leung SF, Zhang J, Chan ATC, et al. Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res 1999; 59: 1188-91 25 Gallagher A, Armstrong AA, MacKenzie J, Shield L, Khan G, Lake A, et al. Detection of Epstein-Barr virus (EBV) genomes in the serum of patients with EBV-associated Hodgkin’s disease. Int J Cancer 1999; 84: 442-8 26 Capone RB, Pai SI, Koch WM, Gillison ML, Danish HN, Westra WH, et al. Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV-associated head and neck squamous cell carcinoma. Clin Cancer Res 2000; 6: 4171-5 27 Pornthanakasem W, Shotelersuk K, Termrungruanglert W, Voravud N, Niruthisard S, Mutirangura A. Human papillomavirus DNA in plasma of patients with cervical cancer. BMC Cancer 2001; 1: 2 28 Lo YMD, Chan ATC, Chan LYS, Leung SF, Lam CW, Huang DP, et al. Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein-Barr virus DNA. Cancer Res 2000; 60: 6878-81 29 Lo YMD, Chan LYS, Chan ATC, Leung SF, Lo KW, Zhang J, et al. Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 1999; 59: 5452-5 30 Esteller M, Sanchez-Cespedes M, Rosell R, Sidransky D, Baylin SB, Herman JG. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res 1999; 59: 67-70


Circulating nucleic acids as a diagnostic tool 31 Hibi K, Taguchi M, Nakayama H, Takase T, Kasai Y, Ito K, et al. Molecular detection of p16 promoter methylation in the serum of patients with esophageal squamous cell carcinoma. Clin Cancer Res 2001; 7: 3135-8 32 Wong IH, Lo YM, Zhang J, Liew CT, Ng MH, Wong N, et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Res 1999; 59: 71-3 33 Silva JM, Dominguez G, Villanueva MJ, Gonzalez R, Garcia JM, Corbacho C, et al. Aberrant DNA methylation of the p16INK4a gene in plasma DNA of breast cancer patients. Br J Cancer 1999; 80: 1262-4 34 Wong IH, Ng MH, Huang DP, Lee JC. Aberrant p15 promoter methylation in adult and childhood acute leukemias of nearly all morphologic subtypes: potential prognostic implications. Blood 2000; 95: 1942-9 35 Lo YMD, Wong IH, Zhang J, Tein MS, Ng MH, Hjelm NM. Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specic polymerase chain reaction. Cancer Res 1999; 59: 3899-903 36 Grunau C, Clark SJ, Rosenthal A. Bisulte genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 2001; 29: E65-5 37 Usadel H, Brabender J, Danenberg KD, Jeronimo C, Harden S, Engles J, et al. Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer. Cancer Res 2002; 62: 371-5 38 Zhong S, Ng MC, Lo YMD, Chan JC, Johnson PJ. Presence of mitochondrial tRNA(Leu(UUR)) A to G 3243 mutation in DNA extracted from serum and plasma of patients with type 2 diabetes mellitus. J Clin Pathol 2000; 53: 466-9 39 Jeronimo C, Nomoto S, Caballero OL, Usadel H, Henrique R, Varzim G, et al. Mitochondrial mutations in early stage prostate cancer and bodily uids. Oncogene 2001; 20: 5195-8 40 Nomoto S, Yamashita K, Koshikawa K, Nakao A, Sidransky D. Mitochondrial D-loop mutations as clonal markers in multicentric hepatocellular carcinoma and plasma. Clin Cancer Res 2002; 8: 481-7 41 Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, et al. DNA fragments in the blood plasma of cancer patients: quantitation and evidence for their origin from apoptotic and necrotic cells. Cancer Res 2001; 61: 1659-65 42 Giacona MB, Ruben GC, Iczkowski KA, Roos TB, Porter DM, Sorenson GD. Cell-free DNA in human blood plasma:length measurements in patients with pancreatic cancer and healthy controls. Pancreas 1998; 17: 89-97 43 Lo KW, Lo YMD, Leung SF, Tsang YS, Chan LYS, Johnson PJ, et al. Analysis of cell-free Epstein-Barr virus associated RNA in the plasma of patients with nasopharyngeal carcinoma. Clin Chem 1999; 45: 1292-4 44 Reddi KK, Holland JF. Elevated serum ribonuclease in patients with pancreatic cancer. Proc Natl Acad Sci USA 1976; 73: 2308-10 45 Kopreski MS, Benko FA, Kwak LW, Gocke CD. Detection of tumor messenger RNA in the serum of patients with malignant melanoma. Clin Cancer Res 1999; 5: 1961-5 46 Chen XQ, Bonnefoi H, Pelte MF, Lyautey J, Lederrey C, Movarekhi S, et al. Telomerase RNA as a detection marker in the serum of breast cancer patients. Clin Cancer Res 2000; 6: 3823-6 47 Silva JM, Dominguez G, Silva J, Garcia JM, Sanchez A, Rodriguez O, et al. Detection of epithelial messenger RNA in the plasma of breast cancer patients is associated with poor prognosis tumor characteristics. Clin Cancer Res 2001; 7: 2821-5

129

48 Fleischhacker M, Beinert T, Ermitsch M, Seferi D, Possinger K, Engelmann C, et al. Detection of ampliable messenger RNA in the serum of patients with lung cancer. Ann N Y Acad Sci 2001; 945: 179-88 49 Lo YMD, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998; 62: 768-75 50 Bianchi DW, Williams JM, Sullivan LM, Hanson FW, Klinger KW, Shuber AP. PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 1997; 61: 822-9 51 Krabchi K, Gros-Louis F, Yan J, Bronsard M, Masse J, Forest JC, et al. Quantication of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques. Clin Genet 2001; 60: 145-50 52 Lo YMD, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 1999; 64: 218-24 53 Honda H, Miharu N, Ohashi Y, Samura O, Kinutani M, Hara T, et al. Fetal gender determination in early pregnancy through qualitative and quantitative analysis of fetal DNA in maternal serum. Hum Genet 2002; 110: 75-9 54 Sekizawa A, Kondo T, Iwasaki M, Watanabe A, Jimbo M, Saito H, et al. Accuracy of fetal gender determination by analysis of DNA in maternal plasma. Clin Chem 2001; 47: 1856-8 55 Zhong XY, Burk MR, Troeger C, Jackson LR, Holzgreve W, Hahn S. Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses. Prenat Diagn 2000; 20: 795-8 56 Wei C, Saller DN, Sutherland JW. Detection and quantication by homogeneous PCR of cell-free fetal DNA in maternal plasma. Clin Chem 2001; 47: 336-8 57 Zhong XY, Hahn S, Holzgreve W. Prenatal identication of fetal genetic traits. Lancet 2001; 357: 310-1 58 Rijnders RJ, van der Schoot CE, Bossers B, de Vroede MA, Christiaens GC. Fetal sex determination from maternal plasma in pregnancies at risk for congenital adrenal hyperplasia. Obstet Gynecol 2001; 98: 374-8 59 Faas BH, Beuling EA, Christiaens GC, von dem Borne AE, van der Schoot CE. Detection of fetal RHD-specic sequences in maternal plasma. Lancet 1998; 352: 1196 60 Lo YMD, Hjelm NM, Fidler C, Sargent IL, Murphy MF, Chamberlain PF, et al. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med 1998; 339: 1734-8 61 Amicucci P, Gennarelli M, Novelli G, Dallapiccola B. Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem 2000; 46: 301-2 62 Saito H, Sekizawa A, Morimoto T, Suzuki M, Yanaihara T. Prenatal DNA diagnosis of a single-gene disorder from maternal plasma. Lancet 2000; 356: 1170 63 Chen CP, Chern SR, Wang W. Fetal DNA in maternal plasma: the prenatal detection of a paternally inherited fetal aneuploidy. Prenat Diagn 2000; 20: 355-7 64 Chen CP, Chern SR, Wang W. Fetal DNA analyzed in plasma from a mother’s three consecutive pregnancies to detect paternally inherited aneuploidy. Clin Chem 2001; 47: 937-9 65 Chiu RWK, Lau TK, Gong ZQ, Cheung PT, Leung TN, Lo YMD. Noninvasive prenatal exclusion of congenital adrenal hyperplasia by maternal plasma analysis: a feasibility study. Clin Chem 2002; 48: 778-80 Ann Clin Biochem 2003; 40: 122–130


130

Chan et al.

66 Lo YMD, Lau TK, Zhang J, Leung TN, Chang AM, Hjelm NM, et al. Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem 1999; 45: 1747-51 67 Lo YMD, Leung TN, Tein MS, Sargent IL, Zhang J, Lau TK, et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999; 45: 184-8 68 Zhong XY, Laivuori H, Livingston JC, Ylikorkala O, Sibai BM, Holzgreve W, et al. Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia. Am J Obstet Gynecol 2001; 184: 414-9 69 Leung TN, Zhang J, Lau TK, Chan LYS, Lo YMD. Increased maternal plasma fetal DNA concentrations in women who eventually develop preeclampsia. Clin Chem 2001; 47: 137-9 70 Swinkels DW, de Kok JB, Hendriks JC, Wiegerinck E, Zusterzeel PL, Steegers EA. Hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome as a complication of preeclampsia in pregnant women increases the amount of cell-free fetal and maternal DNA in maternal plasma and serum. Clin Chem 2002; 48: 650-3 71 Leung TN, Zhang J, Lau TK, Hjelm NM, Lo YMD. Maternal plasma fetal DNA as a marker for preterm labour. Lancet 1998; 352: 19045 72 Lau TK, Lo KW, Chan LYS, Leung TY, Lo YMD. Cell-free fetal deoxyribonucleic acid in maternal circulation as a marker of fetalmaternal hemorrhage in patients undergoing external cephalic version near term. Am J Obstet Gynecol 2000; 183: 712-6 73 Sekizawa A, Jimbo M, Saito H, Iwasaki M, Sugito Y, Yukimoto Y, et al. Increased cell-free fetal DNA in plasma of two women with invasive placenta. Clin Chem 2002; 48: 353-4 74 Ohashi Y, Miharu N, Honda H, Samura O, Ohama K. Correlation of fetal DNA and human chorionic gonadotropin concentrations in second-trimester maternal serum. Clin Chem 2002; 48: 386-8 75 Farina A, Caramelli E, Concu M, Sekizawa A, Ruggeri R, Bovicelli L, et al. Testing normality of fetal DNA concentration in maternal plasma at 10-12 completed weeks’ gestation: a preliminary approach to a new marker for genetic screening. Prenat Diagn 2002; 22: 148-52 76 Costa JM, Ernault P. Automated assay for fetal DNA analysis in maternal serum. Clin Chem 2002; 48: 679-80 77 Chiu RWK, Poon LLM, Lau TK, Leung TN, Wong EMC, Lo YMD. Effects of blood-processing protocols on fetal and total DNA quantication in maternal plasma. Clin Chem 2001; 47: 1607-13 78 van Wijk IJ, de Hoon AC, Jurhawan R, Tjoa ML, Grifoen S, Mulders MA, et al. Detection of apoptotic fetal cells in plasma of pregnant women. Clin Chem 2000; 46: 729-31

79 Poon LLM, Leung TN, Lau TK, Lo YMD. Prenatal detection of fetal Down’s syndrome from maternal plasma. Lancet 2000; 356: 181920 80 van Wijk IJ, de Hoon AC, Grifoen S, Mulders MA, Tjoa ML, van Vugt JM, et al. Identication of triploid trophoblast cells in peripheral blood of a woman with a partial hydatidiform molar pregnancy. Prenat Diagn 2001; 21: 1142-5 81 Poon LLM, Leung TN, Lau TK, Lo YMD. Presence of fetal RNA in maternal plasma. Clin Chem 2000; 46: 1832-4 82 Poon LLM, Leung TN, Lau TK, Chow KCK, Lo YMD. Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma. Clin Chem 2002; 48: 3541 83 Fitzgerald JM, Ramchurren N, Rieger K, Levesque P, Silverman M, Libertino JA, et al. Identication of H-ras mutations in urine sediments complements cytology in the detection of bladder tumors. J Natl Cancer Inst 1995; 87: 129-33 84 Goessl C, Krause H, Muller M, Heicappell R, Schrader M, Sachsinger J, et al. Fluorescent methylation-specic polymerase chain reaction for DNA-based detection of prostate cancer in bodily uids. Cancer Res 2000; 60: 5941-5 85 Utting M, Werner W, Dahse R, Schubert J, Junker K. Microsatellite analysis of free tumor DNA in urine, serum, and plasma of patients: a minimally invasive method for the detection of bladder cancer. Clin Cancer Res 2002; 8: 35-40 86 Zhang J, Tong KL, Li PK, Chan AY, Yeung CK, Pang CC, et al. Presence of donor- and recipient-derived DNA in cell-free urine samples of renal transplantation recipients: urinary DNA chimerism. Clin Chem 1999; 45: 1741-6 87 Botezatu I, Serdyuk O, Potapova G, Shelepov V, Alechina R, Molyaka Y, et al. Genetic analysis of DNA excreted in urine: a new approach for detecting specic genomic DNA sequences from cells dying in an organism. Clin Chem 2000; 46: 1078-84 88 Zhong XY, Hahn D, Troeger C, Klemm A, Stein G, Thomson P, et al. Cell-free DNA in urine: a marker for kidney graft rejection, but not for prenatal diagnosis? Ann N Y Acad Sci 2001; 945: 250-7 89 Mutirangura A, Pornthanakasem W, Theamboonlers A, Sriuranpong V, Lertsanguansinchi P, Yenrudi S, et al. Epstein-Barr viral DNA in serum of patients with nasopharyngeal carcinoma. Clin Cancer Res 1998; 4: 665-9 90 Lui YYN, Chik KW, Chiu RWK, Ho CY, Lam CWK, Lo YMD. Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. Clin Chem 2002; 48: 421-7 Accepted for publication 5 November 2002
