R E V I E W S
`
`Cell-free nucleic acids as biomarkers
`in cancer patients
`
`Heidi Schwarzenbach*, Dave S. B. Hoon‡ and Klaus Pantel*
`
`Abstract | DNA, mRNA and microRNA are released and circulate in the blood of cancer
`patients. Changes in the levels of circulating nucleic acids have been associated with tumour
`burden and malignant progression. In the past decade a wealth of information indicating the
`potential use of circulating nucleic acids for cancer screening, prognosis and monitoring of
`the efficacy of anticancer therapies has emerged. In this Review, we discuss these findings
`with a specific focus on the clinical utility of cell-free nucleic acids as blood biomarkers.
`
`In 1948, Mandel and Métais1 described the presence of
`cell-free nucleic acid (cfNA) in human blood for the first
`time. This attracted little attention in the scientific com-
`munity and it was not until 1994 that the importance
`of cfNA was recognized as a result of the detection of
`mutated RAS gene fragments in the blood of cancer
`patients2,3 (TIMELINE). In 1996, microsatellite altera-
`tions on cell-free DNA (cfDNA) were shown in cancer
`patients4, and during the past decade increasing atten-
`tion has been paid to cfNAs (such as DNA, mRNA and
`microRNAs (miRNAs)) that are present at high concentra-
`tions in the blood of cancer patients (FIG. 1). Indeed, their
`potential value as blood biomarkers was highlighted in a
`recent editorial in the journal Science5.
`Detecting cfNA in plasma or serum could serve as
`a ‘liquid biopsy’, which would be useful for numer-
`ous diagnostic applications and would avoid the need
`for tumour tissue biopsies. Use of such a liquid biopsy
`delivers the possibility of taking repeated blood sam-
`ples, consequently allowing the changes in cfNA to
`be traced during the natural course of the disease or
`during cancer treatment. However, the levels of cfNA
`might also reflect physiological and pathological
`processes that are not tumour-specific6. cfNA yields
`are higher in patients with malignant lesions than in
`patients without tumours, but increased levels have
`also been quantified in patients with benign lesions,
`inflammatory diseases and tissue trauma7. The physi-
`ological events that lead to the increase of cfNA during
`cancer development and progression are still not well
`understood. However, analyses of circulating DNA
`allow the detection of tumour-related genetic and epi-
`genetic alterations that are relevant to cancer develop-
`ment and progression. In addition, circulating miRNAs
`have recently been shown to be potential cancer
`biomarkers in blood.
`
`This Review focuses on the clinical utility of cfNA,
`including genetic and epigenetic alterations that can
`be detected in cfDNA, as well as the quantification of
`nucleo somes and miRNAs, and discusses the relationship
`between cfNA and micrometastatic cells.
`
`Biology of cfNA
`The release of nucleic acids into the blood is thought to
`be related to the apoptosis and necrosis of cancer cells
`in the tumour microenvironment. Secretion has also
`been suggested as a potential source of cfDNA (FIG. 1).
`Necrotic and apoptotic cells are usually phagocytosed
`by macrophages or other scavenger cells8. Macrophages
`that engulf necrotic cells can release digested DNA into
`the tissue environment. In vitro cell culture experiments
`indicated that macrophages can be either activated or
`dying during the process of DNA release8. Fragments
`of cellular nucleic acids can also be actively released9,10.
`It has been estimated that for a patient with a tumour that
`weighs 100 g, which corresponds to 3 × 1010 tumour
`cells, up to 3.3% of tumour DNA may enter the blood
`every day 11. On average, the size of this DNA varies
`between small fragments of 70 to 200 base pairs and
`large fragments of approximately 21 kilobases12.
`Tumour cells that circulate in the blood, and micro-
`metastatic deposits that are present at distant sites, such
`as the bone marrow and liver, can also contribute to the
`release of cfNA13,14.
`Tumours usually represent a mixture of different cancer
`cell clones (which account for the genomic and epig-
`enomic heterogeneity of tumours) and other normal cell
`types, such as haematopoietic and stromal cells. Thus,
`during tumour progression and turnover both tumour-
`derived and wild-type (normal) cfNA can be released
`into the blood. As such, the proportion of cfNA that
`originates from tumour cells varies owing to the state
`
`microRNAs
`Small non-coding RNA
`molecules that modulate the
`activity of specific mRNA
`molecules by binding and
`inhibiting their translation into
`polypeptides.
`
`*Institute of Tumour Biology,
`Center of Experimental
`Medicine, University Medical
`Center Hamburg-Eppendorf,
`Hamburg 20246, Germany.
`‡Department of Molecular
`Oncology, John Wayne
`Cancer Institute, Santa
`Monica, California 90404,
`USA.
`Correspondence to K.P.
`e-mail:
`pantel@uke.uni-hamburg.de
`doi:10.1038/nrc3066
`Published online 12 May 2011
`
`426 | JUNE 2011 | VOLUME 11
`
`00001
`
` www.nature.com/reviews/cancer
`
`EX1061
`
`© 2011 Macmillan Publishers Limited. All rights reserved
`
`
`
`Cell-free nucleic acids as biomarkers
`in cancer patients
`
`Heidi Schwarzenbach*, Dave S. B. Hoon‡ and Klaus Pantel*
`
`Abstract | DNA, mRNA and microRNA are released and circulate in the blood of cancer
`patients. Changes in the levels of circulating nucleic acids have been associated with tumour
`burden and malignant progression. In the past decade a wealth of information indicating the
`potential use of circulating nucleic acids for cancer screening, prognosis and monitoring of
`the efficacy of anticancer therapies has emerged. In this Review, we discuss these findings
`with a specific focus on the clinical utility of cell-free nucleic acids as blood biomarkers.
`
`In 1948, Mandel and Métais1 described the presence of
`cell-free nucleic acid (cfNA) in human blood for the first
`time. This attracted little attention in the scientific com-
`munity and it was not until 1994 that the importance
`of cfNA was recognized as a result of the detection of
`mutated RAS gene fragments in the blood of cancer
`patients2,3 (TIMELINE). In 1996, microsatellite altera-
`tions on cell-free DNA (cfDNA) were shown in cancer
`patients4, and during the past decade increasing atten-
`tion has been paid to cfNAs (such as DNA, mRNA and
`microRNAs (miRNAs)) that are present at high concentra-
`tions in the blood of cancer patients (FIG. 1). Indeed, their
`potential value as blood biomarkers was highlighted in a
`recent editorial in the journal Science5.
`Detecting cfNA in plasma or serum could serve as
`a ‘liquid biopsy’, which would be useful for numer-
`ous diagnostic applications and would avoid the need
`for tumour tissue biopsies. Use of such a liquid biopsy
`delivers the possibility of taking repeated blood sam-
`ples, consequently allowing the changes in cfNA to
`be traced during the natural course of the disease or
`during cancer treatment. However, the levels of cfNA
`might also reflect physiological and pathological
`processes that are not tumour-specific6. cfNA yields
`are higher in patients with malignant lesions than in
`patients without tumours, but increased levels have
`also been quantified in patients with benign lesions,
`inflammatory diseases and tissue trauma7. The physi-
`ological events that lead to the increase of cfNA during
`cancer development and progression are still not well
`understood. However, analyses of circulating DNA
`allow the detection of tumour-related genetic and epi-
`genetic alterations that are relevant to cancer develop-
`ment and progression. In addition, circulating miRNAs
`have recently been shown to be potential cancer
`biomarkers in blood.
`
`This Review focuses on the clinical utility of cfNA,
`including genetic and epigenetic alterations that can
`be detected in cfDNA, as well as the quantification of
`nucleo somes and miRNAs, and discusses the relationship
`between cfNA and micrometastatic cells.
`
`Biology of cfNA
`The release of nucleic acids into the blood is thought to
`be related to the apoptosis and necrosis of cancer cells
`in the tumour microenvironment. Secretion has also
`been suggested as a potential source of cfDNA (FIG. 1).
`Necrotic and apoptotic cells are usually phagocytosed
`by macrophages or other scavenger cells8. Macrophages
`that engulf necrotic cells can release digested DNA into
`the tissue environment. In vitro cell culture experiments
`indicated that macrophages can be either activated or
`dying during the process of DNA release8. Fragments
`of cellular nucleic acids can also be actively released9,10.
`It has been estimated that for a patient with a tumour that
`weighs 100 g, which corresponds to 3 × 1010 tumour
`cells, up to 3.3% of tumour DNA may enter the blood
`every day 11. On average, the size of this DNA varies
`between small fragments of 70 to 200 base pairs and
`large fragments of approximately 21 kilobases12.
`Tumour cells that circulate in the blood, and micro-
`metastatic deposits that are present at distant sites, such
`as the bone marrow and liver, can also contribute to the
`release of cfNA13,14.
`Tumours usually represent a mixture of different cancer
`cell clones (which account for the genomic and epig-
`enomic heterogeneity of tumours) and other normal cell
`types, such as haematopoietic and stromal cells. Thus,
`during tumour progression and turnover both tumour-
`derived and wild-type (normal) cfNA can be released
`into the blood. As such, the proportion of cfNA that
`originates from tumour cells varies owing to the state
`
`microRNAs
`Small non-coding RNA
`molecules that modulate the
`activity of specific mRNA
`molecules by binding and
`inhibiting their translation into
`polypeptides.
`
`*Institute of Tumour Biology,
`Center of Experimental
`Medicine, University Medical
`Center Hamburg-Eppendorf,
`Hamburg 20246, Germany.
`‡Department of Molecular
`Oncology, John Wayne
`Cancer Institute, Santa
`Monica, California 90404,
`USA.
`Correspondence to K.P.
`e-mail:
`pantel@uke.uni-hamburg.de
`doi:10.1038/nrc3066
`Published online 12 May 2011
`
`426 | JUNE 2011 | VOLUME 11
`
`00001
`
` www.nature.com/reviews/cancer
`
`EX1061
`
`© 2011 Macmillan Publishers Limited. All rights reserved
`
`
`
`R E V I E W S
`
` At a glance
`(cid:114)(cid:2)(cid:43)(cid:80)(cid:69)(cid:84)(cid:71)(cid:67)(cid:85)(cid:71)(cid:70)(cid:2)(cid:78)(cid:71)(cid:88)(cid:71)(cid:78)(cid:85)(cid:2)(cid:81)(cid:72)(cid:2)(cid:69)(cid:75)(cid:84)(cid:69)(cid:87)(cid:78)(cid:67)(cid:86)(cid:75)(cid:80)(cid:73)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:75)(cid:69)(cid:2)(cid:67)(cid:69)(cid:75)(cid:70)(cid:85)(cid:2)(cid:10)(cid:38)(cid:48)(cid:35)(cid:14)(cid:2)(cid:79)(cid:52)(cid:48)(cid:35)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:79)(cid:75)(cid:69)(cid:84)(cid:81)(cid:52)(cid:48)(cid:35)(cid:2)(cid:10)(cid:79)(cid:75)(cid:52)(cid:48)(cid:35)(cid:11)(cid:11)(cid:2)(cid:75)(cid:80)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:68)(cid:78)(cid:81)(cid:81)(cid:70)(cid:2)(cid:84)(cid:71)(cid:72)(cid:78)(cid:71)(cid:69)(cid:86)(cid:2)(cid:82)(cid:67)(cid:86)(cid:74)(cid:81)(cid:78)(cid:81)(cid:73)(cid:75)(cid:69)(cid:67)(cid:78)(cid:2)
`(cid:82)(cid:84)(cid:81)(cid:69)(cid:71)(cid:85)(cid:85)(cid:71)(cid:85)(cid:14)(cid:2)(cid:75)(cid:80)(cid:69)(cid:78)(cid:87)(cid:70)(cid:75)(cid:80)(cid:73)(cid:2)(cid:79)(cid:67)(cid:78)(cid:75)(cid:73)(cid:80)(cid:67)(cid:80)(cid:86)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:68)(cid:71)(cid:80)(cid:75)(cid:73)(cid:80)(cid:2)(cid:78)(cid:71)(cid:85)(cid:75)(cid:81)(cid:80)(cid:85)(cid:14)(cid:2)(cid:75)(cid:80)(cid:72)(cid:78)(cid:67)(cid:79)(cid:79)(cid:67)(cid:86)(cid:81)(cid:84)(cid:91)(cid:2)(cid:70)(cid:75)(cid:85)(cid:71)(cid:67)(cid:85)(cid:71)(cid:85)(cid:14)(cid:2)(cid:85)(cid:86)(cid:84)(cid:81)(cid:77)(cid:71)(cid:14)(cid:2)(cid:86)(cid:84)(cid:67)(cid:87)(cid:79)(cid:67)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:85)(cid:71)(cid:82)(cid:85)(cid:75)(cid:85)(cid:16)(cid:2)(cid:38)(cid:87)(cid:84)(cid:75)(cid:80)(cid:73)(cid:2)(cid:86)(cid:74)(cid:71)(cid:85)(cid:71)(cid:2)
`(cid:82)(cid:84)(cid:81)(cid:69)(cid:71)(cid:85)(cid:85)(cid:71)(cid:85)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:75)(cid:69)(cid:2)(cid:67)(cid:69)(cid:75)(cid:70)(cid:85)(cid:2)(cid:67)(cid:84)(cid:71)(cid:2)(cid:85)(cid:74)(cid:71)(cid:70)(cid:2)(cid:75)(cid:80)(cid:86)(cid:81)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:68)(cid:78)(cid:81)(cid:81)(cid:70)(cid:2)(cid:68)(cid:91)(cid:2)(cid:67)(cid:82)(cid:81)(cid:82)(cid:86)(cid:81)(cid:86)(cid:75)(cid:69)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:80)(cid:71)(cid:69)(cid:84)(cid:81)(cid:86)(cid:75)(cid:69)(cid:124)(cid:69)(cid:71)(cid:78)(cid:78)(cid:85)(cid:16)
`(cid:114)(cid:2)(cid:43)(cid:80)(cid:2)(cid:69)(cid:67)(cid:80)(cid:69)(cid:71)(cid:84)(cid:2)(cid:82)(cid:67)(cid:86)(cid:75)(cid:71)(cid:80)(cid:86)(cid:85)(cid:14)(cid:2)(cid:69)(cid:75)(cid:84)(cid:69)(cid:87)(cid:78)(cid:67)(cid:86)(cid:75)(cid:80)(cid:73)(cid:2)(cid:38)(cid:48)(cid:35)(cid:2)(cid:69)(cid:67)(cid:84)(cid:84)(cid:75)(cid:71)(cid:85)(cid:2)(cid:86)(cid:87)(cid:79)(cid:81)(cid:87)(cid:84)(cid:15)(cid:84)(cid:71)(cid:78)(cid:67)(cid:86)(cid:71)(cid:70)(cid:2)(cid:73)(cid:71)(cid:80)(cid:71)(cid:86)(cid:75)(cid:69)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:71)(cid:82)(cid:75)(cid:73)(cid:71)(cid:80)(cid:71)(cid:86)(cid:75)(cid:69)(cid:2)(cid:67)(cid:78)(cid:86)(cid:71)(cid:84)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:85)(cid:2)(cid:86)(cid:74)(cid:67)(cid:86)(cid:2)(cid:67)(cid:84)(cid:71)(cid:2)(cid:84)(cid:71)(cid:78)(cid:71)(cid:88)(cid:67)(cid:80)(cid:86)(cid:2)(cid:86)(cid:81)(cid:2)
`(cid:69)(cid:67)(cid:80)(cid:69)(cid:71)(cid:84)(cid:2)(cid:70)(cid:71)(cid:88)(cid:71)(cid:78)(cid:81)(cid:82)(cid:79)(cid:71)(cid:80)(cid:86)(cid:14)(cid:2)(cid:82)(cid:84)(cid:81)(cid:73)(cid:84)(cid:71)(cid:85)(cid:85)(cid:75)(cid:81)(cid:80)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:84)(cid:71)(cid:85)(cid:75)(cid:85)(cid:86)(cid:67)(cid:80)(cid:69)(cid:71)(cid:2)(cid:86)(cid:81)(cid:2)(cid:86)(cid:74)(cid:71)(cid:84)(cid:67)(cid:82)(cid:91)(cid:16)(cid:2)(cid:54)(cid:74)(cid:71)(cid:85)(cid:71)(cid:2)(cid:67)(cid:78)(cid:86)(cid:71)(cid:84)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:85)(cid:2)(cid:75)(cid:80)(cid:69)(cid:78)(cid:87)(cid:70)(cid:71)(cid:2)(cid:78)(cid:81)(cid:85)(cid:85)(cid:2)(cid:81)(cid:72)(cid:2)(cid:74)(cid:71)(cid:86)(cid:71)(cid:84)(cid:81)(cid:92)(cid:91)(cid:73)(cid:81)(cid:85)(cid:75)(cid:86)(cid:91)(cid:2)(cid:10)(cid:46)(cid:49)(cid:42)(cid:11)(cid:2)(cid:2)
`(cid:67)(cid:80)(cid:70)(cid:2)(cid:79)(cid:87)(cid:86)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:85)(cid:2)(cid:81)(cid:72)(cid:2)(cid:86)(cid:87)(cid:79)(cid:81)(cid:87)(cid:84)(cid:2)(cid:85)(cid:87)(cid:82)(cid:82)(cid:84)(cid:71)(cid:85)(cid:85)(cid:81)(cid:84)(cid:2)(cid:73)(cid:71)(cid:80)(cid:71)(cid:85)(cid:2)(cid:10)(cid:85)(cid:87)(cid:69)(cid:74)(cid:2)(cid:67)(cid:85)(cid:2)TP53(cid:11)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:81)(cid:80)(cid:69)(cid:81)(cid:73)(cid:71)(cid:80)(cid:71)(cid:85)(cid:2)(cid:10)(cid:85)(cid:87)(cid:69)(cid:74)(cid:2)(cid:67)(cid:85)(cid:2)KRAS and BRAF(cid:11)(cid:16)
`(cid:114)(cid:2)(cid:35)(cid:70)(cid:70)(cid:75)(cid:86)(cid:75)(cid:81)(cid:80)(cid:67)(cid:78)(cid:2)(cid:73)(cid:71)(cid:80)(cid:71)(cid:86)(cid:75)(cid:69)(cid:2)(cid:67)(cid:78)(cid:86)(cid:71)(cid:84)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:85)(cid:2)(cid:86)(cid:74)(cid:67)(cid:86)(cid:2)(cid:67)(cid:84)(cid:71)(cid:2)(cid:70)(cid:71)(cid:86)(cid:71)(cid:69)(cid:86)(cid:67)(cid:68)(cid:78)(cid:71)(cid:2)(cid:81)(cid:80)(cid:2)(cid:69)(cid:75)(cid:84)(cid:69)(cid:87)(cid:78)(cid:67)(cid:86)(cid:75)(cid:80)(cid:73)(cid:2)(cid:38)(cid:48)(cid:35)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:87)(cid:85)(cid:71)(cid:70)(cid:2)(cid:67)(cid:85)(cid:2)(cid:68)(cid:75)(cid:81)(cid:79)(cid:67)(cid:84)(cid:77)(cid:71)(cid:84)(cid:85)(cid:2)(cid:75)(cid:80)(cid:2)(cid:69)(cid:67)(cid:80)(cid:69)(cid:71)(cid:84)(cid:2)(cid:75)(cid:80)(cid:69)(cid:78)(cid:87)(cid:70)(cid:71)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)
`(cid:75)(cid:80)(cid:86)(cid:71)(cid:73)(cid:84)(cid:75)(cid:86)(cid:91)(cid:2)(cid:81)(cid:72)(cid:2)(cid:80)(cid:81)(cid:80)(cid:15)(cid:69)(cid:81)(cid:70)(cid:75)(cid:80)(cid:73)(cid:2)(cid:73)(cid:71)(cid:80)(cid:81)(cid:79)(cid:75)(cid:69)(cid:2)(cid:38)(cid:48)(cid:35)(cid:2)(cid:84)(cid:71)(cid:82)(cid:71)(cid:67)(cid:86)(cid:2)(cid:85)(cid:71)(cid:83)(cid:87)(cid:71)(cid:80)(cid:69)(cid:71)(cid:85)(cid:2)(cid:10)(cid:85)(cid:87)(cid:69)(cid:74)(cid:2)(cid:67)(cid:85)(cid:2)ALU(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)LINE1(cid:11)(cid:16)(cid:2)(cid:35)(cid:78)(cid:86)(cid:74)(cid:81)(cid:87)(cid:73)(cid:74)(cid:2)(cid:85)(cid:86)(cid:75)(cid:78)(cid:78)(cid:2)(cid:75)(cid:80)(cid:2)(cid:86)(cid:74)(cid:71)(cid:75)(cid:84)(cid:2)(cid:75)(cid:80)(cid:72)(cid:67)(cid:80)(cid:69)(cid:91)(cid:14)(cid:2)(cid:38)(cid:48)(cid:35)(cid:2)
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`(cid:114)(cid:2)(cid:39)(cid:82)(cid:75)(cid:73)(cid:71)(cid:80)(cid:71)(cid:86)(cid:75)(cid:69)(cid:2)(cid:67)(cid:78)(cid:86)(cid:71)(cid:84)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:85)(cid:2)(cid:75)(cid:80)(cid:2)(cid:73)(cid:71)(cid:80)(cid:71)(cid:85)(cid:2)(cid:10)(cid:85)(cid:87)(cid:69)(cid:74)(cid:2)(cid:67)(cid:85)(cid:2)(cid:73)(cid:78)(cid:87)(cid:86)(cid:67)(cid:86)(cid:74)(cid:75)(cid:81)(cid:80)(cid:71)(cid:2)S(cid:15)(cid:86)(cid:84)(cid:67)(cid:80)(cid:85)(cid:72)(cid:71)(cid:84)(cid:67)(cid:85)(cid:71)(cid:2)(cid:50)(cid:19)(cid:2)(cid:10)GSTP1(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:85)(cid:71)(cid:82)(cid:86)(cid:75)(cid:80)(cid:2)(cid:27)(cid:2)(cid:10)SEPT9(cid:11)(cid:11)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:67)(cid:70)(cid:71)(cid:80)(cid:81)(cid:79)(cid:67)(cid:86)(cid:81)(cid:87)(cid:85)(cid:2)
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`(cid:89)(cid:75)(cid:78)(cid:78)(cid:14)(cid:2)(cid:86)(cid:74)(cid:71)(cid:84)(cid:71)(cid:72)(cid:81)(cid:84)(cid:71)(cid:14)(cid:2)(cid:73)(cid:67)(cid:75)(cid:80)(cid:2)(cid:69)(cid:78)(cid:75)(cid:80)(cid:75)(cid:69)(cid:67)(cid:78)(cid:2)(cid:87)(cid:86)(cid:75)(cid:78)(cid:75)(cid:86)(cid:91)(cid:2)(cid:75)(cid:80)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:70)(cid:71)(cid:86)(cid:71)(cid:84)(cid:79)(cid:75)(cid:80)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:2)(cid:81)(cid:72)(cid:2)(cid:82)(cid:84)(cid:81)(cid:73)(cid:80)(cid:81)(cid:85)(cid:75)(cid:85)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:86)(cid:84)(cid:71)(cid:67)(cid:86)(cid:79)(cid:71)(cid:80)(cid:86)(cid:2)(cid:71)(cid:72)(cid:72)(cid:75)(cid:69)(cid:67)(cid:69)(cid:91)(cid:16)
`
`and size of the tumour. The amount of cfNA is also influ-
`enced by clearance, degradation and other physiological
`filtering events of the blood and lymphatic circulation.
`Nucleic acids are cleared from the blood by the liver and
`kidney and they have a variable half-life in the circulation
`ranging from 15 minutes to several hours7. Assuming an
`exponential decay model and plotting the natural loga-
`rithm of cfDNA concentration against time, serial DNA
`measure ments have shown that some forms of cfNA
`might survive longer than others. When purified DNA
`was injected into the blood of mice, double-stranded
`DNA remained in the circulation longer than single-
`stranded DNA15. Moreover, viral DNA as a closed ring may
`survive longer than linear DNA15. However, regardless
`of its size or configuration, cfDNA is cleared from the
`circulation rapidly and efficiently16. miRNAs seem to be
`highly stable, but their clearance rate from the blood has
`not yet been well studied in cancer patients owing to
`the novelty of this area of research. The nuclease activ-
`ity in blood may be one of the important factors for the
`turnover of cfNA. However, this area of cfNA physiology
`remains unclear and needs further examination.
`
`Circulating cfDNA
`DNA content. In patients with tumours of different histo-
`pathological types, increased levels of total cfDNA, which
`consists of epigenomic and genomic, as well as mito-
`chondrial and viral DNA, have been assessed by different
`fluorescence-based methods (such as, PicoGreen stain-
`ing and ultraviolet (UV) spectrometry) or quantitative
`PCR (such as, SYBR Green and TaqMan). Although
`cancer patients have higher cfDNA levels than healthy
`control donors, the concentrations of overall cfDNA
`
`vary considerably in plasma or serum samples in both
`groups17–19. A range of between 0 and >1,000 ng per ml
`of blood, with an average of 180 ng per ml cfDNA, has
`been measured20–23. By comparison, healthy subjects have
`concentrations between 0 and 100 ng per ml cfDNA of
`blood, with an average of 30 ng per ml cfDNA7. However,
`it is difficult to draw conclusions from these studies, as
`the size of the investigated patient cohort is often small
`and the techniques used to quantify cfDNA vary. A large
`prospective study assessed the value of plasma DNA
`levels as indicators for the development of neoplastic or
`pulmonary disease. The concentration of plasma DNA
`varied considerably between the European Prospective
`Investigation into Cancer and Nutrition (EPIC) centres
`that were involved in the study. This variation was pro-
`posed to be due to the type of population recruited and/or
`the treatment of the samples24. However, the quantifica-
`tion of cfDNA concentrations alone does not seem to be
`useful in a diagnostic setting owing to the overlapping
`DNA concentrations that are found in healthy individuals
`with those in patients with benign and malignant disease.
`The assessment of cfDNA concentration might prove to
`be useful in combination with other blood tumour bio-
`markers. Following surgery, the levels of cfDNA in cancer
`patients with localized disease can decrease to levels that
`are observed in healthy individuals25. However, when the
`cfDNA level remains high, it might indicate the presence
`of residual tumour cells17. Further studies are needed for
`the repeat assessment of quantitative cfNA in large cohorts
`of patients with well-defined clinical parameters. Such
`investigations will be crucial if we are to use cfDNA as a
`prognostic biomarker, as will the isolation and processing
`of cfNA to defined standards (discussed below).
`
`NATURE REVIEWS | CANCER
`
`00002
`
`© 2011 Macmillan Publishers Limited. All rights reserved
`
` VOLUME 11 | JUNE 2011 | 427
`
`
`
`R E V I E W S
`
`cfDNA is composed of both genomic DNA (gDNA)
`and mitochondrial DNA (mtDNA). Interestingly, the
`levels of cell-free mtDNA and gDNA do not correlate in
`some tumour types26,27, indicating the different nature of
`circulating mtDNA and gDNA. In contrast to two copies
`of gDNA, a single cell contains up to several hundred
`copies of mtDNA. Whereas gDNA usually circulates in a
`cell-free form, circulating mtDNA in plasma exists in both
`particle-associated and non-particle-associated forms28.
`Diverging results have been reported regarding whether
`cell-free mtDNA levels are increased and clinically
`relevant in cancer patients.
`The cfDNA can also include both coding and non-
`coding gDNA that can be used to examine microsatellite
`instability, loss of heterozygosity (LOH), mutations, poly-
`morphisms, methylation and integrity (size). In recent
`years, considerable attention has been paid to non-coding
`DNA, particularly repetitive sequences, such as ALU
`(which is a short interspersed nucleic element (SINE)) and
`as long interspersed nucleotide elements such as LINE1
`(REFS 29–31) (discussed below). ALU and LINE1 are dis-
`tributed throughout the genome and are known to be less
`methylated in cancer cells compared with normal cells32.
`
`Tumour-specific LOH. Genetic alterations found in
`cfDNA frequently include LOH that is detected using
`PCR-based assays13,18,33–38 (TABLE 1). Although similar
`plasma- and serum-based LOH detection methods have
`been used, a great variability in the detection of LOH
`in cfDNA has been reported. Despite the concordance
`between tumour-related LOH that is present in cfDNA
`in blood and LOH that is found in DNA isolated from
`matched primary tumours, discrepancies have also been
`found7. These contradictory LOH data that have
`been derived from blood and tumour tissue and the low
`incidence of LOH in cfDNA have partly been explained by
`technical problems and the dilution of tumour-associated
`cfDNA in blood by DNA released from normal cells11,39–41.
`Moreover, the abnormal proliferation of benign cells,
`
`owing to inflammation or tissue repair processes, for
`example, leads to an increase in apoptotic cell death, the
`accumulation of small, fragmented DNA in blood and
`the masking of LOH42.
`Alternative approaches, such as the detection of
`tumour-specific deletions are needed to better address
`the inherent problems of LOH analyses.
`
`Tumour-specific gene mutations. The analysis of
`cfDNA for specific gene mutations, such as those in
`KRAS and TP53, is desirable because these genes have
`a high mutation frequency in many tumour types and
`contribute to tumour progression43. Additionally, clini-
`cally relevant mutations in BRAF, epidermal growth
`factor receptor (EGFR) and adenomatous polyposis coli
`(APC) have now been studied in cfDNA. Several thera-
`peutic agents in clinical trials target the KRAS, BRAF,
`EGFR or p53 pathways44,45, and require the identifica-
`tion of the mutation status of the patient’s tumour to
`predict response to treatment. In this regard, cfDNA
`provides a unique opportunity to repeatedly monitor
`patients during treatment. In particular, in stage IV
`cancer patients, biopsies are not possible or repeat sam-
`pling of primary tumour and metastatic samples is not
`practical or ethical.
`The major problem with this approach has been
`assay specificity and sensitivity. Assays targeting
`cfDNA mutations require that the mutation in the
`tumour occurs frequently at a specific genomic site.
`A major drawback of cfDNA assays is the low frequency of
`some of the mutations that occur in tumours. In general,
`wild-type sequences often interfere with cfDNA muta-
`tion assays. This is due to the low level of cfDNA
`mutations and the dilution effect of DNA fragments
`and wild-type DNA in circulation. In PCR-based assays
`tec
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