REVIEWS
`
`Liquid biopsy: monitoring cancer-genetics
`in the blood
`
`Emily Crowley, Federica Di Nicolantonio, Fotios Loupakis and Alberto Bardelli
`
`Abstract | Cancer is associated with mutated genes, and analysis of tumour-linked genetic alterations is
`increasingly used for diagnostic, prognostic and treatment purposes. The genetic profile of solid tumours
`is currently obtained from surgical or biopsy specimens; however, the latter procedure cannot always be
`performed routinely owing to its invasive nature. Information acquired from a single biopsy provides a spatially
`and temporally limited snap-shot of a tumour and might fail to reflect its heterogeneity. Tumour cells release
`circulating free DNA (cfDNA) into the blood, but the majority of circulating DNA is often not of cancerous origin,
`and detection of cancer-associated alleles in the blood has long been impossible to achieve. Technological
`advances have overcome these restrictions, making it possible to identify both genetic and epigenetic
`aberrations. A liquid biopsy, or blood sample, can provide the genetic landscape of all cancerous lesions
`(primary and metastases) as well as offering the opportunity to systematically track genomic evolution.
`This Review will explore how tumour-associated mutations detectable in the blood can be used in the clinic
`after diagnosis, including the assessment of prognosis, early detection of disease recurrence, and as
`surrogates for traditional biopsies with the purpose of predicting response to treatments and the development
`of acquired resistance.
`
`Crowley, E. et al. Nat. Rev. Clin. Oncol. 10, 472–484 (2013); published online 9 July 2013; doi:10.1038/nrclinonc.2013.110
`
`Introduction
`Biopsies have been used by clinicians to diagnose and
`manage disease for 1,000 years.1 In patients with cancer,
`biopsies allow the histological definition of the disease
`and, more recently, have revealed details of the genetic
`profile of the tumour enabling prediction of disease pro-
`gression and response to therapies. As the techniques that
`have enabled us to analyse a biopsy become ever more
`sophisticated, we have realised the limitations of looking
`at this single snap-shot of the tumour. This single-biopsy
`bias was highlighted in a study by Gerlinger et al.2 in which
`it was demonstrated that a portion taken from different
`parts of a primary tumour and its metastases showed
`extensive intertumoural and intratumoural evolution.
`This tumoural heterogeneity highlights the diffi culty of
`dictating a therapeutic course of action based on a single
`biopsy, as it is likely to underestimate the c omplexity of the
`genomic landscape of the tumour.
`Having established that there is considerable tumour
`heterogeneity, taking multiple biopsies from the patients’
`primary tumour and metastases would seem to be the most
`obvious next step, so why is it not routinely done? There are
`many difficulties in obtaining a tissue biopsy—i ncluding
`the discomfort suffered by the patient, inherent clinical
`risks to the patient, potential surgical complications and
`economic considerations—meaning that multiple or serial
`biopsies are often impractical. In addition, some tumours
`are not accessible for biopsy, the procedure itself might
`
`Competing interests
`The authors declare no competing interests.
`
`increase the risk of the cancer ‘seeding’ to other sites,3 and
`the procedure might not be recommended for patients
`receiving antiangiogenic treatment.4 Even in an ideal
`situation where several meta static sites can be biopsied
`simultaneously, the analysis of the samples can delay the
`initiation of treatment, and might irremediably jeopardise
`it. These limitations are particular restraints in the setting
`of acquired resistance to therapy, as the ability of a clinician
`to detect thera peutic biomarkers at an early stage would
`allow a potentially successful change in treatment course.
`In light of these limitations on the use of single biopsies,
`new ways to observe tumour genetics and tumour dynam-
`ics have evolved. In 1948, the publication of a manuscript
`that described circulating free DNA (cfDNA) and RNA
`in the blood of humans was, without knowing it, the first
`step towards the ‘liquid biopsy’.5 Furthermore, the levels of
`cfDNA were higher in diseased than healthy individuals,
`indicating that it is possible to screen for the presence of
`disease through a simple blood test.6 Indeed, the specific
`detection of tumour-derived cfDNA has been shown to
`correlate with tumour burden, to a change in response
`to treatment or surgery, and to indicate that subpopula-
`tions of tumour cells that are resistant to treatment can
`prolifer ate in response to therapy.7,8 With the development
`of sensitive techniques that enable the detection of rare
`mutations, it is now possible to understand the hetero-
`geneous landscape of the tumour using a blood sample.9
`Nevertheless, key challenges lie ahead and not all results
`consistently support the application of c irculating tumour
`DNA (ctDNA) in the clinic.
`
`Department of
`Oncology, University
`of Turin, Institute for
`Cancer Research
`and Treatment,
`Strada Provinciale
`142 Km 3.95,
`10060 Candiolo,
`Turin, Italy (E. Crowley,
`F. Di Nicolantonio,
`A. Bardelli). Unit of
`Medical Oncology 2,
`Azienda Ospedaliero-
`Universitaria Pisana,
`Via Roma 67,
`56126 Pisa, Italy
`(F. Loupakis).
`
`Correspondence to:
`A. Bardelli
`alberto.bardelli@
`unito.it
`
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`The early diagnosis of cancer is one application for
`cfDNA assessment and has been covered extensively
`in previous reviews.10,11 This Review will explore how
`tumour-associated genetic alterations detectable in the
`blood can be used in the clinic, including the assessment
`of prognosis, early detection of disease recurrence, and
`as surro gates for traditional biopsies with the purpose of
`predicting response to treatments or the development
`of acquired resistance. It will also cover technical and
`logistical challenges that might represent hurdles for
`clinical implementation.
`
`Discovery and detection of ctDNA
`There are at least two potential, but not mutually exclu-
`sive, mechanisms by which DNA can enter into the cir-
`culation. These mechanisms can be broadly categorized
`as passive and active. The passive mechanism suggests
`that apoptotic and necrotic cells release nuclear and
`mitochondrial DNA into the circulation in the process
`of cellular destruction.12 Under normal physiological
`conditions, infiltrating phagocytes clear apoptotic and
`necrotic debris and, therefore, levels of cfDNA in healthy
`individuals are low.13 However, certain conditions, such
`as within a tumoural mass or in the instance of exhaus-
`tive exercise or inflammation, clearance does not happen
`efficiently,13 leading to an accumulation of cellular debris,
`including DNA, that is then released into the blood
`(Figure 1).14,15 Alternatively, active DNA release has been
`reported in studies with cultured cell lines of different
`origins and is believed to involve the spon taneous release
`of DNA fragments into the circulation.16–19 Multiple
`hypotheses have been formulated to explain why living
`cancer cells would actively release DNA into the circu-
`lation, including the possibility that cancer cells release
`oncogenic DNA to affect the transformation of suscep-
`tible cells at distant sites.20,21 Several reviews are available
`that detail the biology and mechanisms of DNA release
`into circulation.10,12,22
`ctDNA might also be released by circulating tumour
`cells (CTCs) present in the blood.23 Definitive evidence
`for this mechanism has not been reported, and there is a
`discrepancy between the number of CTCs and the quan-
`tity of cfDNA in the blood. A single human cell contains
`6 pg of DNA24 and there is an average of 17 ng of DNA
`per ml of plasma in advanced-stage cancers;25 therefore,
`if CTCs were the primary source of ctDNA it would
`require over 2,000 cells per ml of plasma. In reality, there
`are, on average, less than 10 CTCs per 7.5 ml blood.26
`Presently, the most commonly used protocols to obtain
`cfDNA require approximately 1 ml of serum or plasma
`(3 ml of blood) and preparation should not exceed 4–5 h
`following the blood draw. For plasma preparation, blood
`must be collected in a tube treated with an anti coagulant,
`preferably EDTA (ethylenediaminetetraacetic acid). Cells
`are then removed by centrifugation and the super natant,
`or plasma, is removed.27 Serum is collected after the
`blood is allowed to clot and following centrifugation
`the supernatant, or serum, is removed.28 Circulating
`DNA is then extracted from the plasma or serum using
`c ommercially available kits.
`
`REVIEWS
`
`Key points
`
`■ Under representation of the heterogeneity of a tumour and poor sample
`availability means tissue biopsies are of limited value for the assessment
`of tumour dynamics in the advanced stages of disease
`■ Extended periods between sampling and clinical application of the results,
`as well as additional lines of treatment between sampling, might result in
`an altered genetic composition of the tumour
`■ Circulating free DNA can be extracted from the blood and tumour-specific
`aberrations assessed to provide a genetic landscape of the cancerous lesions
`in a patient
`■ Tracking tumour-associated genetic aberrations in the blood can be used to
`assess the presence of residual disease, recurrence, relapse and resistance
`■ Monitoring the emergence of tumour-associated genetic aberrations in
`the blood can be used to detect the emergence of resistant cancer cells
`5–10 months before conventional methods
`■ To implement circulating tumour DNA testing in the clinic, standardization of
`techniques, assessment of reproducibility and cost-effectiveness is required
`as well as prospective validation in clinical trials
`
`The analysis of ctDNA from the blood offers an excit-
`ing clinical application in patients, that is, the detection
`of tumour-specific genetic aberrations (Figure 1). This
`approach has greater dynamic range compared to total
`circulating DNA,29 is more specific, and has many poten-
`tial applications in the clinic. However, it is also tech-
`nically more challenging owing to the presence of high
`levels of DNA originating from tissue of non-tumour
`origin. A proportion of cfDNA is derived directly from
`the tumour (ctDNA) and this fraction can be quanti-
`fied.30–35 Furthermore, an in vivo study has shown a direct
`correlation between tumour burden and the quantity of
`ctDNA released.33
`High-analytical sensitivity and specialized equip-
`ment are required for detection of ctDNA because the
`quantity and quality of tumour-derived DNA can vary
`dramatically. Techniques are available that allow reliable
`monitoring of tumour-associated genetic aberrations,
`including somatic mutations, loss of heterozygosity and
`chromosomal aberrations in the blood (Table 1), at fre-
`quencies as low as 0.01%.8,9,36–38 The fraction of circu-
`lating DNA that is derived from the tumour can range
`between 0.01% and 93%.36,39 Tumour-specific genetic
`aberrations in cfDNA might offer intriguing insights
`into tumour dynamics. Nevertheless, there are clini-
`cal situations, including early stage disease, where it is
`unlikely to have broad applications as well as clinical
`settings where cfDNA levels are below optimal levels for
`the detection of mutations.25,40,41 According to a study by
`Perkins et al.,25 the optimal DNA concentration for the
`detection of mutations (using a Sequenom® MassARRAY®
`platform) is approximately 30 ng/ml in the plasma. As
`certain cancers, including melanoma, have a low average
`total cfDNA level (7 ng/ml in the plasma) the accuracy of
`detection could be suboptimal. However, with improving
`technique sensitivities, the ability to detect genetic aber-
`rations at lower frequencies will undoubtedly improve,
`as exemplified by the improvement in PIK3CA mutation
`detection in studies by Board et al.40 and Higgins et al.,42
`which showed a concordance between primary tumour
`tissue and plasma DNA of 95% and 100%, respectively.
`
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`
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`
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`
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`REVIEWS
`
`Circulating
` tumour cell
`
`Healthy
`tissue
`
`Apoptosis
`or necrosis
`
`Blood plasma or
`serum sample
`containing ctDNA
`
`Apoptosis
`
`In(cid:10)amed
`tissue
`
`Healthy cell
`Phagocyte
`Tumour cell
`Mutation
`Red blood cell
`Endothelial cell
`Chromosome
`
`Figure 1 | Release and extraction of cfDNA from the blood. cfDNA is released from healthy, inflamed or diseased
`(cancerous) tissue from cells undergoing apoptosis or necrosis. cfDNA can be extracted from a blood sample and
`genetic aberrations in the DNA released from cancerous tissue detected and quantified. Tumour-derived genetic alterations
`that can be detected in the blood include point mutations (consecutive purple, red, green and blue DNA strands), copy
`number fluctuations (red portion of chromosomes) and structural rearrangements (green and red DNA strands).
`Abbreviations: cfDNA, circulating free DNA; ctDNA, circulating tumour DNA.
`
`Liquid biopsies in the clinic
`In the past few years there has been considerable focus
`on the need for ‘biomarkers’. These biomarkers should
`be surrogate indicators for a future event, such as disease
`recurrence, disease progression, or death, and should
`indicate if a specific treatment will reduce that risk.43
`Despite extensive investigations, there are no currently
`approved applications for liquid biopsies in the clinical
`setting. Nevertheless, recently published data demon-
`strate that ongoing research holds considerable promise
`for the future of molecular testing of ctDNA—one of the
`most promising future tools for clinical application.
`
`Assessment of prognosis
`Prognosis
`Assessing prognosis for an individual patient involves
`a combination of clinical observations, staging, and
`histopathological and biomolecular characterization
`of different tumour types. This information, derived
`from imaging and biopsy specimens, allows a clini-
`cian to appraise the tumour biology, precisely stage the
`patient’s tumour and differentiate between those patients
`with more-aggressive or less-aggressive disease. In this
`context, liquid biopsies are unlikely to supersede current
`
`methods, but their use might be important in circum-
`stances where a biopsy is not available or genetic analysis
`of archived tumour samples is not possible.44,45
`Assessment of disease stage is one of the most-
`reliable predictors of prognosis and the relationship
`between levels of tumour-specific genetic aberrations
`and stage requires thorough evaluation for all cancer
`types. Studies have revealed a statistically significant
`correlation between disease stage and the presence
`of tumour- associated genetic aberrations (including
`mutations in TP53, KRAS, and APC and allelic imbal-
`ances) in the blood of patients with resectable breast,
`ovarian, pancreatic and colorectal cancer and oral
`squamous-cell carcinoma.27,28,46–53
`Furthermore, following mastectomy as treatment for
`breast cancer, it was reported that patients with tumours
`with vascular invasion, more than three lymph-node
`metastases, and high histological grade at diagnosis
`had persistent tumour-associated microsatellite DNA
`alterations as detected by PCR post-surgery in plasma-
`extracted DNA.46 Moreover, the presence of tumour-
`associated genetic aberrations in the blood, including
`TP53 mutations47 and loss of hetero zygosity,48 correlated
`with overall survival or disease-free survival as assessed
`
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`Table 1 | Tumour-associated genetic aberrations in circulating free DNA
`
`Tumour type
`
`Stage
`
`n
`
`Tumour-specific
`aberration
`
`Tumour
`burden
`or stage*
`
`Source
`
`Technique
`
`Reference‡
`
`REVIEWS
`
`APC
`
`No/Yes
`
`Plasma
`
`BEAMing
`
`Colorectal
`cancer
`
`Breast cancer
`
`Ovarian
`cancer
`
`Early to
`advanced
`Advanced
`
`Early to
`advanced
`Early to
`advanced
`
`Early to
`advanced
`Early to
`advanced
`
`Advanced
`
`Advanced
`
`Early to
`advanced
`
`33
`
`18
`
`104
`
`70
`
`72
`
`34 (retrospective)
`and 51
`(prospective)
`30
`
`38
`
`63
`
`APC, KRAS,
`PIK3CA, TP53
`APC, KRAS, TP53
`
`KRAS
`
`PIK3CA
`
`PIK3CA
`
`PIK3CA, TP53,
`structural variation
`
`TP53, PTEN, EGFR,
`BRAF, KRAS,
`PIK3CA
`
`Yes
`
`NA
`
`NA
`
`Yes
`
`NA
`
`Yes
`
`Yes
`
`Yes
`
`Plasma
`
`BEAMing
`
`Serum
`
`PCR-SSCP
`
`Plasma
`
`ME-PCR
`
`Plasma
`and serum
`Plasma
`
`ARMS-
`Scorpion PCR
`BEAMing
`
`Diehl et al.
`(2005)36
`Diehl et al.
`(2008)27
`Wang et al.
`(2004)28
`Frattini et al.
`(2008)34
`
`Board et al.
`(2010)40
`Higgins et al.
`(2012)42
`
`Plasma
`
`Plasma
`
`Serum
`
`TAm-Seq and
`digital PCR
`
`Dawson et al.
`(2013)29
`
`TAm-Seq
`Digital PCR
`Fluorescent-
`PCR
`
`WGS
`
`Forshew et al.
`(2012)38
`Kuhlmann
`et al. (2012)52
`
`4
`
`21
`
`44
`
`64
`
`20
`
`SNV
`
`KRAS
`
`KRAS
`
`Yes
`
`Yes
`
`Plasma
`
`Plasma
`
`MASA PCR
`
`No/Yes
`
`Plasma
`
`RFLP-PCR
`
`Microsatellite loci
`
`Yes
`
`Microsatellite loci
`
`No
`
`Serum
`
`Serum
`
`PCR
`
`PCR
`
`Chan et al.
`(2013)9
`
`Yamada et al.
`(1998)54
`Castells et al.
`(1999)53
`
`Hamana et al.
`(2005)50
`Kakimoto et al.
`(2008)157
`
`Nygaard et al.
`(2013)59
`
`Hepatocellular
`carcinoma
`
`Early
`
`Early to
`advanced
`Early to
`advanced
`
`Early to
`advanced
`Early to
`advanced
`
`Pancreatic
`cancer
`
`Oral
`squamous-cell
`carcinoma
`
`Non-small-cell
`lung cancer
`
`Breast and
`osteosarcoma
`
`Colorectal and
`breast cancer
`
`Advanced
`
`246
`
`KRAS
`
`Advanced
`
`3
`
`Advanced
`
`10
`
`Genomic
`alterations
`
`Chromosomal
`alterations
`
`Yes
`
`Yes
`
`Yes
`
`Plasma
`
`ARMS-qPCR
`
`Plasma
`and serum
`
`Nested-real
`time PCR
`
`McBride et al.
`(2010)154
`
`Plasma
`
`WGS
`
`Leary et al.
`(2012)37
`
`*This column indicates if the study observed a correlation between tumour-associated genetic aberrations and tumour burden or disease stage. ‡The table
`includes studies in which different tumour-associated genetic aberrations have been detected using a variety of techniques, with different cancer types and at
`different stages. Abbreviations: ARMS, amplification refractory mutation system; BEAMing, beads, emulsion, amplification, magnetics; MASA, mutant allele
`specific amplification; ME-PCR, mutant enriched PCR; NA, not applicable; PCR-SSCP, single-strand conformation polymorphism PCR; qPCR, quantitative PCR;
`RFLP-PCR, restriction fragment length polymorphism PCR; SNV, single nucleotide variants; WGS, whole-genome sequencing.
`
`by multivariate analysis. There are, however, conflict-
`ing studies in which a correlation between stage and
`levels of tumour-associated genetic aberrations (includ-
`ing KRAS) are not observed.34,54 Controversy associ-
`ated with such studies is often due to limited sample
`sizes as well as some of the studies not being designed
`to specific ally address this issue,34 in addition to the
`obvious technical differences that will be discussed later
`in this Review.
`What are the possible applications of liquid biopsies
`in the clinic for the assessment of patient prognosis?
`In the context of resectable tumours, the utility of
`liquid biopsies is likely to be limited. In this setting,
`a sample of the tumour itself is usually available and
`aside from conventional histological, immunohisto-
`chemical and molecular analysis, the development of
`gene- expression classifier assays provide additional
`prognostic information.55,56 In the context of breast and
`colorectal cancer, there are FDA-approved assays that
`
`can discriminate, in specific settings, between high-risk
`and low-risk groups.55–58 Equivalent panels of genetic
`aberrations that can be tested using cfDNA samples to
`accurately stratify patients are not currently available.
`Therefore, liquid biopsies in the resectable setting are
`currently unlikely to have significant clinical utility for
`assessing prognosis.
`What could be the utility of liquid biopsies for
`advanced-stage, unresectable diseases? In a multi-
`variate analysis, KRAS mutations present in the plasma
`of 246 patients with advanced-stage non-small-cell lung
`cancer (NSCLC) was shown to predict poor prognosis
`in patients receiving first-line chemotherapy.59 KRAS
`mutations in circulation have also been assessed in a
`cohort of 44 patients with pancreatic cancer to deter-
`mine patient prognosis and, despite the poor sensitiv-
`ity of the test (27%), it was shown that patients with
`KRAS mutations had a significantly reduced probability
`of survival compared to those patients without KRAS
`
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`

`A different approach involves the quantification of
`the level of multiple tumour-associated genetic muta-
`tions present in the blood—this approach might be
`more sensi tive than the assessment of total cfDNA or
`measuring single tumour-specific aberrations for the
`stratification of patients with cancer according to their
`probability of survival.29 For example, the number of
`copies of DNA harbouring specific somatic muta-
`tions when detected in the plasma were calculated in
`a cohort of 30 women with metastatic breast cancer,
`and an inverse relationship (P <0.001) was identified
`between plasma copy number and survival.29 Whether
`this method can be used to accurately stratify patients
`of all cancer types25 and at different disease stages40,72
`remains to be validated.
`
`Detection of recurrence
`A promising clinical application for liquid biopsies is the
`early detection of relapse after potentially curative treat-
`ments (Figure 2a). After treatment with curative intent,
`patients are monitored for signs of residual disease and
`local or distant recurrences using radiological imaging
`during post-treatment follow-up.73–75 These methods are
`costly, often requiring contrast media (exposing patients
`to doses of radiation), and thus cannot be used for fre-
`quent monitoring. Another important consideration is
`that these imaging techniques have limited sensitivity for
`the detection of micrometastases.76,77
`In a landmark paper, Diehl et al.27 showed that by
`monitoring tumour-specific aberrations (including APC,
`TP53 and KRAS) in the plasma of patients with colorectal
`cancer post-surgery it was possible to identify disease
`recurrence with almost 100% sensitivity and specifi-
`city. Patients with residual disease were also identified
`based on the persistence of tumour-associated genetic
`aberrations in cfDNA immediately after surgery in each
`case of incomplete resection. Other reports, including
`studies in breast and lung cancer, and oral squamous-
`cell carcinoma, demonstrated a consistent relationship
`between disease recurrence and the reappearance of
`certain tumour aberrations, including KRAS, APC and
`TP53 mutations as well as allelic imbalances.34,50,51,78
`Monitoring single tumour-associated aberrations
`in the blood is not only effective for the stratification
`of patients with minimal residual disease and to iden-
`tify patients likely to relapse, but also to identify those
`with dormant disease. Dormancy is a common feature of
`many cancers, including breast, melanoma, renal cancer
`and non-Hodgkin lymphoma, and cannot be detected
`by standard methods.79 In 50 patients with breast cancer,
`tracking tumour-specific copy-number aberrations in
`the plasma pre-operatively and post-operatively demon-
`strated that tumour-specific copy-number aberrations
`persisted up to 12 years post diagnosis.80 As DNA is
`cleared from circulation within 30 min,81 the presence
`of DNA might reflect persistent dormant cells cycling
`between replication and cell death; however, relapse was
`not detected in any of the 50 patients. Nevertheless, pro-
`spective validation in a larger cohort and in other cancer
`models is warranted.
`
`REVIEWS
`
`Surgery
`
`Early relapse
`
`b
`
`Treatment
`
`Resistance
`
`A
`
`A
`
`Time
`
`Time
`
`a
`
`genetic aberation
`Tumour-associated
`
`Figure 2 | Monitoring tumour-specific aberrations to detect recurrence and
`resistance. The figure represents two hypothetical clinical scenarios in which
`genetic alterations in the blood plasma or serum can be tracked following
`a | surgery, to monitor signs of early relapse and b | treatment with a targeted
`agent (such as anti-EGFR monoclonal antibodies in CRCs) to monitor the
`emergence of resistant clones. ‘A’ represents the time of clinically-detectable
`recurrence or relapse. The blue line represents a ‘founder’ (early occurring)
`mutation that is present throughout the bulk of the tumour and reflects overall
`tumour burden. The red and green lines represent genetic aberrations associated
`with the growth and expansion of resistant clones.
`
`mutations (17% versus 41% at 6 months, and 0% versus
`24% at 12 months in KRAS mutant and wild type, respec-
`tively).53 However, a parallel study, conducted in 308
`patients with advanced-stage NSCLC, showed no corre-
`lation between KRAS mutations in the plasma and prog-
`nosis.60 BRAF mutations as assessed in serum samples
`have also been shown to effectively stratify 103 patients
`with melanoma in both early stage and advanced-stage
`settings.61 Nevertheless, results remain contradictory in
`these small patient populations.
`Neuroblastoma is a special case where amplification
`of the gene MYC-related oncogene (MYCN) has been
`identified as a genetic hallmark of aggressive disease,
`and this abnormality provides important information
`for patient treatment.62,63 Patient stratification according
`to MYCN status is important because improved treat-
`ment approaches, such as immunotherapy,64 were shown
`to significantly increase event-free and overall survival
`compared to standard treatments and might improve cur-
`rently unsatisfactory long-term survival rates in patients
`with high risk MYCN amplified neuroblastomas.65
`It is estimated that for 29% of all patients with
`neuro blastoma,65,66 MYCN status is unknown despite
`guidelines recommending genetic testing for patient
`stratification and treatment tailoring.67,68 Liquid biop-
`sies are able to accurately detect MYCN amplification
`in the serum of stage III to IV patients with neuro-
`blastoma with a sensitivity and specificity of 75–85% and
`100%, respectively.69–71
`
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`

`REVIEWS
`
`The sensitivity of detecting single tumour- specific
`genetic aberrations in serum or plasma is very vari-
`able, particularly in settings of low tumour burden.30,40
`Monitoring tumour-specific genetic aberrations using
`next-generation sequencing (NGS) techno logy might
`offer the opportunity to increase such sensitivity.
`Genome-wide sequencing of plasma cfDNA has been
`carried out in patients diagnosed with hepato cellular
`carcinoma,9 and by calculating the fractional concentra-
`tion of tumour-derived DNA (mutations or copy-number
`aberrations) with respect to total DNA, the research-
`ers were able to demonstrate a significant reduction in
`tumour-derived DNA following surgery. This finding
`highlights one of the inherent technical difficulties of
`using NGS platforms in that the study demonstrated
`a reduction (threefold to 60-fold), not eradication, of
`tumour-derived DNA following surgery. As none of the
`patients relapsed during the course of the study it was
`assumed that surgery was curative and persistent tumour-
`associated aberrations were related to sequencing errors.
`Although residual disease is a possible explanation,
`tumour-related mutations have also been observed in
`healthy individuals and smokers suggesting that genetic
`aberrations might be present at low frequencies even in
`the absence of cancer.82–84 This possibility will be clarified
`with the use of increasingly sensitive techniques.
`
`Difficult-to-diagnose cancers
`Liquid biopsies can be used to assist in the clinical
`manage ment of difficult-to-diagnose patients with
`advanced-stage cancer, as is the case for bone metasta-
`ses,29 some pancreatic cancers and deep pelvic masses.38
`For example, in a study in which targeted deep sequenc-
`ing of cancer-related genes (including TP53, PIK3CA
`and KRAS) was carried out on cfDNA in a patient who
`had previously undergone surgery to resect synchro-
`nous cancers of the bowel and ovary, it was shown that
`on relapse the meta stasis was derived from the original
`ovarian cancer (owing to the presence of a R273H TP53
`mutation and absence of other mutations).38 A biopsy
`in this case was not possible and had the informa-
`tion derived from ctDNA been available immediately
`unneces sary delay or uncertainty over treatments might
`have been avoided.
`
`Open issues
`In theory, the use of liquid biopsies to identify early
`relapse or progression is very attractive, but there are still
`some open issues. The most intriguing is the possibility
`that tumour-associated aberrations can be lost or gained
`over the monitoring period or in response to drug pres-
`sure.29,42,85 This variation has been ascribed to changes in
`clonal sub-populations and might occur in response to
`treatment, adaptation to environmental changes or an
`increase in metastatic potential.86 This observation is also
`interesting in cancers that have long latency periods and
`liquid biopsies could be used to establish genetic markers
`of relapse.
`Attempts to monitor early relapse or progression
`require the selection of appropriate ‘founder’ genetic
`
`aberrations (such as APC in colorectal cancer or KRAS in
`pancreatic cancer) that are likely to be present from the
`initiation of tumori genesis, but provide little to no selec-
`tive advantage (so-called ‘passive’ mutations).87–90 For
`example, EGFR mutation frequency decreases follow-
`ing chemotherapy treatment and would, therefore, not
`represent a suitable marker of overall tumour burden in
`a patient receiving chemotherapy.85 The less responsive
`the tumour-specific aberration is to therapy the more
`suitable the marker. The plethora of passive and active
`mutations that accumulate throughout the lifetime of
`a tumour means that selecting multiple candid ates is
`not simple.91 Monitoring relapse or progression using
`tumour-associated genetic aberrations in the blood
`requires detailed knowledge of the genes most frequently
`mutated in each type of cancer. These are now available
`from large cancer genomics projects such as The Cancer
`Genome Atlas92 and the International Cancer Genome
`Consortium.93 A rational way to guide the selection of
`marker lesions might be to choose those that occur early
`during tumour progression, as they would presumably
`be present in the whole population as opposed to a
`clonal sub-population.2
`
`Prediction of response to treatment
`The presence or absence of a single genetic alteration
`in tumour DNA is currently employed to guide clini-
`cal decision making for a number of targeted agents (for
`example, EGFR mutations for gefitinib in NSCLC, BRAF
`mutations for vemurafenib in melanoma, KRAS muta-
`tions for cetuximab or panitumumab in colorectal cancer,
`ALK rearrangements for crizotinib in NSCLC).94–96
`Ever-increasing numbers of genomic alterations are
`being tested as putative predictive biomarkers in clinical
`trials of novel anticancer therapies.97
`Targeted agents are often used or tested in patients
`with advanced-stage disease (often with multiple metas-
`tases). However, the associated molecular test is often
`performed on archived tumour tissues collected years
`before. This situation is suboptimal as it might not reflect
`the genomic landscape of current disease. In the context
`where a new biopsy cannot be obtained, ctDNA might
`provide superior molecular information compared to
`archival tissue DNA for determining the current cancer
`molecular status. Potentially, a ‘liquid biopsy’ could
`obviate the need for tumour tissue DNA in metastatic
`patients. Gevensleben et al.98 have recently described the
`optimization of a digital PCR method to detect HER2
`amplification in ctDNA of patients with metastatic breast
`cancer using a development cohort (65 patients) and an
`independent validation cohort (58 patients). The poten-
`tial clinical use of such a tool would be in the analysis of
`HER2 status in patients who might not have received a
`biopsy of recurrent breast cancer as part of their routine
`care, as is often the case. Such patients with metastatic
`disease that are identified as acquiring HER2-positive
`status might subsequently benefit from HER2 targeted
`therapeutics, such as trastuzumab. The researchers also
`showed that three patients with metastatic breast cancer
`acquired a HER2-positive status on disease recurrence
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`NATURE REVIEWS | CLINICAL ONCOLOGY
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`VOLUME 10