CLB-09002; No. of pages: 5; 4C: Clinical Biochemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Review
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The translational potential of circulating tumour DNA in oncology
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K.M. Patel a,b, D.W.Y. Tsui a,⁎
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Article history: Received 1 December 2014 Received in revised form 2 April 2015 Accepted 3 April 2015 Available online xxxx
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Keywords: Circulating nucleic acids Cancer DNA Oncology Urine Plasma Blood Next generation sequencing
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Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK Department of Academic Urology, University of Cambridge Hospitals, Box 243, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0QQ , UK
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Introduction . . . . . . . . . . . . De novo mutation detection in blood . Understanding resistance mechanisms Other bodily fluids . . . . . . . . . Low volume disease . . . . . . . . . Discussion . . . . . . . . . . . . . Acknowledgements . . . . . . . . . References . . . . . . . . . . . . .
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Introduction
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In 1977, Leon et al. discovered that serum circulating cell-free DNA (cfDNA) levels were higher in patients with cancer [1]. This initial work, focusing on total levels of cfDNA, eventually encouraged the investigation of circulating nucleic acids as a biomarker of cancer. However, total levels of cfDNA were found to be insensitive [2–4] and nonspecific [2,5,6], in part, due to great variability between individuals and, that cfDNA levels are raised in a number of conditions including pregnancy [7] and myocardial infarction [8].
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The recent understanding of tumour heterogeneity and cancer evolution in response to therapy has raised questions about the value of historical or single site biopsies for guiding treatment decisions. The ability of ctDNA analysis to reveal de novo mutations (i.e., without prior knowledge), allows monitoring of clonal heterogeneity without the need for multiple tumour biopsies. Additionally, ctDNA monitoring of such heterogeneity and novel mutation detection will allow clinicians to detect resistant mechanisms early and tailor treatment therapies accordingly. If ctDNA can be used to detect low volume cancerous states, it will have important applications in treatment stratification post-surgery/radical radiotherapy and may have a role in patient screening. Mutant cfDNA can also be detected in other bodily fluids that are easily accessible and may aid detection of rare mutant alleles in certain cancer types. This article outlines recent advances in these areas. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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⁎ Corresponding author. E-mail address: [email protected] (D.W.Y. Tsui).
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Tumour DNA contains specific somatic alterations. DNA fragments which contain these tumour-specific somatic mutations can be detected in the blood, and hence are called circulating tumour DNA (ctDNA) [9]. It is challenging to identify ctDNA fragments, because they are surrounded by multiple copies of normal genomic DNA. One strategy is to use a personalised approached, where one could first identify mutations in tumour and subsequently design mutation specific assays to detect ctDNA in plasma [10,11]. A number of studies have demonstrated that the presence of ctDNA and, the dynamics of ctDNA in plasma, reflect tumour burden. For example, in 2008, Diehl et al. evaluated 162 plasma samples from 18 colorectal cancer patients, to demonstrate that levels of ctDNA increased with tumour burden, and that ctDNA kinetics were more sensitive than serum carcino-embroyonic antigen (CEA) for monitoring disease
http://dx.doi.org/10.1016/j.clinbiochem.2015.04.005 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Patel KM, Tsui DWY, The translational potential of circulating tumour DNA in oncology, Clin Biochem (2015), http:// dx.doi.org/10.1016/j.clinbiochem.2015.04.005
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De novo mutation detection in blood
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Substantial work in the field has demonstrated that ctDNA can be detected by tracking tumour-specific point mutations or structural rearrangements, in many cancer types [14,16–20]. However, it is now apparent that the mutational characteristics of tumours are not static and continually evolve in response to various selection pressures (e.g. initiation of targeted therapy) [21]. In order for assays to remain relevant as tumour clones evolve, a personalised approach would ideally require multi-site biopsies repeated sequentially. Furthermore, due to the extent of intra-tumour heterogeneity as demonstrated by several studies [22–24], a single site biopsy risks missing clinically important mutations from a heterogeneous tumour. For these reasons, a personalised ctDNA detection approach based on the detection of mutations found in tumour samples is challenging. There is therefore a need for non-invasive detection of mutations without prior knowledge of their existence directly from the blood, we term this de novo mutation detection. In 2012 Forshew et al. demonstrated that mutations can noninvasively be detected de novo through targeted plasma re-sequencing [15]. They proposed a targeted sequencing approach termed TAm-Seq, which allowed the re-sequencing of approximately 6000 nucleotides whilst maintaining high depth analysis. In a proof-of-concept example, the authors re-sequenced tumour tissue from a right oophorectomy specimen, taken from a patient with ovarian cancer at the time of debulking surgery. The authors identified a TP53 mutation and tracked its ctDNA levels using TAm-Seq. As the cancer progressed, TAm-Seq analysis revealed the emergence of an EGFR mutation in plasma samples. This mutation was not found in the original oophorectomy specimen. However, on further investigation an identical EGFR mutation was present at low frequencies in samples taken from the omentum at the time of the initial debulking surgery. Forshew et al. hypothesised that as chemotherapy regimes restrained the growth of other clones, the resistant EGFR clone, which was initially present only at low frequency, gained in dominance. They demonstrate that plasma analysis can identify heterogeneous clones from different sites of the body. Though TAm-Seq was able to detect de novo mutations directly from the plasma, Forshew et al. only targeted b 6 Kb of the genome [15]. To detect unselected genetic events that span across the genome, Leary et al. used massively paralleled sequencing (MPS) of the whole genome in 7 patients with colorectal cancer and 3 patients with breast cancer [25]. They developed a method termed personalised analysis of rearranged ends (PARE) [16] and detected at least one chromosomal rearrangement in the plasma of every cancer patient but not in the plasma of healthy volunteers. Similarly, in 2013 Chan et al. used “Shotgun” MPS of the whole genome to identify copy number variations and single nucleotide variants (SN) from the plasma of 4 patients with hepatocellular carcinoma [26]. Furthermore, they demonstrated the ability of MPS to track ctDNA level changes pre- and post-surgery. Interestingly, shotgun MPS of the plasma was also able to distinguish between tumour types in a patient with synchronous breast and ovarian tumours [26]. The above studies illustrate that ctDNA analysis, through de novo mutation
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Understanding resistance mechanisms
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Due to needle biopsy sampling bias and the limited availability of “research biopsies” in advanced cancer patients, the investigation of tumour resistance mechanisms have been challenging. However, its investigation can be of great clinical value as treatment regimens can be tailored to target resistant mechanisms or, treatment intervals introduced as resistant clones are detected and gain dominance. The hypothesis is that plasma is able to capture DNA from all tumour clones. If that holds true, its analysis may demonstrate the evolutionary process tumours undergo to evade targeted therapies and possibly traditional chemotherapies. In 2012, Misale et al. observed the emergence of resistance conferring KRAS mutations in plasma [29]. They analysed ctDNA from the plasma of 10 colorectal cancer patients taking cetuximab before and after progression. Using BEAMing (bead, emulsion, amplification and magnetics), they found KRAS mutations or amplifications in the plasma of 60% of the patients who progressed. Importantly the emergence of resistant clones occurred up to 10 months prior to radiographic confirmation of disease progression. Mutations in the plasma were concordant with KRAS mutations found in subsequent metastatic tumour biopsies. In a parallel study, Diaz et al. described the ctDNA analysis of 28 patients with chemorefractory metastatic colorectal cancer receiving panitumumab. 38% of patients whose initial tumours had wild-type KRAS developed mutant KRAS over the next 6 months. Through ctDNA levels and tumour growth analysis Diaz et al. calculated that KRAS mutants were likely to be present at low quantities prior to initiation of panitumumab [20] and lend weight to the omental EGFR mutant findings of Forshew et al. [15]. In another example, Oxnard et al. used droplet dPCR [30] to analyse plasma ctDNA in 9 patients with NSCLC who were treated with erlotinib therapy [31]. Plasma levels of EGFR mutation T790M were undetectable prior to erlotinib therapy in this group. Initiation of erlotinib led to a
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detection, can continue to track disease burden as tumours evolve, without the need for re-biopsy. However, in the studies outlined above the expense of whole genome MPS would limit analysis to a small number of samples. For this reason, whole-genome sequencing (WGS) approaches for routinely detecting ctDNA in patient populations are currently, prohibitively expensive. Although low depth, and therefore reduced cost, WGS approaches have been successful at detecting copy number variations [26,27], a higher depth of coverage is often required to detect rearrangements at high resolution or SNVs directly from plasma DNA. Furthermore, where low mutant:wild type allele frequencies exist, e.g. in early stage disease, an even higher depth of coverage would be necessary to detect ctDNA fragments. In addition, WGS approaches detect a higher ratio of intronic or passenger mutations than targeted re-sequencing [26]. The clinical significance of passenger mutations is currently unknown and often not targetable. Another approach demonstrated by Murtaza et al. is to perform whole exome sequencing (WES) of the plasma to track tumour evolution in response to therapy [28]. This proof-of-concept study involved 6 patients with metastatic tumours with plasma collected at the beginning of treatment and at the time of relapse. Subsequent re-sequencing and variant analysis revealed that by comparing the relative representation of mutations in pre- and post-relapse samples, one could identify enrichment of mutations that may drive resistance. This work demonstrated that a WES approach would allow the detection of clinically relevant mutations at a lower cost than WGS [28]. WGS can screen a larger spectrum of the genome but is currently too expensive for routine use to detect SNVs, whereas WES approaches allow more in-depth interrogation of multiple regions but is less sensitive to identifying copy number changes. As the cost of sequencing continues to fall, the price of WGS of plasma is likely to become more palatable. At such a time, WGS may become routine for the analysis of de novo mutations in serial plasma samples.
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burden in colorectal cancer [12]. In 2013, Dawson et al. investigated women with metastatic breast cancer using a similar personalised approach. Tumour tissue mutations were identified in 30 women using next generation re-sequencing and subsequently, digital PCR (dPCR) [13,14] and tagged-amplicon deep sequencing (TAm-Seq) [15] were used to detect ctDNA in 29 out of the 30 women. Computed tomograms were compared with levels of ctDNA, cancer antigen 15-3 (CA15-3) and circulating tumour cells (CTCs) taken sequentially. Overall, ctDNA was able to detect changes in tumour burden earlier and with greater sensitivity than the standard biomarker (CA15-3) or CTCs [11]. These results suggest that ctDNA has the potential to be used as a measure of tumour response in a non-invasive way.
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Much ctDNA research to-date focuses on plasma and serum derived samples. However, frequent blood sampling in patients who are already prone to anaemia of chronic disease is not ideal. Interestingly, tumourspecific nucleic acids have been detected in other bodily fluids, including stool [13], urine [33], saliva [34], cerebrospinal fluid [35] and pleural fluid [36]. Particular fluids may concentrate mutant cfDNA from regional drainage and may facilitate low volume disease mutant cfDNA detection. For example, mutated DNA fragments from localised oral cancers are hypothesised to be more concentrated in saliva than in the circulation. Additionally, many bodily fluids are freely available and found in abundance, particularly urine. This may allow more frequent and more voluminous sampling, thus aiding low volume disease detection. The presence of genetic material in the urine has long been established, though initially from urinary cells [37]. In 1999, Zhang et al. demonstrated the presence of SRY gene (found on the Y chromosome) in the urine supernatant of female recipients of male donor renal transplants [38]. By 2000, Botezatu et al. demonstrated that some genomic DNA escaped into the urine through renal filtration [39]. They demonstrated this by subcutaneously injecting mice with radiolabelled human DNA and collecting urine over the next 3 days. Collected urine was determined to contain human cfDNA through its radioactivity and PCR amplification of human specific Alu repeat sequences Subsequently, by performing nested PCR to detect highly repetitive regions located on the Y chromosome, Botezatu et al. demonstrated the presence of male DNA in the urine of pregnant women carrying male foetuses [39]. In 2004, Su et al. demonstrated the presence of mutant cfDNA in the urine of colorectal carcinoma patients [40]. Using restriction enriched PCR with primer pairs targeting a known mutation in KRAS codon 12, they were able to selectively amplify mutant cfDNA in the fractionated urine of colorectal cancer patients. Interestingly, they also performed size analysis on urinary DNA using polyacrylamide gels and found two main bands of cfDNA. The first was a band approximately 150–250 nucleotides long, which they hypothesised was derived from the circulation through renal filtration, the second and much larger band represented DNA N 1 kilobase long, which they hypothesised originated in the lining of the urinary tract [40]. In a subsequent study by Tsui et al., next generation sequencing approaches were used to assess the size of DNA present in the urine of pregnant women. They found that maternal urinary cfDNA had a predominant peak at 29 bp and, that the majority of maternal cfDNA was below 100 bp in length [41]. In addition, next generation sequencing approaches have been used to detect mutant cfDNA derived from the urine of cancer patients. In
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2012, Millholland et al. targeted the known hotspots in FGFR3 exons 7, 10 and 15 to detect FGFR3 mutations in urinary DNA [33]. They reported a concordance of 91% by detecting mutant urinary cfDNA in 10 out of 11 patients with FGFR3 mutations in their primary tumours. In a separate group, the investigators assessed the urine of 43 known bladder cancer patients and found mutated FGFR3 in 24 of 43 cases [33]. As the mutation status of the primary tumour was not reported, no comment can be made on the concordance of their urinary assay with tumour mutations, though it appears proportional to the prevalence of FGFR3 mutations in bladder cancer populations detected in other studies [42,43]. Similar findings were also presented in previous studies relying on molecular assays to analyse FGFR3 mutations [44,45]. The ability of these assays to detect bladder cancer mutations as a whole was, however, low due to the sole focus on FGFR3, despite some studies selecting patients who had FGFR3 aberrations in their initial tumour sample. The findings of Botezatu and Su demonstrate that cfDNA filtered through the kidney is available for mutational analysis in urine, a bodily fluid that is readily available and abundant. However, quantification of peripheral fluid mutant cfDNA may prove difficult to interpret, with additional factors such as the hydration state, renal disease and presence of bladder outflow obstruction in the case of urine, likely to affect mutant cfDNA levels. Further studies would need to take into consideration these factors to systematically evaluate the potential of urine for mutant cfDNA analysis.
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drop in ctDNA levels of the sensitising EGFR mutations in all patients. However, in 6 of the 9 patients, T790M EGFR mutant levels began to increase, 4–24 weeks prior to progression being confirmed by RECIST criteria [31]. As part of the largest ctDNA study reported to date, Bettegowda et al. analysed plasma samples pre- and post-EGFR blockade to detect a broad range of resistance mechanisms in a subgroup of 24 colorectal cancer patients [32]. They showed that the ctDNA of 23 patients had at least one new mutation in mitogen-activated protein kinase pathway genes and that 50% of these new mutations occurred in KRAS codon 12. Currently, only a small number of cases in only a few cancer types have been investigated using methods with high analytical sensitivity (e.g. dPCR, Safe-Seq, and TAm-Seq) and further large-scale studies are required. However, the above studies demonstrate that ctDNA can be utilised to detect tumour resistance mechanisms. Furthermore, the work by Murtaza et al. indicate that less biased, WES approaches for ctDNA analysis could be employed to detect unexpected resistance mechanisms. When coupled with the growing number of targeted therapeutic options, the potential of using ctDNA to guide sequential treatment is promising.
As the sensitivity of ctDNA analysis improves, the detection of ctDNA at lower levels will become feasible. Many cancer patients still present too late for curative therapies and our best chance of improving cancer mortality rates lies in the early detection of cancer [46]. However at present, published studies have largely focused on ctDNA analysis in the advanced cancer setting, where levels of mutant:wild type allele fractions are around 10–50% [10,15,28]. For earlier stages, the mutant:wild type allele fractions are likely to be lower. Despite this, ctDNA has been detected in the plasma of multiple cancers types at an early stage. In Diehl et al.'s seminal work, mutant:wild type allele frequencies ranging from 0.001% to 0.12% were detected in patients with localised colorectal cancer. Of note, ctDNA was also detected from plasma samples in their control group of patients with colorectal adenomas, where mutant:wild type allele frequencies of 0.001–0.02% were reported [10]. The ability to detect ctDNA in patients with adenomatous colorectal polyps has important implications. Colorectal adenomas are precursors to adenocarcinoma and as such, often require further treatment. That ctDNA was detected at this pre-cancerous stage suggests a potential role of ctDNA in early diagnosis or even for population screening. In more recent work, Bettegowda et al. reported a range of 0 (notdetected) to 135,000 mutant fragments/5 mL of blood in 223 patients with 12 tumour types, all with localised disease [32]. By detecting mutations in tumour tissue and subsequently searching for these in matched plasma samples, they detected ctDNA in 55% (122/223) of patients [32]. Furthermore, by using a droplet dPCR assay, Beaver et al. were able to detect PIK3CA mutations in the pre-operative plasma samples of 14/ 15 patients who had low volume breast cancer and PIK3CA mutations in their tumour samples [47]. For 10 patients, blood samples were collected post-operatively and 5 had persistently detectable ctDNA. Indeed, one of these patients went on to have cancer recurrence. If applicable to other cancers, low volume ctDNA analysis could monitor the emergence of early recurrence following surgical resection or other radical therapies and help distinguish between patients who require further adjuvant therapy and patients who are essentially cured. Presently, ctDNA has only been detected in early disease in a small number of cancers [18,32,47]. Early evidence suggests that detecting levels in early disease in certain cancers e.g. glioma, will be extremely challenging [32]. Additionally, it is uncertain whether alerting
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Acknowledgements
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The authors wish to thank the Addenbrooke's Charitable Trust, the Royal College of Surgeons and the Cambridge Cancer Centre for their support. We wish also to thank Dr. Nitzan Rosenfeld, Dr. Tim Forshew and Dr. Florent Mouliere for comments and suggestions.
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The analysis of ctDNA presents many opportunities to improve cancer management. Early detection of ctDNA could alert clinicians to the presence of sinister disease and prompt curative treatment. Indeed, in several cancer types there are known mutations in precancerous states (e.g. APC in colorectal adenomas [51] and BRAF in benign naevi of the skin [52]). However, there is a risk that early detection of some mutations may lead to over-investigation (particularly to localise the source of the mutation) and overtreatment of patients, who may have never progressed to invasive cancer. At present, the focus of ctDNA research remains on advanced cancers where ctDNA fractions in plasma are higher. The ability of ctDNA to track de novo mutations will deliver more precise cancer monitoring and uncover resistant mechanisms. When metastatic tumour biopsy is not available, ctDNA would provide valuable information for therapy stratification. The limitations of tumour re-biopsy were highlighted in a recent study by Andre et al. who from 2011 to 2012, conducted a large-scale multi-centred trial (SAFIR01/UNICANCER) to investigate the feasibility of using genomic information to stratify patients to targeted therapies. Biopsy samples from metastases were sent to 5 centres for analysis [53]. The primary endpoint was to stratify 30% of patients to targeted therapies (including trial drugs) using array CGH and Sanger sequencing. This was not achieved, with only 13% (55 out of 423) of enrolled patients being suitable to receive targeted therapy. Several important limitations for this were highlighted: Firstly, array CGH analysis required a large amount of DNA and low yield from biopsies prevented analysis in 91 cases. Secondly, of 195 patients with targetable results only 55 could receive targeted therapies. Several of the challenges encountered by the SAFIR01 team might be addressed by integrating ctDNA analysis into the trial: firstly, plasma can be collected more regularly and easily. Secondly, methods for ctDNA analysis require less input DNA than traditional Sanger sequencing or array CGH. Thirdly, the ability of ctDNA to display clonal heterogeneity and track clonal progression, could potentially overcome spatial and temporal heterogeneity issues encountered when relying on tumour biopsies. Lastly, blood sampling is safer, and has fewer complications when compared to tumour biopsy and the use of sequential sampling could track response to treatment more closely or may have alerted clinicians to the development of resistant mechanisms. However, several aspects of ctDNA
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analysis require addressing prior to its adoption in the clinic. Plasma processing and data analysis require standardisation to allow largescale, multi-institutional clinical trials. Such trials would need to clearly demonstrate patient benefit before ctDNA analysis can be used to guide treatment decisions. Despite the above, Rothé et al. performed hotspot mutation analysis on 17 breast cancer patients to ascertain whether ctDNA can be used to guide therapeutic decisions [54]. Metastatic tumour and plasma samples were taken at the same time-point and Rothé et al. reported a 76% concordance (13/17 patients) between these samples. Two patients had mutations detected in plasma at relatively high frequencies (12% and 14.3%) but not the tumour samples. The remaining two patients had mutations detected in the tumour but not the plasma. Further analysis of these plasma samples by targeted illumina sequencing revealed mutations identical to that found in the tumour, although present at low frequency. Interestingly, for 9 out of 69 tumour samples, the investigators were unable to perform NGS analysis where as they were able to for all 31 plasma samples received. The investigators conclude that plasma ctDNA analysis is a suitable alternative to metastatic biopsy for molecular screening. Furthermore, in 2014, Douillard et al. published the results of a comparison between ctDNA analysis and tumour biopsy DNA analysis of EGFR mutation status in 1060 advanced lung cancer patients. They found the concordance of EGFR mutations between matched tumour and plasma to be 94.3%, with the test yielding a sensitivity of 65.7% and specificity of 99.8% [55]. Furthermore, patients who have activating EGFR mutations detected in tumour or plasma samples demonstrated similar progression free survival (9.7 vs 10.2 months) after gefitinib treatment [55], supporting the use of ctDNA analysis to initiate targeted therapies. In addition, cell-free tumour DNA is present in alternative bodily fluids, some of which are of much higher abundance than tumour biopsy or plasma, such as urine. These bodily fluids may be more abundant than plasma and maybe more easily collected. Analysis of these fluids may facilitate the detection of rare mutated DNA fragments. The choice of specific fluids to analyse would differ across cancer types, depending on the proximity of the fluid to the tumour sites and clinical context. In summary, there is increasing evidence that ctDNA analysis may have an important role at several stages of disease. Due to their short half-life, ctDNA profiles can present a snapshot of current clonal burden and rapidly assess tumour response to therapies. Indeed, many of the protein based tumour markers currently used to asses treatment response in the clinic (e.g. PSA, CA125, CEA, αFP) have a half-life measured in days [56], whereas the half-life of cfDNA is measured in minutes to hours [12,57]. We envision that, the ability of ctDNA to detect de novo mutations could overcome the problem of mutational profiling in the presence of continual tumour evolution, thus improving our understanding resistance mechanisms and allow us to monitor tumour response in real-time. Furthermore, improvements in the detection of ctDNA present at low volumes in plasma and other bodily fluids, may lead to an earlier diagnosis, where intervention can still lead to cure or improve our ability to stratify patients to adjuvant therapy following radical treatments. What is certain is that as the cost of massively paralleled sequencing reduces, ctDNA analysis will become more readily available and has the potential to improve cancer care.
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physicians to the early presence of cancer through ctDNA analysis will translate to improved outcomes. Indeed, the use of prostate specific antigen (PSA) to detect early prostate cancer has resulted in overtreatment, with disease specific mortality improved in only high-risk groups [48]. Furthermore in the future, ctDNA detection may be “too sensitive” and detect mutations from cancers before they can be localised. Indeed by using restriction enriched PCR Gormally et al. were able to detect KRAS mutations in 13 out of 1098 “healthy volunteers”. However, in this instance, 5 “healthy volunteers” were subsequently diagnosed with bladder cancer and a further volunteer diagnosed with upper aero-digestive tract cancer within 25 months [49], conceivably these tumours may have been present at microscopic level at the time of investigation and had superior localisation techniques been available, may have been detected and treated early. Furthermore, Chan et al. were able to detect Epstein Barr viral DNA in plasma, a known risk factor for nasopharyngeal carcinoma, when screening 1318 asymptomatic volunteers [50]. They detected viral DNA in 69 volunteers and on further investigation found nasopharyngeal cancer in 3 previously asymptomatic individuals, demonstrating the utility of plasma-based cancer screening [50]. It is however, likely that robust ctDNA detection will only be possible in certain cancers and, that early detection may be possible in only a subset of these. Although, where ctDNA is detectable at an early stage, it has the potential to reduce mortality through more patients being amenable to curative surgery.
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[1] Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients 448 and the effect of therapy. Cancer Res 1977;37:646–50. 449
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Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
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The translational potential of circulating tumour DNA in oncology
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K.M. Patel a,b, D.W.Y. Tsui a,⁎
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Article history: Received 1 December 2014 Received in revised form 2 April 2015 Accepted 3 April 2015 Available online xxxx
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Keywords: Circulating nucleic acids Cancer DNA Oncology Urine Plasma Blood Next generation sequencing
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Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK Department of Academic Urology, University of Cambridge Hospitals, Box 243, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0QQ , UK
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Introduction . . . . . . . . . . . . De novo mutation detection in blood . Understanding resistance mechanisms Other bodily fluids . . . . . . . . . Low volume disease . . . . . . . . . Discussion . . . . . . . . . . . . . Acknowledgements . . . . . . . . . References . . . . . . . . . . . . .
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Introduction
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In 1977, Leon et al. discovered that serum circulating cell-free DNA (cfDNA) levels were higher in patients with cancer [1]. This initial work, focusing on total levels of cfDNA, eventually encouraged the investigation of circulating nucleic acids as a biomarker of cancer. However, total levels of cfDNA were found to be insensitive [2–4] and nonspecific [2,5,6], in part, due to great variability between individuals and, that cfDNA levels are raised in a number of conditions including pregnancy [7] and myocardial infarction [8].
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The recent understanding of tumour heterogeneity and cancer evolution in response to therapy has raised questions about the value of historical or single site biopsies for guiding treatment decisions. The ability of ctDNA analysis to reveal de novo mutations (i.e., without prior knowledge), allows monitoring of clonal heterogeneity without the need for multiple tumour biopsies. Additionally, ctDNA monitoring of such heterogeneity and novel mutation detection will allow clinicians to detect resistant mechanisms early and tailor treatment therapies accordingly. If ctDNA can be used to detect low volume cancerous states, it will have important applications in treatment stratification post-surgery/radical radiotherapy and may have a role in patient screening. Mutant cfDNA can also be detected in other bodily fluids that are easily accessible and may aid detection of rare mutant alleles in certain cancer types. This article outlines recent advances in these areas. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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⁎ Corresponding author. E-mail address: [email protected] (D.W.Y. Tsui).
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Tumour DNA contains specific somatic alterations. DNA fragments which contain these tumour-specific somatic mutations can be detected in the blood, and hence are called circulating tumour DNA (ctDNA) [9]. It is challenging to identify ctDNA fragments, because they are surrounded by multiple copies of normal genomic DNA. One strategy is to use a personalised approached, where one could first identify mutations in tumour and subsequently design mutation specific assays to detect ctDNA in plasma [10,11]. A number of studies have demonstrated that the presence of ctDNA and, the dynamics of ctDNA in plasma, reflect tumour burden. For example, in 2008, Diehl et al. evaluated 162 plasma samples from 18 colorectal cancer patients, to demonstrate that levels of ctDNA increased with tumour burden, and that ctDNA kinetics were more sensitive than serum carcino-embroyonic antigen (CEA) for monitoring disease
http://dx.doi.org/10.1016/j.clinbiochem.2015.04.005 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Patel KM, Tsui DWY, The translational potential of circulating tumour DNA in oncology, Clin Biochem (2015), http:// dx.doi.org/10.1016/j.clinbiochem.2015.04.005
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De novo mutation detection in blood
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Substantial work in the field has demonstrated that ctDNA can be detected by tracking tumour-specific point mutations or structural rearrangements, in many cancer types [14,16–20]. However, it is now apparent that the mutational characteristics of tumours are not static and continually evolve in response to various selection pressures (e.g. initiation of targeted therapy) [21]. In order for assays to remain relevant as tumour clones evolve, a personalised approach would ideally require multi-site biopsies repeated sequentially. Furthermore, due to the extent of intra-tumour heterogeneity as demonstrated by several studies [22–24], a single site biopsy risks missing clinically important mutations from a heterogeneous tumour. For these reasons, a personalised ctDNA detection approach based on the detection of mutations found in tumour samples is challenging. There is therefore a need for non-invasive detection of mutations without prior knowledge of their existence directly from the blood, we term this de novo mutation detection. In 2012 Forshew et al. demonstrated that mutations can noninvasively be detected de novo through targeted plasma re-sequencing [15]. They proposed a targeted sequencing approach termed TAm-Seq, which allowed the re-sequencing of approximately 6000 nucleotides whilst maintaining high depth analysis. In a proof-of-concept example, the authors re-sequenced tumour tissue from a right oophorectomy specimen, taken from a patient with ovarian cancer at the time of debulking surgery. The authors identified a TP53 mutation and tracked its ctDNA levels using TAm-Seq. As the cancer progressed, TAm-Seq analysis revealed the emergence of an EGFR mutation in plasma samples. This mutation was not found in the original oophorectomy specimen. However, on further investigation an identical EGFR mutation was present at low frequencies in samples taken from the omentum at the time of the initial debulking surgery. Forshew et al. hypothesised that as chemotherapy regimes restrained the growth of other clones, the resistant EGFR clone, which was initially present only at low frequency, gained in dominance. They demonstrate that plasma analysis can identify heterogeneous clones from different sites of the body. Though TAm-Seq was able to detect de novo mutations directly from the plasma, Forshew et al. only targeted b 6 Kb of the genome [15]. To detect unselected genetic events that span across the genome, Leary et al. used massively paralleled sequencing (MPS) of the whole genome in 7 patients with colorectal cancer and 3 patients with breast cancer [25]. They developed a method termed personalised analysis of rearranged ends (PARE) [16] and detected at least one chromosomal rearrangement in the plasma of every cancer patient but not in the plasma of healthy volunteers. Similarly, in 2013 Chan et al. used “Shotgun” MPS of the whole genome to identify copy number variations and single nucleotide variants (SN) from the plasma of 4 patients with hepatocellular carcinoma [26]. Furthermore, they demonstrated the ability of MPS to track ctDNA level changes pre- and post-surgery. Interestingly, shotgun MPS of the plasma was also able to distinguish between tumour types in a patient with synchronous breast and ovarian tumours [26]. The above studies illustrate that ctDNA analysis, through de novo mutation
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Understanding resistance mechanisms
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Due to needle biopsy sampling bias and the limited availability of “research biopsies” in advanced cancer patients, the investigation of tumour resistance mechanisms have been challenging. However, its investigation can be of great clinical value as treatment regimens can be tailored to target resistant mechanisms or, treatment intervals introduced as resistant clones are detected and gain dominance. The hypothesis is that plasma is able to capture DNA from all tumour clones. If that holds true, its analysis may demonstrate the evolutionary process tumours undergo to evade targeted therapies and possibly traditional chemotherapies. In 2012, Misale et al. observed the emergence of resistance conferring KRAS mutations in plasma [29]. They analysed ctDNA from the plasma of 10 colorectal cancer patients taking cetuximab before and after progression. Using BEAMing (bead, emulsion, amplification and magnetics), they found KRAS mutations or amplifications in the plasma of 60% of the patients who progressed. Importantly the emergence of resistant clones occurred up to 10 months prior to radiographic confirmation of disease progression. Mutations in the plasma were concordant with KRAS mutations found in subsequent metastatic tumour biopsies. In a parallel study, Diaz et al. described the ctDNA analysis of 28 patients with chemorefractory metastatic colorectal cancer receiving panitumumab. 38% of patients whose initial tumours had wild-type KRAS developed mutant KRAS over the next 6 months. Through ctDNA levels and tumour growth analysis Diaz et al. calculated that KRAS mutants were likely to be present at low quantities prior to initiation of panitumumab [20] and lend weight to the omental EGFR mutant findings of Forshew et al. [15]. In another example, Oxnard et al. used droplet dPCR [30] to analyse plasma ctDNA in 9 patients with NSCLC who were treated with erlotinib therapy [31]. Plasma levels of EGFR mutation T790M were undetectable prior to erlotinib therapy in this group. Initiation of erlotinib led to a
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detection, can continue to track disease burden as tumours evolve, without the need for re-biopsy. However, in the studies outlined above the expense of whole genome MPS would limit analysis to a small number of samples. For this reason, whole-genome sequencing (WGS) approaches for routinely detecting ctDNA in patient populations are currently, prohibitively expensive. Although low depth, and therefore reduced cost, WGS approaches have been successful at detecting copy number variations [26,27], a higher depth of coverage is often required to detect rearrangements at high resolution or SNVs directly from plasma DNA. Furthermore, where low mutant:wild type allele frequencies exist, e.g. in early stage disease, an even higher depth of coverage would be necessary to detect ctDNA fragments. In addition, WGS approaches detect a higher ratio of intronic or passenger mutations than targeted re-sequencing [26]. The clinical significance of passenger mutations is currently unknown and often not targetable. Another approach demonstrated by Murtaza et al. is to perform whole exome sequencing (WES) of the plasma to track tumour evolution in response to therapy [28]. This proof-of-concept study involved 6 patients with metastatic tumours with plasma collected at the beginning of treatment and at the time of relapse. Subsequent re-sequencing and variant analysis revealed that by comparing the relative representation of mutations in pre- and post-relapse samples, one could identify enrichment of mutations that may drive resistance. This work demonstrated that a WES approach would allow the detection of clinically relevant mutations at a lower cost than WGS [28]. WGS can screen a larger spectrum of the genome but is currently too expensive for routine use to detect SNVs, whereas WES approaches allow more in-depth interrogation of multiple regions but is less sensitive to identifying copy number changes. As the cost of sequencing continues to fall, the price of WGS of plasma is likely to become more palatable. At such a time, WGS may become routine for the analysis of de novo mutations in serial plasma samples.
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burden in colorectal cancer [12]. In 2013, Dawson et al. investigated women with metastatic breast cancer using a similar personalised approach. Tumour tissue mutations were identified in 30 women using next generation re-sequencing and subsequently, digital PCR (dPCR) [13,14] and tagged-amplicon deep sequencing (TAm-Seq) [15] were used to detect ctDNA in 29 out of the 30 women. Computed tomograms were compared with levels of ctDNA, cancer antigen 15-3 (CA15-3) and circulating tumour cells (CTCs) taken sequentially. Overall, ctDNA was able to detect changes in tumour burden earlier and with greater sensitivity than the standard biomarker (CA15-3) or CTCs [11]. These results suggest that ctDNA has the potential to be used as a measure of tumour response in a non-invasive way.
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Much ctDNA research to-date focuses on plasma and serum derived samples. However, frequent blood sampling in patients who are already prone to anaemia of chronic disease is not ideal. Interestingly, tumourspecific nucleic acids have been detected in other bodily fluids, including stool [13], urine [33], saliva [34], cerebrospinal fluid [35] and pleural fluid [36]. Particular fluids may concentrate mutant cfDNA from regional drainage and may facilitate low volume disease mutant cfDNA detection. For example, mutated DNA fragments from localised oral cancers are hypothesised to be more concentrated in saliva than in the circulation. Additionally, many bodily fluids are freely available and found in abundance, particularly urine. This may allow more frequent and more voluminous sampling, thus aiding low volume disease detection. The presence of genetic material in the urine has long been established, though initially from urinary cells [37]. In 1999, Zhang et al. demonstrated the presence of SRY gene (found on the Y chromosome) in the urine supernatant of female recipients of male donor renal transplants [38]. By 2000, Botezatu et al. demonstrated that some genomic DNA escaped into the urine through renal filtration [39]. They demonstrated this by subcutaneously injecting mice with radiolabelled human DNA and collecting urine over the next 3 days. Collected urine was determined to contain human cfDNA through its radioactivity and PCR amplification of human specific Alu repeat sequences Subsequently, by performing nested PCR to detect highly repetitive regions located on the Y chromosome, Botezatu et al. demonstrated the presence of male DNA in the urine of pregnant women carrying male foetuses [39]. In 2004, Su et al. demonstrated the presence of mutant cfDNA in the urine of colorectal carcinoma patients [40]. Using restriction enriched PCR with primer pairs targeting a known mutation in KRAS codon 12, they were able to selectively amplify mutant cfDNA in the fractionated urine of colorectal cancer patients. Interestingly, they also performed size analysis on urinary DNA using polyacrylamide gels and found two main bands of cfDNA. The first was a band approximately 150–250 nucleotides long, which they hypothesised was derived from the circulation through renal filtration, the second and much larger band represented DNA N 1 kilobase long, which they hypothesised originated in the lining of the urinary tract [40]. In a subsequent study by Tsui et al., next generation sequencing approaches were used to assess the size of DNA present in the urine of pregnant women. They found that maternal urinary cfDNA had a predominant peak at 29 bp and, that the majority of maternal cfDNA was below 100 bp in length [41]. In addition, next generation sequencing approaches have been used to detect mutant cfDNA derived from the urine of cancer patients. In
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2012, Millholland et al. targeted the known hotspots in FGFR3 exons 7, 10 and 15 to detect FGFR3 mutations in urinary DNA [33]. They reported a concordance of 91% by detecting mutant urinary cfDNA in 10 out of 11 patients with FGFR3 mutations in their primary tumours. In a separate group, the investigators assessed the urine of 43 known bladder cancer patients and found mutated FGFR3 in 24 of 43 cases [33]. As the mutation status of the primary tumour was not reported, no comment can be made on the concordance of their urinary assay with tumour mutations, though it appears proportional to the prevalence of FGFR3 mutations in bladder cancer populations detected in other studies [42,43]. Similar findings were also presented in previous studies relying on molecular assays to analyse FGFR3 mutations [44,45]. The ability of these assays to detect bladder cancer mutations as a whole was, however, low due to the sole focus on FGFR3, despite some studies selecting patients who had FGFR3 aberrations in their initial tumour sample. The findings of Botezatu and Su demonstrate that cfDNA filtered through the kidney is available for mutational analysis in urine, a bodily fluid that is readily available and abundant. However, quantification of peripheral fluid mutant cfDNA may prove difficult to interpret, with additional factors such as the hydration state, renal disease and presence of bladder outflow obstruction in the case of urine, likely to affect mutant cfDNA levels. Further studies would need to take into consideration these factors to systematically evaluate the potential of urine for mutant cfDNA analysis.
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drop in ctDNA levels of the sensitising EGFR mutations in all patients. However, in 6 of the 9 patients, T790M EGFR mutant levels began to increase, 4–24 weeks prior to progression being confirmed by RECIST criteria [31]. As part of the largest ctDNA study reported to date, Bettegowda et al. analysed plasma samples pre- and post-EGFR blockade to detect a broad range of resistance mechanisms in a subgroup of 24 colorectal cancer patients [32]. They showed that the ctDNA of 23 patients had at least one new mutation in mitogen-activated protein kinase pathway genes and that 50% of these new mutations occurred in KRAS codon 12. Currently, only a small number of cases in only a few cancer types have been investigated using methods with high analytical sensitivity (e.g. dPCR, Safe-Seq, and TAm-Seq) and further large-scale studies are required. However, the above studies demonstrate that ctDNA can be utilised to detect tumour resistance mechanisms. Furthermore, the work by Murtaza et al. indicate that less biased, WES approaches for ctDNA analysis could be employed to detect unexpected resistance mechanisms. When coupled with the growing number of targeted therapeutic options, the potential of using ctDNA to guide sequential treatment is promising.
As the sensitivity of ctDNA analysis improves, the detection of ctDNA at lower levels will become feasible. Many cancer patients still present too late for curative therapies and our best chance of improving cancer mortality rates lies in the early detection of cancer [46]. However at present, published studies have largely focused on ctDNA analysis in the advanced cancer setting, where levels of mutant:wild type allele fractions are around 10–50% [10,15,28]. For earlier stages, the mutant:wild type allele fractions are likely to be lower. Despite this, ctDNA has been detected in the plasma of multiple cancers types at an early stage. In Diehl et al.'s seminal work, mutant:wild type allele frequencies ranging from 0.001% to 0.12% were detected in patients with localised colorectal cancer. Of note, ctDNA was also detected from plasma samples in their control group of patients with colorectal adenomas, where mutant:wild type allele frequencies of 0.001–0.02% were reported [10]. The ability to detect ctDNA in patients with adenomatous colorectal polyps has important implications. Colorectal adenomas are precursors to adenocarcinoma and as such, often require further treatment. That ctDNA was detected at this pre-cancerous stage suggests a potential role of ctDNA in early diagnosis or even for population screening. In more recent work, Bettegowda et al. reported a range of 0 (notdetected) to 135,000 mutant fragments/5 mL of blood in 223 patients with 12 tumour types, all with localised disease [32]. By detecting mutations in tumour tissue and subsequently searching for these in matched plasma samples, they detected ctDNA in 55% (122/223) of patients [32]. Furthermore, by using a droplet dPCR assay, Beaver et al. were able to detect PIK3CA mutations in the pre-operative plasma samples of 14/ 15 patients who had low volume breast cancer and PIK3CA mutations in their tumour samples [47]. For 10 patients, blood samples were collected post-operatively and 5 had persistently detectable ctDNA. Indeed, one of these patients went on to have cancer recurrence. If applicable to other cancers, low volume ctDNA analysis could monitor the emergence of early recurrence following surgical resection or other radical therapies and help distinguish between patients who require further adjuvant therapy and patients who are essentially cured. Presently, ctDNA has only been detected in early disease in a small number of cancers [18,32,47]. Early evidence suggests that detecting levels in early disease in certain cancers e.g. glioma, will be extremely challenging [32]. Additionally, it is uncertain whether alerting
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The authors wish to thank the Addenbrooke's Charitable Trust, the Royal College of Surgeons and the Cambridge Cancer Centre for their support. We wish also to thank Dr. Nitzan Rosenfeld, Dr. Tim Forshew and Dr. Florent Mouliere for comments and suggestions.
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The analysis of ctDNA presents many opportunities to improve cancer management. Early detection of ctDNA could alert clinicians to the presence of sinister disease and prompt curative treatment. Indeed, in several cancer types there are known mutations in precancerous states (e.g. APC in colorectal adenomas [51] and BRAF in benign naevi of the skin [52]). However, there is a risk that early detection of some mutations may lead to over-investigation (particularly to localise the source of the mutation) and overtreatment of patients, who may have never progressed to invasive cancer. At present, the focus of ctDNA research remains on advanced cancers where ctDNA fractions in plasma are higher. The ability of ctDNA to track de novo mutations will deliver more precise cancer monitoring and uncover resistant mechanisms. When metastatic tumour biopsy is not available, ctDNA would provide valuable information for therapy stratification. The limitations of tumour re-biopsy were highlighted in a recent study by Andre et al. who from 2011 to 2012, conducted a large-scale multi-centred trial (SAFIR01/UNICANCER) to investigate the feasibility of using genomic information to stratify patients to targeted therapies. Biopsy samples from metastases were sent to 5 centres for analysis [53]. The primary endpoint was to stratify 30% of patients to targeted therapies (including trial drugs) using array CGH and Sanger sequencing. This was not achieved, with only 13% (55 out of 423) of enrolled patients being suitable to receive targeted therapy. Several important limitations for this were highlighted: Firstly, array CGH analysis required a large amount of DNA and low yield from biopsies prevented analysis in 91 cases. Secondly, of 195 patients with targetable results only 55 could receive targeted therapies. Several of the challenges encountered by the SAFIR01 team might be addressed by integrating ctDNA analysis into the trial: firstly, plasma can be collected more regularly and easily. Secondly, methods for ctDNA analysis require less input DNA than traditional Sanger sequencing or array CGH. Thirdly, the ability of ctDNA to display clonal heterogeneity and track clonal progression, could potentially overcome spatial and temporal heterogeneity issues encountered when relying on tumour biopsies. Lastly, blood sampling is safer, and has fewer complications when compared to tumour biopsy and the use of sequential sampling could track response to treatment more closely or may have alerted clinicians to the development of resistant mechanisms. However, several aspects of ctDNA
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analysis require addressing prior to its adoption in the clinic. Plasma processing and data analysis require standardisation to allow largescale, multi-institutional clinical trials. Such trials would need to clearly demonstrate patient benefit before ctDNA analysis can be used to guide treatment decisions. Despite the above, Rothé et al. performed hotspot mutation analysis on 17 breast cancer patients to ascertain whether ctDNA can be used to guide therapeutic decisions [54]. Metastatic tumour and plasma samples were taken at the same time-point and Rothé et al. reported a 76% concordance (13/17 patients) between these samples. Two patients had mutations detected in plasma at relatively high frequencies (12% and 14.3%) but not the tumour samples. The remaining two patients had mutations detected in the tumour but not the plasma. Further analysis of these plasma samples by targeted illumina sequencing revealed mutations identical to that found in the tumour, although present at low frequency. Interestingly, for 9 out of 69 tumour samples, the investigators were unable to perform NGS analysis where as they were able to for all 31 plasma samples received. The investigators conclude that plasma ctDNA analysis is a suitable alternative to metastatic biopsy for molecular screening. Furthermore, in 2014, Douillard et al. published the results of a comparison between ctDNA analysis and tumour biopsy DNA analysis of EGFR mutation status in 1060 advanced lung cancer patients. They found the concordance of EGFR mutations between matched tumour and plasma to be 94.3%, with the test yielding a sensitivity of 65.7% and specificity of 99.8% [55]. Furthermore, patients who have activating EGFR mutations detected in tumour or plasma samples demonstrated similar progression free survival (9.7 vs 10.2 months) after gefitinib treatment [55], supporting the use of ctDNA analysis to initiate targeted therapies. In addition, cell-free tumour DNA is present in alternative bodily fluids, some of which are of much higher abundance than tumour biopsy or plasma, such as urine. These bodily fluids may be more abundant than plasma and maybe more easily collected. Analysis of these fluids may facilitate the detection of rare mutated DNA fragments. The choice of specific fluids to analyse would differ across cancer types, depending on the proximity of the fluid to the tumour sites and clinical context. In summary, there is increasing evidence that ctDNA analysis may have an important role at several stages of disease. Due to their short half-life, ctDNA profiles can present a snapshot of current clonal burden and rapidly assess tumour response to therapies. Indeed, many of the protein based tumour markers currently used to asses treatment response in the clinic (e.g. PSA, CA125, CEA, αFP) have a half-life measured in days [56], whereas the half-life of cfDNA is measured in minutes to hours [12,57]. We envision that, the ability of ctDNA to detect de novo mutations could overcome the problem of mutational profiling in the presence of continual tumour evolution, thus improving our understanding resistance mechanisms and allow us to monitor tumour response in real-time. Furthermore, improvements in the detection of ctDNA present at low volumes in plasma and other bodily fluids, may lead to an earlier diagnosis, where intervention can still lead to cure or improve our ability to stratify patients to adjuvant therapy following radical treatments. What is certain is that as the cost of massively paralleled sequencing reduces, ctDNA analysis will become more readily available and has the potential to improve cancer care.
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physicians to the early presence of cancer through ctDNA analysis will translate to improved outcomes. Indeed, the use of prostate specific antigen (PSA) to detect early prostate cancer has resulted in overtreatment, with disease specific mortality improved in only high-risk groups [48]. Furthermore in the future, ctDNA detection may be “too sensitive” and detect mutations from cancers before they can be localised. Indeed by using restriction enriched PCR Gormally et al. were able to detect KRAS mutations in 13 out of 1098 “healthy volunteers”. However, in this instance, 5 “healthy volunteers” were subsequently diagnosed with bladder cancer and a further volunteer diagnosed with upper aero-digestive tract cancer within 25 months [49], conceivably these tumours may have been present at microscopic level at the time of investigation and had superior localisation techniques been available, may have been detected and treated early. Furthermore, Chan et al. were able to detect Epstein Barr viral DNA in plasma, a known risk factor for nasopharyngeal carcinoma, when screening 1318 asymptomatic volunteers [50]. They detected viral DNA in 69 volunteers and on further investigation found nasopharyngeal cancer in 3 previously asymptomatic individuals, demonstrating the utility of plasma-based cancer screening [50]. It is however, likely that robust ctDNA detection will only be possible in certain cancers and, that early detection may be possible in only a subset of these. Although, where ctDNA is detectable at an early stage, it has the potential to reduce mortality through more patients being amenable to curative surgery.
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