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available at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/molonc

Review

Cell-free circulating tumour DNA as a liquid biopsy in breast cancer5 Leticia De Mattos-Arrudaa,b,c, Carlos Caldasa,d,e,* a

Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK Vall d’Hebron Institute of Oncology, Vall d’Hebron University Hospital, Barcelona, Spain c Universitat Aut onoma de Barcelona, Barcelona, Spain d Department of Oncology, University of Cambridge, Cambridge, UK e Cambridge Experimental Cancer Medicine Centre and NIHR Cambridge Biomedical Research Centre, Cambridge, UK b

A R T I C L E

I N F O

A B S T R A C T

Article history:

Recent developments in massively parallel sequencing and digital genomic techniques

Received 19 October 2015

support the clinical validity of cell-free circulating tumour DNA (ctDNA) as a ‘liquid biopsy’

Received in revised form

in human cancer. In breast cancer, ctDNA detected in plasma can be used to non-

26 November 2015

invasively scan tumour genomes and quantify tumour burden. The applications for ctDNA

Accepted 3 December 2015

in plasma include identifying actionable genomic alterations, monitoring treatment re-

Available online 17 December 2015

sponses, unravelling therapeutic resistance, and potentially detecting disease progression before clinical and radiological confirmation. ctDNA may be used to characterise tumour

Keywords:

heterogeneity and metastasis-specific mutations providing information to adapt the ther-

Breast cancer

apeutic management of patients. In this article, we review the current status of ctDNA as a

Circulating cell-free tumour DNA

‘liquid biopsy’ in breast cancer.

Stratification

ª 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Monitoring Resistance Heterogeneity

Review criteria Review of the biomedical literature was conducted in English using PubMed and MEDLINE databases. Articles published before October 2015 were included. Search terms used included “breast cancer”, “circulating cell free DNA”, “circulating tumor DNA”, “plasma”, “liquid biopsies”. Abstracts of studies presented at the ASCO, AACR, ESMO Annual Meetings, 5

and San Antonio Breast Cancer Symposium were also considered.

1.

Introduction

Breast cancer represents a heterogeneous collection of diseases with different biological characteristics and clinical

This is a contribution to the special issue edited by Klaus Pantel and Catherine Alix-Panabieres, Liquid Biopsies. * Corresponding author. Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. Tel.: þ44 1223 769650. E-mail address: [email protected] (C. Caldas). http://dx.doi.org/10.1016/j.molonc.2015.12.001 1574-7891/ª 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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outcomes. Recent advances in massively parallel sequencing technologies have enabled the genomic characterisation of driver genes and actionable mutations in breast tumours (Nik-Zainal et al., 2012; Shah et al., 2009; Stephens et al., 2012) in addition to establishing a refined classification of breast cancers that integrates genomics and transcriptomics (Ali et al., 2014; Curtis et al., 2012; Dawson et al., 2013a; Dvinge et al., 2013). In the era of ‘Precision Medicine’, unravelling this inter- and intra-tumour heterogeneity is paramount for understanding the biology of the disease and providing molecular information to tailor the therapeutic management of patients. Efforts in this direction include massively parallel sequencing of single cells (Navin et al., 2011) and minimally invasive approaches to characterise cancer mutations in blood and other biological fluids (Haber and Velculescu, 2014). The procurement of tumour tissue specimens poses challenges for the development of biomarkers since tumour biopsies are invasive, costly, time consuming and not amenable for repetition. Blood-based circulating biomarkers, including circulating tumour cells (CTCs), cell-free nucleic acids and exosomes, have been studied as ‘liquid biopsies’, that is, surrogates or complementary biomarkers to overcome the drawbacks of invasive tissue biopsies. Plasma is known to carry small amounts of fragmented cell-free DNA of 160e180 base pairs, which is likely to be originated from cancer cells through the process of necrosis and apoptosis (Diaz and Bardelli, 2014; Jahr et al., 2001; Mouliere and Rosenfeld, 2015). The fraction of cell-free DNA that contains specific tumour mutations of a given patient is named cell-free circulating tumour DNA (ctDNA). Hence, ctDNA in plasma constitutes a non-invasive source of material that may allow the identification of the genomic make-up of tumours. Tumour-derived somatic single nucleotide variants (SNVs), copy number alterations (CNA) and structural variants (SVs) have been detected in ctDNA from breast cancer patients (Bettegowda et al., 2014; Dawson et al., 2013b; De MattosArruda et al., 2015; De Mattos-Arruda et al., 2014; Forshew et al., 2012; Leary et al., 2010, 2012; Murtaza et al., 2015; Murtaza et al., 2013; Olsson et al., 2015; Rothe et al., 2014). Hence, ctDNA provides a new means for studying breast cancer patients in terms of monitoring tumour burden, assessing the mechanisms of therapeutic responses and resistance, detecting minimal residual disease, and understanding unresolved biologic challenges posed by tumour heterogeneity and clonal evolution (De Mattos-Arruda et al., 2013). In this manuscript, we review the current status of ctDNA as a ‘liquid biopsy’ in breast cancer, and discuss the translational and clinical relevance for patients with early- and late-stage disease.

2. Genomic characterization of ‘liquid biopsies’ in breast cancers The analysis of ctDNA is challenging because the tumour contribution to cell-free DNA is diluted in the background of normal cell-free DNA. The development of massively parallel sequencing and digital genomic technologies has allowed both screening and validation of genomic alterations in

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ctDNA. These highly sensitive techniques have improved the detection of mutations in patients with breast cancer, including rare mutant variants. They include PCR-based (Board et al., 2010; Chen et al., 2009; McBride et al., 2010), digital PCR-based (Beaver et al., 2014; Bettegowda et al., 2014; Dawson et al., 2013b; Forshew et al., 2012; Garcia-Murillas et al., 2015; Gevensleben et al., 2013; Higgins et al., 2012; Leary et al., 2012; Oshiro et al., 2015) and massively parallel sequencing-based technologies (Bettegowda et al., 2014; Dawson et al., 2013b; De Mattos-Arruda et al., 2014; Forshew et al., 2012; Garcia-Murillas et al., 2015; Guttery et al., 2015; Leary et al., 2010, 2012; Murtaza et al., 2015; Murtaza et al., 2013; Rothe et al., 2014) (Table 1). Massively parallel sequencing has been developed to analyse ctDNA at several levels (i.e., whole genome, whole exome, targeted sequencing) (Figure 1). Cancer genome characterisation efforts have shown that only a few genes are frequently mutated in breast cancer (e.g., TP53 and PIK3CA) and a much large number of cancer genes are rarely mutated (Cancer Genome Atlas, 2012; Nik-Zainal et al., 2012; Shah et al., 2009; Stephens et al., 2012). Targeted sequencing approaches designed to detect somatic mutations in known driver genes are clearly limited to patients that harbour such mutations. Such customised gene panels using target enrichment (hybridization capture or amplicon PCR) have been used to interrogate mutations in both tumour tissue and plasma cell-free DNA (Cheng et al., 2015; Forshew et al., 2012). Digital genomic technologies offer higher sensitivity compared to most massively parallel sequencing technologies and have been used as an orthogonal method for validation of results and for quantification of ctDNA. Cost-effective and fast methods that sample plasma ctDNA of patients with no prior knowledge about the genomic composition of tumour tissues are being developed (Belic et al., 2015), in a way that the tumour genomes of breast cancer patients can be scanned non-invasively. A key point is that ctDNA may be suitable for revealing actionable genomic alterations and informing decisions making processes in the clinic.

3. Translational and clinical applications of ctDNA in breast cancer Potential applications of ctDNA have provided insights for managing patients with breast cancer in terms of i) Patient stratification based on actionable genomic alterations, ii) Longitudinal monitoring of disease burden and the identification of mechanisms of therapeutic resistance iii) Assessing minimal residual disease and early detection of recurrence, and iv) Deciphering tumour heterogeneity (Figure 2).

3.1. Patient stratification based on actionable genomic alterations Plasma ctDNA has the potential for the characterisation of breast cancer mutations and to longitudinally monitor these genomic alterations. Retrospective and prospective studies have demonstrated that assessment of ctDNA is capable of identifying and monitoring tumour-derived genomic alterations of a given breast cancer under targeted therapies (De

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Table 1 e Selected methodologies for the analysis of ctDNA in plasma. Breast cancer stage Early

Advanced

Technology

Tumour-specific aberration detected in plasma DNA

ARMS-Scorpion PCR MAP Digital PCR (droplet)

Hotspot PIK3CA mutations TP53 mutation Rearrangements, PIK3CA mutations

Shallow whole-genome sequencing ARMS-Scorpion PCR Real-time PCR PARE

Rearrangements

BEAMing Digital PCR (microfluid)

PIK3CA mutations SNVs (e.g., PIK3CA and TP53 mutations), copy number alterations and rearrangements HER2 amplification, ESR1 mutations

Digital PCR (droplet)

Hotspot PIK3CA mutations Rearrangements Rearrangements

MSK-IMPACT

SNVs and copy number alterations based on a custom panel

TAm-Seq

SNVs (e.g., PIK3CA and TP53 mutations), copy number alterations and rearrangements

Safe-SeqS PCR-Ligation Modified FAST-SeqS

SNVs SNVs Pre-screening tool for an estimation of ctDNA percentage Selected SNVs (e.g., TP53, PIK3CA, ESR1, PTEN, AKT1, IDH2, FGFR1, FGFR2, SMAD4 mutations) Protein coding SNVs and copy number alterations SNVs, tumour specific rearrangements, copy number changes (e.g., ERBB2 and CDK6 amplifications) Copy number alterations

Ion-AmpliSeq Whole exome sequencing Whole-genome sequencing

Shallow whole-genome sequencing (Plasma-Seq)

References (Board et al., 2010) (Chen et al., 2009) (Beaver et al., 2014; Garcia-Murillas et al., 2015; Olsson et al., 2015) (Olsson et al., 2015) (Board et al., 2010) (McBride et al., 2010) (Bettegowda et al., 2014; Leary et al., 2010) (Higgins et al., 2012) (Dawson et al., 2013b; Forshew et al., 2012; Murtaza et al., 2013) (Gevensleben et al., 2013; Guttery et al., 2015) (Cheng et al., 2015; De Mattos-Arruda et al., 2015; De Mattos-Arruda et al., 2014) (Dawson et al., 2013b; Forshew et al., 2012; Murtaza et al., 2015; Murtaza et al., 2013) (Bettegowda et al., 2014) (Bettegowda et al., 2014) (Belic et al., 2015) (Guttery et al., 2015; Rothe et al., 2014) (Murtaza et al., 2015; Murtaza et al., 2013) (Dawson et al., 2013b; Forshew et al., 2012; Leary et al., 2012) (Heidary et al., 2014)

Abbreviations: ARMS, Amplification Refractory Mutation System allele-specific PCR and Scorpion probes; BEAMing, Beads, Emulsion, Amplification, Magnetics; MAP, MIDI-Activated Pyrophosphorolysis; PARE, Personalised Analysis of Rearranged Ends; Safe-SeqS, Safe-Sequencing System; SNV, single nucleotide variants; TAm-Seq, Tagged-Amplicon deep Sequencing.

Mattos-Arruda et al., 2014; Frenel et al., 2015; Janku et al., 2015). ctDNA has the potential to reveal exceptional responses and also identify the emergence of mutations associated with resistance to these therapies (Dawson et al., 2013b; Janku et al., 2015; Murtaza et al., 2013). The current models of ‘Precision Medicine’ tailor medical treatments to the genomic complexity of each patient’s tumour (De Mattos-Arruda and Rodon, 2013). The analysis of genomic alterations in tumour tissue biopsies aims to select and match patients for specific targeted therapies. In this context, there is evidence showing that plasma can be prospectively surveyed as an alternative to metastatic tumour tissue biopsies in molecular screening programs (Rothe et al., 2014). This would allow a subset of advanced breast cancer patients without accessible metastatic lesions to be enrolled in clinical trials and have access to personalised therapies. In fact, in the context of early phase clinical trials using experimental targeted therapies, tumour-derived genomic alterations have been evaluated by serial ctDNA samples to

massively parallel sequencing (Frenel et al., 2015). The genomic alterations identified on ctDNA (e.g., PIK3CA mutations) have allowed the stratification of patients with solid tumours, including those with breast cancers, and monitoring tumour responses to the administration of experimental targeted therapy. There are a number of prospective programs that aim to personalise the treatment of breast cancers at the genomic level (Andre et al., 2014; Arnedos et al., 2014; Bedard et al., 2013; Meric-Bernstam et al., 2015). The prospective multicentre SAFIR-01 trial exemplifies the feasibility of identifying and targeting both frequent and rare genomic alterations in an era previous to massively parallel sequencing approaches (Andre et al., 2014). For instance, targetable genomic alterations (e.g., PIK3CA, CCND1, and FGFR1) were identified in 46% of the analysed patients, and 13% of them were targeted by anticancer therapies. As an example of a personalised cancer initiative that includes liquid biopsies, the SAFIR-02 Breast trial (Evaluation

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Plasma cell-free DNA

Tumour tissue

Mutant Wild-type

Massively parallel sequencing WGS

WES

Digital genomic technologies

TS

Digital PCR

Increase in Complexity

Increase in Sensitivity

Figure 1 e Schematic for assessing plasma cell-free circulating tumour DNA (ctDNA) and tumour tissue biopsies using massively parallel sequencing and digital genomic technologies. Note that for digital PCR it is essential to know a priori the genomic alteration to be targeted. WGS, whole genome sequencing, WES, whole exome sequencing, TS, targeted sequencing.

A. Stratification

B. Longitudinal Monitoring Genomic alteration ctDNA

PIK3CA mutation ERBB2 amplification

Therapy selection

Anti-PIK3CA Anti-HER2

100 80 Resistance

60

Sensitive alteration 40

Resistant alteration

20 0

0

2

4

6

8

10

time (months)

Adjuvant therapy

Clinic-radiologic detection

20 ctDNA detection

10

Late Relapse Early Relapse

Mutation A 0

0

2

4

6

8

10

12

Mutation B

time (months)

Clonal

30

metastasis

40

D. Intra- and inter-tumour heterogeneity

Follow up

1.0

Subclonal

Genomic alteration ctDNA

C. Early detection

0.6

Plasma cell-free DNA

0.8

0.4 0.2 0.0 0.0

0.2

0.4

Subclonal

0.6

0.8

1.0

Clonal

primary tumour

Figure 2 e The roles of cell-free circulating tumour DNA (ctDNA) in breast cancers. A. Genomic stratification of patients; B. Monitoring of tumour burden and mechanisms of therapeutic resistance; C. Early detection of recurrence; D. Deciphering intra- and inter-tumour heterogeneity. Clonal and subclonal events are depicted in both the x-axis and y-axis for primary tumour and metastasis. The red circle represents clonal events in both the primary tumour and metastasis.

of the Efficacy of High Throughput Genome Analysis as a Therapeutic Decision Tool for Patients With Metastatic Breast Cancer) (NCT02299999) is a randomised trial that takes advantage of high throughput genome analysis as a therapeutic decision tool. The trial includes patients with HER2-negative recurrent and/or metastatic breast cancers. A metastatic site

is biopsied and targeted sequenced, and comparative genomic hybridization array is also performed. Patients are randomised either to a molecularly targeted agent matching the detected genomic alteration or to maintenance chemotherapy. It is expected that only a small proportion of these patients will have targetable genomic alterations. Liquid

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biopsies, however, are anticipated to be relevant for capturing the collection of genomic alterations of a given tumour, monitoring the emergence of treatment-resistant clones over time and providing an efficient means toward personalising therapy. However, caution should be taken when assuming that a specific genomic alteration will lead to a therapeutic benefit for a given patient. Previous studies that assessed the efficacy of molecularly targeted agents in metastatic patients selected on the basis of the molecular profile have failed to show clinical benefit compared with treatment at clinician’s choice (Dienstmann et al., 2012; Le Tourneau et al., 2015).

3.2. Longitudinal monitoring of disease burden and the identification of mechanisms of resistance The analysis of tumour genomes including tumour-specific somatic mutations, copy number alterations, and structural variations in ctDNA has been shown to be sensitive and specific as a non-invasive tool for identifying the genomic portrait of cancer cells in patients with breast cancer. In this context, ctDNA also provides a means for longitudinal analysis of tumour genomes. That included the monitoring of frequent genomic alterations in breast cancer (e.g., TP53 and PIK3CA mutations, copy number alterations, translocations), and the emergence of resistance to systemic therapies (Dawson et al., 2013b; De Mattos-Arruda et al., 2014; Forshew et al., 2012; Garcia-Murillas et al., 2015; Guttery et al., 2015; Leary et al., 2010, 2012; Murtaza et al., 2015; Murtaza et al., 2013). In metastatic breast cancer, a proof-of-concept study demonstrated that ctDNA is a tool to monitor tumour burden dynamics in patients undergoing systemic therapy (Dawson et al., 2013b). The detection of ctDNA using structural variations and TP53 and PIK3CA gene mutations found in the primary tumours was demonstrated to be more sensitive and specific than other circulating blood biomarkers (i.e., CTCs enumeration using the FDA-cleared CellSearch system and CA 15e3 levels) and radiographic imaging of tumours. In addition, the fluctuation in the number of ctDNA copies was associated with response to therapy and there was a significant relationship between amplifiable copies of ctDNA and patient outcome (Dawson et al., 2013b). A universal challenge in metastatic breast cancer patients is the emergence of resistance to therapy. In breast cancer, over two-thirds of patients express oestrogen receptor-alpha (ER-alpha, encoded by ESR1, the oestrogen receptor gene), and the majority of these cases are sensitive to ER inhibition. However, resistance to oestrogen inhibition invariably occurs (Merenbakh-Lamin et al., 2013). Mutations in the ESR1 gene are acquired in approximately 20% of breast cancers patients treated with endocrine agents, and constitute one of the resistance mechanisms to aromatase inhibitors (Merenbakh-Lamin et al., 2013; Robinson et al., 2013; Toy et al., 2013). ESR1 mutations can be robustly identified in ctDNA of ERpositive metastatic breast cancer patients (Chu et al., 2015; Guttery et al., 2015; Schiavon et al., 2015). Given that ESR1 mutation in plasma predicts for resistance to subsequent aromatase inhibitor therapy in ER-positive patients, a potential application for ctDNA is the detection of ESR1 mutations before disease progression occurs. This would allow for an

early change in therapeutic options, with a potential benefit to patients. Exome-wide analysis of ctDNA at different time points during treatment allowed for the identification of mutations associated with acquired drug resistance in advanced breast cancers (Murtaza et al., 2013). Plasma ctDNA samples had their mutant allelic fractions compared at different time points. The increase in abundance of the allele fractions in plasma ctDNA over time could be an indicator of selective pressures due to therapy, and associated with the emergence of resistance. For instance, an ER-positive, HER2-positive breast cancer patient treated with tamoxifen and trastuzumab showed an increase in the levels of a MED1 gene mutation, which is an ER co-activator and known to be involved in tamoxifen resistance (Cui et al., 2012; Nagalingam et al., 2012). After secondary therapy with lapatinib (i.e., an antiHER2 tyrosine-kinase inhibitor) plus capecitabine, the GAS6 gene mutation was identified and linked to activation of the AXL tyrosine-kinase receptor, which is known to be involved with resistance to lapatinib (Liu et al., 2009). Tracing the genomic evolution of tumours in response to therapy, it is therefore possible to detect the emergence of resistant tumour cells in response to selective pressures from specific therapies by monitoring a patient’s plasma for the presence of rare subclonal resistance mutations (McGranahan et al., 2015; Murtaza et al., 2015; Nik-Zainal et al., 2012).

3.3. Assessing minimal residual disease and early detection of recurrence Currently, the comprehensive analysis of tumour genomes is facilitated when plasma DNA has a high fraction of ctDNA. Hence, massively parallel sequencing data of ctDNA in earlystage disease (i.e., breast cancer patients being treated in the neoadjuvant and adjuvant settings) is limited. In patients with non-metastatic breast cancers, an ideal ‘liquid biopsy’ tool would capture and monitor genomic markers of minimal residual disease following curative resection, possibly preceding the development of clinical or radiologic recurrence and providing a tool to assess tumour dormancy. In addition, an ideal ‘liquid biopsy’ would categorise patients who are at high-risk for recurrence and spare low-risk patients from the toxicities of unnecessary systemic therapies. Very few studies have evaluated ctDNA in early-stage breast cancer patients. Because different techniques have been used, the ability to detect ctDNA has varied (Beaver et al., 2014; Board et al., 2010; Chen et al., 2009; GarciaMurillas et al., 2015; Olsson et al., 2015). Recent work has revealed that ctDNA in plasma can be detected in up to 50% of early-stage breast cancers patients (Bettegowda et al., 2014). Tumour-derived PIK3CA mutations in plasma were identified prior to and after breast surgery using digital PCR (Beaver et al., 2014). The assay had a sensitivity and specificity of 90% and 100%, respectively showing the feasibility of detecting DNA mutations in the plasma of early-stage breast cancer patients including those with minimal, clinically undetectable disease (Beaver et al., 2014). Two recent pilot studies demonstrated that the identification of tumour genomic alterations in the plasma of non-

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metastatic breast cancer patients anticipate the diagnosis of clinical metastatic relapses (8e11 months) (Garcia-Murillas et al., 2015; Olsson et al., 2015). Given that the identification of genomic events associated with micrometastatic disease seem to be more sensitive than imaging methods (Beaver et al., 2014; Chen et al., 2009; Garcia-Murillas et al., 2015; Olsson et al., 2015), and may identify patients with higher risk of relapse, longitudinal sampling of genomic alterations in plasma may create opportunities for therapeutic interventions before the development of clinical metastasis (Figure 3). In addition, identifying targetable genomic alterations associated with primary or secondary therapeutic resistance, either during adjuvant therapies or surveillance, may ultimately modulate the long-term adjuvant therapy of such patients.

3.4.

Deciphering tumour heterogeneity

Breast cancers are genomically heterogeneous. In breast, akin to other solid tumours, the collection of genomic alterations found within a given tumour may differ according to the region sampled, between primary tumour and metastatic deposits, and even between distinct metastatic deposits (Aparicio and Caldas, 2013; De Mattos-Arruda et al., 2014; Gerlinger et al., 2012; Yates et al., 2015). The genomic analyses of breast cancers have provided direct evidence of spatial and temporal intra-tumour heterogeneity (NikZainal et al., 2012; Shah et al., 2009; Stephens et al., 2012) and have shown that the range of subclonal heterogeneity is variable among breast cancers (Yates et al., 2015). Currently, clinical and therapeutic decisions are usually based on individual biopsies that may not be representative of the entire tumour burden or not real-time assessments of the tumour tissue. The clinic goals for understanding tumour heterogeneity consist on i) characterising the cancers of patients and guiding their treatment, and ii) monitoring the emergence of drug resistance and selecting tailored therapies. However, these goals cannot be

Genomic alteration ctDNA

Treatment starts

Clinic-radiologic progression

100 Genomic resistence

80 Genomic response

60

Late relapse Clinic-radiologic response

Early relapse

40

Mutation X Mutation Y

20 0

0

5

10

15

time (months)

Figure 3 e Longitudinal monitoring of cell-free circulating tumour DNA (ctDNA) derived from plasma. Detection of early relapse or progression as compare to imaging methods, and the emergence of genomic events associated with therapeutic resistance to chemotherapeutics and targeted therapy. The genomic alterations revealed by ctDNA can anticipate the development of clinical or radiologic relapse or progression in early-stage and advanced-stage breast cancers.

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accomplished with the current mode of analyses of tumour tissue biopsies.

3.4.1. The impact of ctDNA for the identification of genomic alterations among multiregional tumour samples ctDNA in plasma represents a valuable source of tumourderived DNA to decode cancer genomes non-invasively. Given the impracticality of sampling multiple visceral metastatic implants, ctDNA may constitute a representative readout of the collection of genomic alterations of every tumour deposit of a metastatic breast cancer patient (De Mattos-Arruda et al., 2015; De Mattos-Arruda et al., 2014; Forshew et al., 2012; Murtaza et al., 2015, 2013). Until recently the degree to which ctDNA represents intra- and inter-tumour heterogeneity has not been characterised. There is some evidence to demonstrate that ctDNA from plasma may reflect the clonal tumour hierarchy of breast cancers (Figure 4). Murtaza et al. performed an extensive analysis of sequential tumour tissue biopsies and plasma ctDNA samples in an ERþ/HER2þ metastatic breast cancer patient, including the multiple tumour metastatic deposits derived from a warm autopsy of the same patient (Murtaza et al., 2015). Tumour-derived mutations in both tumour tissue and plasma identified by massively parallel sequencing were clustered using a bioinformatic Bayesian method called Pyclone (Murtaza et al., 2015; Shah et al., 2012). Ubiquitous mutations (i.e., present in all tumour samples) had the highest levels in ctDNA followed by mutations present at the metastatic deposits analysed, and finally by private mutations (i.e., present in only one of the metastatic samples). Notably, plasma-based Pyclone mutation clusters reflected the multiregion tumour metastatic deposits without dependence on tumour genomic data, and also captured organ-specific private mutations during targeted therapy. ctDNA from plasma identified the dynamic variety of clonal and subclonal tumour heterogeneity (Murtaza et al., 2015). This has important implications to uncover intra- and inter-metastatic heterogeneity and clonal evolution and to establish the use of ctDNA in the clinic. In the context of assessing multiregional tumour samples, plasma derived-ctDNA has been compared to ctDNA from other body fluids, particularly the cerebro-spinal fluid (CSF) as a surrogate for the brain tumour specific genomic alterations. In patients with primary brain tumours, the presence of ctDNA derived from plasma is minimal probably as a result of the bloodebrain barrier (Bettegowda et al., 2014; Chen et al., 2013; Lavon et al., 2010), although proof-of-principle studies have suggested the release of CTCs into the circulation of glioblastoma patients (Muller et al., 2014; Seoane and De MattosArruda, 2014; Sullivan et al., 2014). Recently, tumour-derived DNA has been isolated from the CSF of patients with brain tumours using massively parallel sequencing (De Mattos-Arruda et al., 2015; Pan et al., 2015). ctDNA-derived from the CSF characterised the genomic alterations of central nervous system disease better than plasma in both primary and secondary brain tumours (De Mattos-Arruda et al., 2015). In a subset analysis of a breast cancer autopsies series, patients with central nervous system restricted disease (i.e., minimal or absent extra-cranial disease), the mutations present in brain metastasis were

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Figure 4 e Clonal hierarchy of mutations captured by plasma ctDNA. A. Schematic representation of a metastatic breast cancer patient subjected to multisampling during warm autopsy. B. Illustrative heatmap showing non-synonymous mutations in metastatic deposits (M1-8) and plasma ctDNA. C. Diagram depicting that ubiquitous and clade mutations are more likely to be detected in plasma ctDNA than private mutations.

captured by ctDNA in CSF and not in plasma. By contrast, in patients with abundant visceral disease, tumour-derived mutations in the CSF and plasma ctDNA were comparable. Notably, CSF-derived ctDNA identified organ-specific private mutations (i.e. brain and meningeal private mutations) in a patient with concurrent two neoplasms (i.e., metastatic breast cancer and esthesioneuroblastoma).

4.

Concluding remarks

Digital genomic PCR and massively parallel sequencing technologies have enabled the genomic characterisation of tumour tissue and blood-based cell-free circulating tumour DNA. The evaluation of cell-free DNA in patients with breast cancer represents a continuum, and ranges from the identification of actionable genomic alterations in a minimally invasive way, monitoring of treatment responses and revealing the emergence of resistance mechanisms, to the detection of early recurrence or progression. The applicability of these approaches is likely to expand in the near future with the developments of massively parallel sequencing techniques (Amant et al., 2015). Sampling breast cancer patients’ blood has been shown to retrieve robust information on the tumour’s genomic make-up for early-stage and late-stage breast cancers, without a need for an invasive biopsy. There are, however, some challenges that need to be overcome. Firstly, breast cancers are composed of a large genomic diversity within and among different tumours, and there are few frequently mutated cancer genes that drive cancer progression. In addition, in breast cancer, there are few genomic

alterations that serve as predictive biomarkers for cancer therapy (e.g., ERBB2 gene amplification or mutation) (Simon and Roychowdhury, 2013). Currently, such analyses are restricted to tumour tissue biopsies. The assessment of the same biomarkers as a ‘liquid biopsy’ has enormous potential in clinical oncology practice. Current challenges include the improvement of massively parallel sequencing technologies, standardization and validation of technologies between laboratories and incorporation of ctDNAbased analyses in clinical trials and routine clinical practice. Moreover, not all patients with progressive metastatic disease appear to release tumour-derived DNA into the bloodstream in measurable quantities with the currently technologies (Dawson et al., 2013b; Heidary et al., 2014; Heitzer et al., 2013; Madic et al., 2015). For example, isolated case reports have showed that CTCs and ctDNA may be discordant in some metastatic breast cancer patients (Heidary et al., 2014; Madic et al., 2015). The advance of massively parallel sequencing techniques and its concurrent use with other digital-based approaches will dictate future improvements in the detection of ctDNA for broader use. Preliminary data in metastatic breast cancer has also provided evidence for the association of ctDNA levels and prognosis (Bettegowda et al., 2014; Dawson et al., 2013b). Quantification of ctDNA levels for prognostication will need to be validated in larger cohorts of patients. In conclusion, ctDNA as a liquid biopsy has enormous potential but widespread clinical application awaits for rigorous studies that will need robust assays (analytical validity) to demonstrate clinical validity and clinical utility.

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Acknowledgements  n Espanola contra el CanThe authors acknowledge Asociacio  n Rafael del cer e Advanced Program in Oncology y Fundacio Pino (for L. De Mattos Arruda). Cancer Research UK Cambridge Institute and University of Cambridge, UK. R E F E R E N C E S

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Cell-free circulating tumour DNA as a liquid biopsy in breast cancer.

Recent developments in massively parallel sequencing and digital genomic techniques support the clinical validity of cell-free circulating tumour DNA ...
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