application of cell-free dna analysis to cancer treatment€¦ · cfdna analysis in patients with...

T h e n e w e ngl a nd j o u r na l o f m e dic i n e

n engl j med 379;18 nejm.org November 1, 20181754

Review Article

From the Massachusetts General Hospital Cancer Center and the Department of Medicine, Harvard Medical School, Boston. Address reprint requests to Dr. Corcoran at Massachusetts General Hospital Cancer Center, 149 13th St., 7th Fl., Boston, MA 02129, or at rbcorcoran@ partners . org.

N Engl J Med 2018;379:1754-65.DOI: 10.1056/NEJMra1706174Copyright © 2018 Massachusetts Medical Society.

Tumor biopsies represent the standard for cancer diagnosis and the primary method for molecular testing to guide the selection of precision therapies. Liquid biopsies, particularly those involving cell-free

DNA (cfDNA) from plasma, are rapidly emerging as an important and minimally invasive adjunct to standard tumor biopsies and, in some cases, even a potential alternative approach. Liquid biopsy is becoming a valuable tool for molecular test-ing, for new insights into tumor heterogeneity, and for cancer detection and monitoring. Here, we review the current and potential clinical applications of cfDNA analysis in patients with cancer (see video).

Liquid Biopsies

Although liquid biopsy has most often referred to the analysis of cfDNA from peripheral blood, this term also encompasses the isolation and analysis of tumor-derived material (e.g., DNA, RNA, or even intact cells) from blood or other bodily fluids1,2 (Fig. 1). For example, intact circulating tumor cells intravasate into the bloodstream at low frequency (often <10 circulating tumor cells per milliliter of blood in patients with metastatic cancer). With specialized technology, circulating tumor cells can be detected and isolated from a background of normal blood cells, facilitating molecular analysis and even implantation and growth of these cells in immunocompromised mice.1-3 Subcellular particles called exosomes, or extracellular membrane-encased vesicles, are also released by tumor cells into the bloodstream and contain tumor-specific proteins and nucleic acids.4 Cell-free nucleic acids, in-cluding cell-free RNA (which is less stable than DNA)5 and cfDNA (the focus of this review), are also released into the circulation.

Blood is not the only bodily fluid that can be used for liquid-biopsy approaches. Urine, stool, cerebrospinal fluid (CSF), saliva, pleural fluid, and ascites are all potential sources of tumor-derived material, including cfDNA.2,6-8 Although this review will focus on the analysis of cfDNA from blood, detection and analysis of cfDNA from other bodily fluids may have applications for specific cancer types (e.g., CSF for cancers of the central nervous system) or for the detection of cancers arising in defined organ systems (e.g., stool for colorectal cancer or saliva for head and neck cancers).

Pl a sm a-Der i v ed cfDNA

The term “cfDNA” refers to fragmented DNA found in the noncellular component of the blood, as first reported by Mandel and Metais in 1948.9 It is thought that cfDNA is released into the bloodstream through apoptosis or necrosis, and cfDNA is typically found as double-stranded fragments of approximately 150 to 200 base pairs in length, corresponding to nucleosome-associated DNA.10 Molecules of cfDNA are rapidly cleared from the circulation, with a half-life of an hour or less.

Frontiers in Medicine

Application of Cell-free DNA Analysis to Cancer Treatment

Ryan B. Corcoran, M.D., Ph.D., and Bruce A. Chabner, M.D.

An illustrated glossary and a

video overview of cfDNA analysis

in cancer are available at

NEJM.org

The New England Journal of Medicine Downloaded from nejm.org at ESCUELA VALENCIANA DE ESTUDIOS on November 12, 2018. For personal use only. No other uses without permission.

Copyright © 2018 Massachusetts Medical Society. All rights reserved.

n engl j med 379;18 nejm.org November 1, 2018 1755

Cell-free DNA Analysis and Cancer Treatment

The cfDNA from normal cells is found in plas-ma at low levels in healthy persons (approxi-mately 10 to 15 ng per milliliter, on average),11

and the level can increase under conditions of tissue stress, including exercise, inflammation, surgery, or tissue injury.12

More than 40 years ago, it was observed that patients with cancer have higher overall levels of cfDNA than persons without cancer.11 In patients with cancer, cfDNA that is released from tumor cells is often referred to as circulating tumor

DNA (ctDNA) and constitutes only a portion of the overall cfDNA. The fraction of ctDNA in overall cfDNA in patients with cancer can vary greatly, from less than 0.1% to more than 90%.12,13 Although the fraction of ctDNA tends to parallel tumor burden within an individual patient, substantial variability has been observed among patients with the same cancer type, possi-bly reflecting biologic differences or differences in rates of cell death in the individual tumors.13

Moreover, patients with different tumor types

Figure 1. Overview of Liquid-Biopsy Approaches.

Liquidbiopsy approaches involving peripheral blood include isolation of circulating tumor cells, which intravasate from tumors into the bloodstream; exosomes, which are membranebound vesicles released by tumor cells; and cellfree DNA (cfDNA), which is released by apoptotic or necrotic tumor cells. Although peripheral blood is the most common source of liquid biopsy, several other bodily fluids have been used for specific liquidbiopsy applications, including isolation and analysis of cfDNA, as shown.

B L O O DV E S S E L

Exosomes

Circulating tumor cells

Tumor

Apoptotictumor cell

cfDNA

Exosomes

Liquid-Biopsy Sources

Cerebrospinal fluid

Tumors of the central nervous system

Saliva

Head and neck tumors

Ascites

Metastatic cancers

Stool

Gastrointestinal tract cancers

Urine

• Urinary tract cancers• cfDNA filtered from blood

Pleural fluid

• Thoracic cancers• Metastatic cancers

Peripheral blood

tumor cellstumor cells





show considerable variation in the frequency of detectable ctDNA. Thus, the detection and analy-sis of ctDNA amidst a background of normal germline cfDNA presents a considerable challenge.

Isol ation a nd A na lysis of cfDNA

Owing to the low levels and short half-life of tumor-derived cfDNA, specialized approaches are needed for both isolation and analysis of cfDNA. Two key issues are the stability of the cfDNA it-self and the potential for lysis of normal blood cells, leading to contamination with normal DNA. To limit these effects, when cfDNA is isolated from blood collected in standard phlebotomy tubes, plasma must typically be centrifuged and separated within 1 to 4 hours after collection. This need for rapid processing creates logistic challenges and the potential for preanalytic vari-ability caused by fluctuations of cfDNA concen-tration and purity due to differences in process-ing times.14,15 Alternatively, specialized cfDNA collection tubes containing fixatives can stabilize both cfDNA and intact cells for up to 7 to 14 days at room temperature, allowing for easy shipping, storage, and batched or centralized processing.16

The fraction of ctDNA within the background of normal cfDNA in patients with cancer is typi-cally small and highly variable from patient to patient. Thus, ultrasensitive methods are required to detect mutations, copy-number changes, or other alterations that are present in cfDNA at very low variant-allele frequencies (i.e., the percentage of variant alleles present among all alleles, in-cluding wild-type alleles) (Fig. 2). For detection of individual point mutations, mutation-specific techniques based on polymerase-chain-reaction (PCR) analysis — such as BEAMing (beads, emulsion, amplification, and magnetics) or drop-let digital PCR (ddPCR) analysis — can identify and quantify alterations present at allele frequen-cies of 0.01% or less in cfDNA.17,18 Next-genera-tion sequencing methods have also been tailored for cfDNA, ranging from whole-genome or whole-exome sequencing to targeted sequencing of a limited gene panel.19-22 However, the sensitivity and specificity of these approaches are limited by the error rate of DNA polymerase and the se-quencing reaction. Thus, modified approaches in-corporating deep sequencing coverage, molecular barcoding methods (in which individual input template DNA fragments are tagged with a unique

nucleotide barcode), and error-suppression algo-rithms have improved the limits of detection.23-25

Clinic a l A pplic ations

The ability to analyze tumor-derived DNA from a routine blood draw without the need for an invasive tumor biopsy represents a critical ad-vance with potentially transformative clinical ap-plications (Fig. 3). In particular, the minimally invasive nature of cfDNA analysis provides a means of molecular profiling for tumors that are difficult or unsafe to biopsy and allows a practical means for monitoring tumor DNA seri-ally over time without the risk and potential complications of standard tumor biopsy. In ad-dition, cfDNA analysis may better capture the molecular heterogeneity harbored by multiple dis-tinct clonal populations in a patient’s tumor, as compared with a needle biopsy of a single tumor lesion. Finally, cfDNA analysis offers the poten-tial for tumor detection or monitoring in patients without clinically evident disease.

Diagnosis and Molecular Profiling

Tumor molecular profiling for the selection of therapy has become a fundamental practice in cancer medicine.26 The potential to assess the molecular profile of a patient’s tumor from a simple blood draw, without the need for an inva-sive biopsy, makes cfDNA analysis an attractive tool. However, a key initial question is whether the mutational profile established through cfDNA testing reliably reproduces the mutational pro-file derived from a direct tumor biopsy, which re-mains the standard of care. Early studies, based on small numbers of patient samples, suggest-ed low concordance between DNA alterations detected in tumor and plasma samples from the same patient.27,28 However, the validity of these studies was compromised by the shortcoming that tumor and plasma samples were often not collected at the same time, yielding potential differences due to molecular evolution of the tumor. In addition, many plasma samples were collected at suboptimal times, such as during therapy, when ctDNA levels are at their lowest.

Nonconcordance of key alterations is most often observed in patients with low ctDNA levels, and low levels make detection of alterations more challenging. For example, one recent study com-pared the performance of two commercial cfDNA sequencing assays on parallel pairs of blood





samples drawn from 40 patients with metastatic prostate cancer.29 Low concordance rates were observed, raising questions about the reproduc-ibility of cfDNA analysis. However, the study had major limitations, including the facts that at least half the patients had normal levels of prostate-specific antigen and that participants were not restricted to those not receiving therapy. Thus, much of the variability may be attributable

to low cfDNA levels at or below the limit of de-tection of these assays. In addition, the way in which true positive or negative results are scored in patients with low levels of cfDNA remains an important challenge.

Larger and more carefully controlled studies have shown high concordance rates of 80 to 90% between plasma and tissue samples obtained simultaneously, particularly for alterations in key

Figure 2. Isolation and Analysis of cfDNA.

As shown on the left, cfDNA is isolated from plasma after centrifugation of peripheral blood to separate the cellular component of the blood, including white and red cells. Circulating tumor DNA (ctDNA) is found (often at low fractions) among a background of normal germline cfDNA released by normal cells throughout the body. The cfDNA can be analyzed to identify several common DNAbased alterations observed in tumors, including mutations, copynumber alterations, gene fusions, and DNA methylation changes. As shown at the upper right, owing to the frequently low fraction of ctDNA in a background of normal cfDNA, specialized methods for cfDNA analysis have been developed, including sequencing and methods based on polymerasechainreaction (PCR) analysis (BEAMing [beads, emulsion, amplification, and magnetics] and droplet digital PCR). Typically, those analyses providing the greatest breadth or nucleotide coverage have lesser sensitivity and require higher fractions of ctDNA in overall cfDNA (percentages in red) for analysis. Conversely, methods that are directed against targeted mutation panels or single mutations offer improved limits of detection. As shown at the lower right, molecular barcoding to tag individual molecules of DNA has allowed nextgeneration sequencing approaches for cfDNA to achieve better limits of detection, allowing discrimination of true mutations present in the original template DNA molecule (and thus present in all sequencing reads corresponding to a specific molecular barcode) from mutations introduced by polymerase error during the sequencing reaction.

Plasma

Normal cfDNA

Mutations Nucleotide coverage

Limit of detection

Copy-number alterations

Next-generation sequencing with molecular barcodes

Gene fusions

DNA methylation

ctDNA

True mutation

• Whole-genome sequencing• Whole-exome sequencing

Targeted next-generation sequencing

Next-generation sequencing with molecular barcodes

• BEAMing• Droplet digital PCR analysis

Wild type

Molecularbarcode tags

Each DNA molecule tagged with barcode

Wild typeMutantBuffy coat (source of germline DNA)

10%

1%

0.01%

0.1%

0.001%

Wild type Mutant Wild type

MolecularMolecular

True mutation

Mutant

Polymerase error

ctDNA

Redundant sequencing; true mutations represented in all reads with given barcode

CH3

CHCH3

CHCH

CH3

CHCH3

CHCH

Polymerase errorPolymerase error





driver genes.20,30-32 Similarly, analyses of large cfDNA sequencing databases have yielded mo-lecular landscapes that closely match mutation frequencies produced by large-scale tumor tissue sequencing compendia, such as the Cancer Ge-nome Atlas.33

Although these data suggest that cfDNA test-ing could be a potential surrogate for or adjunct

to standard-of-care tumor biopsy testing, for now tumor biopsy remains the standard for initial pathological diagnosis and molecular testing. Tumor biopsies allow for histologic interpreta-tion and the assessment of non–DNA-based altera-tions, such as in the expression of hormone re-ceptors or other proteins, which can be important for diagnosis and treatment decisions. Further-

Subclonal resistance alteration ASubclonal resistance alteration A

Subclonal resistance alteration BSubclonal resistance alteration B

Normal germline cfDNA

Baseline clonal alteration in ctDNA

Localized Disease Metastatic Disease

Clinically undetectable

Abu

ndan

ce o

f ctD

NA

Clinically detectable

Minimalresidualdisease

Metastaticdisease

Subsequenttherapy

Treatment response

Diseaseprogression

Surgical resection Systemic therapy 1 Systemic therapy 2

Clinical Application: Early detection Residual-disease

detectionMolecularprofiling

Responsemonitoring

Identification of resistance mechanisms

Monitoring ofclonal dynamics

Resectionlocation





more, large series have shown that approxi-mately 15% of patients with metastatic can-cer may not have sufficient ctDNA levels to allow for mutational profiling from plasma, and these numbers vary depending on tumor type and tu-mor burden.13,31,33 Although improving the tim-ing of blood draws for cfDNA analysis (e.g., before the initiation of therapy) may increase the yield of cfDNA testing, confidence in the pres-ence or absence of key alterations decreases as the ctDNA fraction approaches the limit of de-tection of current technologies. This factor must be taken into account when interpreting clinical cfDNA tests.

Nonetheless, cfDNA testing can play an im-portant role in initial molecular testing, par-ticularly for patients in whom standard tumor biopsies yield insufficient material for clinical sequencing, which can occur in as many as 20 to 25% of needle biopsies.30,34 In such circumstances, cfDNA testing for treatment selection is increas-ingly used as an alternative to repeat invasive biopsy and may reveal actionable mutations that guide treatment decisions in these patients.30,35 Technological advances are likely to allow more rapid and inexpensive testing of cfDNA for rela-tively rare abnormalities that could identify ac-tionable mutations and predict the likelihood of response (e.g., the presence of microsatellite in-stability as a predictor of response to T-cell check-point inhibition).

For repeat or serial testing after one or more lines of therapy, minimally invasive cfDNA analy-sis offers many apparent advantages over repeat invasive tumor biopsies, which are less practical, less safe, and less cost-effective. In particular, liquid biopsy may identify emergent genetic al-terations driving acquired resistance to therapy that can be targetable with newer-generation therapies.22,36-43 One of the earliest and best examples is the use of cfDNA testing to identify the emergence of the epidermal growth factor receptor (EGFR) T790M gatekeeper mutation after EGFR inhibitor therapy in EGFR-mutated non–small-cell lung cancer. Key studies have shown a high degree of concordance of cfDNA analysis with tissue testing in identifying the presence of the T790M mutation, which responds to third-generation EGFR inhibitors, such as osimertinib.35,44 Indeed, the cobas EGFR Muta-tion Test can identify T790M and other EGFR driver mutations in plasma and has been ap-proved by the Food and Drug Administration as a companion diagnostic for choosing specific EGFR therapies.45

Tracking of Therapeutic Response

Although ctDNA levels can vary greatly among patients, ctDNA levels in an individual patient over time correlate well with changes in tumor burden and treatment response. The short half-life of cfDNA in circulation (approximately 1 hour) can be advantageous for measuring real-time tumor burden in response to therapy, in contrast to many standard serum tumor markers in cur-rent clinical use (e.g., carcinoembryonic antigen [CEA] and cancer antigen 125 [CA-125]), which

Figure 3 (facing page). Clinical Applications of cfDNA Analysis.

Analysis of cfDNA has potential applications at multiple points throughout the natural course of cancer development, diagnosis, and treatment. Earlydetection methods that screen for evidence of nascent tumors in cfDNA are currently under development. A liquidbiopsy test capable of identifying earlystage cancers in asymptomatic persons may allow cancers to be identified at a stage when they are more likely to be curable. After surgery with curative intent, cfDNA from postoperative plasma drawn during the weeks after surgery can be analyzed for the persistent presence of mutations or other alterations known to exist in the patient’s resected tumor. Since the halflife of cfDNA is very short (an hour or less), any evidence of persistent tumorderived mutations in cfDNA from postoperative plasma can provide direct evidence of residual disease that may ultimately lead to tumor relapse. Detection of residual disease in cfDNA shortly after surgery may allow patients to be stratified according to risk of recurrence and may offer an opportunity for early intervention to salvage cure. In the context of metastatic disease, clinical sequencing of cfDNA can identify potentially targetable genetic alterations to select precision therapies. Sequencing of cfDNA can identify many of the same target alterations identified by tumor sequencing, which can be particularly useful when insufficient tumor material is available for clinical sequencing. Studies have suggested that ctDNA levels closely parallel overall tumor burden and can be used as an accurate means of monitoring treatment response and the development of resistance. On disease progression, cfDNA analysis has proved effective in identifying emergent genetic alterations that drive therapeutic resistance, which can guide subsequent therapy choice. Owing to the potential for extensive tumor heterogeneity in the context of acquired resistance, cfDNA analysis may identify multiple concurrent resistance alterations residing in distinct tumor metastases that would not be captured by a single tumor biopsy. Analysis of cfDNA has been used to monitor the clonal dynamics of multiple tumor subclones during subsequent therapy, an approach that can provide a molecular basis for mixed clinical responses to therapy.





have half-lives of days to weeks.38 Many standard clinical tumor markers also have limited sensi-tivity and specificity, whereas tumor-specific clonal alterations (i.e., those alterations present in the original tumor clone and thus present in all tumor cells) can be monitored in plasma with high sensitivity and are unique to a patient’s tumor. Some studies have suggested that ctDNA levels may actually increase transiently after the initiation of therapy, as tumor-cell death leads to increased release of ctDNA.6 However, within 1 to 2 weeks after the initiation of therapy, ctDNA levels drop dramatically in patients who have a response to therapy.

Indeed, some studies have suggested that changes in ctDNA can outperform standard tumor markers in the prediction of treatment response.38,42 Moreover, a rise in ctDNA levels may precede radiographic progression by weeks to months.36-38,46 Thus, as technologies for cfDNA monitoring become increasingly available and cost-effective, their potential to detect early evi-dence of response or progression may become important for clinical management.

Monitoring of Resistance and Tumor Heterogeneity

The clinical benefit of precision cancer therapies is limited by the eventual emergence of acquired resistance.26 In general, acquired resistance arises from one or more tumor subclones that harbor preexisting resistance alterations, which emerge under the selective pressure of therapy. Analysis of cfDNA has become an effective tool for the early detection and identification of molecular alterations leading to clinical resistance to ther-apy. A key advantage of cfDNA is the ability to capture molecular heterogeneity associated with resistance (Fig. 4). Acquired resistance is often characterized by the clonal outgrowth of mul-tiple resistant subclones in an individual pa-tient.36,39,40,43,47,48 These resistant subclones may coexist in the same lesion or in distinct meta-static sites. Thus, a single-lesion tumor biopsy may dramatically underestimate the molecular heterogeneity present, whereas the presence of multiple resistance mechanisms can often be captured by cfDNA, which is shed from tumor cells throughout the body.

Indeed, studies incorporating multiple tumor biopsies or autopsy specimens have shown that multiple unique resistance alterations frequently coexist in different metastatic sites in an indi-

vidual patient but can be detected collectively in cfDNA from a single plasma sample.39,40,49 A study involving patients with colorectal cancer after anti-EGFR antibody therapy showed that patients may harbor as many as 13 different re-sistance alterations, as detected in cfDNA, with fewer than 10% of patients having only a single resistance alteration.33 Studies comparing matched tumor biopsies and cfDNA tests at the time of disease progression have suggested that cfDNA testing may reveal additional alterations not identified by a single tumor biopsy in as many as two thirds of cases.50 Thus, targeting a sin-gle resistance mechanism on the basis of the results of a single tumor biopsy may lead to mixed clinical responses as a result of the out-growth of distinct resistant subclones not ob-tained in the biopsy specimen,39 and cfDNA test-ing may help guide treatment decisions.

In addition to emerging as an important dis-covery tool, cfDNA analysis can be applied clini-cally for the management of therapeutic resis-tance. The use of cfDNA to identify the presence of the T790M resistance mutation in EGFR-mutated lung cancer was discussed above. Other studies have shown that cfDNA analysis can identify the coexistence of T790M with other resistance altera-tions, such as MET amplification, and that pa-tients with such coexisting alterations may de-rive less benefit from subsequent therapy with third-generation EGFR inhibitors, such as osimer-tinib.44 Similarly, detection of one or more ESR1 mutations in the cfDNA of patients with estrogen-receptor–positive breast cancer predicted poorer outcome of subsequent hormonal therapy.51 Other studies show that resistance to various targeted therapies can be observed in cfDNA before the clinical detection of progression by standard imaging or tumor markers.36,37,40

Analysis of cfDNA has also been used to track the clonal dynamics of distinct resistant subclones during sequential therapy and even after the discontinuation of therapy. Siravegna et al. found that the RAS mutations emerging in patients with colorectal cancer during EGFR blockade can fall to undetectable levels in cfDNA after withdrawal of EGFR-directed therapy, and many of these patients may then benefit from reinstitution of anti-EGFR therapy.46 Integration of real-time cfDNA analysis into clinical trials and eventually into standard clinical manage-ment has the potential to become a valuable tool for precision medicine.





Detection of Postsurgical Residual Disease

One of the most transformative potential appli-cations of cfDNA analysis may be its ability to detect the presence of tumor in patients with no clinically evident disease — for example, as a screening tool in the early detection of new can-cers or for detection of relapse after surgery or adjuvant therapy. In general, the primary means by which solid tumors can be cured is surgical

resection. If any tumor cells remain postsurgi-cally, they can lead to eventual tumor relapse. In high-risk patients, adjuvant chemotherapy can reduce the risk of relapse. However, it is cur-rently not possible to determine which patients harbor residual disease immediately after sur-gery and which patients have been cured of their disease. In particular contexts, such as patients with stage II colorectal cancer who have low

Figure 4. Tumor Heterogeneity and cfDNA.

Acquired resistance to therapy is often thought to arise from rare tumor subclones that harbor preexisting resistance alterations. Although all cells may harbor the original clonal target alteration, preexisting subclonal alterations that provide a fitness advantage under the selective pressure of therapy may exist in some cells. As therapy is initiated, it may exert a cytotoxic effect on most tumor cells, but an outgrowth of resistant subpopulations may occur, leading to dynamic shifts in clonal abundance and eventual disease progression. Because preexisting resistant subclones may reside in different metastatic lesions or in different subpopulations within a single lesion, the emergence of resistance can be characterized by extensive molecular heterogeneity. Thus, a single tumor biopsy specimen that is obtained during disease progression may reveal only a subset (i.e., green only) of the resistant clones present, and subsequent therapies that are directed against this resistance mechanism only may lead to mixed clinical response and treatment failure due to outgrowth of other coexistent clones. Analysis of cfDNA has the potential to identify multiple concurrent mechanisms of resistance and can monitor clonal dynamics during therapy. Clonal alterations (present in the original tumor clone and thus in all cells) are represented in gray, and resistant subclonal alterations in other colors.

Clonal target alteration

Resistance alteration CResistance alteration DResistance alteration B

Resistance alteration A

Abu

ndan

ce o

f ctD

NA

Clo

nal a

bund

ance

Tumor biopsyTumor

First-line therapy Second-line therapy





clinical risk and no evidence of nodal or distant metastases, adjuvant therapy is not routinely given, although approximately 15% of these pa-tients may eventually have a recurrence. An ef-fective method for the detection of postoperative residual disease could spare patients who have been cured the need to undergo potentially toxic adjuvant chemotherapy or could identify those patients who have residual disease and might benefit from adjuvant therapy (Fig. 5).

In a landmark study, Diehl et al. found that cfDNA analysis just a few weeks after surgery for colorectal cancer could accurately predict those patients who had residual disease and would eventually relapse.12 First, tumor-specific muta-tions were identified in each patient by standard sequencing of resected tumor. Next, highly sen-sitive methods (BEAMing), capable of detecting mutant-allele frequencies as low as 0.01%, were used to determine whether one or more of these tumor-specific mutations persisted in that patient’s plasma when collected approximately 4 weeks after surgery. Detection of even trace levels of these mutations would provide direct evidence of residual tumor cells. In a follow-up study in-volving 178 patients with stage II colon cancer who did not receive adjuvant chemotherapy, the detection of residual tumor-specific mutations in postoperative plasma was associated with a risk of tumor recurrence that was 18 times as high as that among patients with undetectable ctDNA (P<0.001). In addition, ctDNA markedly outperformed clinical risk factors and a standard tumor marker (CEA) for predicting relapse.52

Similarly, several key studies have shown that the postoperative detection of tumor-specific mutations in cfDNA can predict residual disease and tumor relapse in breast cancer, lung cancer, and pancreatic cancer.53-56 This approach has the potential to become a critical tool in the postop-erative management of the care of patients with cancer and is currently being tested in prospec-tive clinical trials that will assess the usefulness of residual postoperative ctDNA detection to guide adjuvant chemotherapy (Fig. 5).

Early Cancer Detection

The holy grail of liquid-biopsy applications is the potential for early cancer detection through a simple blood test in otherwise healthy, asymptom-atic persons. At present, no mature technology exists to achieve that goal, but such an approach

would require a highly sensitive method — to detect trace amounts of cfDNA or other material released by precancerous lesions or early-stage cancers — and would also require high specific-ity to minimize false positive results in the large unaffected population undergoing screening.

Additional challenges complicate this undertak-ing. First, since many cancer types share com-mon mutations in genes such as KRAS, BRAF, or TP53, localizing a cancer to a specific organ after a positive liquid-biopsy test may be difficult.57 In addition, benign lesions may harbor the same mutations seen in cancer and have the potential to shed cfDNA into the circulation. Indeed, benign nevi may harbor the same BRAF muta-tions observed in advanced cancers.58 Mutations

Figure 5 (facing page). Use of cfDNA for the Detection of Residual Disease and Management of Postoperative Therapy.

After surgery with curative intent, there is currently no way to determine which patients are cured of their disease and which have subclinical residual disease that may ultimately lead to recurrence. Thus, decisions about adjuvant chemotherapy are based on clinicalrisk stratification. In the example of colon cancer, patients with stage II disease who are at low clinical risk undergo observation alone and do not receive adjuvant chemotherapy, although approximately 10 to 15% will eventually have recurrent disease. Conversely, patients with stage III disease are treated with adjuvant chemotherapy, even though more than half are cured by surgery alone. Current efforts are focused on using postoperative cfDNA to identify patients with residual disease, which may allow more precise decisions about adjuvant treatment. Briefly, tumorspecific mutations or other alterations are identified in the resected tumor specimen of each patient, and plasma specimens obtained a few weeks after surgery are assessed for the persistent presence of these alterations as direct evidence of residual tumor. With further improvement of this approach, it may be possible in future studies to assign patients who would not have received adjuvant therapy as the standard of care (i.e., with stage II disease) to receive adjuvant therapy if evidence of residual disease is detected in cfDNA. Similarly, as the sensitivity and reliability of these tests improve, it may even be possible to assign patients who would receive adjuvant therapy as the standard of care (i.e., those with stage III disease) to a lowrisk group on the basis of cfDNA testing; active surveillance could be considered in lieu of adjuvant therapy, thus sparing many patients who are cured by surgery alone the toxic effects of chemotherapy. Studies are currently ongoing (e.g., the DYNAMIC trial [Australian New Zealand Clinical Trials Registry number, ACTRN12615000381583]).





Current standard of care Potential cfDNA-guided postoperative management


Cure by surgery alone

Residual disease after surgery


Tumor-specific mutation in ctDNA

No evidence of residual disease in postoperative cfDNA

Evidence of residual disease in postoperative cfDNA

No adjuvant chemotherapy

for all


Active surveillance

Serial cfDNAmonitoring

Adjuvant chemotherapy

~10–15% haverecurrence andmay have benefited fromchemotherapy

Stage II disease and low clinical risk(~10–15% with residual disease)

Stage II disease and low clinical risk

Sequence resected tumor, identify

clonal mutations Detection of tumor-specificmutations in postoperative plasma


Active surveillance

Serial cfDNAmonitoring



for all

>50% cured by surgery alone;chemotherapy unnecessary

~30% haverecurrencedespite chemotherapy and ~15% cured by chemotherapy

Stage III disease(~45% with residual disease)

Stage III disease





detectable in cfDNA can also originate from aberrant and often benign clonal populations in the bone marrow, through a process referred to as clonal hematopoiesis of indeterminate poten-tial (CHIP). The frequency of CHIP increases exponentially with age, with a rate of more than 10% among persons older than 70 years of age.59 CHIP is thus a common source of diverse muta-tions detectable in cfDNA, posing a major chal-lenge for this approach. For this reason, methods other than simple mutation detection in cfDNA — including tumor-associated viral sequences, such as in tumors associated with Epstein–Barr virus (EBV) infection and human papillomavirus infection, and DNA methylation changes — are being explored for early detection.7,60,61

Two recent studies have illustrated the poten-tial of liquid biopsy for the early detection of cancer. Chan et al. used detection of EBV DNA in plasma to screen for nasopharyngeal carcinoma in 20,174 Chinese patients.60 A total of 309 pa-tients (1.5%) had detectable EBV DNA in plasma, as confirmed on two sequential tests, and 34 of these patients (0.17% of the original population) had nasopharyngeal carcinoma on endoscopic evaluation. Conversely, only 1 of the EBV DNA–negative patients presented with nasopharyngeal carcinoma within a year after screening. Overall, the sensitivity and specificity of this approach were 97.1% and 98.6%, respectively. Recently, Cohen et al. developed a screening method inte-grating mutation detection in cfDNA with circu-lating protein markers, termed CancerSEEK.57 In

1005 patients with nonmetastatic, clinically de-tectable tumors across eight common tumor types, the test was positive in a median of 70% of patients, with sensitivities ranging from 69 to 98%. Overall specificity was more than 99%, with only 7 of 812 healthy controls testing posi-tive. Moreover, using supervised machine learn-ing, the investigators used the profile of detected mutations and proteins to localize the cancer to its tissue of origin in 83% of cases.

Though these results are encouraging, evalu-ation in a more representative screening popula-tion of asymptomatic patients will be critical. Although further improvement of liquid-biopsy screening approaches and validation in prospec-tive clinical trials are needed, cfDNA analysis as an early-detection tool offers a potentially trans-formative advance for cancer medicine.

Summ a r y a nd Fu t ur e Dir ec tions

Analysis of cfDNA has rapidly emerged as a tech-nology with many promising clinical applications in oncology. Effective clinical integration of cfDNA analysis will require a careful understanding of the advantages and limitations of this approach for proper interpretation of results to guide clini-cal decision making. Although further prospec-tive study is needed, cfDNA analysis harbors the potential to have a transformative effect on can-cer medicine.

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