Review

Clinical application of circulating tumour DNA in colorectal

cancer
Matthew Loft, Yat Hang To, Peter Gibbs, Jeanne Tie

Liquid biopsies that detect circulating tumour DNA (ctDNA) have the potential to revolutionise the personalised Lancet Gastroenterol Hepatol
management of colorectal cancer. For patients with early-stage disease, emerging clinical applications include the 2023

assessment of molecular residual disease after surgery, the monitoring of adjuvant chemotherapy efficacy, and early
detection of recurrence during surveillance. In the advanced disease setting, data highlight the potential of ctDNA
levels as a prognostic marker and as an early indicator of treatment response. ctDNA assessment can complement
standard tissue-based testing for molecular characterisation, with the added ability to monitor emerging mutations
under the selective pressure of targeted therapy. Here we provide an overview of the evidence supporting the use of
ctDNA in colorectal cancer, the studies underway to address some of the outstanding questions, and the barriers to
widespread clinical uptake.
Introduction
The term cell-free DNA (cfDNA) refers to genetic
fragments in the form of nucleic acid chains found in
many bodily fluids. The presence of cfDNA in the blood
was initially described in 1948 by Mandel and Metais,1
with reports in 1977 of increased serum concentrations in
patients with cancer.2 In patients with malignancy, a
proportion of cfDNA is derived from cancer cells and is
referred to as circulating tumour DNA (ctDNA; figure 1).
Technological advances have addressed many of the
initial issues related to sensitivity and specificity, making
ctDNA a promising clinical tool across cancer types and
stages (figure 2).
In contrast to many standard blood-based biomarkers,
such as carcinoembryonic antigen, ctDNA has a half-life
of only 2 h,3,4 conferring a distinct advantage for ctDNA
as a dynamic biomarker of tumour burden. This shorter
half-life, along with the considerably superior sensitivity
and specificity of ctDNA as a cancer biomarker, and
the ability to characterise tumour-specific genetic and
epigenetic abnormalities, extends the potential clinical
utility of ctDNA far beyond that of traditional markers.
Many ctDNA assays are commercially available, with
many more in development. Beyond the fundamental
differences between a tumour-agnostic (plasma analysis
only) and a tumour-informed (initial genomic profiling of
a patient’s tumour tissue to identify patient-specific
somatic mutations that could be monitored in the plasma)
approach, ctDNA detection methods vary considerably.
Notably, plasma only ctDNA assays developed for
metastatic disease genotyping, typically with a limit of
detection of 0·5–1%, are not suitable for the assessment
of molecular residual disease (MRD).5,6 Also, the clinical
utility of each distinct ctDNA test should be independently
validated through well conducted clinical trials. Further
consideration of ctDNA methods is beyond the scope of
this Review, but for each result reported we will specify
the testing platform used. The potential of ctDNA in
population-based cancer screening is another exciting
application, but is also beyond the scope of this review,
which focuses on patients with colorectal cancer.
Published Online
July 24, 2023
https://doi.org/10.1016/
S2468-1253(23)00146-2
Division of Personalised
Oncology, Walter and Eliza Hall
Institute of Medical Research,
Parkville, VIC, Australia
(M Loft MBBS, Y H To MBBS,
Prof P Gibbs MD, Prof J Tie MD);
Curable disease Department of Medical
MRD detection and monitoring efficacy of adjuvant Biology, University of
Melbourne, Parkville, VIC,
treatment
Australia (M Loft, Y H To,
MRD refers to occult tumour remaining after surgery Prof P Gibbs, Prof J Tie);
with curative intent, typically at a distant site, where the Department of Medical
tumour bulk is below the resolution of radiological Oncology, Western Health,
Footscray, VIC, Australia
detection but measurable by molecular techniques. The
(M Loft, Prof P Gibbs);
possibility of using ctDNA to detect MRD was first Department of Medical
described by Diehl and colleagues4 in a study of Oncology, Peter MacCallum
18 patients with resected colorectal cancer liver Cancer Centre, Parkville, VIC,
Australia (Y H To, Prof J Tie);
metastases, where 15 (94%) of 16 patients with a positive Sir Peter MacCallum
postoperative ctDNA had a disease recurrence, and no Department of Oncology,
recurrences were observed in the two patients with University of Melbourne,
negative postoperative ctDNA.4 Observational studies, Parkville, VIC, Australia
(Prof J Tie)
using various fit for purpose ctDNA assays, have
Correspondence to:
repeatedly shown that a high proportion of postoperative
Prof Jeanne Tie, Sir Peter
patients with detectable ctDNA after surgery for colorectal MacCallum Department of
cancer (approaching 100% in some series) will later Oncology, University of
develop clinical recurrence if they do not receive adjuvant Melbourne, Parkville 3000, VIC,
Australia
chemotherapy.7–10 Completed observational studies are jeanne.tie@petermac.org
presented in table 1. The clinical utility of preoperative
ctDNA assessment for colorectal cancer remains
unproven; most series9,12,21 report a high rate of pre­
operative ctDNA positivity and no statistical prognostic
significance, probably because the dominant source of
ctDNA was the primary tumour. By contrast, few patients
remain ctDNA positive post-operatively,12,21 and ctDNA-
positivity is highly correlated with recurrence risk,
independent of any preoperative ctDNA result.
Patients are currently not monitored during the
3–6 months of adjuvant therapy, with carcinoembryonic
antigen concentrations potentially being falsely elevated
by chemotherapy administration.22 Currently, there are
no biomarkers that can indicate the success (or failure) of
adjuvant treatment in any individual patient. When
adjuvant chemotherapy is administered ctDNA can
become undetectable, correlating with a reduced risk of
recurrence.21,23 Emerging evidence shows that ctDNA is
not only useful as a prognostic biomarker post-surgery,
but that serial ctDNA analysis holds promise as an early
real-time biomarker of adjuvant treatment benefit.

www.thelancet.com/gastrohep Published online July 24, 2023 https://doi.org/10.1016/S2468-1253(23)00146-2 1

 Review
Tumour tissue
Apoptosis
or necrosis
ctDNA
Analysis of ctDNA to detect cancer-related changes
A T G C G A C T
Point mutations Translocations and fusions
Me1
Me1
Chromosomal abnormalities Epigenetic modifications (eg, methylation)
Figure 1: The origin of ctDNA, cancer-related changes in ctDNA, and factors affecting ctDNA concentrations and detection
cfDNA=cell free DNA. CTC=circulating tumour cells. ctDNA=circulating tumour DNA. *The greater-than sign represents differences in ctDNA concentrations by
various factors.
Stage I–III colon cancer
The likelihood of clinical occult disease increases with
advancing tumour stage and the presence of adverse
clinicopathological factors. For stage III colon cancer,
historical data suggest clinical occult disease that can
cause subsequent recurrence is present in around 50%
of patients.24,25 Adjuvant chemotherapy is standard of care
because it can eradicate clinical occult disease in a
proportion of these patients, reducing recurrence risk
and improving survival.24–27 However, adjuvant chemo­
therapy provides no benefit in the 50% of patients
without clinical occult disease and will only prevent
recurrence in about a third of those with clinical occult
disease.26,27 Ultimately, few patients benefit from adjuvant
treatment.
Whether there is a benefit for adjuvant chemotherapy
in patients with stage II colorectal cancer remains
contentious, even for patients with adverse features
associated with a high risk of recurrence, with no clear
overall survival benefit shown in a population with low
overall recurrence risk.28 For patients with low-risk
stage II cancers, and even for patients with stage I cancers,
2 www.thelancet.com/gastrohep Published online July 24, 2023 https://doi.org/10.1016/S2468-1253(23)00146-2
Healthy tissue
Factors affecting ctDNA concentrations and
(eg, haematopoietic cells)
detection:
• Metastatic disease sites (liver > lung,
peritoneum, and bone)*
• Cancer burden
• Disease status (eg, before or after surgery,
responding or stable vs progressive disease)
• Types of variant (clonal > sub-clonal)*
• Timing of blood sampling in relation to
local or systemic treatment
• Non-tumour-related factors
(eg, inflammation, infection, and exercise)
cfDNA
CTC
Amplifications and deletions
DNA fragment lengths
a small number will have clincial occult disease and have
a recurrence. Potentially any patient with clinical occult
disease, regardless of stage, could benefit from adjuvant
chemotherapy.
Current studies in stage III colon cancer are exploring
treatment escalation in patients with detectable ctDNA
and de-escalation in those without detectable ctDNA. For
stage II colon cancer, the focus is on avoiding adjuvant
chemotherapy for patients in whom adjuvant chemo­
therapy is unlikely to be beneficial (no detectable ctDNA)
and exploring treatment benefit in those who are ctDNA
positive. Patients with stage I disease are not the focus of
current interventional trials.
In an initial observational study of 230 patients with
resected stage II colorectal cancer, ctDNA detection (using
the tumour-informed Safe Sequencing System [Safe-SeqS]
assay) was associated with a markedly reduced recurrence-
free survival, with an estimated 3-year recurrence-free
survival of 0% for ctDNA-positive patients versus 90% for
ctDNA-negative patients not receiving adjuvant
chemotherapy (hazard ratio [HR] 18, 95% CI 8–40;
p<0·0001).7 The same authors reported on an observational
 Review

Curable disease Molecular residual disease Adjuvant treatment response Surveillance

ctDNA testing Serial ctDNA testing
ctDNA ctDNA
Positive Negative Positive Negative

Starting Escalate adjuvant De-escalate or avoid Starting Further Surveillance Improved lead time vs CEA
3–4 weeks after treatment adjuvant treatment 2–8 weeks after treatment? or imaging for progression,
surgical completion of all with opportunity to increase
resection with Refined patient selection in addition to curable-intent Opportunity to escalate or consider detection of potentially
Definitive treatment curable intent clinicopathological parameters treatment second-line treatment resectable or ablatable disease

Non-curable Prognostication Treatment selection Response monitoring Personalised

disease At diagnosis Refine upfront prognosis prediction Single agent Identify responses as treatment
in addition to conventional markers Doublet early as second cycle Switch
such as BRAF status Triplet De-escalate
Anti-VEGF Rechallenge
Anti-EGFR Novel therapies
Immunotherapy Trials
Genotyping Anti-BRAF Resistance monitoring
Detection influenced Starting
Alternative to tumour tissue Anti-HER2 Non-invasive and
by disease site: 2–4 weeks
genotyping, especially when tissue Other targets dynamic monitoring
↑Liver after
biopsies are suboptimal or rapid of emerging resistance
↓Lung only treatment
↓Peritoneal only turnaround time is crucial
initiation
Palliative treatment ↓Nodal only

Figure 2: Potential clinical applications of ctDNA assessment for management of colorectal cancer with curative intent (stage I—III or resectable oligometastatic stage IV) or palliative intent
(stage IV)
ctDNA=circulating tumour DNA. CTC=circulating tumour cells.

study of 96 patients with stage III colon cancer that
measured ctDNA using the same assay.8 All patients
underwent surgery and received standard adjuvant
chemotherapy. A positive postoperative ctDNA result was
associated with an inferior 3-year recurrence-free survival
compared with a negative postoperative ctDNA result (HR
3·8, 95% CI 2·4–21·0; p<0·001) Additionally, patients with
detectable ctDNA following chemotherapy similarly had
an inferior 3-year recurrence-free survival (30% vs 77%,
HR 6·8, 95% CI 11·0–157·0; p<0·001). The median time
to recurrence from the post-chemotherapy blood draw was
short at 51 days (range, 9–470 days).8 A separate
observational study of 130 patients with
stage I–III colorectal cancer reported that patients with
positive ctDNA (tumour-informed Signatera assay) after
definitive therapy during surveillance were at a higher risk
of disease recurrence than were ctDNA-negative patients
(HR 43·5, 95% CI 9·8–193·5; p<0·001).9 A later pooled
analysis29 of three cohort studies of 485 patients with stage
II–III colorectal cancer found that 5-year recurrence-free
survival was significantly lower in postoperative patients
testing positive for ctDNA (38·6%, 95% CI
27·7–53·9) compared with those testing negative
(85·5%, 81·8–89·3; HR 7·56, 95% CI 4·85–11·79;
p<0·001) 5-year overall survival was also significantly lower
in patients with detectable (64·6%, 95% CI 47·8–87·4)
versus undetectable (89·4%, 85·9–93·1) postoperative
ctDNA (HR 3·77, 95% CI 1·96–7·25; p<0·001). ctDNA was
a more accurate predictor of recurrence than standard
individual clinicopathological factors.29
Research by Henriksen and colleagues20 assessed
140 patients with stage III colorectal cancer with serial
ctDNA measurements (tumour-informed Signatera assay)
both before and after adjuvant chemotherapy. Post­
operative ctDNA was detected in 20 (14%) patients, of
whom 18 (90%) received adjuvant chemotherapy. Of the
13 patients with assessable ctDNA after adjuvant chemo­
therapy, only three (23%) showed durable ctDNA
clearance and did not relapse at 3 years. However, all ten
patients who had no or only transient ctDNA clearance
relapsed. Correspondingly, persistently detectable ctDNA
after adjuvant chemotherapy was associated with a
significantly lower recurrence-free survival (HR 50·76,
95% CI 15·4–167·0; p<0·001).20 Other observational
studies have shown similar results, with the ability of
adjuvant chemotherapy to convert postoperative ctDNA-
positivity into ctDNA-negativity after chemotherapy in
patients with early-stage colorectal cancer ranging from
17% to 50%.8,9,18
The large observational GALAXY trial reported data on
315 patients with stage II colorectal cancer and 397 patients
with stage III colorectal cancer and analysed ctDNA via
the Signatera assay.21 Postoperative ctDNA detection was
associated with inferior 18-month disease-free survival
compared with no postoperative ctDNA detection for both
stage II (34·7%, 95% CI 15·7–54·7 vs 94·0%, 90·6–96·2;
HR 18·0, 95% CI 8·7–35·0; p<0·0001) and stage III
(49·0%, 38·3–58·9 vs 92·4%, 88·6–95·0; HR 9·6,
5·8–16·0; p<0·0001). Additionally, ctDNA positivity
4 weeks after surgery was the most significant independent

www.thelancet.com/gastrohep Published online July 24, 2023 https://doi.org/10.1016/S2468-1253(23)00146-2 3

 Review

Patient population Sample size ctDNA assay ctDNA sampling Neoadjuvant or Key results
(approach, number timepoints adjuvant
of variants tracked chemotherapy
in plasma) given
Tie et al, 20167 Stage II 230 Safe-SeqS (TI, 1) 4–10 weeks Adjuvant (23%) In patients not treated with adjuvant therapy, presence of ctDNA
postoperative; after surgery was associated with an inferior 3-year RFS
serial follow-up (0% vs 90%; HR 18 [95% CI 8–40]; p<0·0001); ctDNA detection
for 24 months during surveillance identified recurrence with a median lead time
of 5·5 months
Reinert et al, Stage I–III 125 (5 with Signatera (TI, 16) Preoperative; Adjuvant (62%) Preoperative ctDNA detected in 89% of all patients;
20199 stage I; 39 with postoperative postoperative ctDNA-positive patients were significantly more
stage II; 81 with day 30 likely to have disease recurrence than ctDNA-negative patients
stage III) (HR 7·2 [95% CI 2·7–19·0]; p<0·001); ctDNA detection during
surveillance identified recurrence with a median lead time of
8·7 months
Murray et al, Stage I–V 172 (93 with Triplex real-time Within NR Patients with any feature suggestive of residual disease (close
201811 stage I or II; qPCR assay (TA, 12 months resection margins, apical node involved, distant metastases)
79 with stage III 2 methylated postoperative were 5·3-times more likely to be ctDNA-positive (p=0·008);
or IV) markers) postoperative ctDNA-positivity associated with an increased
recurrence risk (HR 3·8; p=0·004).
Tie et al, 201912 Locally advanced 159 Safe-SeqS (TI, 1) Pre-CRT; Neoadjuvant (CRT; Pre-treatment ctDNA detected in 77%; no difference in RFS
rectal cancer 4–6 weeks after 100%); adjuvant between detected and undetected (HR 1·1; p=0·82); 3-year
CRT; 4–10 weeks (36%) RFS was significantly worse if ctDNA was positive post-CRT
postoperative (HR 6·6 [95% CI 2·6–17·0]; p<0·001) or postoperatively (HR 13·0
[5·5–31·0]; p<0·001); 3-year RFS was 33% (95% CI 16–72) for
postoperative ctDNA-positive patients and 87% (79–95) for
postoperative ctDNA-negative patients
Mason et al, Stage IV post-liver 63 Guardant360 (TA, Postoperative, Neoadjuvant (87%); 2-year OS from date of liver resection was significantly worse for
202113 metastasectomy 70 cancer-related median adjuvant (59%) ctDNA-positive vs ctDNA-negative patients (70% vs 100%;
genes) 13 months p=0·005)
Tie et al, 202114 Stage IV CRC with 54 Safe-SeqS (TI, 1) Pre-treatment Neoadjuvant (43%); Pre-treatment ctDNA detected in 85%; no difference in RFS
upfront resectable (surgery or adjuvant (78%) (HR 4·72; p=0·13) or OS (HR 1·39; p=0·66) between detected and
liver metastases chemotherapy); undetected; median 41-fold (p<0·001) decrease in ctDNA mutant
before each cycle allele fraction with neoadjuvant chemotherapy, but ctDNA
of neoadjuvant clearance was not associated with improved RFS; RFS was
treatment; significantly worse if ctDNA was positive postoperatively
4–10 weeks (HR 6·3 [95% CI 2·6–15·2]; p<0·001), as was OS (HR 4·2 [1·5–11·8];
postoperative; p<0·001); RFS after all treatment (surgery with or without adjuvant
after adjuvant chemotherapy) was significantly better in those with a post-
therapy treatment ctDNA-negative result (0% vs 76%; HR 14·9; p<0·001).
Lee et al, 202115 Stage IV post 58 (38 liver; Ultra-deep targeted Preoperative; Neoadjuvant (47%); Preoperative ctDNA detected in 24%; detection rate of ctDNA
metastasectomy 25 lung; sequencing in 50 3–4 weeks adjuvant (NR) was higher in patients with liver metastases (p=0·0045) or
4 peritoneum; cancer-related genes postoperative tumours measuring 1 cm or more (p=0·018); ctDNA was less
2 lymph node) (TI, NR) likely to be detected after a complete or partial response to
chemotherapy.
Chen et al, Stage II–III 240 (112 with Geneseeq Prime (TI, Preoperative; Adjuvant (73%) Preoperative ctDNA detected in 64%; preoperative detection
202116 stage II; 128 with median 6) once between associated with reduced RFS (HR 5·7; p=0·004); postoperative
stage III) days 3–7 ctDNA-positive patients had a much higher recurrence risk
postoperative; (HR 10·98; p<0·001); ctDNA detection during surveillance
serial follow-up identified recurrence with an accuracy of 92% and mean lead
for 24 months time of 5 months
Loupakis et al, Stage IV post-liver 112 Signatera (TI, 16) Postoperative, Neoadjuvant 96·7% of ctDNA-positive patients had progression, with ctDNA-
202117 metastasectomy median 27 days (49%); adjuvant positivity associated with inferior OS (HR 16·0 [95% CI 3·9–68·0];
(44%) p<0·001); in multivariate analysis, ctDNA-positivity was the
most significant prognostic factor for DFS (HR 5·8 [3·34–10·0];
p<0·001); for ctDNA-positive patients that progressed, the
median lead time was 3·2 months
Parikh et al, Stage I–IV 84 (8 with stage Guardant Reveal Preoperative; Neoadjuvant (45%); ctDNA-positivity after definitive treatment (either surgery only or
202118 I; 20 with stage II; (TA, 40 genes plus 4 weeks adjuvant (55%) completion of adjuvant therapy) predicted relapse in 15 (100%)
40 with stage III; methylation panel) postoperative; of 15 patients; 12 (25%) of 49 patients who were ctDNA-negative
16 with stage IV) 4 weeks after after treatment had recurrence
adjuvant
treatment
(Table 1 continues on next page)

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Patient population Sample size
(Continued from previous page)
Taieb et al, Stage III 1017
202119
Henriksen et al, Stage III 160
202220
Kotani et al, Stage I–IV 1039 (97 with
202321 stage I; 315 with
stage II; 397 with
stage III; 230 with
stage IV)
CRC=colorectal cancer. ctDNA=circulating tumour DNA. ddPCR=digital droplet PCR. DFS=disease-free survival. HR=hazard ratio. NR=not reported. OS=overall survival. qPCR=quantitative PCR. RFS=recurrence-
free survival. Safe-SeqS=Safe-Sequencing System. TI=tumour-informed. TA=tumour-agnostic. CRT=chemoradiation.*Including non-metastatic studies if the sample size was 100 or more and oligometastatic
studies if the sample size was 50 or more.
Table 1: Selected observational ctDNA studies assessing molecular residual disease in curatively resected colorectal cancer*
prognostic factor associated with increased risk of
recurrence (HR 10·82, 95% CI 7·07–16·60; p<0·001).21
Among patients with high-risk stage II disease with
adverse risk features (eg, T4 disease, perforation,
obstruction, or lympho­vascular invasion) and all patients
with stage III disease who had detectable ctDNA after
surgery, those who received any adjuvant chemotherapy
had significantly higher 18-month disease-free survival
than those who did not (61·6%, 95% CI 49·0–71·9 vs
22·0%, 10·9–35·5; HR 6·59, 95% CI 3·53–12·30;
p<0·0001), suggesting benefit from adjuvant
chemotherapy in ctDNA-positive patients.21
In a subset of the GALAXY trial consisting of 182 patients
with stage I–III disease and resectable stage IV disease
who had positive ctDNA 4 weeks after surgery and ctDNA
clearance data available, patients who received adjuvant
chemotherapy had a higher ctDNA clearance rate
(63 [68%] of 92) than those who did not (11 [12%] of 90;
HR 8·5, 95% CI 4·2–17·3; p<0·0001).21 Not achieving
ctDNA clearance on adjuvant chemo­ therapy was
associated with inferior disease-free survival compared
with achieving ctDNA clearance (HR 11·0, 5·2–23·0;
p<0·0001). Not receiving adjuvant chemo­ therapy was
found to be the most significant negative prognostic factor
in multivariate analysis for MRD-positive, ctDNA-positive
patients across stages II–IV of disease.21 Preliminary data
from both the GALAXY study and an Australian pooled
analysis29 suggest that postoperative ctDNA concentrations
(MRD) might impact the efficacy of adjuvant treatment.
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Review
ctDNA assay ctDNA sampling Neoadjuvant or Key results
(approach, number timepoints adjuvant
of variants tracked chemotherapy
in plasma) given
Multiplex ddPCR Postoperative Adjuvant (100%) 3-year DFS was 66% for ctDNA-positive patients and 77% for
(TA, 2 methylation ctDNA-negative patients (p=0·015), with ctDNA an independent
markers) prognostic marker for DFS (adjusted HR 1·55 [95% CI 1·13–2·12];
p=0·006) and OS (HR 1·65 [1·12–2·43]; p=0·011)
Signatera (TI, 16) Preoperative; Adjuvant (87%) Preoperative ctDNA detected in 91%; ctDNA-positive patients
postoperative had a much higher recurrence risk both postoperatively (HR 7·0
(within 8 weeks [95% CI 3·7–13·5]; p<0·001) and after adjuvant treatment (HR 51
of surgery, [15·4–167]; p<0·001); ctDNA detection during surveillance
median 2 weeks); identified recurrence with a median lead time of 9·8 months
after adjuvant
treatment
(within
3 months)
Signatera (TI, 16) Preoperative; Adjuvant (45% of Preoperative ctDNA detected in 91% but not associated with RFS
4, 12, 24, 36, 48, high-risk stage II (HR 0·89; p=0·62); postoperative ctDNA-positivity was
and 72 weeks and stage III) associated with increased recurrence risk (HR 10·0 [95% CI
postoperative 7·7–14·0]; p<0·0001); stage II (HR 18·0 [8·7–35·0]; p<0·0001);
stage III (HR 9·6 [5·8–16·0]; p<0·001); stage IV (HR 5·9 [3·9–9·0];
p<0·0001); postoperative ctDNA-positivity identified patients
with stage II or III CRC who derived benefit from adjuvant
chemotherapy (HR 6·6 [3·53–12·3]; p<0·0001)
To our knowledge, the DYNAMIC trial was the first
randomised controlled trial to report on a ctDNA-guided
approach to the initiation of adjuvant chemotherapy in
patients with stage II colon cancer.30 In the trial,
455 patients were randomly assigned (2:1) to have
adjuvant treatment decisions guided by ctDNA results
(using the tumour-informed Safe-SeqS assay) or by
standard clinicopathological features, at the discretion of
the treating clinician. Patients who were ctDNA-positive
at 4 weeks or 7 weeks after surgery received adjuvant
chemotherapy; ctDNA-negative patients were not treated.
Fewer patients in the ctDNA-guided group than in
the standard group received adjuvant chemotherapy
(45 [15%] of 294 patients vs 41 [28%] of 147 patients; relative
risk 1·82, 95% CI 1·25–2·65), without compromising
2-year recurrence-free survival (93·5% vs 92·4%). In the
ctDNA-guided group, 3-year recurrence-free survival was
92·5% among ctDNA-negative patients and 86·4%
among ctDNA-positive patients treated with adjuvant
chemotherapy, suggesting a treatment benefit in this
patient subset. A prespecified analysis of the DYNAMIC
study presented at the European Society for Medical
Oncology 2022 Congress found a ctDNA clearance rate
of 87% (n=34) at 4 weeks after completing adjuvant
chemotherapy in the 39 patients with positive post­
operative ctDNA for whom post-adjuvant chemotherapy
samples were available.23 Patients who had ctDNA
clearance had a far superior outcome to those who had
persistently detectable ctDNA, with an estimated 1-year
 Review
recurrence-free survival from the end of treatment of
100% versus 20% (HR 55·7, 95% CI 5·8–532·2;
p<0·001).23 To our knowledge, the DYNAMIC study
represents the first evidence from a randomised trial
that a ctDNA guided approach to adjuvant treatment
selection can reduce adjuvant chemotherapy admin­
istration without compromising survival outcomes.
Locally advanced rectal cancer
The treatment approach for locally advanced rectal
cancer is rapidly evolving, with data supporting the use
of total neoadjuvant therapy (the delivery of both
systemic chemotherapy and chemoradiotherapy before
surgery).31,32 Patients who have a clinical complete
response to neoadjuvant therapy (per endoscopy and
clinical imaging) are increasingly being offered an
organ preservation approach as opposed to proceeding
with surgery, although mature survival data are absent
and there are no reliable biomarkers to inform this
decision.
Most ctDNA data are from observational studies of
patients undergoing standard long-course chemo­
radiation followed by surgery. In the largest series to date
of 159 patients with locally advanced rectal cancer, 3-year
recurrence-free survival was significantly lower when
ctDNA (using the Safe-SeqS assay) was detectable
(50%, 95% CI 28–88) compared with undetectable
(85%, 79–93) following long-course neoadjuvant chemo­
radiation (HR 6·6, 95% CI 2·6–17·0; p<0·001) and also
post-surgery (33%, 16–72 vs 87%, 79–95; HR 13·0,
5·5–31·0; p<0·001).12 Post-surgery ctDNA detection
predicted recurrence independently of conventional
clinicopathological risk factors, and ctDNA-positive
patients not treated with adjuvant chemotherapy had
the highest recurrence risk.12 Patients who were
ctDNA-negative after neoadjuvant chemoradiation had
numerically higher rates of pathological complete
response than patients who were ctDNA-positive after
neoadjuvant chemoradiation (28 [21%] of 132 patients vs
one [8%] of 12; p=0·46). In an analysis of 60 patients with
locally advanced rectal cancer treated on the STELLAR
trial, by use of a tumour-informed approach tracking up
to 22 variants, detectable ctDNA following neoadjuvant
therapy (pre-surgery) was associated with an increased
risk of recurrence versus undetectable ctDNA (HR 27·38,
95% CI 8·61–87·06; p<0·0001), but the absence of ctDNA
was not associated with pathological complete response.33
Notably, patients who had clearance of ctDNA (a ratio
of post-neoadjuvant therapy ctDNA fraction to baseline
ctDNA fraction of <2%) were more likely to have either a
pathological complete response or a clinical complete
response than were those who had ctDNA persistence
(seven [44%] of 16 patients vs one [5%] of 19 patients;
p=0·013).
The GEMCAD 1402 trial analysed 72 patients
undergoing total neoadjuvant therapy for locally
advanced rectal cancer, detecting ctDNA (Guardant
6 www.thelancet.com/gastrohep Published online July 24, 2023 https://doi.org/10.1016/S2468-1253(23)00146-2
Reveal assay) in 43 (83%) of 52 baseline samples and in
seven (16%) of 45 samples following total neoadjuvant
therapy.34 Although no association was found between
ctDNA status before and after total neoadjuvant therapy
and pathological complete response, ctDNA positivity
after total neoadjuvant therapy but before surgery was
associated with increased recurrence risk (HR 4·0,
95% CI 1·0–16·2; p=0·033) and reduced overall survival
(HR 23·0, 2·4–212·0; p<0·0001).34 A study by Wang and
colleagues35 combined ctDNA assessment (tumour-
agnostic next-generation sequencing panel) with MRI,
showing that patients with locally advanced rectal
cancer who cleared their ctDNA following neoadjuvant
chemoradiotherapy had a low probability of residual
tumour (odds ratio 0·11, 95% CI 0·01–0·60; p=0·04).
Conversely, detectable ctDNA after neoadjuvant therapy
was associated with decreased recurrence-free survival
(HR 9·29, 95% CI 3·74–23·10; p<0·001) and those with
detectable ctDNA and high-risk MRI features of the
primary tumour at baseline had the highest recurrence
risk (HR 90·29, 17·01–479·26; p<0·001).35 Overall, these
data suggest that ctDNA clearance after chemoradiation
or total neoadjuvant therapy is a positive prognostic
marker for distant recurrence but does not predict patho­
logical complete response. A potential application might
be to identify those who should not be recommended
for organ preservation if ctDNA does not clear after
neoadjuvant therapy.
Neoadjuvant immunotherapy is an emerging strategy
in the small number of patients with colorectal cancers
with deficient mismatch repair.36 In a study of
35 patients (27 with colorectal cancer), Ludford and
colleagues37 reported pathological complete response in
11 (79%) of 14 patients with deficient mismatch repair
and microsatellite instability high colorectal cancer
who received neoadjuvant pembrolizumab. Among
19 patients in the overall study with serial ctDNA samples
at baseline and after 3 weeks of pembrolizumab (assessed
with a 70-gene liquid biopsy panel), 15 (79%) had a
decrease in serial ctDNA measurements. Three (75%)
of four patients with increasing or stable ctDNA
concentrations between baseline and after 3 weeks of
treatment showed progression, suggesting that early
serial ctDNA assessment might identify patients with
immunotherapy-resistant tumours.
Resectable colorectal liver metastases
For patients with metastatic colorectal cancer who present
with liver-only disease, surgery with curative intent might
be possible either initially or after a response to
neoadjuvant therapy. The utility of adjuvant chemo­
therapy after surgery remains uncertain, irrespective of
whether neoadjuvant therapy is given.38
The potential use of ctDNA analysis at different
timepoints in the management of oligometastatic liver
disease has been explored by several groups. Tie and
colleagues14 analysed serial samples from 54 patients

with upfront, resectable colorectal cancer liver
metastases, of whom 23 (43%) received neoadjuvant
chemotherapy, 42 (78%) received adjuvant chemotherapy,
and 16 (30%) received both. ctDNA (using the Safe-SeqS
assay) was detectable in 46 (85%) of 54 patients prior to
any treatment and in 12 (24%) of 49 patients after
surgery. Neoadjuvant chemotherapy resulted in a
median 41-fold decrease (19·10–87·3, p<0·001) in ctDNA
mutant allele fraction, but ctDNA clearance during
neoadjuvant therapy was not associated with improved
recurrence-free survival. Patients with detectable versus
undetectable ctDNA after surgery had a significantly
lower recurrence-free survival (HR 6·3, 95% CI
2·6–15·2; p<0·001) and overall survival (HR 4·2,
1·5–11·8; p<0·001). Among the 11 (27%) patients
with detectable with detectable postoperative ctDNA and
available post-adjuvant chemotherapy samples, 5-year
recurrence-free survival was longer for patients who
cleared their ctDNA after adjuvant chemotherapy than
for those with persistently positive ctDNA (66·7% vs
0·0%; HR 7·87, 0·95–63·70; p=0·056).
Other studies have also consistently shown inferior
progression-free survival associated with ctDNA
detection after surgery.21,39,40 Loupakis and colleagues17
analysed a cohort of 112 patients with metastatic
colorectal cancer who had undergone metastatic
resection with curative intent, detecting ctDNA
(Signatera assay) in 61 (54%) patients, 59 (97%) of whom
ultimately had progression. Postoperative ctDNA
detection was associated with inferior recurrence-free
survival (HR 5·8, 95% CI 3·5–9·7; p<0·001), as well as
overall survival (HR 16·0, 3·9–68·0; p<0·001).17 In
multivariate analysis, MRD status based on ctDNA result
was the most significant prognostic factor associated
with disease-free survival (HR 5·78, 3·34–10·00; p<0·001).
In a prospective ancillary study to the Prodige-14 trial
(droplet digital PCR assay for KRAS mutation), in which
patients with potentially resectable colorectal cancer liver
metastases were treated with first-line triplet or doublet
chemotherapy combined with targeted therapy, those
who had clearance of ctDNA after 4 weeks of preoperative
treatment had higher R0/R1 resection rates compared
with those who did not have clearance (11 [85%] of
13 vs eight [36%] of 22; p=0·01), but ctDNA clearance did
not correlate with significantly improved survival.41
Molecular recurrence monitoring
Following completion of treatment with curative intent
for colorectal cancer, patients are followed up with
serial carcinoembryonic antigen tests and CT imaging,
with the intent of detecting any recurrence before
clinical symptoms emerge, the expectation being that
earlier intervention could improve survival outcomes.
Multiple observational studies have shown that during
this surveillance period, ctDNA might become
detectable months before radiological recurrence or a
carcino­embryonic antigen increase (median lead time
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Review
1·7–11·5 months).7–9,20,42–45 The range of lead times
reported probably reflect several factors including the
variable timing and frequency of ctDNA sampling,
imaging protocols, and assay sensitivities. Prospective
studies showing the clinical effect of adding ctDNA
analysis to standard surveillance are needed before
clinical adoption.
Non-curable disease
ctDNA concentrations are elevated at presentation in the
great majority of patients with metastatic colorectal
cancer,46 but it should be noted that the sites of metastatic
disease can affect ctDNA burden and detection rate.47–49
Bando and colleagues48 showed that in patients with
metastatic colorectal cancer with single organ metastatic
disease, the ctDNA fraction (expressed as the maximum
variant allelic fraction by Guardant360 assay) was
significantly lower in patients with lung-only or peritoneal-
only disease compared with those with liver metastases.
Prognostication
An elevated ctDNA at diagnosis of metastatic colorectal
cancer is associated with a poorer prognosis. Research by
Manca and colleagues49 used variant allele frequency to
quantify a high or low ctDNA burden in patients with
RAS wild-type (RASWT) metastatic colorectal cancer from
the Valentino study. Manca and colleagues showed that a
higher pre-treatment ctDNA was associated with poorer
progression-free survival and markedly shorter overall
survival than lower pre-treatment ctDNA (median overall
survival 21·8 months vs 36·5 months; HR 1·82, 95% CI
1·20–2·76, p=0·005).49 Carcinoembryonic antigen and
radiological measurement of initial disease burden were
not prognostic. Other studies have identified that high
pre-treatment ctDNA concentrations are independently
associated with poorer survival.50,51 However, there
remains no standard definition of what constitutes a
high ctDNA, or guidance as to how this result should
inform therapy.
Tumour genotyping at diagnosis
Anti-EGFR therapy, either in combination with chemo­
therapy or as mono­therapy, is an important part of the
treatment for left-sided RASWT metastatic colorectal
cancer.52,53 Multiple studies have shown excellent con­
cordance between RAS status in plasma and in tumour
tissue, consistently reporting agreement rates exceeding
90%.54–61 However, lower concordance rates were noted in
patients with peritoneal-only (14 [56%] of 25) and lung-
only (29 [66%] of 44) metastases, in whom RASmut is less
likely to be detectable in the circulation.48 Importantly,
progression-free survival on anti-EGFR therapy appeared
similar whether RASWT status was determined by tissue
analysis or by ctDNA.55 Some studies suggest that low-
level RAS mutations, measured either in tissue or ctDNA,
might not exclude a benefit from an EGFR inhibitor, but a
threshold remains to be confirmed.61
 Review
Data suggest that the benefit of first-line anti-EGFR
therapy is confined to patients with metastatic
colorectal cancer with a left-sided RASWT primary
tumour. However, a ctDNA biomarker sub-study of
PARADIGM (a randomised phase 3 trial comparing
FOLFOX [leucovorin, fluorouracil, and oxaliplatin] plus
panitumumab vs FOLFOX plus bevacizumab as first-
line treatment in patients with tissue-confirmed RASWT
metastatic colorectal cancer) found that hyperselection
of patients on the basis of the absence for genomic
alterations in cfDNA in KRAS, NRAS, BRAFV600E, PTEN,
the ectodomain (ECD) of EGFR, HER2 (also known as
ERBB2), MET, ALK, RET, or NTRK1 might define
patients with RASWT metastatic colorectal cancer who
could benefit most from anti-EGFR therapy regardless
of tumour sidedness.62,63 These negatively hyperselected
patients also had longer survival times compared with
patients who had at least one of the genetic alterations.
The ongoing LIBImAb trial (NCT04776655) uses both
tissue and ctDNA analysis to identify patients with
metastatic colorectal cancer who are most likely to derive
benefit from cetuximab therapy.
ctDNA analysis has also shown high concordance with
results for tissue-based testing of other molecular
targets.64 A ctDNA substudy from the SWOG S1406 trial
using a Guardant 68-gene next-generation sequencing
panel showed a concordance rate of 88% in 160 patients
with metastatic colorectal cancer and tumour-confirmed
BRAFV600E mutation.65 The prospective TRIUMPH study
(using Guardant360) enrolled patients with either tissue-
confirmed or ctDNA-confirmed HER2 amplification,
reporting an agreement rate of 73%.66 Patients treated
with pertuzumab and trastuzumab had similar response
rates and progression-free survival whether they had
tissue-confirmed or ctDNA-confirmed HER2-positive
disease.66 Similarly, the Guardant360 assay was able to
detect HER2 amplification with 97·9% sensitivity (95% CI
87·2–99·8) in the HERACLES-A study of patients with
metastatic colorectal cancer.67 Clinical response correlated
with increased initial plasma HER2 and increased HER2
copy numbers.67 Exploratory analysis of patients recruited
to the DESTINY trial also observed increased response
rates associated with high ctDNA HER2 copy number but
noted decreased response rates associated with high
blood tumour mutational burden.68 In a large study
involving 3808 patients with metastatic colorectal cancer,
ctDNA analysis using the Guardant360 assay identified a
potentially actionable fusion, such as in RET, FGFR3, or
ALK, in 40 (1·1%) patients.69
In a pan-cancer study, results indicated that ctDNA
analysis with Guardant360 identified 71 (87%) of 82 cases
with high tissue microsatellite instability, but sensitivity
was highly dependent on ctDNA tumour fraction, and
with markedly reduced sensitivity at tumour fraction less
than 0·2%.70 Although immune checkpoint inhibitor
treatment is usually reserved for patients with tumours
with high microsatellite instability, ctDNA might
8 www.thelancet.com/gastrohep Published online July 24, 2023 https://doi.org/10.1016/S2468-1253(23)00146-2
potentially identify patients with microsatellite-stable
tumours who will benefit from this treatment. The
CO.26 trial randomly assigned patients with advanced
colorectal cancer with treatment-refractory microsatellite-
stable tumours to dual CTLA-4 plus PD-L1 blockade or
best supportive care, and showed that, in patients treated
with immune checkpoint inhibitors, elevated ctDNA-
assessed tumour mutational burden (28 variants per
megabase or more) was associated with the greatest
overall survival benefit (HR 0·34, 90% CI 0·18–0·63;
p=0·004).71 The ARETHUSA study examined the efficacy
of priming patients with microsatellite-stable, RASmut
metastatic colorectal cancer with temozolomide to induce
response to subsequent pembrolizumab therapy.72 Plasma
tumour mutational burden analysis of patients with
microsatellite-stable disease was similar to tissue
analysis and reflected an increase in tumour mutational
burden values after temozolomide priming, potentially
identifying those most likely to benefit from immune
checkpoint inhibitor treatment.72 However, successful
analysis was largely limited to patients with liver-
predominant disease.72
Assessing early treatment response
Serial carcinoembryonic antigen testing is cheap and
widely accessible but is not a reliable marker of therapy
response in metastatic colorectal cancer. The reason
for this unreliability is because carcinoembryonic
antigen is not initially elevated in many patients, and
early elevations on treatment (a carcinoembryonic
antigen surge) can occur in patients responding to
chemotherapy.22 Decreasing carcinoembryonic antigen
concentrations are evidence of response, but knowing
this will not change management and carcinoembryonic
antigen changes are not necessarily proportional to the
RECIST-defined response seen on imaging.
Tie and colleagues73 reported that significant reductions
reductions in ctDNA concentrations could be observed
as early as before the second cycle of first-line systemic
chemotherapy in patients treated for metastatic colorectal
cancer. A major reduction in ctDNA mutant allele
fraction (≥10-fold from baseline to after one treatment
cycle) compared with pre-treatment concentrations was
also significantly associated with radiological response
8–10 weeks after treatment initiation.73 Carcinoembryonic
antigen did not show any statistically significant change
in concentration between baseline and before cycle
two and correlation with radiological response was
statistically inferior to that of ctDNA. Other studies have
consistently shown improved outcomes associated with
ctDNA reduction; however, the timing of post-treatment
assessment has varied from 7 days to 60 days post-
treatment.51,61,74–80 Longitudinal studies with more frequent
ctDNA testing have also indicated that serial monitoring
of ctDNA is more predictive of survival than changes in
carcinoembryonic antigen or imaging.76,77 During second-
line treatment, an absence of a reduction in ctDNA

concentration as early as before cycle two correlated with
poorer survival.79,80
The optimal protocols for ctDNA testing during
treatment of metastatic colorectal cancer are yet to be
defined. Serial ctDNA analysis should have maximum
utility in later-line therapy, given the increased rates of
early treatment failure. Here, identifying treatment
failure within the first month of treatment could allow an
early switch to an alternative therapy that might be more
active, while also minimising the toxicity and financial
cost of persisting with a futile treatment. Such an
approach is being explored in the TACT-D and
Rapid 1 trials (table 2).
Detecting resistance to targeted therapy and timing of
treatment re-challenge
The non-invasive nature of ctDNA testing lends itself
well to serial testing to identify the emergence of resistant
clones. Two landmark studies concurrently showed the
ability of serial ctDNA testing to detect mutations
associated with resistance in patients with RASWT
colorectal cancer treated with anti-EGFR therapy. A study
by Diaz and colleagues81 reported that nine (38%) of
24 patients developed KRAS mutations at various
timepoints, as determined by ctDNA analysis. In a
similar study, Misale and colleagues82 described
acquisition of secondary KRAS mutations in six (60%) of
ten analysed cases and the emergence of KRAS
amplification in an additional case.82 Other studies have
used ctDNA to identify other molecular mechanisms
of resistance to targeted therapies, predominantly
in the MAPK pathway, such as KRAS, NRAS,
BRAF, MAP2K1, MAP2K2, EGFR-ECD, and MET and
HER2 amplifications.83–90 Among acquired EGFR-ECD
mutations, Ser492Leu, Ser464Leu, Gly465Arg, Gly465Glu,
Val441Asp, and Val441Gly are common targets used in
trials examining anti-EGFR treatment rechallenge, such
as Sym00405, CHRONOS, and CITRIC.88,91,92
Patterns of genomic alteration can also vary depending
on treatment factors. A 2023 study screened for
mechanisms of resistance (including KRAS, NRAS,
BRAF, MAP2K1, and EGFR-ECD) in paired plasma
samples of patients with RASWT colorectal cancer treated
with anti-EGFR as monotherapy in the third-line setting,
or in combination with chemotherapy in the first-line
setting.93 Acquired mutations were more likely in those
treated with third-line single-agent anti-EGFR (181 [46%]
of 395) than in those treated with the combination in
the first line (seven [9%] of 77).93 A preclinical model
showed that transcriptional mechanisms of resistance
predominate when anti-EGFR therapy is combined
with chemotherapy, whereas acquired MAPK pathway
resistance mutations did not affect sensitivity to cyto­
toxic chemotherapy.93 Similarly, Raghav and colleagues94
showed that fewer patients acquired genomic alterations
(RAS, BRAF, and EGFR-ECD mutations, or HER2 or
MET amplifications) following first-line anti-EGFR
www.thelancet.com/gastrohep Published online July 24, 2023 https://doi.org/10.1016/S2468-1253(23)00146-2 9
Review
therapy in combination with chemotherapy (four [6·6%]
of 61) compared with later lines (62%).
Results from prospective studies of rechallenge with an
anti-EGFR antibody in the third-line setting have shown
the utility of ctDNA-based RAS assessment to identify
a patient’s likelihood of benefitting from treatment.
The CHRONOS single-arm phase 2 study screened
52 patients with metastatic colorectal cancer for a panel
of KRAS, BRAF, and EGFR-ECD mutations before
considering a panitumumab rechallenge.91 Using a
digital droplet PCR-based assay, in an initial screen,
36 (69%) of the 52 patients did not have detectable
resistant mutations. Of the 27 patients with no detectable
resistant mutations proceeding to treatment, partial
responses were observed in eight (30%), with a further
11 (41%) having stable disease. Median duration of
response was 17 weeks and median overall survival was
55 weeks. In the CRICKET study, a combination of
cetuximab and irinotecan was given as third-line
treatment in patients with metastatic colorectal cancer
who initially had responded well to first-line
cetuximab-based and irinotecan-based therapy and
had progressed on second-line treatment.95 No RAS
mutations were detected in the ctDNA of patients who
had a partial response to the rechallenge, and patients
with ctDNA RASWT disease had an improved progression-
free survival compared with patients with ctDNA RASmut
disease.95 The phase 2, single-arm CAVE trial rechallenged
patients with RASWT metastatic colorectal cancer with
third-line cetuximab and avelumab following previous
complete or partial response to first-line anti-EGFR
therapy.96 Exploratory biomarker analysis showed that
patients with RASWT and BRAFWT ctDNA before treatment
had improved survival compared with those with RASmut
or BRAFmut ctDNA, showing a clear benefit of anti-EGFR
rechallenge in patients with wild-type genes. Of these
studies, only the CHRONOS trial enrolled patients on
the basis of ctDNA screening results, showing the
potential utility of ctDNA analysis in identifying patients
likely to benefit from anti-EGFR rechallenge.
Mathematical modelling of resistant subclonal pop­
ulation expansion and mutant allele decay might provide
insight into the ideal frequency of ctDNA monitoring and
the appropriate timing of anti-EGFR rechallenge. Khan
and colleagues97 developed a predictive model based on
ctDNA-assessed evolution of acquired subclonal RAS
pathway mutations in patients with metastatic colorectal
cancer treated with cetuximab. The model suggested that
ctDNA testing every 4 weeks or more frequently provided
the greatest predictive power in forecasting time to
radiologically evident progression.97 However, validation of
this model in prospective trials is needed. Regarding the
timing of anti-EGFR rechallenge, analysis of post-
progression ctDNA profiles of treated patients with
metastatic colorectal cancer showed that acquired RAS or
EGFR-ECD mutant alleles exponentially decayed with a
half-life of 4·4 months.98 Validation based on retrospective
 Review

Patient population Sample size ctDNA assay Intervention Primary objective

ctDNA-guided anti-EGFR rechallenge
PULSE KRASWT or NRASWT mCRC, as 84 Guardant360 Control arm: physician’s choice of To assess overall survival in
(NCT03992456) assessed by ctDNA after regorafenib or TAS-102; experimental molecularly selected patients
randomised phase 2 progression on previous arm: panitumumab rechallenge with mCRC receiving
anti-EGFR therapy panitumumab rechallenge vs
standard therapy (TAS-102 or
regorafenib)
PARERE RASWT or BRAFWT mCRC, as 214 Idylla ctKRAS- Control arm: panitumumab followed Overall survival
(NCT04787341) assessed by ctDNA after NRAS-BRAF by regorafenib; experimental arm:
randomised phase 2 progression on previous mutation test regorafenib followed by panitumumab
anti-EGFR therapy
CAPRI II GOIM RASWT or BRAFWT mCRC 200 NR Dynamic and longitudinal ctDNA To assess response rates for
(NCT05312398) prior to starting treatment assessment over three lines of each treatment line over
single-arm phase 2 treatment; second-line and third-line three treatment lines
treatment guided by ctDNA assessment
of RAS and BRAF status; first-line:
FOLFIRI plus cetuximab; second-line:
FOLFIRI plus cetuximab (RASWT or
BRAFWT) or FOLFOX plus bevacizumab
(RASmut or BRAFmut); third-line: irinotecan
plus cetuximab (RASWT or BRAFWT) or
regorafenib or TAS-102 (RASmut or
BRAFmut)
CAVE II GOIM RASWT or BRAFWT, as 173 NR Control arm: cetuximab; experimental Overall survival
(NCT05291156) assessed by ctDNA after arm: cetuximab and avelumab
randomised phase 2 progression on anti-EGFR
therapy
A phase 2 study using RASWT or BRAFWT CRC 60 NR ctDNA testing following progression at Objective response rates and
cfDNA in the first line to assess emerging mutations; progression-free survival
detection of RAS second-line therapy determined
mutations in patients by physician; upon second-line
with advanced CRC progression, ctDNA will be tested again;
(NCT04775862) RASWT: investigator choice of either
anti-EGFR or third-line chemotherapy;
RASmut: investigator choice of third-line
chemotherapy
CITRIC (EudraCT RASWT, BRAFWT or EGFR- 66 NR Control arm: investigator’s choice of Objective response rate
2020–000443–31) ECDWT, as assessed by third-line therapy; experimental arm:
phase 2 ctDNA following cetuximab and irinotecan
progression on two
previous regimens of
standard therapy,
including anti-EGFR
monoclonal antibodies
Other ctDNA-guided treatments
Rapid-1 mCRC after progression 78 Signatera Both groups treated with a prespecified Overall survival
(NCT04786600) on oxaliplatin-based sequence of FDA-approved drugs and
randomised phase 2 therapy drug combinations; patients moved to
next-line treatment determined by
treatment arm; control arm (scan-
guided): results of imaging scans show
progressive disease; experimental arm
(ctDNA-guided): substantial increase in
ctDNA concentration
TACT-D mCRC with at least 100 Guardant 360 Both groups receive either regorafenib Assessment of early change
(NCT03844620) two previous therapies or TAS 102; control arm (SOC): assigned in ctDNA as a predictor of
randomised phase 2 treatment as per SOC; experimental radiographic progression
arm (ctDNA): patients undergo ctDNA (in control arm); evaluate
testing and receive either regorafenib differences in clinically
or TAS-102 on the basis of the result significant treatment-related
adverse events between SOC
and ctDNA arms
(Table 2 continues on next page)

10 www.thelancet.com/gastrohep Published online July 24, 2023 https://doi.org/10.1016/S2468-1253(23)00146-2


Patient population Sample size ctDNA assay
(Continued from previous page)
LIBImAb RASWT or BRAFWT mCRC on 280
(NCT04776655) tumour tissue
randomised phase 3
COLOMATE Treatment-refractory 500
(NCT03765736) mCRC
cohort
ctDNA=circulating tumour DNA. cfDNA=cell-free DNA. ECD=ectodomain. FDA=US Food and Drug Administration FOLFIRI=leucovorin, fluorouracil, and irinotecan.
FOLFOX=leucovorin, fluorouracil, and oxaliplatin. mCRC=metastatic colorectal cancer. mut=mutant-type. NR=not reported. SOC=standard of care. TAS-102=trifluridine plus
tipiracil. WT=wild-type.
Table 2: Ongoing ctDNA-based prospective trials in non-resectable metastatic colorectal cancer
analysis of 80 rechallenged patients showed that objective
response rates were highest in patients who waited for
more than two half-lives (32%) from previous anti-EGFR
compared with those who waited for less than one (16%) or
one to two half-lives (20%), but these differences were not
statistically significant.98
Regarding other clinically relevant molecular targets,
serial ctDNA monitoring of patients with BRAFV600E
metastatic colorectal cancer receiving BRAF-targeted
treatment has also uncovered distinct patterns of
convergent genomic evolution associated with acquired
treatment resistance. The frequent emergence of MAPK
pathway alterations could help to rationalise future
combination treatment.99–101 Longitudinal analyses of
patients treated with HER2 dual inhibitors have identified
the emergence of resistant mutations, such as HER2
amplification and PIK3CA mutations.67,68,102 Following
treatment targeting KRAS12G→C, plasma analysis after
progression has shown acquired resistance due to
alterations in the RTK pathway or secondary RAS
mutations.103,104 In cases of tyrosine kinase receptor
alterations, further studies are needed to explore the
efficacy of upstream inhibitors, such as those targeting
SHP2.103
Barriers to real-world adoption of ctDNA guided
decision making
The DYNAMIC study30 in stage II colon cancer is a
landmark trial as it represents the world’s first randomised
controlled trial in any solid tumour to directly compare a
ctDNA-guided approach to adjuvant treatment selection
with standard of care. However, many questions remain,
including the role of ctDNA analysis in guiding treatment
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Review
Intervention Primary objective
Oncomine Cohort 1: patients with incongruent Progression-free survival in
Pan-Cancer RAS status (RASWT on tumour tissue and patients with RASWT or BRAFWT
Cell-Free assay RASmut on ctDNA) will be randomly on tumour tissue and RASmut on
assigned to either control (bevacizumab ctDNA
plus FOLFIRI) or experimental
(cetuximab plus FOLFIRI) groups; in
cohort 2, patients with congruent RAS
status will be treated with 8 weeks of
cetuximab plus FOLFIRI; repeat ctDNA
testing will be completed with RASmut
patients subsequently randomly
assigned to control (continuing
cetuximab) or experimental (switching
to bevacizumab) arms
Guardant 360 Blood-based genomic profiling on Evaluate proportion of patients
patients with treatment-refractory who have an actionable
mCRC to facilitate accrual to genomic profile; evaluate
molecularly assigned therapies subsequent proportion of
enrolment into companion trial
of the higher risk subset of patients with T4 staging.
Further randomised trials are needed to explore various
management scenarios based on ctDNA status for each
tumour stage, and to address key clinical management
questions, such as how therapy should optimally be
escalated (if at all) in ctDNA-positive patients or de-
escalated (if at all) in those with undetectable ctDNA. The
value of repeated ctDNA-based MRD testing in increasing
the negative predictive value of this assay is also being
explored in ongoing trials (table 3).
Current consensus guidelines recommend that
validated ctDNA assays can be used to genotype advanced-
stage cancers and to select patients for targeted therapies
in scenarios in which rapid results are paramount, or
tissue is unavailable.105,106 The most substantial limitations
are the lower sensitivity for fusion events and copy
number changes,107,108 and the potential for non-
informative ctDNA results in certain patient subsets (with
low disease burden, brain, peritoneal, or lung metastases,
or locoregional recurrence).109–111 For patients who are at
high risk of a non-informative result, tumour tissue-
based genomic analysis is still advisable where possible.
Further work is also needed to address the health
economics effect of ctDNA-informed management, given
the large associated costs. Securing reimbursement in
some jurisdictions will usually require cost-effectiveness
studies. This approach is feasible in scenarios in which
ctDNA-based testing can lead to avoiding treatment
and the associated financial and other costs without
compromising outcomes. This possibility was explored
in an analysis of an observational study of patients with
stage II colorectal cancer,112 and a health economics
analysis is planned for the DYNAMIC study. An EGFR
 Review
Patient population
Postoperative ctDNA-guided adjuvant management
DYNAMIC-III Stage III colon cancer
(ACTRN12617001566325)
DYNAMIC-RECTAL Locally advanced rectal
(ACTRN12617001560381) cancer
CIRCULATE-US Stage II and III colon
(NCT05174169) cancer
CIRCULATE AIO-KRK-0217 ctDNA-positive stage II
(NCT04089631) colon cancer
CIRCULATE PRODIGE 70 ctDNA-positive stage II
(NCT04120701) colon cancer
COBRA (NRG GI-005; Stage IIA colon cancer
NCT04068103)
VEGA (UMIN000039205) ctDNA-negative high-
risk stage II and low-risk
stage III colon cancer
TRACC (NCT04050345) High-risk stage II colon
cancer, stage III colon
cancer, and locally
advanced rectal cancer
MEDOCC-CrEATE Stage II colon cancer
(NL6281/NTR6455)
CIRCULATE-SPAIN ctDNA-positive stage II
(EudraCT 2021–000507– and III colon cancer
20)
OPTIMISE Curative-intent stage IV
(NCT04680260) colon cancer
Risk-stratified adjuvant Patients with stage IV
therapy (NCT05062317) colon cancer undergoing
elective curative-intent
hepatectomy after
neoadjuvant
chemotherapy
12 www.thelancet.com/gastrohep Published online July 24, 2023 https://doi.org/10.1016/S2468-1253(23)00146-2
Sample size ctDNA assay Intervention Primary objective
1000 Safe-SeqS Control arm: SOC (clinician-determined); To assess the effect of chemotherapy
experimental arm: ctDNA-positive, de-escalation and escalation strategies guided
escalation of chemotherapy; ctDNA- by ctDNA, including: acceptable rate of
negative, de-escalation of chemotherapy de-escalation in the ctDNA-informed negative
cohort (phase 2); non-inferior recurrence with
ctDNA-guided management in the de-escalated
ctDNA-informed negative cohort (phase 3);
superior recurrence with ctDNA-informed
management in the escalated ctDNA-informed
positive cohort (phase 3)
450 Safe-SeqS Control arm: SOC (clinician-determined); Show that ctDNA results plus pathological nodal
experimental arm: ctDNA-positive, adjuvant status can reduce the number of patients
chemotherapy; ctDNA-negative (ypN0), receiving adjuvant chemotherapy without
surveillance; ctDNA-negative (ypN+), compromising RFS
surveillance or adjuvant chemotherapy
(clinician’s discretion)
1912 Signatera If ctDNA-negative (cancer stage IIIA and IIIB To assess the effect of chemotherapy
only): control arm, SOC chemotherapy de-escalation and escalation strategies guided
(3–6 months’ FOLFOX or 3 months’ CAPOX); by ctDNA
experimental arm, no chemotherapy;
if ctDNA-positive (cancer stage II or III):
control arm, 6 months’ FOLFOX or CAPOX;
experimental arm, 6 months’ FOLFOXIRI
2310 (total); 231 NR Control arm: surveillance; experimental arm: To assess the effect of chemotherapy on DFS in
(ctDNA-positive) adjuvant chemotherapy (capecitabine or patients with ctDNA-positive stage II colon
CAPOX [clinician’s choice]) cancer
1980 (total); 198 ddPCR Control arm: surveillance; experimental arm: Show superior DFS for patients with ctDNA-
(ctDNA-positive) (2 methylated adjuvant 6 months’ FOLFOX positive stage II colon cancer treated with
markers WIF1 and adjuvant FOLFOX
NPY)
1408 Guardant LUNAR-1 Control arm: surveillance; experimental arm: To assess clearance of ctDNA for ctDNA-positive
if ctDNA-positive, adjuvant FOLFOX or patients and the effect of ctDNA-guided
CAPOX; if ctDNA-negative, surveillance treatment on RFS
1240 Signatera Control arm: 3 months’ CAPOX; Show non-inferiority of observation vs adjuvant
experimental arm: surveillance; patients CAPOX in patients with ctDNA-negative high-
who become ctDNA-positive at 3 months risk stage II and low-risk stage III colon cancer
subsequently enrolled in ALTAIR trial
1000 Next-generation Control arm: 6 months’ capecitabine Show non-inferiority of DFS between SOC and
sequencing-based or 3 months’ CAPOX; experimental arm: ctDNA-guided adjuvant treatment
22-gene colorectal if ctDNA-positive, standard adjuvant
panel chemotherapy (as in control condition);
if ctDNA-negative, de-escalation of
chemotherapy (but re-escalate if ctDNA
becomes positive at 3 months)
1320 PDGx elio Control arm: surveillance; experimental arm: Investigate willingness of patients to be treated
if ctDNA-positive, 6 months’ CAPOX or with chemotherapy guided by ctDNA testing
FOLFOX; if ctDNA-negative, surveillance and the effect on recurrence rate
164 NR Control arm: SOC CAPOX; experimental arm: To assess the proportion of patients who clear
FOLFOXIRI ctDNA and the effect on DFS with standard vs
intensified adjuvant treatment
350 NR Control arm: SOC adjuvant therapy; To assess the effect of chemotherapy
experimental arm: if ctDNA-positive, de-escalation and escalation strategies guided
escalation of treatment to 4 months’ by ctDNA on RFS
FOLFOXIRI followed by 2 months’
fluorouracil monotherapy; if ctDNA-negative,
de-escalation based on shared decision
making (either monotherapy or observation)
120 NR Two experimental arms; if ctDNA- To assess RFS of both ctDNA-negative and
negative, de-escalation of chemotherapy ctDNA-positive patients who received risk-
(monotherapy); if ctDNA-positive, stratified adjuvant chemotherapy
escalation of chemotherapy (resumption
of neoadjuvant platinum-based doublet
with or without bevacizumab)
(Table 3 continues on next page)
 Review

Patient population Sample size ctDNA assay Intervention Primary objective

(Continued from previous page)
ctDNA-guided second-line adjuvant therapy
ALTAIR (NCT04457297) ctDNA-positive stage III 240 Signatera Control arm: surveillance; experimental arm: Show benefit of TAS-102 over surveillance in
colon cancer after second-line TAS-102 patients who remained ctDNA-positive after
3 months of CAPOX standard adjuvant chemotherapy
ACT-3 (NCT04259944) ctDNA-positive stage III 500 Guardant LUNAR-1 Control arm: surveillance; experimental arm: Show benefit of FOLFIRI over surveillance in
colon cancer after for MSS or BRAFWT, 6 months’ FOLFIRI; patients who remained ctDNA-positive after
standard adjuvant for BRAFmut, 6 months’ encorafenib– standard adjuvant therapy
chemotherapy binimetinib–cetuximab; for MSI-H,
6 months’ nivolumab
Postoperative ctDNA-guided adjuvant and second-line adjuvant management
CTAC (NCT05529615) High-risk stage II and 2684 NR Patients diagnosed with high-risk stage II Determine non-inferior recurrence risk of
stage III colon cancer and low-risk stage III cancer: if ctDNA- surveillance vs adjuvant chemotherapy in
negative, surveillance; if ctDNA-positive: patients with ctDNA-negative low-risk cancer
control arm, surveillance; experimental arm: and whether second-line chemotherapy can
CAPOX for 3 months; for patients diagnosed improve the DFS of ctDNA-positive patients vs
with high-risk stage III cancers, all receive standard chemotherapy in the high-risk group
CAPOX for 3 months; at 3 months: if ctDNA-
negative, surveillance; if ctDNA-positive:
control arm, further CAPOX for 3 months
(total 6 months); experimental arm, second-
line treatment decided by clinician
ERASE-CRC ctDNA-positive high- 300 NR Part 1: control arm, 6 months’ FOLFOX or Part 1: evaluate the effect of escalating adjuvant
(NCT05062889) risk stage II and stage III CAPOX as adjuvant treatment; experimental chemotherapy in ctDNA-positive patients on
colon cancer arm, FOLFOXIRI for 6 months as adjuvant ctDNA clearance rates and DFS; part 2: evaluate
treatment; part 2: if ctDNA-positive whether further adjuvant treatment with
after adjuvant treatment: control arm, TAS-102 in patients who remained ctDNA-
surveillance; experimental arm, six cycles of positive after adjuvant treatment can increase
TAS-102 ctDNA clearance rates and improve DFS
ctDNA-guided surveillance
IMPROVE-IT2 High-risk stage II and 254 ddPCR (colorectal Control arm: surveillance; experimental arm: Show combination of ctDNA-guided and high-
(NCT04084249) stage III colon cancer panel) 3-monthly PET-CT for ctDNA-positive intensity radiological surveillance could result in
patients earlier detection of recurrent disease and hence
more eligible patients for curative treatment
CAPOX=capecitabine and oxaliplatin. ctDNA=circulating tumour DNA. ddPCR=droplet digital PCR. DFS=disease-free survival. FOLFIRI=folinic acid, fluorouracil, and irinotecan. FOLFOXIRI=folinic acid,
fluorouracil, irinotecan, and oxaliplatin. FOLFOX=leucovorin, fluorouracil, and oxaliplatin. MSI-H=microsatellite instability-high; MSS=microsatellite stable. NR=not reported. RFS=recurrence-free survival.
Safe-SeqS=Safe-Sequencing System. SOC=standard of care. TAS-102=trifluridine plus tipiracil. ypN0=pathologic node negative disease following neoadjuvant systemic or radiation therapy. ypN+=pathologic
node positive disease following neoadjuvant systemic or radiation therapy.

Table 3: Ongoing ctDNA-based randomised trials in curatively resected colorectal cancer

inhibitor treatment rechallenge is another scenario in
which ctDNA-based testing might be cost-saving, in that
avoiding futile treatment in patients with a RAS mutation
would probably save sufficient money to offset the cost of
a ctDNA test.
Ongoing trials
Multiple ongoing studies are further exploring the utility
of ctDNA in informing the curative intent management
of colorectal cancer (table 3), including after surgery,
during and at the completion of adjuvant therapy, and
during surveillance. Studies also continue in the
metastatic disease setting (table 2).
Of particular interest are studies in the MRD setting
exploring additional therapy when patients have
completed standard adjuvant therapy without clinical
recurrence but have detectable ctDNA as a marker of
persistent MRD—the new concept of second-line
adaptive-adjuvant therapy. Trials exploring the benefit of
early introduction of systemic therapy for patients for
whom ctDNA becomes detectable during surveillance
(molecular recurrence) without detectable recurrence on
imaging would also be appealing.
It is notable that many different ctDNA assays are
being developed. Analytical performance has the
potential to differ between ctDNA MRD assays, which
should preclude data from one ctDNA assay being used
to validate the use of a different assay. The clinical use of
each individual ctDNA assay should be independently
validated in carefully designed prospective studies that
cover each specific clinical scenario.
Conclusion
ctDNA has the potential to change the treatment frame­
works for early-stage through to late-stage colorectal
cancer, allowing a higher degree of personalisation. For
early disease, robust evidence currently supports ctDNA
as a marker of recurrence risk, adjuvant treatment
efficacy, and impending clinical recurrence. In the
metastatic setting, ctDNA testing can also aid in

www.thelancet.com/gastrohep Published online July 24, 2023 https://doi.org/10.1016/S2468-1253(23)00146-2 13

 Review
Search strategy and selection criteria
Data for this Review were identified by searches of MEDLINE
and PubMed, and references from relevant articles using the
search terms “colorectal neoplasm” and “circulating tumour
DNA”. Abstracts and reports from meetings were included
only when they related directly to previously published work.
Only articles published in English were included with no
restriction on year of publication.
molecular characterisation, such as identifying patients
who might benefit from an anti-EGFR rechallenge, and
in early monitoring of treatment response. With the
results of multiple randomised trials on the horizon, the
use of ctDNA informed therapy will become increasingly
well defined.
Contributors
All authors contributed to conceptualisation and design. ML and
YHT conducted the literature review and wrote the first draft.
All authors contributed to editing the initial and subsequent revisions
of the manuscript and approved the final version.
Declaration of Interests
PG has received consulting fees from Haystack Oncology and
honoraria from Amgen, Roche, Servier, Merck, Pierre-Fabre, and MSD.
JT has received consulting fees from Haystack Oncology and Seres
Therapeutics, honoraria from Astra Zeneca, Amgen, and Servier,
support for attending meetings from Roche, participated on advisory
boards for Beigene, Novartis, Astra Zeneca, Merck, Serono, MSD,
Pierre Fabre, BMS, Daichii Sankyo, Takeda, Illumina, and Gilead, and
has a leadership role on AGITG and ESMO. All other authors declare no
competing interests.
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