ctDNA As A Predictor of Outcome After Curative Resection For Locally Advanced Rectal Cancer Systematic Review and Meta-Analysis

Received: 10 February 2024 | Revised: 26 April 2024 | Accepted: 3 May 2024
DOI: 10.1111/codi.17039
M E T A - A N A L Y S I S
ctDNA as a predictor of outcome after curative resection for

locally advanced rectal cancer: systematic review and
meta-analysis
Ahmed Nassar1,2 | Noha E. Aly2 | Zhaohui Jin3 | Emad H. Aly1,2
Abstract
1
University of Aberdeen, Aberdeen,
Scotland, UK
2 Aim: To assess the efficacy of ctDNA measurement at different time intervals in predict-
Aberdeen Royal Infirmary, Aberdeen,
Scotland, UK ing response and prognosis in patients diagnosed with locally advanced rectal cancer
3
Department of Medical Oncology, Mayo (LARC) who underwent neoadjuvant treatment prior to curative resection.
Clinic, Rochester, Minnesota, USA
Method: English language randomized controlled trials and observational studies, pub-
Correspondence lished from 1946 to January 2024, comparing outcomes between ctDNA-positive and
Ahmed Nassar, University of Aberdeen
and Aberdeen Royal Infirmary, Foresterhill
ctDNA-negative patients with LARC undergoing neoadjuvant treatment prior to cura-
Road, Aberdeen, AB25 2ZN, Scotland, UK. tive surgical resection were included in the search. The search included Ovid MEDLINE,
Email: [email protected]
Embase, Cochrane Central Register of Controlled Trials (CENTRAL), and the Cochrane
Database of Systematic Reviews (CDSR).
Results: Data for 1022 patients were analysed. Patients with positive ctDNA in the pre-
operative period had more than five times the risk of developing distant metastasis (RR
[95% CI] 5.03 [3.31–7.65], p < 0.001), while those with positive ctDNA in the postopera-
tive period had more than six times the risk (RR [95% CI] 6.17 [2.38–15.95], p < 0.001).
There was no significant relationship between ctDNA status at baseline, pre-, or postop-
erative periods and achievement of pCR (RR [95% CI] 1.21 [0.86–1.7], 1.82 [0.94–3.55],
1.48 [0.78–2.82], p = 0.27, 0.08, and 0.23, respectively). However, patients with positive
ctDNA in the pre- and postoperative periods had more than 13 and 12 times the risk of
overall disease relapse after curative-intent treatment (RR [95% CI] 13.55 [7.12–25.81],
12.14 [3.19–46.14], p < 0.001), respectively.
Conclusion: ctDNA could potentially guide treatment and follow-up in LARC, predicting
high-risk patients for disease relapse, allowing individualized surveillance and treatment
strategies. Prospective studies are needed for standardization.
KEYWORDS
advanced, circulating DNA, ctDNA, rectal cancer
I NTRO D U C TI O N 100 years [1]. Several clinical trials have attempted to elucidate the
role of neoadjuvant chemotherapy (nCT), neoadjuvant chemora-
The debate regarding the optimal treatment for rectal cancer con- diotherapy (nCRT), and total mesorectal excision (TME) [2]. There
tinues despite the major developments in this field over the last has also been increasing interest in opting for a “watch and wait”
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2024 The Author(s). Colorectal Disease published by John Wiley & Sons Ltd on behalf of Association of Coloproctology of Great Britain and Ireland.
1346 | wileyonlinelibrary.com/journal/codi
Colorectal Disease. 2024;26:1346–1358.
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NASSAR et al. 1347
approach in patients who achieve complete clinical response (cCR) response on MRI [5]. (6) Low neoadjuvant rectal (NAR) score as de-
following neoadjuvant treatment or total neoadjuvant treatment termined by the primary study [10, 11]. Standardized definitions are
(TNT) [3]. provided in Table S1.
However, unresolved challenges remain which relate to the The search was conducted on 25 January 2024, by a senior
ability to predict which patients are likely to achieve cCR following information specialist from the Royal College of Surgeons of
nCRT, and the availability of accurate surveillance tools for residual England. Database searches were performed in Ovid MEDLINE,
disease for those who have achieved cCR. It is well recognized that Embase, Cochrane Central Register of Controlled Trials
the accuracy of clinical and radiological surveillance remains subop- (CENTRAL), and the Cochrane Database of Systematic Reviews
timal [4]. (CDSR) using the patient intervention control outcome (PICO)
Circulating tumour DNA (ctDNA) has been increasingly used framework. Table S2 provides the PICO framework and search
as a noninvasive biomarker for the monitoring of tumour burden, terms, while Table S3 demonstrates the complete electronic
predicting prognosis and monitoring treatment response. ctDNA de- search strategy. To ensure comprehensive coverage, a search in
rives from apoptosis or necrosis of the tumour cells, which release the grey literature was conducted through the library depart-
DNA into the blood stream. It can be detected in the presence of ment of the Royal College of Surgeons of England, supplemented
primary, recurrent or metastatic cancer [5]. by manual searches of reference lists of the included studies.
The aim of this study was to assess the efficacy of ctDNA as a Additionally, the Citation Chaser tool was utilized for reference
tool of minimal residual disease (MRD) detection, in predicting re- harvesting [12] (Table S4).
sponse to treatment and identifying those who were at higher risk Two independent reviewers (AN and NEA) conducted the ab-
of recurrence in patients diagnosed with locally advanced rectal stract screening and resolved conflicts through mutual agreement.
cancer (LARC) and underwent neoadjuvant therapy prior to surgical Full-text review and data collection was conducted by one reviewer,
resection. with results verified by a second reviewer.
Observational studies were assessed using the Mathes and
Pieper criteria [13] and the Joanna Briggs Institute (JBI) appraisal
M E TH O D tool for risk of bias assessment [14]. The overall risk of bias for each
specific study was determined based on the number of questions
This review followed the Preferred Reporting Items for Systematic answered with “yes,” “no,” or “unclear.” A study was considered to
reviews and Meta-analyses (PRISMA) statement and was registered have a low concern of bias if no questions were answered unfavour-
in PROSPERO (registration no.: CRD42022375380). ably, some concern if one to three questions were answered unfa-
English-language studies published from inception of literature vourably, and high concern if four or more questions were answered
(1946) to January 2024 were included in the search. Eligible stud- unfavourably.
ies included randomized controlled trials (RCTs) and observational The data included study details, demographics, treatment type,
studies comparing ctDNA and standard surveillance methods as disease characteristics, ctDNA analysis methods, outcomes based
prognostic tools in LARC patients or comparing outcomes between on ctDNA status, and follow up period.
ctDNA-positive and ctDNA-negative patients. Only studies involv-
ing patients with LARC who received nCRT before curative re-
sectional surgery were included. Studies that did not differentiate Statistical analysis
between rectal and colonic cancer data or included patients with
metastatic rectal cancer where curative treatment was not the in- Count, percentages, and ratios represented noncontinuous varia-
tention were excluded. bles, whilst median (range) represented continuous data as reported
This study aimed to assess the ability of ctDNA measurements at in each individual study. When combining data across studies, the
different treatment stages (baseline, pre-, and postoperative) to pre- median and range of median values were used to represent continu-
dict response to treatment, measured by the achievement of patho- ous variables.
logical complete response (pCR) and a good response to treatment Categorical variables within the meta-
analysis were assessed
(as defined in the next paragraph). Additionally, the study evaluated using risk ratios (RR) and their corresponding 95% CIs. The analysis
the ability of ctDNA to predict tumour recurrence by assessing the incorporated the number of events and the total number of patients
development of distant metastasis, locoregional recurrence, and for each variable examined. Survival data were analysed using RR
relapse-free survival (RFS). and 95% CIs. For the meta-analysis of survival outcomes (RFS and
A good response to treatment was considered if at least one of MFS), log RR and standard error (SE) were utilized [15]. When RR and
the following criteria were met: (1) Tumour regression grade (TRG) 95% CI were not directly provided in primary studies, survival curves
of 1 or 2 on pathological examination of the resected tumour [6]. (2) were digitized using WebPlotDigitizer software to extract raw data
MRI TRG (mrTRG) of 1 or 2 on follow-up MRI [7]. (3) R0 resection and such as number of patients and number of events at each timepoint
negative lymph node involvement (R0NN) of the resected specimen [16]. Numbers at risk and censored data were utilized whenever
[8]. (4) Complete endoscopic response to treatment [9]. (5) Complete available in the primary studies. Subsequently, all extracted data
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1348 NASSAR et al.
were entered into Excel spreadsheets to generate log RR and SE The median age (range) of the included patients, calculated from
values, following the methodology established by Parmer et al. and reported median values, was 61 (54–68). Among the 1022 patients,
Williamson et al. [17, 18]. 678 (66.3%) were males and 344 (33.7%) were females.
Meta-analysis was performed using RevMan (Review Manager)
software, version 5 [19]. Heterogeneity was assessed using the I2
and Tau [2] tests. If I2 was greater than 50%, indicating significant Disease characteristics
heterogeneity, the Mantel–Haenszel (M-H) random-effects model
was used [20]. A p-value less than 0.05 was considered statistically All studies included patients with LARC without metastasis, ex-
significant. cept for the study by Truelsen et al. [25], which included 10 pa-
tients with liver metastasis but who were suitable for curative
resection [25]. Two patients in another study developed liver
Handling of confounding factors and other bias metastasis during neoadjuvant chemoradiotherapy [8]. One study
specified that only LARC patients with a performance status of
Patient and disease characteristics were compared between ctDNA 0–2 were included [21].
positive and negative patients at different treatment stages to ad- MRI was the primary modality used for local assessment of
dress confounding factors and potential bias. Studies with a high risk rectal tumours, and computer tomography (CT) was used for
of bias were excluded from the meta-analysis. Funnel plots were uti- systemic staging and detection of disease progression during fol-
lized to assess the risk of publication bias for each outcome included low-up in all studies, except for one [27] that did not specify the
in the meta-analysis. imaging method.
Among seven studies, 448 out of 509 patients (88%) had T3 or
T4 rectal tumours before treatment initiation [5, 7, 11, 24–27]. Out
R E S U LT S of these patients, 205 out of 310 (66%) had positive ctDNA accord-
ing to data from five studies [5, 7, 11, 26, 27]. Additionally, 304 out
A total of 275 records were identified and 259 underwent abstract of 386 (79%) patients had nodal involvement across six studies [5, 7,
screening, resulting in 32 studies for full-text review. After full-text 11, 25–27], and among those with nodal involvement, 199 out of 310
review, 14 cohort studies were included. Of these, 11 were prospec- (64%) patients had positive ctDNA at baseline based on data from
tive and three were retrospective [8, 21, 22]. No additional studies five studies [5, 7, 11, 26, 27].
meeting the inclusion criteria were identified through the grey litera- Four studies reported evidence of extramural vascular invasion
ture search or manual searches of reference lists. The selection pro- (EMVI) at MRI prior to treatment initiation in 147 (44.8%) patients [6,
cess is depicted in a PRISMA diagram in Figure 1. Table 1 provides a 7, 9, 26]. Among patients with EMVI, 88 (77.9%) had positive baseline
summary of the included studies. ctDNA based on data from three studies [7, 9, 26].
Quality and risk of bias assessment Neoadjuvant chemoradiotherapy (Table 1)
One study had high concern [23], and 11 had some concern All studies, except Murahashi et al. [9], included fluoropyrimidine
[5–9, 11, 21, 22, 24–26] regarding bias. The remaining two stud- agents (either 5-fluorouracil or capecitabine) in standard nCRT [9].
ies had low concern [27, 28] (Table 1 and Table S5). Funnel plots A total of 10 studies used only long-course CRT, where external
showed no significant publication bias for the reported outcomes beam radiotherapy (EBRT) was given in fractions with a total dose of
(Figures S1–S6). 45–54 Gy [5–8, 11, 21–24, 26].
Patient characteristics Curative surgical resection
Initially, 1050 patients were included from all studies. However, 16 The time interval between completion of nCRT and curative surgery
patients in the study by Wang et al. [6] were excluded due to incom- ranged from 4 to 17 weeks in eight studies [5, 7, 21–25, 28]. However,
plete sample collection at all time points [6]. In the study by Vidal all surgical patients had surgery within 10 weeks in six studies [5, 22–
et al. [11], 10 patients were excluded due to inadequate samples for 25, 28]. Out of 85 patients included in one study, nine patients who
ctDNA extraction [11]. Additionally, two patients were excluded due completed short-
course radiotherapy underwent surgery within
to the lack of qualified tissue biopsy for next-generation sequencing 10 days [9]. Time to surgery was not specified in five studies [6, 8,
(NGS) [26]. After excluding these patients, the final analysis included 11, 26, 27]. All studies mentioned that surgical patients had under-
1022 patients. gone total mesorectal excision (TME). In one study, intraoperative
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NASSAR et al. 1349
Identification of studies via databases and registers
Records idenfied from databases

(n = 275):
Identification
Records removed before screening:

1- Ovid Medline (n = 92)
- Duplicate records removed:
2- Embase (n = 242)
(n = 16)
3- Cochrane Central Register
of Controlled Trials
CENTRAL (n = 62)
Records screened Records excluded

(n = 259) (n = 227)
Reports sought for retrieval Reports not retrieved

(n = 32) (n = 0)
Screening
Reports assessed for eligibility Reports excluded (n = 18):

(n = 32)
- No clear separaon between colon and rectal

cancer data (n = 16)
- Wrong populaon (colonic cancer only) (n = 2)
Included
Studies included in review

(n = 14)
F I G U R E 1 PRISMA diagram.
sampling of the inferior mesenteric vein for ctDNA was performed methods were employed, including direct fluorescent assay (DFA)
during surgery [5]. [24, 25] and NGS [5, 8, 9, 11, 22, 26]. The timing of ctDNA meas-
urement varied among studies, with most including baseline,
post-chemoradiotherapy, and postoperative samples. Some stud-
Measurement of ctDNA ies did not report postoperative sample results [11, 24, 25, 27],
and the collection timing ranged from postoperative day 1 [22] to
The included studies used various methods to detect ctDNA in 12 weeks [7, 9] after surgery. Similarly, there was high variability in
peripheral blood, including: the measurement of specific cancer reporting timing post-CRT ctDNA sample. However, three studies
mutations detected in biopsies [8, 27, 28], panels of genetic muta- reported post-CRT samples were taken within 48 h before surgery
tions [7, 26], Arthrobacter luteus (ALU)115 measurement, and the [6, 9, 11]. Serial samples were collected in a long-term follow-up
ratio of ALU115 primer segments [21]. Different measurement study [24], whilst a “watch and wait” approach involved periodic
|
1350 NASSAR et al.
TA B L E 1 Summary of the studies included in the systematic review.
Study, year No. of Method of ctDNA

(country) Design Risk of bias patients Type of ctDNA studied measurement
Agostini et al., Retrospective, Some 67 Two primers (DNA fragments): qPCR

2011 (Italy) [21] single centre concern (1) ALU 115 (short fragment), (2) ALU 247 integrity index
(long fragment), (3) β globin (ALU 247/115 ratio)
Ji et al., 2021 Retrospective, Some 46 Mutation signatures were based on 30 NGS
(China) [22] single centre concern signatures verified in Catalogue of Somatic
Mutations in Cancer (COSMIC) [29]
Khakoo et al., Prospective, single Some 47 KRAS, NRAS, BRAF, PIK3CA, TP53, and ddPCR
2020 (UK) [7] centre concern APC
Liu et al., 2022 Prospective, Low 60 (1) Personalized assay targeting tumour- Targeted sequencing PCR
(China) [27] multicentre concern informed mutations,
(2) universal panel of genes frequently
mutated in colorectal cancer (CRC),
(3) low depth sequencing for copy number
alterations (CNAs)
McDuff et al., Retrospective, Some 29 Tumour specific mutations (personalized) ddPCR/NGS
2021 (USA) [8] single centre concern
Murahashi et al., Prospective, single Some 85 Cell-free DNA panel covering 14 genes with NGS, amplicon-based deep
2020 (Japan) [9] centre concern over 240 hotspots sequencing
Pazdirek et al., Prospective, single High 36 Panel of selected mutations commonly Denaturing capillary
2020 (Czech centre concern found in rectal cancer. If tissue mutations, electrophoresis
Republic) [23] these mutations are to be examined in
plasma
Roesel et al., Prospective, two Some 25 50 genes, including the most relevant and NGS
2022 centres concern frequently mutated genes in rectal cancer
(Switzerland) [5]
Schou et al., Prospective, single Some 123 DNA standards were prepared from Human Direct fluorescent assay
2018 (Denmark) centre concern Control Genomic DNA
[24] (Life Technologies)
Tie et al., 2019 Prospective, Low 159 Personalized assay: tissue sample examined qPCR
(Australia) [28] multicentre concern for 15 genetic mutations commonly
encountered in CRC. For each patient,
plasma examined for the specific mutation
found in tissue sample
Truelsen et al., Prospective, single Some 76 DNA standards were prepared from Human Direct fluorescent assay
2022 (Denmark) centre concern Control Genomic DNA
[25] (Life Technologies)
Vidal et al., 2021 Prospective, Some 72 In vitro diagnostic assay validated for Single-sample NGS
(Spain) [11] multicentre concern patients with colorectal cancer
|
NASSAR et al. 1351
Times of ctDNA measurement Neoadjuvant CRT regimen Follow up Outcomes
(1) Pre-CRT: 15 days before CRT EBRT (at least 50 Gy in total) and 5-FU NA Pathological tumour
(2) Post-CRT: 15 days before surgery based chemotherapy response: TRG [1–5]
Baseline, after CRT and 1 day after Capecitabine alone and total of 50 Gy Up to 40 months RFS, pCR, distant metastasis
surgery radiotherapy.
Postoperative chemotherapy:
capecitabine alone, mFOLFOX6 or CapeOx
(1) Pretreatment: within 4 w prior Capecitabine for 6 weeks and total of Median: 26.4 months Tumour response: mr TRG,
to CRT, 50.4–54 Gy radiotherapy RECIST and pathological
(2) Mid-CRT: 3–4 w from the start TRG/MFS, metastasis, DFS,
of CRT, LRFS, and OS
(3) Completion of CRT: 4–12 w from
completion,
(4) Postop: within 4–12 w after
surgery. For W&W: 3–6 m from the
end of CRT until within 3 months of
regrowth
Baseline, during CRT and after CRT Randomly assigned in a 1:1 ratio to either 3 years 3-year RFS/3-year local
short-course CRT (5 Gy × 5 sessions with RFS/3-year distant MFS/
4 cycles of capecitabine plus oxaliplatin OS/PCR
regimen) or long-course CRT (2 Gy × 25
with capecitabine)
Baseline, pre-, and postoperative Long-course: total EBRT of 50.4 Gy with From enrolment to NAR, margin negative and
(1–5 months after surgery) concurrent capecitabine or 5 FU infusion. recurrence or death/median node negative (R0, NN),
Three of the patients who received 5 FU (IQR): 20 (14–43) months PCR, PFS, Recurrence
infusion also received midostaurin
Baseline, pre-and postoperative 33 patients received standard CRT with Up to 100 weeks Response to treatment, PCR,
(12 weeks after surgery) tegafur/gimeracil/oteracil over 4 weeks, recurrence, development of
while 9 patients received SRT. Four metastasis and RFS
patients received neoadjuvant CRT with
CapeOx + Bmab, and 23 patients received
FOLFOX + Bmab followed by standard CRT
Not specified Total of 50.4 Gy EBRT and concurrent 3 years DFS, OS, TRG
capecitabine
7 time points and from IMV Long course of 5 FU based CRT plus total Median (IQR): 14 (8–14) TRG, pathological complete
intraoperatively of 50.4 Gy radiotherapy months response
Included baseline, pre-and postop
baseline, after induction Long course capecitabine +/− oxaliplatin Median: 55 months Recurrence, time to
chemotherapy, and after CRT. Then, and total dose of 54 Gy EBRT recurrence and DFS
serial samples 5 years after surgery
Before CRT, after CRT and Fluoropyrimidine -based chemotherapy Median: 24 months RFS, PCR
4–10 weeks after surgery
Baseline, mid-therapy and at the Short-course (25 Gy) or long-course Median: 32 months PCR, TRG
end of CRT (50.4 Gy) radiotherapy with or without
concomitant capecitabine
Baseline and after completion of Randomly assigned in a 2:1 ratio to either Median (range): 38 PCR, 3-year DFS, OS, NAR
TNT (within 48 h prior to surgery) arm A (aflibercept plus mFOLFOX6) or arm (2.3–51.5) months score
B (mFOLFOX6 alone). Both treatments
were administered for six cycles, followed
by 5 weeks of CRT with capecitabine.
(Continues)
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1352 NASSAR et al.
Study, year No. of Method of ctDNA

(country) Design Risk of bias patients Type of ctDNA studied measurement
Wang et al., 2022 Prospective, single Some 119 422 cancer related genes 5′-end motif profiles
(China) [6] centre concern
Zhou et al.,2021 Prospective, Some 106 Panel of 1021 genes NGS

(China) [26] multicentre concern
Abbreviations: 5-FU, 5 fluorouracil; Bmab, bevacizumab; CRC, colorectal cancer; CRT, chemoradiotherapy; ddPCR, droplet digital PCR; DFS,
disease-free survival; EBRT, external beam radiotherapy; IMV, inferior mesenteric vein; IQR, interquartile range; LRFS, local recurrence-free survival;
MFS, metastasis-free survival; mrTRG, magnetic resonance TRG; NAR score, neoadjuvant rectal score; NRS, neoadjuvant rectal score; NGS,
next-generation sequencing; OS, overall survival; pCR, pathological complete response; PFS, progression-free survival; qPCR, quantitative
polymerase chain reaction; RFS, recurrence-free survival; SRT, short course radiotherapy; TNT, total neoadjuvant treatment; TRG, tumour
regression grade; W&W, watch and wait approach.
sampling after chemoradiotherapy was utilized in another study Meta-analysis indicated slightly higher risk of positive EMVI on
[7] (Table 1). pretreatment MRI in patients with positive baseline ctDNA (RR [95%
CI] 1.4 [1.01–1.95], p = 0.04) [7, 9, 26] (Figure S7). Zhou et al. [26]
found positive preoperative ctDNA associated with positive EMVI
Follow up on presurgery MRI (p < 0.002) [26]. No correlation was observed
between ctDNA status and circumferential resection margin (CRM)
The median (range) follow-up period was 25.2 (14–55) months in involvement [7, 26], or tumour differentiation [26].
eight studies [5, 7, 8, 11, 24–26, 28]. During follow-up, serial imaging
using CT was performed [7, 8, 11, 22–24, 26, 28]. In three studies,
carcinoembryonic antigen (CEA) levels were monitored in addition ctDNA as a tool to assess response to treatment
to CT during follow-up [8, 11, 28].
Correlation between ctDNA status and
achievement of pCR
Correlation between ctDNA status and clinical
characteristics Pooled results showed no correlation between baseline [5, 9, 11,
22, 26–28] or postoperative [5, 8, 9, 22, 26, 28] ctDNA status and
A total of 10 studies found no age-related differences in ctDNA sta- achievement of pCR (RR [95% CI] 1.21 [0.86–1.7] and 1.48 [0.78–
tus at any timepoint [5, 7, 9, 11, 22, 24–28]. Eight studies showed no 2.82], p = 0.27, and 0.23, respectively). While preoperative ctDNA
difference in ctDNA status taken at any timepoint based on gender suggested a trend towards achieving pCR in ctDNA-negative pa-
[7, 9, 11, 22, 25–28]. However, in Schou et al. [24], males had a higher tients, this did not reach statistical significance (RR [95% CI] 1.82
median baseline ctDNA level (1.04 ng/μL) compared to females [0.94–3.55], p = 0.08) [5, 8, 9, 11, 22, 26–28] (Figure 2).
(0.88 ng/μL) (p = 0.03) [24].
No correlation between ctDNA status at any timepoint and
pretreatment T stage was found in seven studies [5, 7, 9, 11, 24– Correlation between ctDNA status and good response
26]. Liu et al. [27] reported more negative post-t reatment ctDNA to treatment
in T3–4 versus T0–2 patients (p = 0.04) [27]. Nodal involvement
[5, 7, 11, 25–27] and clinical staging [7, 9, 24, 26, 28] showed no There was no relationship between ctDNA status at baseline and
association with ctDNA status. Primary tumour location and dis- the likelihood of a good response to treatment (RR [95% CI] 1.03
tance from the anal verge were unrelated to ctDNA status [5, 7, [0.76–1.38], p = 0.86) [5, 7–9, 11, 26]. Although patients who tested
9, 11, 22, 24, 25], except for one study showing higher positive negative for ctDNA in the preoperative period tended to have a
ctDNA in high rectal tumours (>10 cm vs. middle rectal tumours, good response to treatment, this was not statistically significant (RR
p = 0.01) [28]. [95% CI] 1.63 [1–2.66], p = 0.05) [5–9, 11, 26]. Patients who achieved
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NASSAR et al. 1353
TA B L E 1 (Continued)
Times of ctDNA measurement Neoadjuvant CRT regimen Follow up Outcomes
Before CRT (T1 or baseline), before Long-course EBRT 50 Gy over 5 weeks Not specified PCR, TRG, mrTRG
the 15th (T2) and the 25th (T3) with concurrent capecitabine plus
fractions of nCRT, 0 to 1 day before irinotecan, followed by one cycle of
surgery (T4), and 1 week after interval chemotherapy (capecitabine plus
surgery (T5) irinotecan)
4 time points: before CRT (baseline), Randomly assigned in a 1:1 ratio to receive Median: 18.8 months TRG, distal metastasis, PCR
one cycle after the initiation of CRT either capecitabine or CapeOx regimen. All
(On-CRT), about 7 weeks after CRT received long-term radiotherapy (45–50 Gy
and before surgery (preop), and over 5 weeks)
within 1 month after surgery
F I G U R E 2 Relationship between ctDNA status and pathological complete response (pCR).

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1354 NASSAR et al.
a good response to treatment were more than two times more likely [7, 9, 22, 26, 28] (RR [95% CI] 5.03 [3.31–7.65], 6.17 [2.38–15.95],
to have negative postoperative ctDNA (RR [95% CI] 2.18 [1.07–4.43], p < 0.001), respectively (Figure 3).
p = 0.03) [5, 7–9, 26] (Figure S8). Moreover, pooled results from survival data regarding metastasis-
free survival (MFS) showed that patients were about 11 times more
likely to develop metastasis if they had positive preoperative ctDNA
ctDNA as a prognostic factor after treatment (RR [95% CI] 11.04 [4.99–24.42], p < 0.001) [7, 26, 27] (Figure S9).
Development of distant metastasis

Development of locoregional recurrence
Baseline ctDNA had no relationship to future development of dis-
tant metastasis (RR [95% CI] 1.39 [0.76–2.57], p = 0.29) [7, 9, 22, Patients who had positive ctDNA status preoperatively had more
26, 28]. However, there was a strong correlation between ctDNA than four times the risk of developing locoregional recurrence (RR
positivity in the pre-and postoperative periods and development of [95% CI] 4.72 [1.15–19.38], p = 0.03) [8, 11, 27, 28]. Similarly, positive
distant metastasis, where patients had more than five and six times postoperative ctDNA status was associated with about eight times
the risk of developing distant metastasis if they had positive ctDNA the risk compared to ctDNA-negative patients (RR [95% CI] 8.07
in the preoperative [7, 9, 11, 22, 26, 28] and postoperative period [2.52–25.86], p < 0.001) [8, 28] (Figure 10).
F I G U R E 3 Relationship between ctDNA status and development of distant metastasis.

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NASSAR et al. 1355
F I G U R E 4 Relationship between ctDNA status and relapse-free survival (RFS).
Relapse-free survival (RFS) patients who are more likely to have cCR at the time of initial di-
agnosis, assurance that cCR accurately correlates with pCR, and
Pooled results from survival data showed a strong association be- early detection of recurrence. Current practice uses cCR as a
tween positive pre- and postoperative ctDNA and relapse during marker to identify those patients who are also likely to have pCR.
follow-up. Patients with positive ctDNA at the preoperative [7, 11, However, it is increasingly acknowledged that cCR poorly cor-
26–28] and postoperative [7–9, 22, 24, 26, 28] periods had more than relates with pCR [8]. It has also been recognized that the use of
13 and 12 times the risk of relapse after treatment (RR [95% CI] 13.55 MRI following nCRT is mainly useful in excluding cCR rather than
[7.12–25.81], 12.14 [3.19–46.14], p < 0.001), respectively. There was confirming it [30]. As such, there has been an increasing interest
no significant association between baseline ctDNA and disease relapse to explore if ctDNA can help address these challenges in the man-
(RR [95% CI] 1.77 [0.71–4.44], p = 0.22) [7, 11, 22–24, 28] (Figure 4). agement of rectal cancer.
This systematic review revealed no relationship between pre-
operative ctDNA status and the achievement of pCR. Notably,
DISCUSSION patients who exhibited good responses to treatment were more
likely to have negative postoperative ctDNA (p = 0.03). This high-
The major challenges encountered in the currently evolving treat- lights the limitation of ctDNA in predicting pCR, and therefore,
ment options for rectal cancer are the ability to predict those further research is needed to identify additional tools that could
|
1356 NASSAR et al.
predict those patients who are more likely to be suitable for organ test to identify colorectal cancer such as that demonstrated by the
preservation. Eclipse study [35]; de-escalate or escalate adjuvant systemic treat-
This meta-
a nalysis also found that positive postoperative ment as studied in the Circulate-Japan/Circulate-US studies [36, 37];
ctDNA was associated with increased risk, up to eight times, of or modify systemic treatment based on subclonal evaluation based
developing locoregional recurrence compared to ctDNA-n egative on ctDNA tests [38–40].
patients (p < 0.001). Additionally, patients were at a higher risk We suggest that future studies on ctDNA should aim at prospec-
of developing distant metastasis if they had positive ctDNA in tive muticentre studies with standardization in preoperative stag-
the pre-and postoperative periods (p < 0.001). Patients with pos- ing, patient selection for neoadjuvant therapy, restaging scans, time
itive ctDNA at the pre- and postoperative stages had more than delay between completing neoadjuvant therapy and surgery and
13 and 12 times the risk of relapse after treatment, respectively. follow-up.
These findings could potentially be used to identify patients who
would potentially benefit from closer surveillance, such as addi-
tional image studies to identify early recurrent/metastatic dis- CO N C LU S I O N
ease, or adjuvant therapy after surgical resection of rectal cancer.
The strength of this study is that it is the largest systematic re- ctDNA could potentially be a useful tool for guiding treatment strat-
view and meta-analysis in the literature to assess the role of ctDNA egy in patients with LARC and follow-up after cCR. It could help to
in the management of LARC. It included a number of good quality predict high-risk patients who are at a greater risk of disease relapse,
cohort studies with consistent results regarding role of pre-and facilitating individualized surveillance and treatment and potentially
postoperative ctDNA when compared to other published reviews improving long-term outcomes. However, further studies, especially
on this topic [31–33]. prospective studies, are needed to standardize the use of ctDNA in
Two recent systematic reviews by Chang et al. [32] and Mi et al. clinical practice.
[33] evaluated the role of ctDNA in predicting treatment response
and prognosis, and reported similar findings [32, 33]. However, AU T H O R C O N T R I B U T I O N S
the largest number of primary studies were incorporated in this Ahmed Nassar: Investigation; writing – original draft; methodol-
review. Additionally, we scrutinized more specific prognostic out- ogy; writing – review and editing; formal analysis; software; data
comes, including the development of locoregional recurrence and curation; visualization; validation. Noha E. Aly: Writing – review
distant metastasis. Our findings highlight that there is insufficient and editing; validation; visualization; data curation; investigation;
support for the role of ctDNA measurement at any time point in methodology. Zhaohui Jin: Writing – review and editing; visualiza-
predicting pCR. tion; validation; supervision; investigation; methodology. Emad H.
The main limitation of this study was the use of data from various Aly: Conceptualization; investigation; writing – original draft; writ-
publications with heterogenicity of the timing and techniques used ing – review and editing; methodology; validation; visualization;
in ctDNA measurement, study designs and data collection. Among supervision.
these factors, the test modality is possibly the most important com-
ponent. Despite this, the findings from this meta-analysis provide a AC K N OW L E D G E M E N T S
summary of the currently available evidence from the existing lit- With thanks to Janet Clapton, Information Specialist, and Camila
erature that would help guide the current use of ctDNA and future Garces-Bovett, Senior Information Specialist, The Royal College of
research until further data become available to better define the role Surgeons of England Library and Archives Team, for conducting the
of ctDNA in the management of LARC. literature searches.
Liu et al. [27] highlighted the superior performance of preoper-
ative ctDNA measurement using a personalized assay over a uni- F U N D I N G I N FO R M AT I O N
versal panel in predicting recurrence risk. Patients testing positive No funding was obtained for this study.
for ctDNA with the personalized assay had a 27-fold higher risk of
recurrence (RR [95% CI] 27.38 [8.61–87.6], p < 0.0001), compared C O N FL I C T O F I N T E R E S T S TAT E M E N T
to only a five-fold higher risk with the universal panel (RR [95% A Nassar, N E Aly, E H Aly: The authors have no competing in-
CI] 5.18 [1.78–15.2], p = 0.00086). This underscores the promising terests to declare that are relevant to the content of this article.
potential of personalized assays in clinical practice [27]. The early- Zhaohui Jin COI link: https://coi.asco.org/share/V5Z-TR68/Zhaoh
determination of the phase III Cobra study which used a blood only ui%20Jin.
tumour-agonistic panel again raised the concern of the sensitivity
and specificity of this blood only testing approach [34]. DATA AVA I L A B I L I T Y S TAT E M E N T
ctDNA as a small fraction of cell-free DNA is now recognized to Data can be accessed upon request and through this link:https://
be a powerful tool throughout the whole spectrum of our colorec- onedrive.live.com/?authkey=%21AB0aoLIx8p%5FWONQ&id=762
tal cancer care. Patients may benefit from ctDNA-based screening DAE049CA2DD5C%2112978&cid=762DAE049CA2DD5C 2 Items.
|
NASSAR et al. 1357
Res. 2021;27(10):2890–8. https://doi.org/10.1158/1078-0 432.

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ctDNA As A Predictor of Outcome After Curative Resection For Locally Advanced Rectal Cancer Systematic Review and Meta-Analysis

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Received: 10 February 2024 | Revised: 26 April 2024 | Accepted: 3 May 2024

ctDNA as a predictor of outcome after curative resection for

Ahmed Nassar1,2 | Noha E. Aly2 | Zhaohui Jin3 | Emad H. Aly1,2

Quality and risk of bias assessment Neoadjuvant chemoradiotherapy (Table 1)

Patient characteristics Curative surgical resection

Identification of studies via databases and registers

Records idenfied from databases

Records removed before screening:

Records screened Records excluded

Reports sought for retrieval Reports not retrieved

Reports assessed for eligibility Reports excluded (n = 18):

- No clear separaon between colon and rectal

Studies included in review

TA B L E 1 Summary of the studies included in the systematic review.

Study, year No. of Method of ctDNA ­

Agostini et al., Retrospective, Some 67 Two primers (DNA fragments): qPCR

Times of ctDNA measurement Neoadjuvant CRT regimen Follow up Outcomes

Study, year No. of Method of ctDNA ­

Zhou et al.,2021 Prospective, Some 106 Panel of 1021 genes NGS

Times of ctDNA measurement Neoadjuvant CRT regimen Follow up Outcomes

F I G U R E 2 Relationship between ctDNA status and pathological complete response (pCR).

Development of distant metastasis

F I G U R E 3 Relationship between ctDNA status and development of distant metastasis.

F I G U R E 4 Relationship between ctDNA status and relapse-­free survival (RFS).

Res. 2021;27(10):2890–8. https://​doi.​org/​10.​1158/​1078-­​0 432.​

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Records idenfied from databases

- No clear separaon between colon and rectal

Study, year No. of Method of ctDNA

Study, year No. of Method of ctDNA

F I G U R E 4 Relationship between ctDNA status and relapse-free survival (RFS).

Res. 2021;27(10):2890–8. https://doi.org/10.1158/1078-0 432.