ORIGINAL ARTICLE

Circulating tumor DNA and radiological tumor volume identify patients at

risk for relapse with resected, early-stage non-small-cell lung cancer
H. T. Tran1y , S. Heeke1y , S. Sujit2 , N. Vokes1 , J. Zhang1 , M. Aminu2 , V. K. Lam3 , A. Vaporciyan4 , S. G. Swisher4,
M. C. B. Godoy5, T. Cascone1 , B. Sepesi1 , D. L. Gibbons1 , J. Wu1,2z & J. V. Heymach1z
Departments of 1Thoracic Head & Neck Medical Oncology; 2Imaging Physics, The University of Texas MD Anderson Cancer Center, Houston; 3Sidney Kimmel
Comprehensive Cancer Center, Johns Hopkins, Baltimore; Departments of 4Thoracic and Cardiovascular Surgery; 5Thoracic Imaging, The University of Texas MD
Anderson Cancer Center, Houston, USA

Available online 21 November 2023

Background: Predicting relapse and overall survival (OS) in early-stage non-small-cell lung cancer (NSCLC) patients
remains challenging. Therefore, we hypothesized that detection of circulating tumor DNA (ctDNA) can identify
patients with increased risk of relapse and that integrating radiological tumor volume measurement along with
ctDNA detectability improves prediction of outcome.
Patients and methods: We analyzed 366 serial plasma samples from 85 patients who underwent surgical resections and
assessed ctDNA using a next-generation sequencing liquid biopsy assay, and measured tumor volume using a computed
tomography-based three-dimensional annotation.
Results: Our results showed that patients with detectable ctDNA at baseline or after treatment and patients who did
not clear ctDNA after treatment had a significantly worse clinical outcome. Integrating radiological analysis allowed the
stratification in risk groups prognostic of clinical outcome as confirmed in an independent cohort of 32 patients.
Conclusions: Our findings suggest ctDNA and radiological monitoring could be valuable tools for guiding follow-up care
and treatment decisions for early-stage NSCLC patients.
Key words: MRD, early-stage NSCLC, ctDNA, tumor volume, liquid biopsy

INTRODUCTION relapse in multiple tumor entities, including colorectal

In contrast to the dismal prognosis of advanced-stage non-
small-cell lung cancer (NSCLC), the 5-year overall survival
(OS) rate of early-stage NSCLC (I-IIIa) is 33%-60% with sur-
gery with curative intent.1 Recent studies indicate that the
introduction of immunotherapy, given either as neo-
adjuvant chemoimmunotherapy or as monotherapy in the
adjuvant setting, has improved survival outcomes, but
relapse still occurs frequently. Thus, predicting the risk of
relapse after standard-of-care (SOC) treatment remains a
major challenge for these patients.
Because relapse has been associated with the presence
of minimal residual disease (MRD),2 many studies have
evaluated circulating tumor DNA (ctDNA) monitoring in
plasma as a method to detect MRD and predict early
*Correspondence to: Dr John V. Heymach, Departments of Thoracic/Head and
Neck Medical Oncology and Cancer Biology, University of Texas MD Anderson
Cancer Center, Unit 432, Houston, Texas 77230, USA. Tel: þ01-713-792-6363
E-mail: jheymach@mdanderson.org (J. V. Heymach).
y
Contributed equally.
z
Co-senior authors.
0923-7534/© 2023 The Author(s). Published by Elsevier Ltd on behalf of
European Society for Medical Oncology. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
cancer3,4 and lung cancer.5-7
In those studies, however, ctDNA was assessed using a
customized or ‘bespoke’ assay informed by genomic
profiling of tumor. In contrast, tissue-agnostic assays are the
same, independent of the patient, and might thus present
advantages for implementation in routine and timely clinical
care, particularly when whole exome sequencing is not
available from the tumor.
Interestingly, while ctDNA shedding is associated with
tumor volume,8 we recently highlighted that in patients
with advanced NSCLC, ctDNA levels also vary depending on
the tumor genotype and location.9 Furthermore, some pa-
tients with no detectable ctDNA ultimately relapse post-
operatively, presumably because their tumors shed lower
or undetectable levels of DNA into the circulation. Conse-
quently, we hypothesized that integrating ctDNA analysis
with radiologically measured tumor volume may also
improve prediction of relapse.
We therefore retrospectively analyzed a cohort of 85 pa-
tients with early-stage NSCLC who underwent plasma sam-
pling before surgery and shortly after surgery as well as
longitudinal treatment samples collected during follow-up to
detect MRD and predict early relapse using a blood-based,

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Annals of Oncology H. T. Tran et al.

tissue-agnostic assay for robust ctDNA detection. We further
integrated this analysis with radiological tumor volume
assessed before surgery to highlight how integrating ctDNA
and radiological data can improve prediction of relapse-free
survival (RFS) and OS in this clinical setting and confirmed
our results in an independent cohort of 32 NSCLC patients.
PATIENTS AND METHODS
From April 2016 to August 2018, 117 patients with early-
stage NSCLC were consented and enrolled in the Immuno-
genomic Profiling of NoneSmall Cell Lung Cancer protocol
(ICON) study that was approved by The University of Texas
MD Anderson Cancer Center Institution Review Board and
in accordance with the Code of Ethics of the World Medical
Association. All patients in this study received SOC therapies
for early-stage NSCLC (Table 1).
Blood collection
Blood was collected in cell-free DNA (cfDNA) blood collec-
tion tube tubes (Streck Corp, La Vista, NE) at the following
time points: before surgery and 4 weeks, 7 months, 10
months, and 13 months after surgery. Blood samples were
processed within 24 h after collection and cell-free plasma
were frozen at 70 C until analysis. All samples were de-
identified using unique sample identification assignment
by our institution’s biospecimen inventory system. Across
both cohorts, 561 samples have been analyzed.
ctDNA detection
For cohort 1, cfDNA was extracted from 366 banked plasma
samples (median volume: 5.9 ml; range 2-10 ml) and
analyzed with a 500-kb plasma-only assay prototype (Lunar
version 1.2, Guardant Health, Redwood City, CA) that eval-
uates tumor-derived genomic and methylation signals from
an w500-kb panel without the need for separate tumor
tissue or peripheral blood mononuclear cell analysis. Briefly,
extracted cfDNA is subjected to automated methylation
partitioning followed by unique molecular barcoding for
single-molecule tracking. PCR-amplified libraries are then
enriched for the panel covering common truncal oncogenic
mutations (w45 kb, 37 genes including ALK, BRAF, EGFR,
ERBB2, KRAS, MET, NF1, NRAS, PIK3CA, PTEN RB1, STK11,
TP53, and others) and a subset of w450 kb of epigenomic
regions that undergo differential methylation in lung cancer.
Extracted cfDNA fragments were partitioned based on the
level of methylation, uniquely barcoded, and then recom-
bined for library preparation and next-generation sequencing
(NovaSeq 600 System, Illumina, San Diego, CA). Sequencing
reads are analyzed using a proprietary bioinformatics algo-
rithm software trained on cancer and cancer-free control
samples for the binary detection of ctDNA (ctDNA detected
versus ctDNA not detected). The software algorithm iden-
tifies genomic mutations (single-nucleotide variants and
Indels, aligned to the reference human genome, hg19) and
filters only for mutations likely to be tumor derived (i.e. fil-
ters germline variants as well as variants associated with
clonal hematopoiesis) based on a pre-defined threshold. The
bioinformatics algorithm simultaneously determines the
presence of tumor-derived differential methylation by eval-
uating the distribution of cfDNA molecules across the
methylation partitions. A statistical likelihood that the
observed patterns across all regions in a sample are likely to
be tumor derived is generated, and the samples must pass a
pre-defined threshold to result in a ‘methylation-detected’
call. Detection of ctDNA was defined by the presence of a
somatic mutation or by the detection of a methylation
signal. This prototype assay has undergone partial analytic
validation with >90% specificity among 74 self-declared
cancer-free controls (96.6% for epigenomic caller, 95.9%-
100% for genomic caller depending on mutation type,
combined caller 93.2%) and 100% detection at the lowest
dilution limit tested in the limit of detection study of 0.3%
mutant allele fraction.
Additionally, 195 samples from 32 patients in cohort 2
have been analyzed using an earlier iteration of the assay
that omits the inclusion of DNA methylation (Lunar version
1.1, Guardant Health).
Radiological tumor volume assessment
The tumor was contoured on computed tomography (CT)
with the guidance of [18F]2-fluoro-2-deoxy-D-glucose posi-
tron emission tomography (18F-FDG PET) using the MIM
software (MIM Software Inc, Cleveland, OH, USA) by a
thoracic radiologist. The baseline CT and PET Digital Imaging

Table 1. Patient characteristics

Cohort 1 (n [ 85) Cohort 1 with recurrence (n [ 33; 39%) Cohort 2 (n [ 32)

Age Median (range) 69 (38-85) 68 (38-85) 65 (44-85)
Gender Female (%) 41 (48%) 15 (45%) 20 (63%)
Male (%) 44 (52%) 18 (55%) 12 (38%)
Histology Adenocarcinoma 58 (68%) 22 (67%) 25 (78%)
Squamous cell carcinoma 21 (25%) 7 (21%) 5 (16%)
other 6 (7%) 4 (12%) 2 (6%)
Clinical stage I 41 (48%) 10 (30%) 16 (50%)
II 29 (34%) 13 (39%) 12 (38%)
III 15 (18%) 10 (30%) 4 (13%)
Other therapies Neoadjuvant chemotherapies 24 (28%) 14 (42%) 6 (19%)
Adjuvant chemotherapies 23 (27%) 9 (27%) 13 (41%)
Post-operative radiation therapies 16 (19%) 10 (30%) 6 (19%)
Follow-up (months) Median (range) 51 (2-73) 43 (2-73) 65 (5-74)

184 https://doi.org/10.1016/j.annonc.2023.11.008 Volume 35 - Issue 2 - 2024

H. T. Tran et al.
and Communications in Medicine images were curated
from picture archiving and communication system. The
Digital Imaging and Communications in Medicine scans
were converted to Neuroimaging Informatics Technology
Initiative by dcm2nii with the use of the MRIcron tool
(www.nitrc.org). The three-dimensional (3D) manual con-
tours were computationally extracted for subsequent ana-
lyses. The visual quality control of segmentations was done
by superimposing tumor on imaging using ITK-SNA (version
4.0, www.itksnap.org).10,11 Images closest to surgery date
were used, with a mean time of images before surgery of 59
days (range: 3-183 days before surgery). For the partial-solid
tumors, ground-glass components and solid components of
the tumor were delineated separately by using the lung
window on CT. The overall tumor volume is computed as
the aggregate of the volumes associated with both the GGN
and solid nodule regions.
Data analysis
Data were analyzed using R (v4.2.1). Survival including log-
rank test and Cox proportional hazard ratio (HR) was
calculated using the R packages survival (v3.4) and surv-
miner (v0.4.9). Receiver operator characteristics was
assessed using pROC (v1.18) by associating the presence of
ctDNA at baseline before surgery with the tumor volume as
assessed by radiographic 3D reconstruction. Cut-offs for
optimal stratification of tumor volume were calculated
based on Youden’s J and the closest distance to the top left
corner using pROC (Supplementary Table S1, available at
https://doi.org/10.1016/j.annonc.2023.11.008). Differences
in RFS and OS from the time of surgery were analyzed using
Cox proportional hazards models and KaplaneMeier anal-
ysis. Patients were further stratified by ctDNA clearance,
which was defined by a pre-surgery (baseline) ctDNA-
positive test result followed by a ctDNA-negative test
result at any subsequent time point after completion of SOC
therapy. For the analysis of cohort 2 in the combination of
ctDNA plus radiological tumor volume, Cox proportional HR
with Firth’s penalized likelihood has been used for estima-
tion of HR and confidence intervals (CIs).
RESULTS
Patient characteristics
Patient characteristics are summarized in Table 1. Among
the 85 included patients, 58 (68%) had adenocarcinoma,
and 41 (48%) were diagnosed with stage I disease. Twenty-
eight percent of patients received neoadjuvant therapies,
and 27% received post-surgical adjuvant therapies. With a
median follow-up of 4.3 years (range: 0.2-6.1 years) for this
cohort, 39% of patients had a relapse of disease.
Detection of ctDNA before surgery
Before surgery, 80/85 samples were analyzed successfully,
and ctDNA was detected in 48 (60%) of these samples
overall, including 49%, 58%, and 100% of patients with
stage I, II, and III carcinoma that did not receive
Volume 35 - Issue 2 - 2024
Annals of Oncology
neoadjuvant therapies, respectively (Supplementary
Figure S1A, available at https://doi.org/10.1016/j.annonc.
2023.11.008). Before surgery, ctDNA was detected in 18/
24 (75%) patients who received neoadjuvant therapies and
30/56 (54%) patients who did not receive neoadjuvant
therapies (Supplementary Figure S1A and B, available at
https://doi.org/10.1016/j.annonc.2023.11.008). Detection
rates in squamous cell carcinoma were highest (87%), fol-
lowed by other histologies (67%) and adenocarcinoma
(39%; Supplementary Figure S1C, available at https://doi.
org/10.1016/j.annonc.2023.11.008). In patients with stage
I disease, detection of ctDNA was 42% for adenocarcinoma
(n ¼ 11/26) and 78% for squamous cell carcinoma (n ¼ 7/9;
Supplementary Figure S2A, available at https://doi.org/10.
1016/j.annonc.2023.11.008). Clinical stage and lung cancer
histologies were balanced in this cohort (Supplementary
Figure S2B and C, available at https://doi.org/10.1016/j.
annonc.2023.11.008) and no differences between geno-
types on the detection of ctDNA were observed
(Supplementary Figure S3, available at https://doi.org/10.
1016/j.annonc.2023.11.008). Additionally, ctDNA detection
rates increased with histological grading (G1: 38%; G2: 52%,
and G3þ: 85%; Supplementary Figure S4A, available at
https://doi.org/10.1016/j.annonc.2023.11.008). Further-
more, lung adenocarcinoma histological subtypes demon-
strated equal detection rates for acinar versus non-acinar
subtypes (both 44%; Supplementary Figure S4B, available at
https://doi.org/10.1016/j.annonc.2023.11.008). Lastly,
detection of ctDNA at baseline before surgery did not differ
between patients with extrapulmonary versus pulmonary
relapse (67% versus 64%; Supplementary Figure S4C,
available at https://doi.org/10.1016/j.annonc.2023.11.008).
Importantly, the presence of ctDNA before surgery was
associated with a numerically shorter RFS (HR 2.14, 95% CI
0.95-4.81, P ¼ 0.06) and a statistically significant decrease
in OS (HR 3.29, 95% CI 0.95-11.45, P ¼ 0.047) (Figure 1A
and B), suggesting detectability of ctDNA before surgery
may have prognostic value.
ctDNA detected after standard-of-care therapy is
associated with worse outcome
We next investigated whether the detection of ctDNA after
definitive SOC therapy, defined as surgery and neoadjuvant
or adjuvant chemotherapy, and/or post-operative radio-
therapy (PORT) if administered, was also associated with
risk of relapse and OS. We observed that after SOC therapy,
the presence of detectable ctDNA was associated with
statistically significant decreases in RFS (HR2.80, 95% CI
1.23-6.37, P ¼ 0.01) and OS (HR3.99, 95% CI 1.40-11.40,
P ¼ 0.0053) compared to patients without detectable
ctDNA after SOC therapy (Figure 1C and D).
ctDNA clearance defines patient with lower risk or
recurrence
We observed that some patients ‘cleared’ ctDNA after SOC
therapy, defined by a positive ctDNA detection at baseline
followed by a negative ctDNA sample during follow-up after
https://doi.org/10.1016/j.annonc.2023.11.008 185
Annals of Oncology H. T. Tran et al.

A RFS - ctDNA detection before to surgery B OS - ctDNA detection before to surgery

1.00 1.00
Median RFS P = 0.06 + + + + Median OS P = 0.047
+ +++++++++++++ ++
++++++ +
+ + + + HR = 2.14 (0.95-4.81) ++ HR = 3.29 (0.95-11.45)
0.75 + ++++ +++++++ + ++++ + 0.75 +++
+++ +++

Survival probability
++++ +++++++++++++ +
RFS probability

+ +++
+++
++++ +++
++
0.50 +++++++ 0.50

0.25 0.25

0.00
0 20 40
+ no ctDNA
60
+ ctDNA detected
80
0.00
0 20 40
+ no ctDNA
60
+ ctDNA detected
80
ctDNA detected 48 32 28 12 0 48 45 36 14 0
no ctDNA 32 27 23 7 0 32 31 27 9 0
0 20 40 60 80 0 20 40 60 80
Months Months

C RFS - ctDNA detection after SOC D OS - ctDNA detection after SOC

1.00 1.00
Median RFS P = 0.01 + + ++ + Median OS P = 0.0053
+++++
+++++++++++++ + ++++++++ +++ +
+ + HR = 2.80 (1.23-6.37) HR = 3.99 (1.40-11.40)
0.75 + + 0.75

Survival probability
+++++
+++ ++ +++++ + ++++
RFS probability

+++ ++ +
0.50 0.50 ++ + +++ +
++ + + +
0.25 0.25

0.00
0 20 40
+ no ctDNA
60
+ ctDNA detected
80
0.00
0 20 40
+ no ctDNA
60
+ ctDNA detected
80
ctDNA detected 14 7 6 2 0 ctDNA detected 14 14 9 3 0
no ctDNA 50 42 34 11 0 no ctDNA 50 48 41 12 0
0 20 40 60 80 0 20 40 60 80
Months Months

Figure 1. Detection of ctDNA before surgery and/or after completion of standard of care (SOC) therapies are associated with worsen clinical outcomes. (A) RFS and
(B) OS based on ctDNA detection before surgery. (C) RFS and (D) OS based on ctDNA detection after SOC. RFS was calculated from time of surgery to relapse, death
or censoring and OS was calculated from time of surgery to death or censoring. Log-rank tests were used to compute P values, and Cox proportional HRs were
calculated in comparison to patients with detectable ctDNA.
ctDNA, circulating tumor DNA; HR, hazard ratio; OS, overall survival; RFS, relapse-free survival; SOC, standard-of-care.

SOC therapy. We hypothesized that ctDNA clearance at any with risk of RFS and OS, there remained a substantial
point during treatment would be associated with better number of patients with non-detectable ctDNA that
prognosis. Indeed, patients who had detectable ctDNA recurred, as well as ctDNA-positive patients who did not
before treatment and cleared ctDNA after completion of recur. Given the established association between tumor size
SOC treatment had outcomes that were not significantly and recurrence,10 we investigated whether the integration
different than patients with undetectable ctDNA of radiological tumor measurement with ctDNA could
throughout the study for RFS (HR 1.5, 95% CI 0.49-4.7, P ¼ improve prediction of RFS and OS. Radiological tumor vol-
0.478) and OS (HR 4.6, 95% CI 0.48-44, P ¼ 0.186). Patients ume was larger in patients with detectable ctDNA at
with persistent ctDNA, by contrast, had significantly shorter baseline (P ¼ 0.009; Supplementary Figure S7A, available at
RFS and OS compared to patients with undetectable ctDNA https://doi.org/10.1016/j.annonc.2023.11.008) and was
throughout the study [RFS HR persistent compared to never associated with ctDNA detection (area under the receiving
detected ¼ 4.0 (1.46-10.8), P ¼ 0.007; OS HR ¼ 16.5 (2.09- operating characteristic curve ¼ 0.682; Supplementary
131), P ¼ 0.008; Figure 2; Supplementary Figure S5, avail- Figure S7B, available at https://doi.org/10.1016/j.annonc.
able at https://doi.org/10.1016/j.annonc.2023.11.008]. We 2023.11.008). We used Youden’s J to calculate the optimal
furthermore observed a trend toward shorter RFS (HR 2.50, cut-off of tumor volume for the detection of ctDNA in our
95% CI 0.91-6.87, P ¼ 0.075) and OS (HR 3.51, 95% CI 0.95- cohort (Supplementary Table S1, available at https://doi.
13.0, P ¼ 0.061) for persistent ctDNA compared to patients org/10.1016/j.annonc.2023.11.008) to further stratify pa-
who cleared ctDNA (Supplementary Figure S5, available at tients into small-tumor-volume and large-tumor-volume
https://doi.org/10.1016/j.annonc.2023.11.008). Clearance groups and created three risk groups: low risk for patients
for patients with neoadjuvant and those without neo- with both small tumor volume and undetectable ctDNA at
adjuvant therapies are highlighted in Supplementary baseline before surgery; medium risk for patients who
Figure S6, available at https://doi.org/10.1016/j.annonc. either have larger tumor volume or detectable ctDNA at
2023.11.008. Median time to clearance was 20 days baseline before surgery; and high risk for patients with
(range: 2-181 days). both, larger tumor volume and detectable ctDNA at base-
line before surgery (Supplementary Figure S8, available at
https://doi.org/10.1016/j.annonc.2023.11.008). In order to
Combination of ctDNA detection and radiological tumor confirm the results in an independent cohort, we included a
volume assessment predicts clinical outcome second cohort (cohort 2) of 32 patients that have been
While these data provided evidence that ctDNA levels, analyzed with an earlier iteration of the assay. Patients in
particularly after definitive SOC therapy, were associated the high-risk group had a significantly higher risk of relapse

186 https://doi.org/10.1016/j.annonc.2023.11.008 Volume 35 - Issue 2 - 2024

H. T. Tran et al. Annals of Oncology

A RFS - ctDNA clearance B OS - ctDNA clearance

1.00 1.00 + + ++
+ +++++++++ +++++
++ +
Median RFS P = 0.0094 ++ Median OS P = 0.00076
+ +
++ ++
+ + ++ + + +++
0.75 0.75

Survival probability
+ ++ +
++ ++ ++++ + ++++++ +
RFS probability

0.50
+++ 0.50 +
+++ ++ +
+
+++ +
0.25 0.25

0.00 + Persistent + Cleared + Never Detected 0.00 + Persistent + Cleared + Never Detected
0 20 40 60 80 0 20 40 60 80
Persistent 16 8 6 1 0 Persistent 16 16 9 2 0
Cleared 17 13 11 5 0 Cleared 17 15 13 4 0
Never Detected 23 20 16 6 0 Never Detected 23 23 20 8 0
0 20 40 60 80 0 20 40 60 80
Months Months

Figure 2. Failure to clear ctDNA after completion of SOC therapies are associated with greater risks for disease recurrence and shortened overall survival. (A) RFS
and (B) OS in patients without detectable ctDNA throughout the study (Never Detected), patients who cleared ctDNA during treatment (Cleared), and patients
with persistent ctDNA detectable (Persistent). Log-rank tests were used to compute P values.
ctDNA, circulating tumor DNA; OS, overall survival; RFS, relapse-free survival.

compared to patients in the low-risk group (Nlow risk ¼ 19, 1016/j.annonc.2023.11.008), highlighting a robust predic-
Nhigh risk ¼ 32; HR 3.0, 95% CI 1.0-9.1, P ¼ 0.049) but not tion of relapse and survival in two independent cohorts.
patients in the medium-risk group who demonstrated Prediction of relapse was maintained after refining the
comparable outcome (Nmedium risk ¼ 20; HR 1.5, 95% CI radiological tumor analysis to the solid component only
0.43-5.4, P ¼ 0.518) in cohort 1, as confirmed in cohort 2 (Supplementary Figure S9, available at https://doi.org/10.
(Nlow risk ¼ 7, Nmedium risk ¼ 18, Nhigh risk ¼ 7; Figure 3A and 1016/j.annonc.2023.11.008; Supplementary Table S3,
B; Supplementary Table S2, available at https://doi.org/10. available at https://doi.org/10.1016/j.annonc.2023.11.008).
1016/j.annonc.2023.11.008). Likewise, combining ctDNA
and radiology demonstrated statistically significant differ- DISCUSSION
ences in OS in both cohorts (Figure 3C and D; While detection of ctDNA in longitudinal analysis is clearly
Supplementary Table S2, available at https://doi.org/10. able to track tumor evolution and recurrence,12 uncertainty

A RFS - combining ctDNA with radiology (cohort 1) B RFS - combining ctDNA with radiology (cohort 2)
1.00 1.00 ++++ ++
+ + Median RFS P = 0.072 Median RFS P = 0.00067

0.75 + + ++ ++++ ++ ++++ + 0.75

++ ++ + +
RFS probability
RFS probability

+ +++
++
0.50 + ++++ 0.50
+ +++++ ++

0.25 0.25

+
0.00 + high + medium + low 0.00
0 20 40 60 0 20 40 60 80
high 32 20 17 8 high 7 1 1 1 0
medium 20 16 14 4 medium 18 11 11 10 0
low 19 17 15 4 low 7 7 7 7 0
0 20 40 60 0 20 40 60 80
Months Months

C OS - combining ctDNA with radiology (cohort 1) D OS - combining ctDNA with radiology (cohort 2)
1.00 + + 1.00 ++++ ++
++ + ++++++++++ + +++
+ +++++++ ++ ++++Median OS P = 0.033 Median OS P = 0.0015

0.75
++ 0.75
Survival probability

+++
++ ++
RFS probability

+ +++++ ++
++ + +++++ ++++
0.50 0.50
++

0.25 0.25
++

0.00 + high + medium + low 0.00

0 20 40 60 80 0 20 40 60 80
high 32 30 22 9 0 high 7 2 2 2 0
medium 20 19 18 6 0 medium 18 17 15 14 0
low 19 19 17 5 0 low 7 7 7 7 0
0 20 40 60 80 0 20 40 60 80
Months Months

Figure 3. Validation of a risk model with combination of ctDNA and radiologic tumor volume. (A) RFS in patients with detectable ctDNA and radiological tumor
volume above the computed threshold (high risk), patients with undetectable ctDNA and radiological tumor volume below the computed threshold (low risk),
patients with detectable ctDNA but tumor volume below the calculated threshold, or patients with undetectable ctDNA but tumor volume above the calculated
threshold (medium risk) as well as in (B) an independent cohort (cohort 2) of 32 patients. (C) OS for cohort 1 as well as (D) in the independent cohort (cohort 2).
Log-rank tests were used to compute P values.
ctDNA, circulating tumor DNA; OS, overall survival; RFS, relapse-free survival.

Volume 35 - Issue 2 - 2024 https://doi.org/10.1016/j.annonc.2023.11.008 187

Annals of Oncology
persists in the appropriate time point for ctDNA detec-
tion.13 In line with previously published data,14,15 detection
of ctDNA before surgery was associated with worse prog-
nosis which might enable stratification to treatment inten-
sification. However, additional time points within the first 1-
4 months of treatment initiation with immune checkpoint
blockers have been proposed.12,16,17 Here we further
demonstrated that clearance of ctDNA has comparable
prognosis to patients with a baseline negative ctDNA, in line
with recently published results from both early-stage16,17
and advanced-stage NSCLC.18 Using clearance as an
endpoint for future clinical studies is of high interest since it
would allow inclusion of patients where a baseline sample
could not be obtained and thus would facilitate treatment
stratification based on MRD detection.
Interestingly, in our study detection of ctDNA after
definitive SOC therapy was highly associated with worse
clinical outcome and this pragmatic endpoint might there-
fore facilitate comparison across treatment regimens and
would allow a more personalized ctDNA-guided therapy.
We furthermore confirmed that tumor volume was
associated with ctDNA detectability.8 However, both ctDNA
absence and smaller radiological tumor volume were
independently associated with prolonged RFS. This high-
lights that both measurements are at least partially inde-
pendent predictors of relapse. Thus, the integration of
ctDNA detection with radiological volumetric tumor mea-
surement might further enhance the stratification of
patients.
Importantly, the assay used in this study is a laboratory-
developed assay that detects ctDNA for MRD assessment
and does not require baseline tissue profiling, which is a
clear logistical and practical advantage compared to assays
with similar performance that require customization for
each patient.17 The availability of tumor-agnostic ap-
proaches should facilitate access to MRD testing for pa-
tients who do not have sufficient tumor tissue available for
tissue-informed assays and those who require a short test
turnaround time following definitive treatment.14,19 How-
ever, use of such an assay might be more prone to influence
by clonal hematopoeisis,20 and additional studies are
required to further investigate specificity of ctDNA detec-
tion in this setting. However, detection rates at baseline
were comparable to other published assays21 with higher
ctDNA detection in squamous cell carcinoma. Furthermore,
tumor staging is another important factor to be considered
for treatment selection in early-stage NSCLC, independent
of ctDNA status, and prospective studies are needed to
better understand how tumor staging, ctDNA, and radio-
logical analysis can be integrated.
The SOC at the time of sample collection (before 2021)
was adjuvant or neoadjuvant chemotherapy with platinum-
based doublet therapy for resected NSCLC (stage IB-IIIA).
Recently, however, neoadjuvant chemoimmunotherapy7
and adjuvant immunotherapy22 have both been approved
by the Food and Drug Administration in this setting, with
recently published data supporting the efficacy of a regimen
combining the two approaches.23 In this setting, analysis of
188 https://doi.org/10.1016/j.annonc.2023.11.008
H. T. Tran et al.
ctDNA has been proven useful to predict worse clinical
outcomes7,16,24 while the upcoming stage 2 of the Br.36
clinical trial17 will address how ctDNA detection can guide
intensification in patients receiving adjuvant pem-
brolizumab to further establish ctDNA detection as a critical
biomarker in early-stage NSCLC.
Conclusions
In this study, we demonstrated that ctDNA persistence,
detected using an integrated genomic and methylation
assay from plasma ctDNA, is highly predictive of clinical
outcome in early-stage NSCLC and that the integration of
radiographic tumor volume can improve prediction. Such
biomarkers may help tailor-adjuvant therapies or surveil-
lance strategies in resected NSCLC patients.
ACKNOWLEDGEMENTS
We thank Ashli Nguyen-Villarreal, MS, Associate Scientific
Editor, and Sarah Bronson, Scientific Editor, in the Research
Medical Library at The University of Texas MD Anderson
Cancer Center, for editing this article. The authors would like
to acknowledge the Thoracic medical and surgical clinical
team for their research support. We would also like to
acknowledge the generous philanthropic contributions to
the University of Texas MD Anderson Cancer Center Moon
Shots Program, the Mugnaini fund, and Guardant Health for
their support and collaboration on this project.
FUNDING
This work was supported by The University of Texas MD
Anderson Lung Moon Shot Initiative, UT MD Anderson
Cancer CenterdNational Institute of Health [grant numbers
CCSG CA016672, R01 CA262425, R01 CA276178], Guardant
Health, Inc.
DISCLOSURE
HTT reports consulting for Abion Bio. SH reports consulting/
speaker fees from Guardant Health, Qiagen, Boehringer-
Ingelheim, and AstraZeneca. NV reports consulting fees
from Sanofi Genzyme, and Oncocyte. JZ reports personal fees
from AstraZeneca, Geneplus, Hengrui, Innovent, Merck, and
Roche as well as grants and personal fees from Johnson and
Johnson and Novartis, and grants from OrigMed. VKL reports
advisory roles and/or consulting fees from Seagen, Bristol
Myers Squibb, AstraZeneca, Guardant Health, Anheart
Therapeutics, and research funding from Seagen, Bristol
Myers Squibb, Merck, and AstraZeneca. AV reports fees from
AstraZeneca. MCBG reports research funding from Siemens
Healthcare. TC reports speaker fees and honoraria from the
Society for Immunotherapy of Cancer, Bristol-Myers Squibb,
Roche, Medscape, and PeerView; travel, food, and beverage
expenses from Dava Oncology and Bristol Myers Squibb;
advisory roles and/or consulting fees from MedImmune/
AstraZeneca, Bristol Myers Squibb, EMD Serono, Merck &
Co., Genentech, Arrowhead Pharmaceuticals, and Regen-
eron; and institutional research funding from MedImmune/
AstraZeneca, Bristol Myers Squibb, Boehringer-Ingelheim,
Volume 35 - Issue 2 - 2024
H. T. Tran et al.
and EMD Serono. BS reports personal fees from AstraZeneca,
Medscape, and PeerView. DLG reports consulting or advisory
role: Sanofi, GlaxoSmithKline, Janssen Research & Develop-
ment, Eli Lilly, and Menarini Ricerche; research funding:
Janssen Research & Development, Takeda, AstraZeneca,
Astellas, Ribon Therapeutics, and NGM Biopharmaceuticals;
travel, accommodations, expenses: AstraZeneca/MedI-
mmune, BerGenBio, and Takeda. JW and JVH report advisory
roles and consulting fees from AstraZeneca, EMD Serono,
Boehringer-Ingelheim, Catalyst, Genentech, GlaxoSmithK-
line, Hengrui Therapeutics, Eli Lilly, Spectrum, Sanofi, Takeda,
Mirati Therapeutics, BMS, BrightPath Biotherapeutics, Jans-
sen Global Services, Nexus Health Systems, Pneuma Respi-
ratory, RefleXion, and Chugai Pharmaceuticals; institutional
research funding from AstraZeneca, Boehringer-Ingelheim,
Spectrum, and Takeda; and royalties and licensing fees
from Spectrum. All other authors have declared no conflicts
of interest.
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