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Clinical utility of ctDNA by
amplicon based next generation
sequencing in rst line non small
cell lung cancer patients
Valerio Gristina1,4, Tancredi Didier Bazan Russo1,4, Nadia Barraco1,4, Andrea Gottardo1,
Francesco Pepe2, Gianluca Russo2, Fabio Fulfaro1, Lorena Incorvaia1,
Giuseppe Badalamenti1, Giancarlo Troncone2, Umberto Malapelle2, Antonio Russo1,
Viviana Bazan3,5 & Antonio Galvano1,5
The assessment of ctDNA has emerged as a minimally invasive avenue for molecular diagnosis and
real-time tracking of tumor progression in NSCLC. However, the evaluation of ctDNA by amplicon-
based NGS has been not endorsed by all the healthcare systems and remains to be fully integrated
into clinical routine practice. To compare tissue single-gene with plasma multiplexed testing, we
retrospectively evaluated 120 plasma samples from 12 consecutive patients with advanced non-
squamous NSCLC who were part of a prospective study enrolling treatment-naïve patients and in
which tissue samples were evaluated using a single-gene testing approach. While the plasma ctDNA
detection of EGFR and BRAF mutations had an acceptable level of concordance with the archival tissue
(85%), discordance was seen in all the patients in whom ALK alterations were only detected in tissue
samples. Among six responders and six non-responders, early ctDNA mutant allelic frequency (MAF)
reduction seemed to predict radiologic responses and longer survival, whereas increasing MAF values
with the emergence of co-mutations like BRAFV600E, KRASG12V or TP53M237I seemed to be an early
indicator of molecular and radiologic progression. This report using an amplicon-based NGS assay on
ctDNA underscores the real-life need for plasma and tissue genotyping as complementary tools in the
diagnostic and therapeutic decision-making process.
Keywords NSCLC, Liquid biopsy, CtDNA, NGS, Monitoring
Despite the expanding adoption of targeted and immunotherapy-based interventions, the prognosis of patients
with advanced non-small-cell lung cancer (NSCLC) remains regrettably grim1. In the era of precision oncology,
the introduction of liquid biopsy has enabled a paradigmatic transformation in the care of such patients,
oering a promising solution to the limitations of traditional tissue biopsies and establishing itself as a valuable
diagnostic tool in current clinical practice2. Beyond its clinical applicability for diagnostic purposes, the
integration of liquid biopsy testing holds the potential to serve as a valuable tool in monitoring clinical outcomes
and prognostication3,4. Specically, the assessment of circulating tumor DNA (ctDNA), a part of cell-free DNA
(cfDNA) shed from tumor sites into the bloodstream of cancer patients, has emerged as a minimally invasive
avenue for molecular diagnosis and real-time tracking of tumor progression at the time of acquired resistance,
with ctDNA kinetics holding promise as an indicator of treatment ecacy especially in patients with oncogene-
driven NSCLC5. Despite the mounting body of evidence within the scientic literature, the serial monitoring
of ctDNA for predicting radiological responses to conventional treatments has been not endorsed by all the
healthcare systems and remains to be fully integrated into clinical routine practice6.
Even the most recent clinical trials have only adopted polymerase chain reaction (PCR)- and
immunohistochemistry (IHC)-based single-gene testing techniques for assessing the molecular status of tissue
1Department of Precision Medicine in Medical, Surgical and Critical Care (Me.Pre.C.C.), University of Palermo,
Palermo, Italy. 2Department of Public Health, University of Naples Federico II, Naples, Italy. 3Department
of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Palermo, Italy. 4These
authors contributed equally: Valerio Gristina, Tancredi Didier Bazan Russo and Nadia Barraco. 5These authors
jointly supervised this work: Viviana Bazan and Antonio Galvano. email: giuseppe.badalamenti@unipa.it;
antonio.russo@usa.net
OPEN
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samples7–9. Such targeted methodologies employ specic probes to identify known mutations, do not encompass
the entire spectrum of oncogene addictions, and thus fail to detect less prevalent yet clinically signicant
genomic alterations. Furthermore, these methods have limited multiplexing capabilities, thereby constraining
the concurrent analysis of other emerging biomarkers10. To address such limitations, the adoption of plasma
next-generation sequencing (NGS) proves promising, as it saves tissue while facilitating the sequencing of
extensive genomic regions or multiple exons on ctDNA samples11. Despite several research groups have reported
results on the prognostic signicance of ctDNA in NSCLC while many pan-cancer liquid biopsy panels are
commercially available, however, liquid biopsies remain not widely adopted or reimbursed12, while only two
hybrid capture-based cfDNA technologies, such as Guardant360® CDx (Guardant Health, Inc.; Redwood, CA,
USA) and FoundationOne® Liquid CDx (Foundation Medicine, Inc.; Cambridge, MA, USA), have granted the
FDA approval13. Target enrichment, generally achieved by hybrid capture- or amplicon-based approaches,
represents a crucial step in the targeted NGS sequencing workow, signicantly inuencing the success,
eciency, and accuracy of variant detection14. To date, no multiplex amplicon-based liquid biopsy assays have
yet received full FDA approval.
Hence, there is a pressing need for additional data to validate the role of ctDNA by amplicon-based NGS
in forecasting and tracking clinical outcomes in the real-life context of lung cancer. is real-world report,
presented herein, conducts the diagnostic evaluation along with the retrospective assessment of longitudinal
plasma samples by amplicon-based NGS, compared to baseline tissue single-gene testing, to explore the potential
of ctDNA as a predictor of response and survival at the time of rst disease restaging in treatment-naive patients
with advanced NSCLC undergoing standard rst-line treatments.
Materials and methods
Patient samples and study design
To compare tissue single-gene with plasma multiplexed testing, we retrospectively evaluated 12 consecutive
patients with advanced non-squamous lung cancer who were part of a prospective study enrolling treatment-
naïve patients at the Paolo Giaccone University Hospital, Palermo (Italy) and in which formalin-xed paran‐
embedded (FFPE) tissue samples were evaluated according to clinical practice using a targeted single-gene
testing approach (real time-PCR and IHC for the detection of EGFR/BRAF hotspot mutations and ALK/ROS1
alterations, respectively) by a distinct referring pathology unit, as previously described15. Real time-PCR was
performed on FFPE specimens by amplication of 15–30 ng of extracted DNA using the EasyPGX® Ready EGFR
and BRAF kits on EasyPGX® qPCR (Diatech Pharmacogenetics), according to the manufacturer’s instructions.
ese tests allowed the detection of the most clinically relevant hotspot alterations, as reported in Supplementary
Table 1. Data were automatically analyzed as positive or negative results using the EasyPGX® analysis soware
version 4.0.10 (Diatech Pharmacogenetics)16. Paired blood samples were collected at baseline (T0) and following
the rst radiologic evaluation of disease within 12 ± 1 weeks (T1 or W12) during the treatment course. e
collected plasma samples were used to isolate, quantify, and analyze cfDNA using a DNA/RNA-based NGS testing
approach both at T0 and T1. All the patients underwent a computerized tomography scan at T0-T1 and were
classied as radiologic responders (complete (CR) or partial response (PR)) or non‐responders (stable disease
(SD) or progressive disease (PD)) according to the Response Evaluation Criteria in Solid Tumors (RECIST)
version 1.117. Patients with oligo-progressive disease (oligo-PD), dened as limited metastatic areas progressing
on rst-line treatment and treated using local radiation therapy followed by continued targeted agents according
to clinical practice, were labeled as non-responders18. Plasma molecular response or progression was evaluated
according to the reduction/clearance or increase/persistence of the maximal ctDNA mutant allelic fraction
(MAF), respectively. e study was conducted following the Declaration of Helsinki, and the protocol was
approved by e Ethics Committee Palermo I (AIFA code CE 150109).
Plasma separation, cfDNA quantication, and molecular analysis
According to the standard procedure6, blood samples (5 mL) were collected into K2 EDTA tubes for times
ranging from 15min to less than 2h at room temperature and centrifuged twice (10min at 1,200 x g; 10min
at 16,000 x g) using a refrigerated centrifuge (4°C) for plasma collection. e collected plasma samples were
stored at -80°C until further processing or immediately used to extract cfDNA. We extracted cfDNA from
2 mL of plasma using a QIAamp Circulating Nucleic Acid Kit (Qiagen) and quantied it in terms of ng/µl
using a Qubit™ dsDNA HS Assay Kit. Namely, 20 ng of isolated cfDNA was analyzed using Oncomine™ Lung
cfTNA Research Assay while, according to the manufacturer’s recommendations, we accepted an input range
of 1–50 ng of extracted cfDNA to create a successful library. According to the manufacturer’s instructions ant
external quality assessment for our laboratory, a contrived analytic positive control was used to monitor each
batch for quality assurance. e analytical performance of each sequencing run was inspected by evaluating the
technical parameters (reads, medium coverage depth, uniformity of coverage). Quality control check for single
nucleotide variant/indel target regions was based on molecular coverage. As regards the detection of fusion and
exon skipping amplicons, the panel provided ve assays to perform the quality check: two non-fused process
control genes (HMBS and TBP) consistently detected in cell-free nucleic acid (cfNA) extracts and other three
assays (one with the skipping between exon 13 and 15, and two wild type assays) were used to inform the variant
call quality check of fusions and MET exon 14 skipping, respectively. At least one control from each group must
have passed a molecular count > 2. e libraries were quantied using an Ion Library TaqMan™ quantication
kit on a QuantStudio7 Pro Real-Time PCR System (Applied Biosystems) using Design and Analysis Soware
v2.4.3. e libraries were diluted to 30 ng and pooled together. e pool was charged on Ion 510 and Ion 520
and Ion 530 Chef reagents (ermo Fisher Scientic); then, an automatic system (Ion Chef instrument, ermo
Fisher Scientic) was used to automatically charge the Ion 530 chip with the pooled libraries according to the
manufacturer’s instructions.
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Using 20 ng of cfNAs, the specicity of this kit was 99.0% at 0.1% of the limit of detection (LoD). e data were
tested on an amplicon-based sequencing platform Ion Torrent S5™ System. Oncomine TagSeq Lung v2 Liquid
Biopsy‐w2.5‐Single Sample was the workow applied for the analysis of cfNAs samples. To test the reliability of
the data for cfNA sequencing, we used the following thresholds: total mapped reads > 3M, median read coverage
Avg 40,000 – Min > 25,000, median molecular coverage > 2500. e data of DNA sequencing were analyzed
with Ion Torrent TorrentSuite™ (TS, version 5.18) using the Coverage Analysis and Variant Caller plugins. e
LoD of single nucleotide variants/indels detected was calculated by the level of molecular amplicon coverage
and displayed for each variant call. Molecular coverage had to be at least 2 with a minimum detection cuto
frequency of 0.035%. To be reported, fusion and exon skipping amplicons must have > 2 molecular counts. e
sequencing data were categorized by relevance with the related percentage of allelic frequency as annotated by
Ion Reporter Soware v5.18 applying the Variant Matrix Summary (5.18) lter chains for default use.
Statistical considerations
e categorical clinical-pathological variables of the population enrolled in the study were described as absolute
numbers (N) and percentages (%). To describe the treatment ecacy, progression-free survival (PFS) was
computed as the time from treatment start to disease progression or death from any cause; overall survival (OS)
was computed as the period from treatment initiation to death from any cause. To assess the diagnostic accuracy
of liquid biopsy, contingency tables were constructed to describe the results of overall baseline tissue and plasma
testing and subsequently for each gene of interest (EGFR, ALK, and BRAF). e genomic status of tumor tissue
was considered as a gold standard whereas ctDNA evaluation was considered as an experimental group. All
analyses were performed using SPSS soware (ver 27.0). For the diagnostic accuracy analyses, the following
denitions were considered: true positive (TP) as the number of patients with a mutation discovered in both
tissue and liquid biopsy, true negative (TN) as the number of patients with a mutation not discovered in either
the tissue or liquid biopsy, false positive(FP) as the number of patients with a mutation not found in the tissue
but found in liquid biopsy, and nally false negative (FN) as the number of patients with a positive tissue biopsy
and negative liquid biopsy. Consequently, sensitivity and specicity were calculated as the ratio between TP and
the sum of TP and FN × 100 (TP/[TP + FN] × 100) and the ratio between TN and the sum of TN and FP × 100
(TN/[TN + FP] × 100) respectively. Lastly, concordance between ctDNA and tissue was evaluated as ([TP + TN]/
[TP + FN + TN + FN]) × 100.
Results
Among 73 patients prospectively enrolled in the real-world cohort, in this report, we retrospectively focused
on consecutive non-squamous lung cancer patients who received baseline single-gene testing on archival tissue
and had sucient circulating biospecimens. Briey, a total of 120 liquid biopsy plasma samples were collected
isolating cfDNA from 12 patients at baseline with paired available plasma samples at disease radiologic re-
evaluation. Systemic treatment was performed according to clinical indication and routine practice. Clinical-
pathological characteristics of patients included in our analysis are listed in [Supplementary Table 2].
Diagnostic accuracy of NGS plasma ctDNA at baseline
In our patients’ cohort, the molecular landscape determined by tissue single-gene testing identied four distinct
proles: six patients presented with EGFR mutations (LEXO14, LEXO33, LEXO42, LEXO51, LEXO53, and
LEXO65), three patients with ALK IHC positivity (LEXO16, LEXO27, and LEXO70), one patient with a BRAF
mutation (LEXO54), and two classied as non-oncogene addicted (LEXO37 and LEXO44) ([Table1], [Fig.1]).
At baseline, genomic testing showed a tissue-plasma concordance of 85% in the overall population, with a
sensitivity and positive predictive value of 85% whereas presenting with a specicity and negative predictive
value of 75%, respectively. According to genomic subgroups, EGFR and BRAF mutations showed the best
tissue-plasma concordance (85%) whereas ALK alterations presented with a weaker concordance (75%)
([Supplementary Tables 3–6]).
Baseline amplicon-based NGS testing on ctDNA conrmed the presence of tissue EGFR mutations in all
patients except for LEXO51 who presented with intrathoracic disease only ([Figure 1]). Namely, compared to the
canonical exon 19 in-frame deletions identied by tissue RT-PCR, LEXO33 presented on plasma a distinct and
less frequently detected EGFR variant (L747_P753delinsS), whereas LEXO14 exhibited an additional de novo
EGFRT790M along with a TP53 point mutation. Moreover, we successfully detected a classical BRAFV600E both on
tissue and plasma. While the plasma ctDNA detection of EGFR and BRAF point mutations had an acceptable
level of concordance with the archival tissue, discordance was seen in all the patients in whom ALK alterations
were only detected in tissue samples by IHC ([Figure 1], [Table1] [Supplementary Tables 3-6]).
Prognostic signicance of longitudinally monitoring NSCLC using ctDNA
In the overall cohort, we identied six responders and six non-responders according to RECIST 1.1. radiologic
evaluation. PFS and OS according to radiologic and molecular response are shown in [Fig.2].
Among radiologic responders, four patients (the EGFR-positive LEXO42, LEXO53, LEXO65 and the BRAF-
mutant LEXO54) experienced a detectable ctDNA MAF reduction showing a durable and ongoing response
([Figs.1 and 2]). Signicantly, a ctDNA response was not evaluable in two tissue ALK-positive patients (LEXO27
and LEXO70) that, however, had a favorable radiologic response paralleled by signicantly decreasing cfDNA
levels ([Figs.2 and 3]). Of note, patient LEXO70, despite showing a radiologic partial response with no detectable
molecular assessment, unfortunately, died soon because of disseminated intravascular coagulation.
Among radiologic non-responders, LEXO14 had a systemic PD on afatinib and received second-line
osimertinib whereas LEXO33 on rst-line osimertinib experienced an oligo-PD disease that was treated
according to clinical practice (Table1). Intriguingly, in these patients, ctDNA monitoring unveiled increasing on-
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target allelic frequencies and additional o-target alterations (such as BRAF, KRAS and TP53 point mutations)
that, following a sequential single-gene approach, were not initially detected on tissue at baseline ([Figure 1]).
Likewise, three patients (the non-oncogene addicted LEXO37 and LEXO44 together with the EGFR-mutant
LEXO51) experienced molecular progression with the detection of additional KRAS and TP53 mutations at T1,
progressing on standard treatments and presenting with very poor long-term survival compared to the other
cohort patients ([Figs.1 and 2]).
Considering the tissue ALK-positivity, patient LEXO16 started an ALK inhibitor but rapidly presented a
clinically symptomatic and radiologic progression before the planned radiologic restaging at W12. Surprisingly,
the retrospective evaluation of plasma ctDNA at baseline revealed a classical EGFR exon 19 E746_A750 deletion
that was not previously detected by tissue RT-PCR. Of note, the patient harbored an impressive EGFR ctDNA
MAF of 1.42% at baseline ([Figure 1]) and, therefore, was eligible to receive an EGFR inhibitor. e patient
responded favorably to osimertinib at rst restaging, thus conrming the clinical utility and the diagnostic
robustness of plasma NGS compared to tissue single-gene testing.
Although ctDNA and radiologic responses were overall concordant, however, the dynamics of cfDNA
showed some notable exceptions such as patients LEXO54 and LEXO65 showing radiologic and ctDNA
Fig. 1. Overview of the predictive molecular pathology characterization of the enrolled patients including the
mutant allelic frequencies (in brackets) of liquid biopsy ctDNA variants detected by NGS. FFPE, formalin-
xed, paran-embedded tissue; LB, liquid biopsy ctDNA; T0, baseline; T1, disease re-staging; WT, wild-type;
N.D., not detected.
ID
Tissue single-gene
testing (RT-PCR,
IHC) Treatment ctDNA T0
(NGS) MAF T0
(%) ctDNA T1
(NGS) MAF T1
(%) CT
SCAN cfDNA T0
(ng/µl)
cfDNA
T1
(ng/
µl)
LEXO 14 p.E746_A750del,
EGFR afatinib
p.E746_A750del,
EGFR
p.T790M, EGFR
p.R175H, TP53
13.1%
10.3%
2.33%
p.E746_A750del, EGFR;
p.T790M, EGFR;
p.R175H, TP53
0%
89.5%
45.76% PD 0.28 0.42
LEXO 16 ALK+alectinib p.E746_A750del, EGFR 1.42% ND 0% PD 0.23 0.43
LEXO 27 ALK+alectinib ND N.A. ND N.A. PR 0.47 0.34
LEXO 33 p.E746_A750del,
EGFR osimertinib p.L747_P753delinsS,
EGFR 0.43% p.L747_P753delinsS, EGFR;
p.V600E, BRAF;
p.G12D, KRAS
15.13%
8.8%
2.02% PD 0.84 0.54
LEXO 37 -CT + IO ND N.A. p.G12V, KRAS 7.60% PD 0.63 4.01
LEXO 42 p.L861Q, EGFR osimertinib p.L861Q, EGFR 0.4% ND 0% PR 0.59 0.38
LEXO 44 -CT + IO ND N.A. p.M237I, TP53 0.21% PD 0.37 0.78
LEXO 51 p.E746_A750del,
EGFR osimertinib ND N.A. p.G12C, KRAS;
p.G245S, TP53 5.03%
0.43% PD 0.24 0.46
LEXO 53 p.L858R, EGFR osimertinib p.L858R, EGFR 1.5% ND 0% PR 0.92 0.46
LEXO 54 p.V600E, BRAF dabrafenib+
trametinib p.V600E, BRAF 2.76% ND 0%. PR 0.61 0.91
LEXO 65 p.L858R, EGFR osimertinib p.L858R, EGFR 0.55% ND 0% PR 0.45 0.57
LEXO 70 ALK+alectinib ND N.A. ND N.A. PR 8.07 2.74
Tab le 1. Predictive molecular pathology of the included patients at baseline (T0) and rst disease restaging
(T1) undergoing rst-line treatments. RT-PCR, reverse transcriptase-polymerase chain reaction; IHC,
immunohistochemistry; ctDNA, circulating tumor DNA; cfDNA, circulating cell-free DNA; NGS, next-
generation sequencing; MAF, mutant allelic frequency; CT, computed Tomography; CHT + IO, platinum
doublet chemotherapy (carboplatin and pemetrexed) plus pembrolizumab; -, negative single-gene testing by
both RT-PCR and IHC; PD, radiologic progressive disease; PR, radiologic partial response; ND, not detected;
N.A., not available.
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response together with cfDNA increasing levels or patient LEXO33 having radiologic and ctDNA progression
with cfDNA decreasing levels ([Table1], [Fig.3]).
Discussion
Despite being strongly recommended by scientic agencies19,20, the full implementation of tissue NGS in
routine clinical practice remains limited whereas basic single-gene testing is widely available3,21. Further,
the use of liquid biopsy to track cancer response remains challenging in the real-world setting with not yet
universal reimbursement and uptake by all the healthcare systems22. Here, we described the analytical and
clinical performance of a ctDNA multiplex amplicon-based assay that, comparing to the hybrid capture-based
technique, features a quicker and less complex workow while using low quality and quantity of nucleic acid
input oen present in the real-life clinic. Our case series highlighted the use of ctDNA NGS for conrming the
standard tissue ndings of conventional single-gene testing while further revealing additional plasma genomic
alterations with signicant implications in a real-world clinical setting. In this study, we retrospectively evaluated
the plasma of patients who were part of a prospective study, showing that the ctDNA evaluation improved the
baseline detection of actionable alterations (LEXO14, LEXO16, LEXO33) while enabling the eective tracking
of clonal resistance (LEXO33, LEXO37, LEXO44, LEXO51) that would allow prompt patients enrollment in
clinical trials.
In this report, the reliable diagnostic accuracy of plasma ctDNA using an amplicon-based NGS assay for
DNA-based alterations such as EGFR and BRAF point mutations rearmed the performance of this technique
on liquid biopsy in such oncogene-driven settings23. e inability to detect the EGFR mutation on plasma in
one patient (LEXO51) with pleural eusion echoes ndings from the literature, suggesting the notably lower
sensitivity of ctDNA in patients with non-shedding intra-thoracic disease compared to those with distant
metastases24. Conversely, in line with other recent discouraging results, detecting ALK fusions from plasma using
an amplicon-based NGS assay remained challenging, even in high-volume cancers, suggesting the preferred use
of hybrid capture-based sequencing in such cases25.
Consistently with literature26,27, compared to tissue single-gene evaluation, NGS applied to ctDNA oered
a more nuanced view of the genomic landscape, enhancing our understanding of tumor heterogeneity and
pinpointing clinically actionable targets, such as in the seminal case of LEXO16. is patient presented ALK-
positive IHC staining on tissue but rapidly progressed on ALK inhibitor, while showing a plasma ctDNA EGFR
deletion that was not previously detected by RT-PCR but promptly responded to osimertinib. us, ctDNA
may play a role in replacing tissue tumor sampling and single-gene testing in some circumstances, as outlined
by international recommendations, especially in oncogene-addicted patients28. In this case, since the detection
of an impressive EGFR ctDNA MAF of 1.42% in lung cancer patient represented the example of a very unlikely
false-positive nding, the liquid biopsy evaluation was valuable to prevent ineective therapy and avoid
unnecessary side eects, suggesting that in the real-world setting monitoring ctDNA molecular status could
potentially reect response before clinical progression or radiologic imaging29.
We then investigated whether ctDNA clearance or a certain degree of ctDNA kinetics reected by on-
treatment variations of MAF values would better correlate with radiologic response. Mostly in the resistance
Fig. 2. Swimmer plot depicting survival of the included patients according to radiologic (lines) and molecular
(circles) response. PFS, progression-free survival; OS, overall survival; SD, stable disease; PR, radiologic partial
response; PD, radiologicprogressive disease; M.R., molecular response; N.D., not detected.
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setting, dynamic molecular proles captured by the serial monitoring of ctDNA using NGS revealed complexities
in tumor evolution and therapeutic responses that would not have been identied by conventional single-gene
techniques detecting only known hotspot variants on tissue. Here, early ctDNA MAF reduction during rst-
line standard treatments seemed to predict radiologic responses and longer survival, whereas increasing MAF
values with the emergence of co-mutations like BRAFV600E, KRASG12V or TP53M237I seemed to be an early
indicator of molecular and radiologic progression, as clinically corroborated by the later aggressive behavior.
Notably, concomitant mutations in NSCLC typically portend a poorer prognosis30–32, suggesting the earlier
use of ctDNA as a minimally invasive and robust tool for providing crucial insights into potential diagnostic
and therapeutic adjustments in the clinic. Notably, considering the negative prognostic impact of co-mutations
and the adoption of only single-gene testing on tissue in randomized clinical trials, one could argue about the
real-life need for monitoring and adapting cancer treatments using NGS on ctDNA to signicantly improve
clinical outcomes33. Of note, both LEXO14 and LEXO33 experienced a radiographic progression that matched
Fig. 3. Dynamics of cfDNA among responders and non-responders according to radiologic restaging. cfDNA,
circulating cell-free DNA; T0, baseline; T1, rst disease restaging; PD, radiologic progressive disease; PR,
radiologic partial response.
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increasing on- and o-target MAF values at T1, despite showing a relatively long survival that was eventually
inuenced by second-line treatments. In LEXO54, the sensitivity for the detection of BRAFV600E and monitoring
of response to dabrafenib and trametinib rearms the diagnostic accuracy of ctDNA for such patients. However,
the increase in cfDNA levels, despite a partial radiologic response, further suggests that cfDNA levels might
not specically reect tumor burden, possibly indicating that other biological processes like apoptosis, necrosis
and active secretion are at play, as oen described34. Since all the molecular responders showed an ongoing and
responding disease whereas molecular non-responders presented with a progressing or high burden disease,
these results demonstrated the analytical and clinical validity of an amplicon-based NGS plasma assay in the
real-world setting while further conrming the clinical utility of liquid biopsy for the longitudinal monitoring
of patients with advanced NSCLC receiving rst-line treatments. Hence, this approach can signicantly impact
the real-world patient management by adding broader molecular proling and early prognostics for treatment
stratication and early access to actively enrolling clinical trials6.
While the exploratory nature of our analyses was hindered by the absence of NGS-based tissue testing, these
results underscore the practical challenges and opportunities associated with implementing a liquid biopsy-
informed approach for treatment choice and response assessment. While our study emphasizes the potential of
liquid biopsy to detect a broader spectrum of genomic variants, it’s important to acknowledge certain limitations
that provide direction for future research. First, the retrospective nature and the small sample size of the study
necessitate further larger, multi-center validation cohorts. Secondly, the phenomenon of clonal hematopoiesis,
which can lead to the presence of non-tumor-related mutations in the bloodstream, poses a challenge to liquid
biopsy accuracy, potentially resulting in false-positive results35. In this context, plasma tumor fraction analysis
could serve as a potential prognostic and predictive tool to tailor therapy intensity based on individual tumor
biology, reducing false-positive ctDNA results while obviating the need for conrmator y tissue testing in selected
patients36–38.
Conclusions
ese ndings accentuate the diagnostic and monitoring prowess of liquid biopsy, which in this instance
provided an early indication of on-treatment tumor evolution using an amplicon-based NGS assay, thereby
informing potential shis in therapeutic strategy. is report would add compelling insights into the evolving
landscape of advanced NSCLC, underscoring the need for plasma ctDNA analysis and tissue genotyping as
complementary tools in the diagnostic and therapeutic decision-making process.
Liquid biopsy can complement existing tissue biomarker testing, particularly for identifying more patients
who could benet from rst-line targeted treatment by increasing the number of patients with a proper and
well-informed molecular diagnosis. Liquid biopsy may also help identify patients for appropriate second-line
targeted therapy, especially through detection of circulating markers of resistance or in patients who did not
receive frontline biomarker testing.
is study strengthens the application of ctDNA molecular response assessment as an enrichment strategy.
By early identifying patients exhibiting molecular disease progression, this approach has the potential to
mitigate the heterogeneity inherent to clinical trials, creating a more homogenous target population and thereby
opening a therapeutic window of opportunity. is window would facilitate earlier intervention and potentially
overcome primary therapeutic resistance, ultimately leading to improved clinical outcomes.
Data availability
Data could be available upon reasonable request to the authors.
Received: 29 May 2024; Accepted: 12 September 2024
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Acknowledgements
V.G., A.Go., and TD.BR. contributed to the current work under the Doctoral Programme in Experimental On-
cology and Surgery, University of Palermo. e authors thank Mrs Marzia D’alessandro for the English language
revision.
Author contributions
Conceptualization: V.G., T.D.B.R., N.B., V.B.;Data curation: T.D.B.R., F.P., G.T.; Formal analysis: V.G., N.B.,
A.Go., G.R.;Investigation: V.G., N.B., A.Ga.;Methodology: V.G., N.B.;Project administration: V.G., N.B., A.R.,
V.B.;Resources: V.G., N.B., A.R., V.B.;Soware: V.G., A.Ga.;Supervision: L.I., G.B., F.F., G.T., U.M., A.R., V.B.,
A.Ga.;Validation: V.G., N.B., G.R., F.F., U.M., V.B.;Visualization: V.G., N.B., A.Go.;Writing – original dra: V.G.,
T.D.B.R., N.B., A.Go.;Writing – review & editing: T.D.B.R., G.R., L.I., G.B., F.F., G.T., U.M., A.R., V.B.; All authors
have read and agreed to the published version of the manuscript.
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Funding
L.I., G.B., A.R. and A.Ga. were supported by the Piano Nazionale di Ripresa e Resilienza (PNRR) project -
Italian Network of excellence for advanced diagnosis (INNOVA) - PNC-E3-2022-23683266 PNC-HLS-DA
(C43C22001630001). e remaining authors have not declared a specic grant for this research from any fund-
ing agency in the public, commercial, or not-for-prot sectors.
Declarations
Competing interests
F.P. received personal fees (as speaker bureau or advisor) from Menarini.G.T. received personal fees (as
speaker bureau or advisor) from Roche, MSD, Pzer, Boehringer Ingelheim, Eli Lilly, BMS, GSK, Menarini,
AstraZeneca, Amgen and Bayer, all unrelated to the current work.U.M. received personal fees (as consultant
and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, ermo Fisher Scientic, Eli Lilly,
Diaceutics, GSK, Merck and AstraZeneca, Janssen, Diatech, Novartis and Hedera, all unrelated to the current
work.e remaining authors declare that they have no known competing nancial interests or personal
relationships that could have appeared to inuence the work reported in this paper.
Institutional review board
e study was conducted according to the guidelines of the Declaration of Helsinki and approved by Palermo
1 Institutional Ethic Review Board (Statement No. 02/2020, approved on 19 February 2020, AIFA code CE
150109).
Informed consent
Informed consent was obtained from all subjects involved in the study.
Additional information
Supplementary Information e online version contains supplementary material available at https://doi.
org/10.1038/s41598-024-73046-y.
Correspondence and requests for materials should be addressed to G.B. or A.R.
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