LIBELULE: A Randomized Phase III Study to Evaluate the Clinical Relevance of Early Liquid Biopsy in Patients With Suspicious Metastatic Lung Cancer

Aurelie Swalduz, MD, PhD, ^ { { a , b , * } } Camille Schiffler,c Hubert Curcio, MD,d Bana Ambasager,e Gabriel Le Moel, MD,f Didier Debieuvre, MD,s Jean-Marc Dot, MD,h Michael Duruisseaux, MD, PhD,' Pierre Fournel, MD,J Luc Odier, MD,t Sylvie Demolombe, MD,' Acya Bizieux-Thaminy, MD,m Annie Peytier, MD," Roland Schott, MD, Stephane Hominal, MD,P Claire Tissot, MD,9 Pierre Bombaron, MD, Severine Metzger,c Mathilde Donnat,c Sandra Ortiz-Cuaran, Nitzan Rosenfeld, PhD,s,t,u Christodoulos Pipinikas,c Pierre Saintigny, MD, PhD,a,b Maurice Perol, MDa

aDepartment of Medical Oncology, Centre Leon Berard, Lyon, France
bUniv Lyon, Claude Bernard Lyon 1 University, INsERM 1052, cNRS 5286, Centre Leon Berard, Cancer Research Center oj Lyon, Lyon, France
cDepartment of Clinical Research and Innovation, Centre Leon Berard, Lyon, France
dDepartment of Medical Oncology, Centre Frangois Baclesse, Caen, France
eNeoGenomics, Babraham Research Campus, Cambridge, United Kingdom
fDepartment of Pneumology, Centre Hospitalier du Cotentin Louis Pasteur, Cherbourg, France
SDepartment of Pneumology, Groupe Hospitalier de la Region Mulhouse Sud-Alsace, Hopital Emile Muller, GHRMSA - Mulhouse, Mulhouse, France
hDepartment of Pneumology, Medipole, Lyon Villeurbanne, France
'Respiratory Department, Louis Pradel Hospital, Hospices Civils de Lyon Cancer Institute, Lyon, France
jDepartment of Pneumology and Thoracic Oncology, Hopital Nord, Saint-Etienne, France
KDepartment of Pneumology, I'Hopital Nord-Ouest Villefranche sur Saone, Villefranche-sur-Saone, France
'Department of Medical Oncology, Infirmerie Protestante, Caluire et Cuire, France
mDepartment of Pneumology, CH Departemental Vendee, La Roche-sur-Yon, France
nDepartment of Medical Oncology, Centre Hospitalier de Bayeux, Bayeux, France.
Department of Medical Oncology, Institut de Cancerologie Strasbourg Europe (ICAns), Strasbourg, France
PDepartment of Pneumology, Centre Hospitalier Annecy-Genevois, Epagny-Metz Tessy, France
qDepartment of Oncology, Hopital Prive de la Loire, Saint-Etienne, France
"Department of Medicine, Hopital Prive Jean Mermoz, Lyon, France
$Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom
tCancer Research UK Cambridge Centre, Cambridge, United Kingdom.
"Barts Cancer Institute, Queen Mary University of London, London, United Kingdom

Received 30 July 2024; revised 12 October 2024; accepted 8 December 2024
Available online - 16 December 2024

ABSTRACT

Objectives: Genomic profiling is a major component for first-line treatment decisions in patients with NSCLC and the timeliness of biomarker testing is essential to improve time to treatment initiation (TTI) or avoid inappropriate treatment.

Methods: The phase III LIquid Biopsy for the Early detection of LUng cancer Lesion trial (NCT03721120) included patients with radiological suspicion of advanced lung cancer. They were randomized (1:1), the control arm receiving diagnostic procedures according to each center's practice, and the liquid biopsy arm with additional testing performed at the first visit using the InVisionFirst-Lung assay. Treatment initiation and type were defined according to the European Society for Medical Oncology guidelines. Primary endpoint was the time from randomization to initiation of appropriate treatment on the basis of informative genomic and pathological results in the intention-to-treat population.

Results: A total of 319 patients were enrolled (liquid biopsy [LB]: 161; control: 158). The median age was 68 years, 2 8 . 8 % were non-smokers, 1 8 . 1 % had a performance status of 2 or higher, and 5 6 . 7 % had adenocarcinoma. In the LB arm, 8 1 % of patients had circulating tumor DNA findings. The mean TTI was not significantly reduced (LB: 2 9 . 0 { ~ d ~ } control 34 d ( p ~ = ~ 0 . 2 6 ) ). Sensitivity analyses found a shorter TTI in patients from the LB arm who received systemic treatment (LB: 2 9 . 1 { d } ; control: 38.9 d, p = 0 . 0 1 { * } J, in patients with advanced non-squamous NSCLC (LB: 2 9 . 5 { { ~ d } } _ { * } control: 4 0 . 3 { ~ d } . p = 0 . 0 1 \dot { } , and in patients with first-line targetable alterations (LB: 21d; control 37.4 d) ( p \ = 0.004). Time to contributory genomic results was significantly reduced (LB: 1 7 . 9 { { ~ d } } _ { \ i } control: 25.6 d, p < 0 . 0 0 1 \AA -

Conclusion: Early liquid biopsy testing did not significantly shorten the TTI in unselected patients referred for suspected advanced lung cancer. Nevertheless, it could reduce the TTI in patients eligible for systemic treatment, particularly for those with actionable alterations.

⊚ 2024 International Association for the Study of Lung Cancer. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

Keywords: Non-small cell lung cancer (NscLC); Liquid biopsy; Circulating tumor DNA (ctDNA); Next-generation sequencing (NGs); Precision oncology; Treatment initiation

Introduction

Genomic profiling is a major component in supporting the first-line treatment decision process in patients with advanced NscC. Access to the most complete molecular profile of the tumor before treatment initiation is critical for several reasons: (1) to appropriately define and initiate the most efficient treatment, considering favorable outcomes observed in first-line treatment using tyrosine kinase inhibitors (TKIs) when compared with chemotherapy in patients with oncogenic addiction1,2; (2) to avoid inappropriate treatment exposure, frontline immunotherapy either alone or in combination with chemotherapy not being recommended in those patients;3 and (3) to avoid TKIs-related toxicities after immunotherapy-based treatment.4-6

The European Society for Medical Oncology (EsMO) Precision Medicine Working Group recommends a multigene next-generation sequencing (Ngs) approach (RNA- or DNA-based) also capturing fusions, to detect ESMO Scale for Clinical Actionability of molecular Targets level I alterations in advanced NscLc.7 Even if molecular profiling performed on tumor tissue is the standard at diagnosis in advanced patients with NscLC, plasma-based genotyping may circumvent certain limitations of tissue genotyping notably minimizing the need for repeated invasive procedures, extended delays for molecular analysis on tissue samples and may better reflect tumor heterogeneity.8-12 Nevertheless, in the current EsmO and International Association for the Study of Lung Cancer guidelines,13,14 liquid biopsies still remain an alternative approach to diagnosis limiting their applications and reducing their impact on improving the time to treatment initiation (TTI). Despite growing evidence for their clinical relevance, data mainly relies on retrospective ancillary studies or longitudinal cohort studies.15-21

Clinical observation from a large retrospective French practice assessment reported that the mean time-toappropriate frontline treatment initiation from the first consultation with a lung cancer oncologist was 42 days.22 We aimed to investigate whether liquid biopsy in patients with suspicious metastatic or locally advanced lung cancer at the time of first consultation by a lung specialist may reduce TTI. We, therefore, designed a "real-world" randomized study, including different types of centers routinely managing patients with lung cancer, to evaluate the feasibility and clinical relevance of liquid biopsies to decrease TTI.

Materials and Methods

Patients

LIBELULE (LIquid Biopsy for the Early detection of LUng cancer Lesion) is a multicenter, randomized, comparative, open-label phase III study (NCT03721120). Eligible patients had clinico-radiological suspicious presentations of lung cancer, metastatic or locally advanced disease not amendable to loco-regional treatment. Additional patient eligibility criteria included lack of prior biopsy or cytology testing, no prior systemic treatment for advanced Nsclc diagnosis, and no contraindication to systemic lung cancer treatment. Patients were 18 years of age or older, with an Eastern Cooperative Oncology Group performance status of 0 to 2, adequate hepatic, renal, and bone marrow function (as defined in the trial protocol), and no prior history of malignancies (except for basal cell or squamous cell carcinoma of the skin or carcinoma in situ of the cervix) unless the patient has been free of the disease for at least three years. Patients were enrolled in cancer centers, university hospitals, general hospitals, and private practice centers, to better reflect the diversity of recruitment and practices of the different types of centers.

Study Design and Endpoints

Patients were randomly assigned (1:1) to the experimental liquid biopsy arm (LB) or control arm (Appendix Fig. 1). In the LB arm, a liquid biopsy was performed at the first visit using the InVisionFirst-Lung assay (NeoGenomics Laboratories, Inc.), an ampliconbased NGS panel covering 37 NSCLC-associated genes, including fusions (Appendix Fig. 2),16,20 and diagnostic procedures (cytological or histological sampling with genomic analysis when indicated) were planned according to each center practice (DNA-based or RNA-based NGS, or polymerase chain reactionbased assay). As previously described, the InVisionFirst-Lung panel demonstrated a sensitivity of over 9 0 % for allele fractions greater than 0 . 2 5 % approximately 7 0 % for allele fractions between 0 . 1 3 % and 0 . 1 6 % and 3 7 . 4 1 % for allele fractions between 0 . 0 6 % and 0 . 0 8 % whereas maintaining a specificity of nearly 9 9 . 9 9 9 7 % 23 In the control arm, diagnostic procedures were planned according to each center practice and local liquid biopsies were allowed. Randomization was stratified according to the type of center (comprehensive cancer centers, university hospitals, general hospitals, and other private not-forprofit hospitals). On the basis of the high positive predictive value of liquid biopsy testing and the significant rate of false negatives due to low circulating tumor DNA (ctDNA) shedding depending on clinical presentations, genomic alterations were grouped into three categories24,25 defined according to ESMO guidelines and French health authorities reimbursement policy.11 Category 1 included alterations defined by the availability of first-line targeted therapies: EGFR, BRAF V600E mutations, ALK- or ROS1-rearrangements. Category 2 alterations were considered when reflecting the contributory nature of the liquid biopsy but without targeted therapies available in first-line: MET exon 14 skipping mutations, mutations in ERBB2, KRAS, BRAF other than V600 or LKB1 and rearrangements involving RET or NTRK. Other alterations (category 3), were considered as non-specific. When a category 1 alteration was identified in ctDNA, first-line targeted therapies could be initiated regardless of the availability of pathological and molecular results from tissue analysis. For category 2 alterations identified in ctDNA, pathological reports including PD-L1 status on the basis of tissue analysis were mandatory to initiate treatment. For other alterations, pathological results and molecular status from tissue analysis were required to start first-line treatment (Appendix Fig. 1).

When several alterations were identified, category 1 prevailed over category 2, which itself prevailed over category 3 alterations.

The primary endpoint was the time from randomization to initiation of appropriate treatment on the basis of informative genomic (on liquid or tissue biopsy) and pathological results (TTI). Appropriate treatment had to be based on informative results defined as contributory pathological results and contributory molecular results for patients with non-squamous Nsclc. When results were informative, treatment was considered appropriate if it was initiated according to EsmO guidelines and French health authorities' reimbursement policy.11 In the case of non-informative genomic results (no molecular results available on tissue or liquid biopsy), any treatment initiated by an investigator was considered appropriate. If treatment was initiated when informative results were available, the date of appropriate treatment initiation was recorded as the date of treatment initiation. If treatment was initiated when informative results were not available, i.e. before obtaining the genomic profile results on tissue or liquid biopsy, TTI was calculated using the date when informative results or non-contributory results were obtained. When no systemic therapy for lung cancer was initiated, TTI used the date of physician decision (surveillance, local therapy), the date of pathological results for diagnosis other than lung cancer or death, or patient refusal date (Appendix Fig. 3).

Secondary endpoints included time to availability of informative genomic results, percentage of treatments initiated before obtaining molecular results, progressionfree survival (PFS), and safety of diagnostic procedures. Incremental costs between both strategies will be compared with incremental health improvement measured in quality-adjusted life-years gained and in progression-free life years gained. A cost-effectiveness analysis is pending and will not be reported herein.

Statistical Considerations

On the basis of a French retrospective study conducted on around 250 patients with advanced NscLC, the mean TTI was 42 days (associated SD: 22.5 d).22 The expected decrease of the mean TTI in the LB arm was on the basis of the following hypotheses: a 21-day reduction in patients with category 1 alterations (expected to represent 1 3 % of the population) and a 17-day reduction in patients with category 2 alterations (expected to represent 3 6 % of the population). This resulted in a mean expected TTI in the experimental group of 33 days - 2 1 % TTI reduction). On the basis of a non-parametric, two-sided Wilcoxon Mann and Whitney test with an alpha value of 0.05 and 9 0 % power, 286 patients were required to reject the null hypothesis (HO): the TTI distributions are not different between the LB arm and control arm.

Efficacy analyses were performed in the intention-totreat (ITT) population (all randomly assigned patients) and in patients with category 1 and 2 alterations. A TTI and sensitivity analyses were performed considering only patients who had received systemic treatment for lung cancer and patients with advanced non-squamous NSCLC. Quantitative criteria were compared using the Wilcoxon test. PFS was measured from randomization to radiographic evidence of disease progression (as assessed by investigators or death), using the Kaplan-Meier method and described in terms of median, with the associated two-sided 9 5 % confidence interval (CI). Patients with no cancer or other cancer were censored. For patients who were event-free at the time of analysis, PFS was censored at the date of the last tumor evaluation.

SAS software version 9.4 was used for all statistical analyses.

Ethical Considerations

The protocol (NcT03721120) was conducted in accordance with the Declaration of Helsinki principles, the current International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines for Good Clinical Practice, and in compliance with French and European laws and regulations in force, as well as any applicable guidelines (ID-RCB number: 2018-A00810-55). All patients provided written informed consent before enrolment. The trial was designed by investigators and supported by the "Programme Hospitalier de Recherche Clinique national en cancerologie (PHRc-K)- 2 0 1 8 ^ { \prime \prime } and LYon Recherche Innovation contre le CANcer ( { L Y R I C A N + } ) ,INCa-DGOS-INSERM-ITMOcancer_18003. Liquid biopsy analysis by InVisionFirst-Lung was provided by NeoGenomics Laboratories, Inc. The investigators had full access to all the data collected and contributed to analysis and interpretation.

Results

From April 2019 to August 2022, a total of 319 patients with suspicion of advanced NscLc from 15 participating French centers (four comprehensive cancer centers, two university hospitals, six general hospitals, and three other private not-for-profit hospitals) were randomized 1:1 (LB arm: 161; control: 158). Ten patients were excluded after consent withdrawal or lost to follow-up before initiation of any study procedures and the ITT population included 154 patients in the LB arm and 155 in the control arm (Fig. 1). At the time of data cut-off (September 30, 2023), the median follow-up duration was 26.4 months (range: 16.2-33.7).

Demographic, Clinical, and Pathological Characteristics

Baseline characteristics are summarized in Table 1 and were well balanced between the two arms. The median age across the entire study cohort was 68 years (range: 39-97); 5 5 . 7 % were male; 1 8 % had Eastern Cooperative Oncology Group performance status of 2 or higher; 2 8 . 8 % had no smoking history. Histological diagnosis was obtained for 9 6 . 1 % of the patients. As patient inclusion into the study was based on the suspicion of advanced lung cancer, histological diagnoses were distributed as follows: 7 5 . 1 % of NSCLC, including 5 7 . 9 % adenocarcinoma and 1 1 . 3 % squamous cell carcinoma, 1 0 . 4 % SCLC, and 1 0 . 7 % patients having diagnosis other than lung cancer. Those included 17 patients ( 5 . 5 % ) with no cancer (including sarcoidosis, aspergillosis and 12 patients without tumor cells identified on the diagnostic biopsy) and 16 patients ( 5 . 2 % ) with other cancers (including melanoma, breast, gynaecological, and digestive tract cancers) (Appendix Table 1). In the ITT population, 8 5 . 5 % had lung cancer; 1 1 . 7 % had locally advanced disease not eligible for local treatment and 7 2 . 2 % had metastatic disease with a median number of metastatic sites being three in both arms (range: 1-8). Among them, 3 . 1 % of patients presented with the brain as a unique metastatic site, whereas 2 0 . 6 % were found with brain and/ or thoracic metastases only. Patients were mainly enrolled in comprehensive cancer centers and general hospitals (Table 1).

Genomic Profiling Results

In the LB arm, ctDNA was detected in 1 2 5 / 1 5 4 ( 8 1 . 2 % ) of patients and 87/101 ( 8 6 . 1 % ) of patients with advanced non-squamous NSCLC. Among the 27 patients with CNS or thoracic-only disease included in the LB arm, 23 had alterations detected, and the InvisionFirstLung assay was able to detect 19 of them. In the LB and control arms, tumor tissue contributed to genomic profiling in 8 1 . 2 % and 8 9 . 6 % of patients with nonsquamous NScLC, respectively. Of note, in the control arm, 1 5 % of patients had a liquid biopsy test performed out of protocol. Combined use of tissue and ctDNA allowed in the LB arm the detection of category 1 alterations in 2 9 . 9 % of the ITT population, that is 4 2 . 7 % of the patients with non-squamous NscLC, whereas in the control arm, tissue profiling identified category 1 alterations in 2 3 . 9 % of the ITT population, that is 3 6 . 5 % of the patients with non-squamous NSCLC (Fig. 2, Appendix Table 2). EGFR mutations were the category 1 alterations most frequently found, in 2 2 . 7 % and 2 0 . 6 % of the ITT population and 3 2 . 7 % and 3 2 . 3 % of the patients with non-squamous NSCLC in the LB and the control arm, respectively (Fig. 2, Appendix Table 2). EGFR mutations were found in one patient with squamous cell carcinoma in each arm. ALK-fusions, BRAF V600-mutations, and ROS1-fusions were reported in 3 . 2 % _ { i } 2 . 6 % and 1 . 3 % respectively in the LB arm and 2 . 6 % , 1 . 3 % and 0 % respectively, in the ITT population of the control arm. Category 2 alterations were detected in 2 7 . 9 % and 2 2 . 6 % of the ITT population and 3 4 . 7 % and 3 1 . 3 % of the patients with non-squamous NSCLC in the LB arm and the control arm, respectively, with KRAS mutations being the most frequent alteration detected ( 1 9 . 5 % and 1 5 . 5 % including 7 . 1 % and 9 % of KRAS G12C in the LB and control arms of the ITT population, respectively) (Appendix Table 2). In the LB arm, category 3 mutations or no alteration were detected in 2 9 . 2 % and 4 . 6 % of the ITT population respectively, combining tissue and ctDNA analysis. In the control arm, category 3 alterations only or no alterations were detected in 1 5 . 5 % and 3 . 2 % of the ITT population, respectively and for those who have liquid biopsy performed out of protocol, 8 % (12 patients) harbored a category 1 or category 2 mutation. Interestingly, among patients with brain or thoracic metastases exclusively included in the LB arm ( { n } = 2 7 ) . category 1 and 2 were identified in 5 and 8 patients, respectively (Appendix Fig. 4).

Figure 1. Flowchart.

Finally, in the LB arm, tumor tissue analysis identified a category 1 or 2 alteration, whereas liquid biopsy did not in 8 . 4 % of patients, whereas liquid biopsy identified a category 1 or 2 alteration, whereas tissue analysis did not in 1 3 . 6 % of patients in the ITT population. Among these patients with category 1 or 2 alteration identified in ctDNA but not in tissue, 7 6 . 1 % had insufficient quality of tissue DNA for molecular analysis. Complete molecular results of the InVisionFirst-Lung assay obtained in the LB arm are described in Appendix Figure 4.

First-Line Treatment Description

Systemic treatment was initiated in 120/154 patients ( 7 7 . 9 % ) and 1 0 4 / 1 5 5 patients ( 6 7 . 1 % ) in the LB and control arm, respectively. The main reasons for not initiating treatment were diagnosis other than cancer, local treatments, and palliative care (Fig. 1). Among patients who initiated a systemic treatment in the LB arm { { ( n ~ = ~ } } 1 2 0 { { ) } } - 108/120 ( 9 0 % ) patients initiated the appropriate treatment on the basis of informative results, whereas 1 2 / 1 2 0 patients ( 1 0 % ) started treatment without informative results available, including five patients for whom molecular characterization was not obtained at all during the course of the study (non-contributory tissue and/or liquid biopsy). No patients received an inappropriate treatment in the LB arm. In the control arm, 83/104 patients ( 7 9 . 8 % ) initiated appropriate treatment on the basis of informative results, whereas 19/104 patients ( 1 8 . 3 % ) initiated treatment without informative results available, including six patients for whom molecular characterization was not obtained at all. In the control arm, two patients initiated inappropriate first-line treatment with chemo-immunotherapy instead of targeted therapy in patients with category 1 (common EGFR mutations) genomic alteration.

Time to Treatment Initiation

In the ITT population, the mean TTI was 29.0 days - 9 5 % CI: 25.9-32.1) in the LB arm versus 34 days 9 5 % CI: 28.4-39.5) in the control arm ( p = 0 . 2 6 4 ) . The mean TTI was significantly shorter for patients who received first-line systemic treatment in the LB arm (29.1 d versus 3 8 . 9 { ~ d ~ } in the control arm, 9 5 % CI: 29.7-37.6, p = 0 . 0 1 1 { \ i } , reducing mean TTI by 9.8 days. Mean TTI was significantly reduced in patients with advanced non-squamous NSCLC with a reduction of 10.8 days (LB arm: 29.5 d versus control arm: 4 0 . 3 { ~ d ~ } . 9 5 % CI: 3 0 . 2 \AA ^ { - } 39.3, p ~ = ~ 0 . 0 1 0 ] . Mean TTI was also significantly reduced by 16.6 days (LB arm: 21 d versus control arm: 37.6 d) in patients receiving first-line targetable treatment for category 1 alterations ( 9 5 % CI: 19.0-37.7, p = 0.003). Finally, the time to contributory genomic analysis was significantly reduced in the LB arm compared with the control arm (17.9 d versus 2 5 . 6 { ~ d ~ } 9 5 % CI: 19.5-24.1; p < 0 . 0 0 1 \AA ) (Fig. 3). Of note, excluding the 12 patients of the control arm with a liquid biopsy performed out of protocol from the analysis, the median TTI remains the same at 34.6 days (versus 34.0 d) with no significant difference compared to arm A ( p \ = 0.211).

LB arm

Control arm

Figure 2. Pathological and molecular repartition. Molecular alterations on the table on the left are described on the basis of the patients with non-squamous NSCLC, excluding "patients with no lung cancer" and patients with SCLC and SCC. The solid sections of the graph represent category 1 alterations, whereas the sections with "small dots" represent category 2 alterations. \*T/BL- are distributed among T - / L B - ( 2 % ) A T - / L B _ { N C } ( 3 % ) 3 T _ { N C } / L B - ( 8 % ) , and T _ { N C } / L B _ { N C } ( 3 % ) . LB, liquid biopsy; \angle B + , LB with category 1 and/or 2 alterations; L B - , LB without category 1 and/or 2 alterations; NC, non-contributory; SCC, squamous cell carcinoma; { \mathsf { T } } + , tissue with category 1 and/or 2 alterations; { \mathsf { T } } - { \mathsf { \Omega } } tissue without category 1 and/or 2 alterations.

The median turn-around time (TAT) to obtain pathological results was six and seven days in LB and control arms, respectively. The median TAT between tissue biopsy and molecular results on the basis of tissue analysis was 22 days in both arms. In the LB arm, the median TAT was nine days for ctDNA-based analysis, and liquid biopsy results were obtained before pathological results in 98/ 154 ( 6 3 . 6 % ) of patients, allowing earlier initiation of an appropriate treatment. In the LB arm, in patients with category 1 alterations (Fig. 4A), liquid biopsy molecular results were obtained before those coming from tissue analysis in 31/45 ( 6 8 . 9 % ) patients. In patients with category 2 alterations (Fig. 4B), histological results were obtained by a median of 2.0 days (range: - 1 3 . 0 to 106.0) after those of liquid biopsy. In both arms, the mean time between obtaining informative results and treatment initiation was 12.9 days (range: 0-346), similar for category 1 alterations (14.1 d [range: 0-346]) and category 2 alterations (12.5 d [range: 0-61]); nevertheless, 36/120 ( 3 0 . 0 % ) of patients initiated treatment 15 days or more after receiving the informative results, including 1 3 / 1 2 0 ( 1 0 . 8 % ) who started after more than 30 days (Fig. 4).

Figure 3. Time to treatment initiation. TTI is presented on the ITT population, patients receiving a systemic treatment in first-line, patients with advanced non-squamous NSCLC, and patients with category 1 alteration. Ad-nsq NSCLC, advanced non-squamous NSCLC; ITT, intention-to-treat; TTI, time to treatment.

We also evaluated the mean TTI and the time to contributory genomic analysis according to the type of centers in each arm. In the LB arm and the control arm, the mean TTI was 28.5 days versus 37.7 days in comprehensive cancer centers, with the time to contributory genomic analysis being 18.1 days versus 26.3 days, respectively. In general hospitals, the mean TTI was 34.9 days compared with 28.2 days, with a time to contributory genomic analysis of 18.5 days versus 27.9 days in the LB and the control arms, respectively. Data from other types of centers are not presented owing to the small sample sizes in these subgroups.

Progression-Free Survival

No significant difference in PFS as assessed by investigators was observed in the ITT population between the LB arm and the control arm (median: 8 . 4 ~ { { m o } } versus 7 . 0 { \ m o } ) (Fig. 5A), as well as in the treated population (median: 8 . 1 ~ { m o } versus 8 . 8 { \ m o } ) (Fig. 5B) and in subgroup analysis according to categories of genomic alterations.

Safety

No severe adverse events related to liquid biopsy procedures were reported in the LB arm.

Discussion

LIBELULE trial is, to our knowledge, the first prospective multicenter randomized clinical to evaluate the clinical relevance of performing early liquid biopsies in case of suspicious advanced NscLC. Early liquid biopsy did not significantly reduce the TTI in the ITT population (including all patients with a suspicion of advanced NSCLCJ, with a mean TTI of 29 days in the LB arm versus 34 days in the control arm ( p = 0 . 2 6 ) , despite a reduction in time to contributory tumor genomic profiling (17.9 versus 25.6 days). Nevertheless, exploratory sensitivity analyses found that early liquid biopsy shortened the mean TTI by 9.8, 10.8, and 16.6 days in patients receiving first-line systemic treatment, patients with advanced non-squamous NscLC, and patients with first-line targetable oncogenic alteration, respectively. In addition, ctDNA analysis identified category 1 or 2 alterations not found on tissue profiling in 1 3 . 6 % of patients in the LB arm (ITT population).

Several studies have explored the potential benefits of upfront liquid biopsy in order to reduce the time to obtain molecular results and subsequently initiate treatment. These studies have been either retrospective or prospective, typically conducted at single expert centers, and non-randomized, often involving either a single-arm design or comparison to a historical control arm.21,26-28 Many of these studies reported positive outcomes (TTI, PFS), particularly focusing on nonsquamous metastatic patients, who are of interest for molecular analysis.28 For example, Garcia-Pardo et al.28 evaluated the same population of patients with suspicion of advanced NscLC than in our study, but patients were selected on tumor board, in a unique expert cancer center. In this study, TTI was improved in patients with non-squamous NsCLC. Instead, to evaluate the strategy of early liquid biopsy upon suspicion of NSCLC, we chose to assess the TTI across the entire patient population and different types of institutions. Exploratory sensitivity analyses revealed a significant reduction in TTI when considering all patients receiving first-line treatment, as well as in subgroups of patients with nonsquamous metastatic NSCLC and those with targetable alterations in first-line therapy. The fact that reducing the time needed to obtain genomic characterization did not result in a significant reduction in TTI in the ITT population may be the consequence of several factors. The lack of first-line systemic therapy in 2 8 % of patients was not expected. The main reason for not receiving systemic treatment was the initiation of sole palliative care in this study conducted in real-life conditions. Sensitivity analyses reported that immediate liquid biopsy resulted in a clinically relevant reduction of the TTI in patients who initiated a systemic treatment and in patients eligible for frontline targeted therapy, which holds critical importance in symptomatic patients.

Figure 4. Time to contributory genomic profile and treatment initiation in patients with category 1 and category 2 alterations. (A) Patients with category 1 alterations. (B) Patients with category 2 alterations. Red bars represent the TTl in the LB arm and blue bars represent the TTl in the control arm. Dots represent the time when pathological (blue), InVisionFirst-Lung LB (red), and out-of-protocol LB (orange) results were available. LB, liquid biopsy; TTI, time to treatment.

Furthermore, tissue molecular characterization was left to the discretion of the investigator according to local practices which could be very different according to each center and associated reimbursement procedures as our study encompassed a diverse range of clinical practices, including academic and non-academic centers. Frequent molecular determination only using EGFR rapid polymerase chain reaction detection assays has probably led to underestimating the frequency of other molecular alterations in the control arm and also may have reduced the time to obtain genomic results on tissue as compared with more comprehensive but more time-consuming NGS panels.

Another point may have contributed to longer TTI in the LB arm. Namely, 3 0 % of the patients required more than 15 days after the availability of informative genomic results to initiate treatment including 1 0 . 8 % of the patients who started treatment more than 30 days after genomic results. This may be related to the initiation of palliative care before starting systemic treatment in some cases but also the TTI could have probably been improved in others. In our study conducted in real-life conditions, treatment initiation exclusively based on ctDNA, without confirmation on tissue was an innovative approach; in some instances, physicians tended to wait for the tissue genomic analysis report before initiating treatment even though liquid biopsy detected a category 1 alteration, therefore leading to an increase in the TTI. This may be because of a lack of knowledge or confidence in the results of molecular analyses, particularly in the results of targeted NGS, as demonstrated in two dedicated surveys in thoracic oncology.29,30 False positive ctDNA rates are extremely low and several studies have previously demonstrated similar response rates when targeted therapies are initiated on the basis of driver identification in tissue or ctDNA.31 One of the key features of any assay used for ctDNA analysis is the sensitivity and the need to capture all recommended genes including ALK-, ROS1-, RET- and NTRK-fusions. As previously described, for targetable genes, predictive positive value is over 9 8 % even at extremely low allelic fractions for high-fidelity ctDNA molecular profiling assays.2,32,33 Our assay was also sensitive in patients known to be "low-shedders" as most alterations found on tissue were found on ctDNA in patients with CNS or thoracic-only disease. False positives resulting from clonal hematopoiesis of indeterminate potential may emerge, as most commercial assays only use filters to reduce such mutations but do not sequence DNA from paired white blood cells. The current study addressed this issue by selecting category 1 and 2 alterations relevant to NsCLC treatment decisions, which are not common clonal hematopoiesis of indeterminate potential genes.34

Nevertheless, even if performing an early liquid biopsy did not directly translate into a significant reduction in the TTI, obtaining a complete and rapid molecular characterization is crucial as it reduced the number of patients without contributory tissue molecular profile before initiating a systemic treatment by 5 0 % including two patients with EGFR mutations receiving chemoimmunotherapy in the control arm. Optimizing appropriate first-line treatment is equally important to avoid ineffective treatment (i.e., immunotherapy in patients with oncogenic addiction such as EGFR, ALK, or ROs1) and also potentially toxic strategies in the case of immunotherapy rapidly followed by TKIs. Previous reports identify a median TAT of eight to 10 days with liquid biopsies versus 20 to 31 days with tissue.21,31,35,36 In LIBELULE, results were comparable with a median TAT of nine days from blood draw to molecular results (including blood shipment from France to the United States), resulting in a significant improvement in the time to contributory results (17.9 d versus 25.6 d).

As expected, although molecular characterization directly impacts patient survival,37 reduction of TTI did not translate into a frontline PFS improvement either; shortening treatment initiation times is unlikely to be sufficient to alter the natural history of the disease under treatment, especially in cases of slowly progressive disease38 but may be more important in cases of extensive, symptomatic or rapidly progressive disease.

In addition to the prognostic impact of positive ctDNA detection (FLAURA2), this approach of adding ctDNA analysis in patients with suspicious advanced NsCLC also increases the number of guideline-recommended biomarkers detected. Most often, only small biopsies are obtained at diagnosis of NsCLC therefore limiting the molecular characterization process in about 2 5 % of the patients,32,39 and even more when sequential approaches are used (rapid testing first or DNA-sequencing followed by RNA sequencing for detection of fusions).21,32,40 False negatives may represent an important issue. It can be related to variations of ctDNA shedding, especially in certain clinical presentations (such as low tumor burden, brain as only metastatic site, or exclusive thoracic disease),41 technical failures, or insufficient assay sensitivity. In our study, 8 . 4 % of patients had a non-informative liquid biopsy result whereas tissue was contributory, similar to previous reports with sensitive assays.42 Meanwhile, 1 3 . 6 % of patients had category 1 and 2 alterations detected in ctDNA whereas tissue was negative. It is possible to use an algorithmic determination of the circulating DNA (cDNA) tumor fraction to improve the negative predictive value of the liquid biopsy and avoid the need for reflex genomic analysis of tissue when the cDNA tumor fraction is 1 % or higher.43

Our study has certain limitations. It reflects the management of patients outside clinical trials and the diagnostic procedures and access with a French perspective which can only partly be generalized to the rest of the world, as shown in the International Society for Study for the Lung Cancer survey on molecular testing worldwide.30 This strategy of early ctDNA analysis when advanced lung cancer is suspected, has to be evaluated from a cost-effectiveness perspective, considering that in our scenario ctDNA profiling was unnecessarily performed in approximately 3 0 % of the patients with squamous NSCLC and other cancers not currently benefiting from molecular profiling. Early ctDNA analysis could avoid the need for tissue rebiopsy, with limitations in terms of feasibility and timescales.' Previously, Ezeife et al.44 demonstrated that performing a liquid biopsy in patients with advanced NscLC and a low-smoking history was not associated with extra costs, through a decreased number of patients who may have received immunotherapy inappropriately due to ignorance of an actionable genomic alteration. The negative predictive value of ctDNA analysis can also be improved by the determination of cDNA tumor fraction, therefore avoiding the need for additional genomic analysis on tissue. The cost-effectiveness analysis of our study is pending.

In conclusion, our study is the first large prospective randomized trial to evaluate the impact of early liquid biopsy on TTI reduction in patients with suspicion of advanced NscLc. This trial did not meet its primary endpoint in a poorly selected population; nevertheless, sensitivity analyses revealed a clinically relevant reduction in TTI in patients initiating systemic treatment, especially in those with targetable alterations in the first-line setting, as well as an increase in the proportion of patients with detectable targetable genomic alterations. To achieve these benefits in this setting, using a sensitive assay that encompasses all recommended targetable genes in NSCLC is imperative. Moreover, these results tend to show that optimizing the management of liquid biopsies relies not only on assay performance but also on the interpretation of results and their application and likely requires better education of clinicians given the constantly evolving technological innovations.

CRediT Authorship and Contribution Statement

Aurelie Swalduz: Conceptualization, Investigation, Methodology, Project administration, Writing - original draft, Writing - review & editing.

Camille Schiffler: Conceptualization, Formal analysis, Methodology, Writing - original draft, Writing - review & editing.

Hubert Curcio: Investigation, Writing - review & editing.

Bana Ambasager: Writing - review & editing.

Gabriel Le Moel: Investigation, Writing - review & editing.

Didier Debieuvre: Investigation, Writing - review & editing.

Jean-Marc Dot: Investigation, Writing - review & editing.

Michael Duruisseaux: Investigation, Writing - review & editing.

Pierre Fournel: Investigation, Writing - review & editing.

Luc Odier: Investigation, Writing - review & editing.

Sylvie Demolombe: Investigation, Writing - review & editing.

Acya Bizieux-Thaminy: Investigation, Writing - review & editing.

Annie Peytier: Investigation, Writing - review & editing.

Roland Schott: Investigation, Writing - review & editing.

Stephane Hominal: Investigation, Writing - review & editing.

Claire Tissot: Investigation, Writing - review & editing.

Pierre Bombaron: Investigation, Writing - review & editing.

SeverineMetzger:Conceptualization, Project administration, Writing - review & editing.

Mathilde Donnat: Data curation.

Sandra Ortiz-Cuaran: Conceptualization, Writing - review & editing.

Nitzan Rosenfeld: Writing - review & editing.

Christodoulos Pipinikas: Writing - review & editing.

Pierre Saintigny: Conceptualization, Investigation, Methodology, Writing - review & editing.

Maurice Perol: Conceptualization, Investigation, Methodology, Project administration, Writing - original draft, Writing - review & editing.

Disclosure

Dr. Swalduz is a consultant for Amgen, AstraZeneca, Roche, Pfizer, Sanofi, Janssen, Pfizer, and Takeda; reports personal fees from AstraZeneca, Roche, Pfizer, MSD, Boehringer-Ingelheim, Daiichi Sankyo, Sanofi, Takeda, Janssen, and Amgen; has received support for meetings or travel from Roche, Janssen, Pfizer, and Takeda. Dr. Debieuvre has received research funding from Roche, AstraZeneca, Lilly, BMs, Boehringer-Ingelheim, Chiesi, Chugai, Pfizer, MSD, Novartis, GSK, Sandoz, Takeda, OSE Immunotherapeutics, Bayer, Janssen, Sanofi, Aventis, Amgen; is a consultant for Roche, BMs, Pfizer, OSE Immunotherapeutics, Amgen; reports personal fees from AstraZeneca, Chugai, Roche, Novartis, Pfizer, MSD, BMS, Boehringer-Ingelheim, Gilead, Amgen; has received support for meetings or travel from Roche, Boehringer-Ingelheim, Novartis, Pfizer, BMS, AstraZeneca, MSD, GSK; and declare Participation on a Data Safety Monitoring Board or Advisory Board for Roche, Boehringer-Ingelheim, Pfizer, MSD, BMS, Novartis, AstraZeneca, Amgen. Dr. Duruisseaux has received research funding from Pfizer, AstraZeneca, Takeda, Blueprint, Merus, and BMs; is a consultant for Roche, BMS, Pfizer, Amgen, Boehringer Ingelheim, abbvie, Takeda, MSD, Novartis, Gamamabs Pharma, GSK, Guardant, and AstraZeneca; reports personal fees from AstraZeneca, Roche, Novartis, Pfizer, MSD, BMS, Boehringer-Ingelheim, Amgen, Guardant, Janssen; has received support for meetings or travel from Roche. Dr. Fournel reports personal fees from Sanofi, MsD, AstraZeneca; has received support for meetings or travel from Takeda; and declares participation on the Data Safety Monitoring Board or Advisory Board for Sanofi, MSD

Roland Schott is a consultant for Roche, BMs, and AstraZeneca; reports personal fees from Roche, BMS, and

AstraZeneca; has received support for meetings or travel from Roche, Takeda, MsD, Pfizer, Ipsen. Dr. Tissot is a consultant for Amgen, AstraZeneca, BMs, MSD, and Sanofi; and reports personal fees from BMS, AstraZeneca, MSD, Boehringer-Ingelheim, Sanofi, and Amgen. Dr. Rosenfeld declares to receive royalties or licenses, consulting fees, support for meetings or travel, Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid; Stock or stock options by Inivata Ltd., NeoGenomics Inc.; he declares to receive Patents planned, issued or pending from Inivata Ltd., NeoGenomics Inc and Cancer Research UK and University of Cambridge. Dr. Saintigny declares to have support by Inivata for funded ctDNA analysis of the LIBELULE trial. Dr. Perol is a consultant for BMS, MSD, AstraZeneca, Roche, Daiichi Sankyo, Janssen, Ipsen, Esai, GSK, eli Lilly, Pfizer, Takeda, Novocure; reports personal fees from AstraZeneca, Pfizer, MSD, BMs, Janssen, AnHeart Therapeutics, Sanofi, and Takeda; has received payment for expert testimony from BMs, AstraZeneca, Roche, Janssen; has received support for meetings or travel from Roche, BMs, MSD, AstraZeneca, Pfizer, Takeda; and declare Participation on a Data Safety Monitoring Board or Advisory Board for Roche and Pharmamar. The remaining authors declare no conflict of interest.

Acknowledgments

This research was supported by a PHRC-K 2018 and LYon Recherche Innovation contre le CANcer (LYRICAN + - INCa-DGOS-INSERM-ITMO cancer_18003 Inivata.

Supplementary Data

To access the supplementary material accompanying this article, visit the online version of the Journal of Thoracic Oncology at www.jto.org and at https://doi.org/10.1 016/j.jtho.2024.12.011.

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