Paving the way for ctDNA-guided trials in lung cancer: insights from the LIBELULE study

The management of advanced non-small cell lung cancer (NSCLC) has been revolutionized by the identification of actionable genomic alterations and the development of targeted therapies (1). International guidelines such as those from the European Society for Medical Oncology (ESMO) (1) recommend broad molecular profiling using next-generation sequencing (NGS) to optimize treatment selection, particularly in non-squamous histologies. However, traditional tissue-based genotyping often faces logistical and biological barriers, including insufficient tissue, invasive procedures, and delays in result availability. In the ever-evolving landscape of thoracic oncology, the advent of liquid biopsy (LB), particularly the use of circulating tumor deoxyribonucleic acid (ctDNA), has opened new frontiers for precision medicine, challenging these limitations (2). LB, specifically through analysis of ctDNA, offers a rapid, non-invasive, and comprehensive approach to molecular diagnosis. It provides real-time insights into tumor heterogeneity and allows for dynamic monitoring of tumor evolution (3). Its use has expanded in recent years from a complementary tool to a potential first-line diagnostic strategy, especially when tissue is inadequate or when rapid therapeutic decisions are required (4).

Including panels with both DNA and ribonucleic acid (RNA) analysis in LB for lung cancer is crucial for comprehensive detection of mutations, fusions, and amplifications (5). DNA analysis is essential for identifying genetic mutations such as EGFR or KRAS, which are critical for guiding targeted therapies, but RNA analysis, despite the challenge posed by RNA instability, is invaluable for detecting gene fusions and exon-skipping events that DNA analysis might miss (6). Recent advancements in RNA-based NGS have demonstrated high sensitivity and specificity, ensuring reliable results (6). Proper sample processing and optimized experimental workflows have further mitigated issues related to RNA quality, making RNA panels a robust component of molecular diagnostics (7). Platforms such as Guardant360^®, FoundationOne Liquid CDx^®, and InVisionFirst-Lung^® have been validated for clinical use, offering high sensitivity and specificity in detecting actionable mutations. Beyond ctDNA, emerging technologies such as direct NGS on cytology specimens obtained through endobronchial ultrasound-guided fine-needle aspiration (EBUS-FNA), either through fresh or frozen tissue, represent a promising complementary approach. Recent comparative studies have demonstrated that endoscopic ultrasound with bronchoscope-guided fine-needle aspiration (EUS-B-FNA, a transesophageal variant of EBUS-FNA) provides high DNA and RNA yields suitable for comprehensive NGS analysis, with success rates comparable or superior to other bronchoscopic techniques (8). This approach may be particularly valuable when ctDNA analysis is inconclusive or cannot be performed. Furthermore, applying parallel sequencing to both plasma and EBUS-FNA-derived samples could reduce discordant findings and increase diagnostic confidence.

Beyond targeted therapies, LB has demonstrated its utility as a tool for assessing the biological and pathological response in NSCLC. As an example, in the neoadjuvant setting, exploratory analyses from clinical trials such as AEGEAN (9) or NADIM (10) have shown significant findings regarding ctDNA clearance and its correlation as a predictive biomarker for pathologic complete response (pCR). A systematic review of multiple trials revealed that ctDNA clearance had a pooled sensitivity of 0.98 and a diagnostic odds ratio (DOR) of 57.36 for predicting pCR, although specificity was limited (11). Furthermore, clinical trials have utilized ctDNA as a guide for randomizing patients, such as DYNAMIC study were the investigators demonstrated the feasibility of using ctDNA to stratify patients, enhancing the precision of treatment allocation and improving clinical outcomes. The study found that patients with ctDNA clearance had significantly better progression-free survival (PFS) and overall survival (OS) compared to those with persistent ctDNA [hazard ratio (HR) for PFS, 0.32; 95% confidence interval (CI): 0.20–0.52; HR for OS, 0.22; 95% CI, 0.12–0.39] (12).

The LIBELULE trial (13) represents a landmark effort to assess the clinical utility of early LB in patients with suspected metastatic NSCLC. This prospective, randomized phase III study not only highlights the growing maturity of ctDNA-based platforms but also raises critical questions regarding implementation, clinical decision-making, and future directions. The trial was conducted across 15 French institutions with 319 enrolled patients. Patients with radiological suspicion of advanced lung cancer were randomized (1:1) to undergo either standard diagnostic procedures (control arm) or standard diagnostics plus early LB using the InVisionFirst-Lung^® assay (LB arm) (3,6,14). The primary endpoint was time-to-treatment initiation (TTI) based on informative pathological and molecular results. Secondary endpoints included time to availability of molecular results, appropriateness of first-line therapy, and PFS. The InVisionFirst-Lung^® assay is a plasma-based NGS panel covering 37 genes relevant to NSCLC. It was performed at first consultation, with results used to guide first-line therapy when category 1 (targetable) alterations were detected. TTI did not show significant differences between arms but a trend (29.0 days in the LB arm vs. 34.0 days in the control arm; P=0.26). However, sensitivity analyses revealed that early LB significantly reduced TTI in patients receiving systemic therapy (29.1 vs. 38.8 days; P=0.01), patients with advanced non-squamous NSCLC (29.5 vs. 40.3 days; P=0.01) and patients with targetable alterations (21.0 vs. 37.6 days; P=0.004). Of note, the time to informative genomic results was significantly shorter in the LB arm (17.9 vs. 25.6 days; P<0.001), and LB alone identified category 1 or 2 (without targeted therapies available in first-line) alterations in 13.6% of patients not detected by tissue assessment (8% of detection rate for category 1/2 alterations when the tissue was not informative), mainly due to tissue scarcity. These findings suggest that early LB may not benefit the entire unselected population, nonetheless it provides substantial clinical utility in molecularly enriched or treatment-eligible subsets. Indeed, LB reduced 10 days TTI for non-squamous NSCLC patients and 16 days for a front-line targeted treatment. It should highlight that the study cohort might not represent the general lung cancer population, with almost 30% being non-smokers and nearly half being women. This finding could overvalue the 42.7% of the NSCLC population with Category 1 molecular alterations found, indicating a selection bias. Approximately 30% of patients with metastatic NSCLC are diagnosed with a molecular alteration with a targeted available drug in the first- or second-line setting, more frequent among non-smoker patients (15).

As ctDNA testing becomes increasingly embedded in clinical practice and trial design, the selection of an appropriate assay is critical to ensure reliable results, particularly in terms of sensitivity and specificity. Patients enrolled into LIBELLULE study were evaluated with an early LB using the InVisionFirst-Lung^® assay. The panel demonstrated a sensitivity of over 90% for variant allele fractions (VAFs) above 0.25%, approximately 70% for VAFs between 0.13% and 0.16%, and 56.25% for VAFs between 0.06% and 0.08%, while maintaining a specificity of almost 99.999%. This high sensitivity and specificity place the InVisionFirst-Lung^® panel as a reliable tool for detecting actionable mutations in NSCLC, as supported by current literature. The most commonly used commercial tests for LB assessment in NSCLC might be FoundationOne Liquid CDx^® and Guardant360^®. FoundationOne Liquid CDx^®, a pan-cancer cfDNA-based comprehensive genomic profiling assay, showed a sensitivity of over 90% for VAFs above 0.25%, approximately 70% for VAFs between 0.13% and 0.16%, and 56.25% for VAFs between 0.06% and 0.08%. The specificity of this assay is nearly 99.9997%, making it highly reliable for detecting genomic alterations (16). Those results are consistent with other genomic panels validated for LB assessment. FoundationOne Liquid CDx^® assay demonstrated high analytical performance. The test showed a positive percent agreement of 96.3% and a negative percent agreement greater than 99.9% when compared to orthogonal NGS methods. The limits of detection were 0.40% VAF for substitutions and indels, 0.37% VAF for rearrangements, 21.7% tumor fraction (TF) for copy number amplifications, and 30.4% TF for copy number losses. The false positive rate was extremely low at 0.013% and the assay demonstrated reproducibility of 99.59%. These findings led to the Food and Drug Administration (FDA) approval and confirmed the test as a reliable and accurate tool for detecting genomic alterations in a broad range of solid tumors. In this setting, Guardant360^® has shown a sensitivity of 85.8% for detecting guideline-recommended biomarkers in NSCLC, with a specificity of over 98.2% for FDA-approved targets (17).

Despite the promising potential of LB in cancer management, several challenges and practical barriers hinder its routine implementation. One significant issue is low ctDNA shedding, particularly in early-stage disease or tumors with low metastatic burden, which can lead to false negatives and necessitate reflex tissue testing (18). Assay performance varies due to differences in panel composition, gene coverage, and analytical sensitivity, affecting diagnostic accuracy; thus, selecting a platform and interpreting results should be carefully assessed by expert molecular boards. Although the specificity of ctDNA assays is generally high, isolated case reports or small series have highlighted the potential for false-positive findings in LB, particularly in oncogene-driven NSCLC. These occurrences, although infrequent, should prompt caution when initiating treatment based solely on LB findings in the absence of tissue confirmation (19). Moreover, mutations arising from hematopoietic clones may be detected in plasma and incorrectly attributed to tumor-derived ctDNA (20,21). Additionally, limited reimbursement for LB in many healthcare systems restricts access and hinders equitable implementation, highlighting the need for cost-effectiveness and budget impact data to support wider adoption (22). Furthermore, turnaround time delays could be a key challenge in this setting. LIBELULE results could be an example, as despite obtaining results in an average of 18 days with the InVisionFirst-Lung^® panel, the first systemic treatment was initiated in an average of 29 days, indicating other opportunities for improvement in the patient pathway, particularly in optimizing clinical workflows in general hospitals compared to comprehensive cancer centers (CCCs) (23). Although the LIBELULE study does not explicitly report comparative outcomes between CCCs and smaller hospitals, the authors acknowledge that such variability may exist. Differences in infrastructure, resource availability, and clinical coordination could impact the implementation and timeliness of treatment decisions. This highlights the need for targeted strategies to optimize real-world integration of LB across diverse healthcare settings. Finally, it is necessary to know the cost-effectiveness, cost-utility ratio or opportunity cost of the experimental procedure of the LIBELULE trial. These aspects are essential for broader policy decisions and the integration of LB into national diagnostic pathways. Future economic analyses should assess the value of early LB, particularly in healthcare systems with constrained resources (24).

There is growing consensus that we might now be technically ready to integrate LB into hospital workflows for the molecular diagnosis of patients with suspected or confirmed advanced lung cancer. However, its implementation must be standardized across both CCCs and general hospitals to ensure equal access and diagnostic opportunity for all patients, regardless of care setting. Several clinical trials are currently underway to explore the utility of LB not only in initial molecular diagnosis but also as a tool for early detection of relapse and disease monitoring (Table 1). The results of these studies, together with the ongoing technological evolution of NGS platforms, which are becoming increasingly more sensitive and specific, will likely provide the scientific foundation to support widespread clinical adoption. Moreover, as these technologies continue to improve, they are expected to become more cost-effective, facilitating broader integration into routine practice. Nonetheless, there is still significant work warranted to optimize clinical pathways, eliminate disparities, and realize the full potential of LB in thoracic oncology. The LIBELULE study represents a major step forward in the field of NSCLC, supporting the role of LB in precision oncology. Alongside trials such as DYNAMIC in colorectal cancer, LIBELULE provides valuable clinical data; its distinct advantage lies in being one of the first randomized trials in this setting. It demonstrates the utility of LB in reducing the time to initiation of systemic therapy, particularly in non-squamous NSCLC. Nevertheless, further studies are needed, especially in broader, less selected populations not enriched for patients with targetable molecular alterations, and across diverse healthcare systems. Such efforts will be essential to validate these findings and inform broader implementation strategies.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the Editorial Office, Translational Lung Cancer Research. The article has undergone external peer review.

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-427/prf

Funding: None.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-427/coif). A.R.D. reports lecture fees and/or consulting fees from MSD, Roche, BMS and Takeda. The other author has no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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