Unlocking the future of immunotherapy with circulating tumor DNA (ctDNA)-based liquid biopsy in non-small cell lung cancer

The advent of immune checkpoint inhibitors (ICIs) has initiated a new era in the management of non-small cell lung cancer (NSCLC), fundamentally altering treatment strategies and offering hope for improved survival outcomes and durable responses in a subset of patients (1). These agents, by targeting inhibitory pathways such as programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4), have not only reactivated antitumor immunity but also challenged longstanding assumptions about cancer progression and resistance (2). However, despite these advances, a majority of patients do not respond to the treatment (3,4). Thus, significant hurdles remain in predicting which patients will derive the greatest benefit from immunotherapy, as well as in the early detection of resistance mechanisms and the management of immune-related adverse events (irAEs).

Traditional imaging techniques, while indispensable in clinical oncology, often fail to capture the subtleties of immune-mediated tumor control—particularly in scenarios of stable disease where tumor dimensions remain unchanged despite effective biological responses. A promising strategy that has emerged to address these challenges is the utilization of liquid biopsy approaches, particularly the analysis of circulating tumor DNA (ctDNA) (5). Noteworthy, ctDNA provides a quantitative and temporal readout of tumor burden that can precede radiographic changes. Unlike traditional tissue biopsies, which are invasive and provide only a snapshot of the tumor’s molecular landscape, liquid biopsies offer a minimally invasive means to monitor the dynamic interplay between tumor evolution and the host immune response over time. This real-time molecular monitoring is especially valuable in NSCLC, where heterogeneity and rapid clonal evolution can complicate treatment assessment (6).

In a recent study by Murray et al. published in 2024 in Clinical Cancer Research, the investigators explored the dynamic changes in ctDNA and immune repertoire among patients with metastatic NSCLC undergoing immunotherapy (7). Employing targeted error-correction sequencing in combination with matched sequencing of both white blood cells and tumor tissue, the study achieved a high degree of sensitivity and specificity in detecting molecular alterations (8). One of the key innovations was the quantification of cell-free tumor load (cfTL), where the complete clearance of the maximum mutant allele fraction of the most abundant tumor-derived mutation at each serial plasma sample was used as a surrogate for a molecular response. This molecular response was found to be significantly correlated with improved progression-free survival (PFS) and overall survival (OS), suggesting that ctDNA may serve as a more immediate and accurate biomarker of treatment efficacy than conventional radiographic imaging. The authors showed that in patients with radiographically stable disease, dynamic changes in ctDNA more accurately reflect survival outcomes and the extent of therapeutic benefit. While radiographic changes in tumor burden showed some correlation with ctDNA trends, 30% of patients exhibited molecular responses despite stable or increasing tumor burden on imaging. This underscores the clinical utility of ctDNA assessments in capturing treatment response heterogeneity and providing a more precise evaluation of disease dynamics beyond conventional imaging. However, it should be noted that various definitions of ctDNA molecular response have been proposed in the literature, due to differences in particular next-generation sequencing assay, shaped by their sensitivity and negative predictive value (9).

Beyond tumor burden and survival assessment, the study by Murray et al. focused on the predictive value of immune repertoire reshaping with a focus on T-cell receptor (TCR) dynamics. The researchers observed significant clonotypic expansions and regressions in peripheral T-cell populations, occurring on average five months before the clinical onset of irAEs. Such early immune perturbations may serve as a prognostic indicator, effectively functioning as an “early warning system” that could allow clinicians to implement preemptive strategies to mitigate severe toxicity. Longitudinal monitoring of the peripheral T-cell repertoire combined with immunoproteomic profiling enabled the identification of patients at increased risk of developing immune-related toxicities. This approach facilitates early intervention strategies, potentially mitigating the severity and morbidity associated with irAEs. This is of paramount importance, given that irAEs can be both life-threatening and a major cause of treatment discontinuation, thereby adversely affecting patient outcomes (10). However, it has to be stressed that while significant reshaping of the TCR repertoire was linked to irAE development, the study does not fully address whether these changes precede or merely coincide with toxicity onset. In addition, the specificity of TCR clonotype expansions for irAEs versus anti-tumor immune responses remains unclear (11). Last, but not least, introducing these sophisticated assays into clinical practice might be impossible, due to significant cost and necessary lab infrastructure.

The implications of these findings extend well beyond the immediate study. Historically, biomarkers such as PD-L1 expression and tumor mutational burden (TMB) have been employed to predict responses to immunotherapy, yet both have limitations in terms of sensitivity and specificity (12). The dynamic, real-time assessment of ctDNA coupled with immune profiling offers a more nuanced view by capturing the bidirectional interactions between the tumor and the immune system, as well as their evolution over time. This integrative approach aligns seamlessly with the principles of precision oncology, which advocate for tailoring treatment strategies to the molecular and immunological profile of each patient’s disease (13,14).

Notwithstanding these promising advances, several challenges must be addressed to facilitate the routine clinical adoption of ctDNA and immune profiling. The study by Murray et al. was conducted on a relatively small retrospective cohort of 30 patients with NSCLC, which, while providing valuable insights, underscores the need for validation in larger, multicenter trials to ensure that the findings are robust and generalizable. This cohort included patients receiving both ICIs alone and chemoimmunotherapy combinations, making it difficult to delineate whether ctDNA and TCR dynamics differ based on treatment type. Moreover, the standardization of ctDNA analysis poses a significant technical challenge. Currently, multiple sequencing platforms and analytical approaches are used, leading to variability in sensitivity, specificity, and reproducibility across studies. Variabilities in sequencing depth, bioinformatics algorithms, and pre-analytical sample handling can introduce discrepancies that undermine the reproducibility of ctDNA quantification (15). Concerted efforts in establishing technical standards and inter-laboratory quality controls are thus imperative (16). Noteworthy, a subset of patients (13%) had undetectable ctDNA at baseline, preventing molecular response assessment and while tumor-informed sequencing strategies were used, tumor heterogeneity and low-shedding tumors may still result in false-negative ctDNA assessments (17).

Even after standardization, the economic considerations, including the cost of high-throughput sequencing and the requisite bioinformatics infrastructure, represent significant barriers, particularly in resource-limited settings and need to be taken into account (18). Moreover, the integration of such sophisticated molecular monitoring tools into everyday clinical workflows will require extensive training and a fundamental shift in the way oncologists approach treatment planning. Collaborative efforts between clinicians, researchers, and industry partners will be crucial in overcoming these obstacles and in establishing standardized protocols that ensure both accuracy and cost-effectiveness. Integrating ctDNA monitoring into existing clinical workflows will also require a careful re-examination of current treatment standards. For instance, a critical question remains as to how clinicians should respond to the detection of persistent ctDNA. Should such findings prompt an immediate alteration in therapeutic strategy, or is there a need to corroborate ctDNA data with other biomarkers and clinical indicators before making treatment decisions? Similar questions arise in the context of immune repertoire profiling for predicting irAEs. Although the study clearly demonstrates an association between TCR dynamics and toxicity risk, the underlying molecular mechanisms remain incompletely understood. Future investigations must aim to elucidate the signaling pathways that drive these immune changes, as well as to determine whether interventions aimed at modulating TCR dynamics could mitigate toxicity without compromising antitumor efficacy (19).

Expanding the scope of ctDNA-based monitoring beyond standard immune checkpoint blockade also holds significant promise. While the study primarily focused on patients receiving conventional ICIs, the principles of ctDNA dynamics could be extrapolated to novel immunotherapeutic strategies, including combination regimens that integrate targeted therapies, adoptive T-cell transfers, and cancer vaccines. Comparative analyses of ctDNA kinetics across different treatment modalities might reveal distinct patterns of tumor and immune responses, thereby enabling the optimization of combination approaches that maximize therapeutic efficacy while minimizing adverse effects (20).

Furthermore, the interplay between tumor evolution and immune escape mechanisms remains an area of intense investigation (21). The observed correlation between molecular response and improved clinical outcomes suggests that ctDNA clearance may not only reflect tumor regression but also indicate a sustained, effective immune surveillance. Conversely, the persistence or resurgence of ctDNA could signify either inherent tumor resistance or the activation of adaptive immune escape pathways. A deeper understanding of these processes could reveal novel therapeutic targets and strategies to overcome immune resistance, a major obstacle in the long-term management of NSCLC (22).

As research continues to evolve, one of the most intriguing prospects lies in the development of ctDNA-guided adaptive treatment strategies. The real-time tracking of tumor evolution via liquid biopsy heralds the possibility of truly personalized cancer therapy. For example, patients demonstrating early ctDNA clearance might be candidates for treatment de-escalation, thereby reducing the cumulative burden of therapy-associated toxicities. Conversely, persistent or rising ctDNA levels could serve as a trigger for early therapeutic intensification or a switch to alternative modalities. Such adaptive strategies represent a significant transformation—from the traditional one-size-fits-all approach to a dynamic, response-driven model of care (23).

The broader implications of these advancements are profound, particularly in the context of precision oncology. Integrative biomarker strategies that combine ctDNA analysis with immune profiling and other omics technologies (such as proteomics and metabolomics) offer a multidimensional view of tumor biology and host responses. This holistic approach is expected to yield more accurate predictive models, not only for treatment response but also for the early detection of relapse and the emergence of drug resistance. The synergy between liquid biopsy technologies and computational analytics—including machine learning and artificial intelligence—further enhances our ability to decode complex biological data, ultimately leading to more informed clinical decision-making (24).

Looking ahead, the future of ctDNA-guided therapy in NSCLC is replete with opportunities for further research. Large-scale, prospective multicenter studies are needed to validate the prognostic and predictive utility of ctDNA and immune profiling across diverse patient populations. Concurrently, mechanistic studies aimed at dissecting the molecular underpinnings of ctDNA dynamics and TCR repertoire shifts will be critical in identifying new therapeutic targets. In parallel, advancements in sequencing technologies and bioinformatics pipelines promise to further enhance the sensitivity and specificity of these assays, bringing us closer to the goal of real-time, personalized cancer care (25).

In summary, the study by Murray et al. offers a valuable contribution to the ongoing developments in the application of liquid biopsy for the management of NSCLC, while having relevant methodological limitations. By integrating ctDNA-based tumor monitoring with comprehensive immune profiling, the authors provide a robust framework for refining the assessment of immunotherapy response and for predicting treatment-associated toxicities. While challenges related to sample size, technical standardization, and clinical integration remain, the potential impact of these findings on personalized oncology is substantial. As liquid biopsy technologies continue to evolve, they are poised to transform the landscape of cancer treatment—leading our community to an era of truly individualized therapy that is responsive to the dynamic interplay between tumor biology and the immune system.

Acknowledgments

None.

Footnote

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

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-326/prf

Funding: None.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-326/coif). B.T. receives lecture fees from Pfizer and MSD, and honoraria from Roche. 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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