Emerging role of circulating tumor DNA as a predictive biomarker in consolidation immunotherapy for unresectable stage III NSCLC

Lung cancer remains one of the most prevalent and deadly forms of cancer worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all lung cancer cases (1). Stage III NSCLC, in particular, presents a significant clinical challenge due to its heterogeneous nature, often involving locally advanced disease that is unresectable. The advent of consolidation immunotherapy has revolutionized the treatment landscape for unresectable stage III NSCLC. Following the groundbreaking results of the PACIFIC trial in 2018, the use of immune checkpoint inhibitor (ICI) durvalumab for up to 12 months after chemoradiotherapy (CRT), has significantly improved progression-free survival (PFS) and overall survival (OS) compared to standard CRT alone (2). However, shorter treatment durations may also be effective. This was shown by the CheckMate-816 trial (3), where only three cycles of chemotherapy combined with nivolumab [programmed cell death protein 1 (PD-1) inhibition, ICI] neoadjuvant therapy ICI for resectable stage II/III, demonstrated a significantly longer event-free survival and a higher percentage of complete pathological responses compared to neoadjuvant chemotherapy alone.

ICIs are particularly recognized for their long-lasting responses also for advanced disease stage, with some subgroups [programmed cell death ligand 1 (PD-L1) high] achieving over 32% 5-year survival (4). However, the overall percentage of patients who benefit from this treatment remains relatively low. While PD-L1 expression is commonly used as a tumor biomarker, it is not always a reliable predictor of response to immunotherapy. ICIs have shown efficacy even in NSCLC patients with minimal or no PD-L1 expression, and conversely, patients with high PD-L1 levels may not always respond to the therapy (5). To reduce serious side effects and costs, it is crucial to develop tumor-specific biomarker-based methods to identify patients who are unlikely to benefit from ICI and to determine the optimal duration and timing of treatment. Additionally, patients who do not respond should be promptly shifted to an alternative therapy. Therefore, there is an urgent need for real-time, minimally invasive (molecular) methods to monitor therapeutic responses and inform decision-making in precision immuno-oncology.

The potential applications of liquid biopsies are extensive, ranging from the early detection of cancer and monitoring of minimal residual disease (MRD) to predicting treatment responses, identifying actionable driver mutations, and detecting resistance mutations (6,7). Especially plasma-based circulating tumor-derived DNA (ctDNA) analysis is becoming increasingly important in immuno-oncology due to its ability to quickly and accurately assess clinical responses (8-10). Recent studies have demonstrated that monitoring of (changes in) the fraction of ctDNA within total DNA derived from cell-free plasma samples serves as a non-invasive surrogate for biological tumor responses and clinical outcomes across various cancer types (11-14).

The study by Dr. Jun and coworkers in Journal of Thoracic Oncology (June 2024) (15) builds on this growing body of evidence by investigating the role of ctDNA as a prognostic biomarker in the context of consolidation immunotherapy for unresectable stage III NSCLC. The study enrolled a cohort of patients with unresectable stage III NSCLC who had completed standard CRT and were eligible for consolidation immunotherapy. Blood samples were collected at baseline (post-definitive CRT), during immunotherapy and at subsequent follow-up visits to measure ctDNA levels. They used a previously described Monte Carlo-based ctDNA detection index to monitor for somatic single-nucleotide variants (16). The primary endpoints were PFS and OS, with secondary endpoints including the correlation of ctDNA dynamics with these clinical outcomes. The study’s results are compelling. Detection of ctDNA was associated with significantly inferior PFS at all measured time points. Specifically, patients with detectable ctDNA after CRT had a 24-month PFS of 29% compared to 65% for those without detectable ctDNA. Detection of ctDNA before the second cycle of ICI resulted in a 24-month PFS of 0% versus 72% for patients without detectable ctDNA. At the end of ICI, the 24-month PFS was 15% for patients with detectable ctDNA compared to 67% for those without. Moreover, patients with decreasing or undetectable ctDNA levels after one cycle of ICI had significantly better outcomes (24-month PFS) than those with increasing ctDNA levels (72% compared to 0%). Remarkably, patients who achieved ctDNA clearance after ICI had a 74% probability of remaining progression-free at 24 months, highlighting the potential of ctDNA as a powerful prognostic biomarker for clinical outcomes. Overall, this study shows that ctDNA dynamics may serve as a prognostic biomarker for clinical outcomes in patients undergoing consolidation immunotherapy for unresectable stage III NSCLC, potentially guiding more personalized treatment strategies and improving patient prognosis. For example, if one cycle of ICI results in ctDNA clearance, treatment duration could potentially be reduced or stopped there. Conversely, persistent ctDNA after treatment completion might necessitate additional interventions.

These findings align with other studies that show ctDNA as a potential valuable biomarker associated with patient outcomes of consolidation ICI treatment. For example, Raja et al. [2018] were among the first to demonstrate that early reductions in ctDNA variant allele frequencies (VAF) in patients with advanced NSCLC treated with durvalumab, strongly correlated with a favorable clinical outcome, including longer PFS and OS. Importantly, changes in VAF preceded radiographic responses, suggesting the potential for ctDNA to guide early therapeutic decision-making and identify patients unlikely to benefit from continued treatment (17). Ricciuti et al. showed that for patients treated with first-line immunotherapy, a decrease was associated with significantly higher response rates (60.7% vs. 5.8%) and longer median PFS (8.3 vs. 3.4 months) and median OS (26.2 vs. 13.2 months) compared with cases with ctDNA increase (18). van der Leest et al. showed that a 30% decrease in mutant ctDNA levels was associated with superior median PFS (43 weeks) and OS (125 weeks) compared with that of the combined patient group with increasing or stable ctDNA levels (PFS 6 weeks; OS 29 weeks) (8). Goldberg et al. found that a ctDNA response (a>50% decrease in mutant allele fraction from baseline) was associated with superior PFS [hazard ratio (HR) 0.29] and superior OS (HR 0.17) (19). In another study, similar findings were found when monitoring ctDNA changes using plasma-based ctDNA next-generation sequencing (NGS) analysis in advanced NSCLC patients; a 50% molecular response improved PFS (10 vs. 2 months) and OS (18.4 vs. 5.9 months) compared with patients not achieving this end point (10). Anagnostou et al. showed that a ctDNA response (clearance of maximal mutant allele fraction from baseline) was associated with longer PFS (5.03 vs. 2.6 months) and OS (not reached vs. 7.23 months) (20).

Advantages of ctDNA in resectable stage II–III NSCLC patients

While the majority of studies have concentrated on advanced and metastatic disease, ctDNA also offers significant advantages for patients with resectable stage II–III NSCLC. For instance, the AEGEAN trial demonstrated that perioperative durvalumab combined with neoadjuvant chemotherapy significantly improved pathological complete response and event-free survival in patients with resectable NSCLC (21). Additionally, a correlation between ctDNA clearance and pathological response was observed (22), suggesting that ctDNA could potentially be a marker to decide to eliminate the need for invasive surgery (in cases of pathological complete response), where, for example, radiology fails to do so. Furthermore, ctDNA has shown potential as an early marker to assess treatment efficacy, which may provide critical guidance for tailoring therapy in real time. Early ctDNA clearance could also help identify patients who are achieving optimal outcomes with current treatment strategies, while those without clearance may require further interventions. These findings highlight the potential of ctDNA in redefining treatment strategies, particularly for patients in the perioperative or post-treatment monitoring settings (23).

Uncertainty in the optimal duration of ICI consolidation therapy after a radical treatment and the relevance of ctDNA monitoring

Currently, the optimal duration of consolidation ICI treatment after chemo-radiotherapy (CRT) for unresectable stage III NSCLC remains an open question. While the Food and Drug Administration (FDA)-approved regimens based on the PACIFIC trial recommend 12 months of consolidation durvalumab, real-world data from a Veterans Health Administration study showed a median treatment duration of only 7.1 months due to toxicity, particularly pneumonitis (24). Since then, several perioperative clinical trials, such as CheckMate 77T (25), Keynote 671 (26) and the AEGEAN trial (21), have demonstrated that four cycles of neoadjuvant ICI combined with platinum-based chemotherapy and 1 year of adjuvant treatment significantly improved disease-free survival in resectable NSCLC. Similar observations were made in the CheckMate 816 trial, which is a neoadjuvant-only trial. Additional clinical trials are needed to determine that ctDNA may have an important role in deciding whether neoadjuvant treatment only instead of perioperative treatment is sufficient, as well as to optimize treatment duration. One such initiatives is the GUIDE.MRD consortium that aims to set a standard for ctDNA to measure residual disease in stage III NSCLC, pancreatic cancer and colorectal cancer after a radical treatment (27). Although Jun’s study did not directly compare different durations of ICI treatment (all patients were planned to receive six months, with a median treatment duration of four months), it provided evidence that this timeframe was sufficient to improve PFS and OS. Possibly, patients with ctDNA clearance after six months likely would not have benefited from longer ICI, as they demonstrated excellent long-term outcomes. This suggests that shorter treatment durations may be effective for certain patient groups, even though the study’s small sample size limits broader generalization. In addition, patients with undetectable ctDNA may achieve favorable outcomes with CRT alone or potentially benefit from a shorter course of immunotherapy. Conversely, patients whose ctDNA is not cleared by consolidation immunotherapy represent a high-risk subgroup. These individuals may require novel therapeutic approaches or may not benefit from any further intervention, which is an area worth studying.

The temporal response advantage of ctDNA

Additionally, ctDNA responses are often observed significantly earlier than radiographic responses. For instance, recent research in early-stage colorectal cancer demonstrated that ctDNA detection preceded radiographic recurrence by over 5 months, underscoring its potential for early intervention (28). Jun’s study found a median lead time of 4.8 months between ctDNA detection and radiologic progression. Goldberg et al. reported a median detection time of ~1 month using ctDNA, compared to almost 2.5 months by imaging. Anagnostou et al. found a median time to ctDNA response was 2.1 months. These findings suggest that ctDNA could serve as an early and more sensitive indicator of treatment response.

Despite the substantial progress made, liquid biopsies still face several challenges before being fully integrated into clinical decision-making for stage III NSCLC. For one, there is no standard ctDNA test yet that serves as a reference, “gold standard”. Another major issue is the lack of consensus on what constitutes a ctDNA molecular response. Various measures, such as mean or maximum mutant allele frequency of tumor-derived alterations, have been proposed, but a standardized definition is still missing. Additionally, the definitions of ctDNA molecular response often depend on the NGS assay used, which varies in sensitivity and negative predictive value. The heterogeneity in cohort composition, treatments administered, time points analyzed, and ctDNA methodologies used in studies further complicates the interpretation of results. These challenges are illustrated in Jun’s study, which observed that ctDNA status and response were associated with differing clinical outcomes when using a small cohort. However, it also revealed limitations, as 45% of patients (5 out of 11) who experienced disease progression had undetectable ctDNA after CRT and before consolidation ICI. This finding underscores that while ctDNA shows promise, it is not yet reliable enough for clinical use. The ctDNA assays used in the study had a detection limit of approximately 0.01%. Fortunately, newer, more sensitive ctDNA MRD assays can detect mutations at levels as low as one part per million (29), offering 100 times greater sensitivity compared to the first generation, which failed to detect ctDNA in stage III patients. However, increasing sensitivity may also lead to higher ctDNA positivity in patients who are responding well, as seen in Jun’s study and others, where some patients with a prolonged response exhibited persistent ctDNA positivity. Therefore, ctDNA positivity alone may not be the decisive factor in guiding treatment. The primary value of ctDNA MRD in unresectable NSCLC patients lies in its potential to guide de-escalation strategies, sparing patients from the unnecessary burden of costly and toxic intensive treatments. Hopefully, the next-generation assays can provide better refinement in patient selection and potentially allow for more tailored immunotherapy approaches. Beyond cancer, plasma-based liquid biopsies hold significant potential in other fields. In prenatal testing, cell-free DNA (cfDNA) analysis has revolutionized non-invasive screening for chromosomal abnormalities, such as Down syndrome, with remarkable accuracy (30). Additionally, in organ transplantation, donor-derived cfDNA has shown promise as a biomarker for early detection of organ rejection, enabling timely intervention and improved outcomes (31). These applications highlight that cfDNA’s utility extends far beyond oncology, offering transformative possibilities in diverse clinical settings.

Examining multiple markers alongside ctDNA may further enhance the predictive power of ctDNA testing. For instance, van der Leest et al. demonstrated that combining ctDNA with PD-L1 expression improved prognostic value eightfold in PFS and more than doubled in OS (8). Patients with a reduction in circulating mutant tumor DNA and a PD-L1-expressing tumor showed improved PFS, OS, and durable clinical benefit. The data suggest that the combination of tumor-tissue PD-L1 expression and tumor-specific mutant ctDNA reduction is a more effective tool for monitoring response to ICI than either PD-L1 expression or ctDNA changes alone. In Jun et al.’s study, the population is relatively small and incorporating multiple markers in this study would not yield reliable conclusions. Further research is needed to explore the role of ctDNA in specific subgroups, for example, patients with targetable EGFR mutations or ALK fusions (32) and patients with tumors harboring KEAP1 or STK11 mutations (10,33), who are known to have different and often worse responses to immunotherapy.

The study by Jun et al. underscores the potential of ctDNA as a predictive biomarker for MRD in the context of consolidation immunotherapy for unresectable stage III NSCLC by demonstrating that ctDNA levels correlate with treatment outcomes. In addition to these findings, ctDNA may also serve a valuable role alongside traditional radiographic imaging in improving diagnostic accuracy. Its ability to detect molecular changes often months before visible radiologic progression highlights ctDNA as an early and more sensitive indicator of treatment response. This complementary use of ctDNA with imaging could enhance real-time monitoring and enable more personalized treatment adjustments for patients with stage III NSCLC, optimizing therapeutic outcomes and potentially reducing unnecessary treatment-related toxicity. To build on these promising results, future research should focus on prospective studies with large cohorts, well-defined and numerous blood draws, and the inclusion of additional blood-based and tumor-derived biomarkers.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, AME Clinical Trials Review. The article has undergone external peer review.

Peer Review File: Available at https://actr.amegroups.com/article/view/10.21037/actr-24-201/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://actr.amegroups.com/article/view/10.21037/actr-24-201/coif). E.S. reports grants from Abbott, Biocartis, Astrazeneca, Invitae/Archer, Bayer, Bio-Rad, Roche, Agena Bioscience, CC Diagnostics, MSD/Merck, SNN/EFRO; consulting fees from MSD/Merck, AstraZeneca, Roche, Novartis, Bayer, BMS, Lilly, Amgen, Illumina, Agena Bioscience, CC Diagnostics, Janssen Cilag (Johnson&Johnson), Astellas Pharma, GSK, Sinnovisionlab, Sysmex, Protyon; travel expenses and honoria to UMCG institution from Bio-Rad, Roche, Biocartis, Lilly, Agena Bioscience; honoria paid to UMCG institution from Seracare, Illumina; travel expenses from BioRad, Biocartis, Ageno Sciences, Illumina; travel/hotel/registration expenses from Roche/Foundation Medicine, QCMD; serves as an unpaid board member of Dutch Society of Pathology, European Society of Pathology, European Liquid Biopsy Society, and Committee for Clinical Essential Targets (cieKNT); and is a member of national guideline advisory committee and a committee member for assessment of molecular diagnostics (cieBOD). T.J.N.H. reports grants from Roche, BMS, Astra Zeneca; has participated in the advisory boards of BMS, Roche, MSD, Pfizer; has leadership or fiduciary role in CieBOM; and has conducted PI pharma studies for AstraZeneca, GSK, Novartis, Merck Serono, Roche, BMS, Amgen. The other authors have 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.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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