Purpose/Objective(s)
Mid-chemoRT circulating tumor DNA (ctDNA) measurement could be a powerful tool for assessing early therapeutic response and informing treatment strategies in patients with inoperable stage IIB-IIIC non-small-cell lung cancer (NSCLC). We performed Tempus xF, a commercially available liquid biopsy assay that detects cancer-associated mutations across 105 genes in plasma cell-free DNA, to explore the potential for ctDNA levels to predict treatment outcomes, with the goal of integrating these biomarkers into radiation oncology clinical practice.

Materials/Methods
We conducted a prospective single-arm phase II clinical trial evaluating MRI-guided hypofractionated RT with intermittent adaptation combined with concurrent chemotherapy and consolidation durvalumab in 26 NSCLC patients. ctDNA analysis was performed across the full cohort with Tempus xF. Pre- and mid-chemoRT ctDNA levels were quantified by measuring the maximum variant allele frequency (max VAF) of nonsynonymous variants, and then further stratified into ctDNA high vs. low groups by median-splitting by max VAF levels. ctDNA-based stratification was then correlated with patient survival by Kaplan-Meier analysis, and separately by Cox regression models that incorporated ctDNA as a continuous variable.

Results
Median follow-up time from study enrollment was 26 months. ctDNA max VAF levels ranged from 0.3% to 10.9% (median=1.4%) at baseline, and from 0.2% to 28.7% (median=1.5%) at the mid-chemoRT timepoint (median=14 days after XRT start). After median-splitting based on ctDNA levels, Kaplan-Meier survival analysis demonstrated that patients with elevated ctDNA max VAF had significantly worse overall survival (OS) at baseline (median=12 months vs. not reached; HR=4.3, p=0.04) and again when measured at the mid-chemoRT timepoint (median=13 vs. 38 months; HR=4.3, p=0.04). Additionally, patients with elevated ctDNA at baseline had worse time to progression (TTP) (median=7 vs. 39 months; HR=3.8, p=0.04) and disease-specific survival (DSS) (median=14 months vs. not reached; HR>10, p=0.03). Furthermore at the mid-chemoRT timepoint, patients with elevated ctDNA exhibited worse DSS (median=13 months vs. not reached; HR>10, p=0.005). Cox regression models with ctDNA included as a continuous variable again demonstrated the prognostic value of ctDNA max VAF at both the pre-treatment timepoint (p=0.02 for TTP, p=0.05 for OS, p=0.02 for DSS) and the mid-treatment timepoint (p=0.008 for TTP, p=0.04 for OS, p=0.01 for DSS).

Conclusion
Mid- and pre-chemoRT application of the Tempus xF assay to plasma cell-free DNA followed by max VAF analysis of non-synonymous variants enables accurate risk stratification of inoperable stage IIB-IIIC NSCLC treated with MRI-guided chemoRT. Our prospective data suggest that early quantification of ctDNA using a commercially available ctDNA assay may help guide risk- and response-adapted radiotherapy and consolidation therapy personalization in future clinical practice.

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