ctDNA-based detection of molecular residual disease in stage I-III non-small cell lung cancer patients treated with definitive radiotherapy

Background Sensitive and reliable biomarkers for early detection of recurrence are needed to improve post-definitive radiation risk stratification, disease management, and outcomes for patients with unresectable early-stage or locally advanced non-small cell lung cancer (NSCLC) who are treated with definitive radiation therapy (RT). This prospective, multistate single-center, cohort study investigated the association of circulating tumor DNA (ctDNA) status with recurrence in patients with unresectable stage I-III NSCLC who underwent definitive RT. Methods A total of 70 serial plasma samples from 17 NSCLC patients were collected before, during, and after treatment. A personalized, tumor-informed ctDNA assay was used to track a set of up to 16 somatic, single nucleotide variants in the associated patient’s plasma samples. Results Pre-treatment ctDNA detection rate was 82% (14/17) and varied based on histology and stage. ctDNA was detected in 35% (6/17) of patients at the first post-RT timepoint (median of 1.66 months following the completion of RT), all of whom subsequently developed clinical progression. At this first post-RT time point, patients with ctDNA-positivity had significantly worse progression-free survival (PFS) [hazard ratio (HR): 24.2, p=0.004], and ctDNA-positivity was the only significant prognostic factor associated with PFS (HR: 13.4, p=0.02) in a multivariate analysis. All patients who developed clinical recurrence had detectable ctDNA with an average lead time over radiographic progression of 5.4 months, and post-RT ctDNA positivity was significantly associated with poor PFS (p<0.0001). Conclusion Personalized, longitudinal ctDNA monitoring can detect recurrence early in patients with unresectable NSCLC patients undergoing curative radiation and potentially risk-stratify patients who might benefit most from treatment intensification.


Introduction
Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer-related diagnoses and deaths (1).The current standard-ofcare for patients with inoperable NSCLC is definitive curative radiotherapy (RT) including stereotactic body radiotherapy (SBRT) for early-stage disease and concurrent chemoradiation (CRT) followed by durvalumab for locally-advanced disease (2).
For early-stage disease, SBRT has shown excellent long-term primary tumor control rates, with nodal and distant recurrences representing the most common failure pattern (3).In patients with locally advanced NSCLC, consolidation durvalumab significantly improves the progression-free survival (PFS) compared to CRT alone, yet in-field recurrence and distant metastases present a challenge post-CRT and durvalumab.Thus, careful long-term surveillance is necessary for early detection of recurrence before the onset of disease-related symptoms and at a time when therapy might provide greater clinical benefit.The current surveillance protocol includes computed tomography (CT) of the chest every 3 months for 2 years, every 6 months during years 3 and 4, and annually thereafter (2).Radiographic surveillance is associated with several challenges such as low sensitivity, detection of macroscopic disease, and difficulties in interpretation of results due to posttreatment effects, such as inflammatory changes, radiation fibrosis, or reactive lymph nodes in cases with local recurrences (4).Therefore, a need exists for a sensitive, blood-based biomarker for early detection of molecular residual disease (MRD), postdefinitive therapy.
Circulating tumor DNA (ctDNA) has emerged as a prognostic biomarker to assess MRD and predict recurrence (5,6).In this study, we assessed the prognostic value of a tumor-informed ctDNA assay for longitudinal monitoring of patients with stage I-III NSCLC undergoing definitive radiotherapy to detect recurrence and identify patients who might benefit from intensification of systemic therapy.

Subjects and study design
All patients had a pathologically confirmed diagnosis of lung cancer.Blood samples (n=70) serially collected (before and after SBRT as well as before, during, and after conventional RT with/ without concurrent systemic therapy and adjuvant durvalumab) from a prospective clinical cohort of patients (N=17) with stage I-III NSCLC diagnosed between 2017 and 2020 were used for ctDNA analysis.All patients were staged according to American Joint Committee on Cancer (AJCC) 8 th Edition.Patients with stage I disease were treated with SBRT in 4 or 5 fractions (10 -12 Gy per fraction).Patients with stage II and stage III disease were treated with conventional fractionation (2 Gy per fraction) with or without concurrent and adjuvant systemic therapy.Post-treatment plasma was collected generally concurrently with the standard-of-care imaging at the discretion of the treating clinician.Light, moderate, and heavy smokers were defined as <20 packs/year, 20-40 packs/year, and >40 packs/year, respectively.The longitudinal setting was defined as serial ctDNA testing of patients after discontinuation of RT (during systemic therapy, if given, and during surveillance), wherein patients had sample collection for ctDNA tests at regular intervals or as determined by the treating physician.This study was approved by the Memorial Sloan Kettering Institutional Review Board.The study was conducted in accordance with the principles of the Declaration of Helsinki 2013.All patients provided informed consent.

Personalized ctDNA assay workflow
Personalized, tumor-informed ctDNA assays were designed for all patients as previously described (7).Briefly, a set of 16 highconfidence, patient-specific, somatic, clonal single nucleotide variants (SNVs) were selected for multiplex polymerase chain reaction (mPCR) testing from whole-exome sequencing of formalin-fixed paraffin-embedded (FFPE) tumor tissue and matched normal blood samples.The mPCR primers targeting the patient-specific SNVs were designed, synthesized, and used for tracking ctDNA in patients' longitudinal plasma samples.Plasma samples were considered ctDNA-positive when at least 2 SNVs were detected above a predefined confidence threshold.ctDNA concentration was expressed as mean tumor molecules (MTM)/ mL of plasma.

Statistical analysis
Fisher's exact test was used to evaluate the statistical significance of the association between ctDNA detection rates at baseline and categorical variables.Using the Kaplan-Meier method, PFS was assessed as the primary outcome between the date of RT initiation and clinical recurrence using post-RT ctDNA status for patient stratification.Log-rank test or Cox proportional hazards model was used for comparing two survival distributions with p ≤ 0.05 being considered significant.Statistical analyses were carried out in STATA v16.1.

Results
Patient demographics, baseline characteristics, and treatment regimens are presented in Table 1.Patients were followed for a median of 26 months (range: 4-54).ctDNA assays were successfully designed for all patients.

Discussion
Definitive radiation is the standard-of-care for patients with inoperable localized lung cancer.Our study demonstrates that tumor-informed ctDNA monitoring is an effective tool to detect MRD among patients treated with definitive RT. ctDNA monitoring preceded clinical recurrence by a median of 5.4 months, providing a critical window for early therapeutic intervention.
ctDNA monitoring is a promising technology to personalize therapy selection among patients with localized lung cancer.In the early-stage setting, the determination of adjuvant therapy after definitive RT is based on high-risk clinical and pathologic features (2).We observed that all patients with detectable MRD developed clinical recurrence, suggesting utilization of ctDNA may identify a group of patients at high risk of relapse who are likely to derive benefit from adjuvant therapy.In the locally advanced setting, one year of adjuvant durvalumab is standardof-care for patients following definitive chemoradiation.MRD monitoring may allow for personalization of adjuvant therapy by identifying patients benefiting from consolidative durvalumab versus those who may benefit from an alternative approach (6).For example, in our cohort, patients who failed to clear ctDNA while receiving adjuvant durvalumab developed clinical recurrence.It is possible this cohort would have benefited from an alternative or intensified systemic therapy.An ongoing study (NCT04585490) evaluating personalized escalation of therapy for patients with locally advanced lung cancer treated with chemoradiation therapy will be an important contribution to patient care and demonstration of the clinical utility of ctDNA-based MRD analysis.
MRD monitoring may also identify patients with a low risk of recurrence for whom adjuvant therapy could be de-intensified.For example, among patients receiving adjuvant durvalumab in the PACIFIC trial, 19% who received placebo remained disease-free at 5 years, suggesting there are patients who do not derive clinical benefit but are exposed to toxicity of one year of adjuvant durvalumab (8).In a study by Monding et al, one patient with undetectable ctDNA died from pneumonitis related to immune checkpoint inhibition, highlighting the importance of identifying patients most likely to benefit from a therapy which poses a risk of high-grade toxicities (6).
Other studies in locally-advanced NSCLC utilizing different ctDNA technologies have demonstrated the utility of MRD detection at first timepoint (9), 1-month (10), or within 2 weeks to 4 months (11) of post-definitive treatment to be prognostic of clinical outcomes.In our study, ctDNA-positive patients at first post-RT timepoint were 24 times more likely to experience disease progression.Nonetheless, sensitivity was improved with longitudinal monitoring, which has been reported in lung cancer (9,12) and other solid tumors (7,13,14).
In our cohort, we observed a baseline (pre-RT) ctDNA detection rate of 82% which was associated with stage and histology, consistent with prior analyses (6,(10)(11)(12).ctDNA detection is challenging in lowvolume disease with limited ctDNA shedding, and further efforts are required to optimize detection in this patient population; however, it is important to note that the patients with baseline ctDNA-negativity had favorable outcomes.
Although blood samples were collected prospectively in this study, the correlation between ctDNA status and PFS was analyzed retrospectively, which precluded real-time assessment of risk of progression based on ctDNA status of patient at a given time point.Additionally, our study is limited by the small cohort size, heterogeneous disease stages and treatment regimens, and limited clinical follow-up for some patients.Nonetheless, our study demonstrated high sensitivity and specificity for ctDNA with for detection of recurrence with serial monitoring after completion of RT.Currently, the determination of adjuvant therapy in early-stage NSCLC patients after definitive RT is based on the presence of highrisk pathologic features (poorly differentiated tumor, vascular invasion, and visceral pleural involvement).However, our results highlight the potential utility of a personalized and tumor-informed ctDNA testing approach to risk-stratify patients for treatment decision-making.Prospective studies with larger cohorts are warranted to establish the clinical utility of ctDNA, particularly to determine the optimal interval for ctDNA testing, to validate the prognostic performance of longitudinal ctDNA monitoring, and to evaluate the benefits and risks of ctDNA-guided adjuvant treatment decision-making in NSCLC patients receiving curative RT.
FIGURE 1 (A) Overview plot depicting treatment regimen, longitudinal ctDNA analysis and clinical outcomes for each patient in the cohort.(B, C).Pre-RT ctDNA detection rate based on (B) histology, and (C) stage.(D, E).Pre-RT ctDNA levels (MTM/mL) in 14 patients with detectable ctDNA based on (D) histology, and (E) stage.RT, radiotherapy; ctDNA, circulating tumor DNA; NED, no evidence of disease; MTM, mean tumor molecules.

TABLE 1
Patient demographics, baseline characteristics, treatment regimen, and outcome at the last follow-up.