Introduction

Curative radiotherapy (RT) is offered to patients with early stage or locoregional non-small cell lung cancer (NSCLC) not fit or eligible for, or not wanting surgery, either as stereotactic body radiation therapy (SBRT) or as long-course RT. Despite the curative intent, relapse of lung cancer is seen within the first year, and the 5-year survival in Denmark for all lung cancer stages is only around 27% [1]. Better means of providing a reliable prognosis for these patients could help select the patients needing extra treatment, and earlier detection of relapse could enable more patients to receive a curative treatment for their relapse.

A patient with a solitary lung tumor of up to 5 cm and World Health Organization performance status (WHO PS) 3 or better can be offered SBRT, with a dose/fractionation of 66 Gray (Gy) in three fractions being the standard in Denmark [2]. This treatment offers 90% or better local control after 2 years and only a few side effects; however, it requires that the tumor has an appropriate distance from organs at risk [3]. Patients with larger tumors and/or locoregional lymph-node involvement can be offered long-course RT, with a dose/fractionation of 66 Gy in 33 fractions being the standard in Denmark [2]. Concomitant chemotherapy with a platinum-based drug and/or vinorelbine is offered to most patients (PS 0–2) along with the long-course RT. Patients with a histologic PD-L1 expression > 1% and PS 0–1 are also offered adjuvant immune checkpoint inhibitor treatment with Durvalumab for 1 year following the completion of chemoradiotherapy. Chemoradiotherapy has a poorer rate of local control compared to SBRT, and relapses frequently occur, often during the first few years after the treatment [4].

Blood-based biomarkers are obtained through minimally invasive procedures, are readily available, and have been studied intensively in recent years. Previous studies have indicated that such markers may predict the effect of treatment or indicate better/worse prognosis for patients with lung cancer [5]. The molecular biology technique droplet digital polymerase chain reaction (ddPCR) uses partitioning of samples into thousands of nanoliter-sized droplets. The polymerase chain reaction takes place in each of these droplets, allowing detection of molecular targets in very low abundance with very high sensitivity [6]. With the use of newer multiplex systems, several targets can be analyzed simultaneously, allowing faster analyses and higher sensitivity for detecting molecular targets [7]. Methylated Homeobox A9 (HOXA9) circulating tumor DNA (ctDNA) measured with ddPCR has been demonstrated as a valuable prognostic marker in patients with advanced NSCLC [8], but it is not known whether HOXA9 alone or in combination with other tumor-agnostic methylated ctDNA markers offers any prognostic value in curatively irradiated patients with NSCLC.

Soluble Programmed Death-Ligand 1 (sPD-L1) has been tested as a prognostic biomarker in patients with NSCLC, and the previous results indicate a correlation between the level of sPD-L1 and risk of recurrence and overall survival (OS) [9, 10]. It is not known whether combining sPD-L1 with other biomarkers can enhance the prognostic value.

NK-cell activity (NKA) as measured with the NKVue® assay using interferon-γ (IFN-γ) as a surrogate for NKA has shown reliable and reproducible results in an earlier study from our group [11]. Patients showing an abnormally low level of NKA during palliative systemic therapy have been shown to have a poorer prognosis across different cancer types [12]. It is not known whether the baseline NKA itself may hold prognostic value in NSCLC.

Aim

The aim of this study was to evaluate the prognostic and relapse-detecting value of the biomarkers NKA, sPD-L1, and methylated ctDNA measured at baseline and during 12 months of follow-up in NSCLC patients treated with curative RT.