Patients who underwent thoracic radiotherapy combined with ICIs treatments at the Central Hospital Affiliated to Shandong First Medical University (Jinan, China) between January 2019 and May 2022 were reviewed. The sites targeted for radiotherapy encompassed intrapulmonary or mediastinal tumors, such as primary lung cancer, metastatic lung cancer and esophageal cancer, among others. All patients were treated with intensity-modulated radiotherapy using a linear accelerator (Synergy/Infinity, Elekta, Sweden). The purpose of radiotherapy included radical or palliative therapy. The present study was approved (approval no. R202303060092) by the Ethical Committee of Central Hospital Affiliated to Shandong First Medical University (Jinan, China). Oral and written informed consents were obtained from patients and their surrogates in person.

Herein, ICIs agents included antibodies targeting programmed cell death protein 1 (PD-1) or programmed death-ligand 1. There exist two distinct classifications of sequential approach. The first category bore resemblance to the study of Antonia et al (13), wherein patients were administered ICIs subsequent to the completion of a radiotherapy course. The second category entailed the suspension of ICIs during the initiation of radiotherapy. Meanwhile, the concurrent treatment approach followed that of the ETOP NICOLAS trial (14) and Keynote-799 study (15), with ICIs being used during radiotherapy.

Patients and treatment characteristics were retrospectively collected from each patient's medical records, including patient demographics, smoking history, tumor types and Eastern Cooperative Oncology Group Performance Status (ECOG PS). Lung V20 (the percentage of lung volume received >20 Gy), the mean lung dose (MLD) and total dose were evaluated on the treatment planning workstation.

The patients underwent evaluation one month following radiotherapy, with subsequent evaluations conducted concurrently with tumor assessment. Additional examinations were conducted in patients exhibiting respiratory disease-related symptoms. TRP was diagnosed by experienced pulmonologists and radiologists. TRP was defined as new-onset infiltrates on thoracic imaging and/or clinical symptoms such as cough, shortness of breath, or wheezing, while excluding other etiologies (disease progression, infection, or heart failure). Pneumonitis severity was graded using the Common Toxicity Criteria for Adverse Events version 5.0 (https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/ctcae_v5_quick_reference_5x7.pdf). NLR was collected from a patient's blood routine at the time of diagnosis of TRP.

A total of 16 male C57BL/6 mice (6-8 weeks old, weighing ~18-22 g) were purchased from SiPeiFu Biotechnology Co., Ltd (Beijing, China). Mice were housed under standard light-dark cycle (12/12-h light/dark cycle) and temperature (22±2˚C) conditions with sterilized food and water provided ad libitum, following institutional and office of laboratory animal welfare guidelines. All animal experiments were approved (approval no. JNCHIACUC2021-26) by the Ethical Committee of Central Hospital Affiliated to Shandong First Medical University (Jinan, China).

Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). The acute radiation pneumonitis model was established following established protocols (16). Mice were anesthetized and exposed to whole thorax radiation by X-ray at a dose rate of 600.00 cGy/min and a cumulative radiation dose of 18 Gy from a linear accelerator (Elekta Synergy, Sweden) at our institution. Mice were randomly assigned to two groups prior to irradiation initiation: The concurrent group and the sequential group (n=8 in each group).

The two dosing strategies used in the present study are demonstrated in Fig. 1. In the concurrent group, the anti-PD-1 antibody (200 µg/mouse; Bio X Cell, cat. no. BE0146; 1:12.5 resuspended with fresh PBS) was administered intraperitoneally on days 1, 3 and 5 after irradiation. In the sequential group, the anti-PD-1 antibody was injected intraperitoneally on days 14, 16, and 18 after irradiation.

Mice were sacrificed by cervical dislocation on the 28th day after irradiation. The lungs were then removed and immersed in 4% paraformaldehyde for 48 h before being embedded in paraffin. Peripheral blood samples were collected from the mice after euthanasia via the orbital sinus using ethylenediaminetetraacetic acid tubes. All mice were euthanized by dislocating cervical vertebra under general anesthesia at the end of experiments or if a humane endpoint was reached. Humane endpoint was defined as the occurrence of severe dyspnea, vomiting, inability to ambulate or rise for food and water, or a loss of >15% of body weight. However, none of the animals reached these humane endpoints.

Histopathological changes were evaluated following H&E staining. Alveolar congestion, hemorrhage, aggregation of inflammatory cells in airspaces or vessel walls and the thickness of the alveolar walls were assessed using a 0-4-point semi-quantitative histological analysis method (17) (4: Extremely serious; 3: Serious; 2: Middle; 1: Slight; 0: Normal). In total, five fields of view were randomly selected, and the histology score of each sample was determined using an average of all the scores.

Neutrophil counts were determined by quantifying Ly6G⁺ CD11b⁺ cells in peripheral blood using flow cytometric analysis, following established methodologies (18). For the analysis of lymphocyte populations in peripheral blood, a lymphoid gate (low-side scatter) was applied to exclude cells of monocytic origin (19). The following antibodies were utilized: APC-Cy7 anti-mouse CD45 (cat. no. 557659; BD Biosciences), PE anti-mouse CD11b (cat. no. 24965) and PerCP-cy5.5 anti-mouse ly6G (cat. no. 63460) both from Cell Signaling Technology, Inc.). Cell counts were analyzed using a BD Canto II flow cytometer, and the analysis was performed with FACS Diva software (version 6.1.2; BD Biosciences).

Univariate and multivariate logistic regression analyses were conducted to identify the independent risk factors for TRP. In addition, the proportional hazard ratio (HR) and 95% confidence intervals (CIs) were also calculated. Receiver operating characteristic (ROC) curves were employed to assess the effects of lung V20, total dose, MLD and NLR on TRP. Propensity score matching (PSM) was adopted to match subjects in the concurrent and sequential groups. A paired Student's t-test was performed after matching. All data represent the mean ± standard error of Mean (SEM) from at least three independent experiments. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were conducted using SPSS 27.0 software (IBM Corp.) and/or Prism GraphPad 8.0 (Dotmatics).

The clinicopathological characteristics of the 80 patients included in the present study are listed in Table I. Among them, 31 patients were <70 years old, while the remaining were >70 years old. There were 21 (26.25%) patients with an ECOG PS of 0, 42 (52.5%) patients with a PS of 1, and 17 (21.25%) patients with a PS of 2. In terms of tumor categories, a total of 31 instances of intrapulmonary tumors were observed, constituting 38.75% of the cases, predominantly manifesting as lung metastases. Additionally, there were 29 occurrences of mediastinal tumors, encompassing esophageal cancer, gastroesophageal junction tumors and mediastinal lymph node metastases. A total of 20 cases involved both intrapulmonary and mediastinal lymph node metastases, accounting for 25.00% of the total. In total, three dose-volumetric parameters were selected: Whole lung V20, total dose and MLD. Of the total, 59 patients had V20 <20% (73.75%), while 21 patients had V20 ≥20% (26.25%). For MLD, 63 patients (78.75%) had MLD <10 Gy, and 17 patients (21.25%) had MLD ≥10 Gy. Regarding treatment approach, 14 patients (17.50%) received concurrent ICIs and radiotherapy, while 66 patients did not undergo concurrent administration of thoracic radiotherapy and ICIs. On peripheral blood testing onset of pneumonitis (Mindary Blood Cell Analyzer), 44 (55.00%) had NLR <5 and 36 (45.00%) had NLR ≥5. During follow-up period, a total of 14 patients (17.50%) developed grade ≥2 pneumonitis.

The relationships between TRP and clinical characteristics are revealed in Fig. 2. In the univariate analysis, V20, total dose, MLD, sequence of administration and NLR were found to be independent predictive factors for TRP. Univariate logistic regression identified that the HR value of whole lung V20 was 1.16 (95% CI, 1.05-1.27; P=0.002), the HR value of MLD was 1.004 (95% CI, 1.002-1.006; P<0.001), the HR value of total dose was 1.13 (95% CI, 1.01-1.26; P=0.031), the HR value for administration sequence was 0.14 (95% CI, 0.04-0.5; P=0.002) and the HR value of NLR was 1.38 (95% CI, 1.12-1.68; P=0.002).

In the multivariate analysis, V20, MLD, total dose, administered sequence and NLR remained independent predictive factors for TRP. Multivariate logistic regression revealed that the HR value of whole lung V20 was 1.23 (95% CI, 1.02-1.46; P=0.03), the HR value of MLD was 1.004 (95% CI, 1.002-1.008; P=0.039), the HR value of total dose was 1.36 (95% CI, 1.02-1.81; P=0.04), the HR value of administration sequence was 0.17 (95% CI, 0.002-0.47; P=0.01) and the HR value of NLR was 1.47 (95% CI, 1.004-2.15; P=0.048).

ROC curve analyses were constructed to determine the area under the ROC curve (AUC) for each variable (Fig. 3). Based on the results of the ROC curve test, the AUC for V20 was 0.769 (22.87% was used as the cutoff value). The AUC for total dose was 0.683, with 54 Gy used as the cutoff value. The AUC for MLD was 0.786, with 10.47 Gy used as the cutoff value. The AUC for NLR was 0.796, with 6.08 used as the cutoff value.

To evaluate pathological changes, H&E staining was utilized in mouse models (Fig. 4). The staining was quantified by pathologists to assess lung injury. Notably, the concurrent treatment group exhibited more severe lung tissue injury compared with the sequential treatment group, with a score of 3.2±0.2 in the concurrent treatment group vs. 2.2±0.3 in the sequential treatment group (P<0.05).

In the patients reviewed, baseline characteristics were not initially balanced. To address this, PSM was performed using SPSS software (version 27.0; IBM Corp.). Ultimately, 26 patients were successfully matched. A paired Student's t-test revealed that the NLR in the concurrent treatment group was significantly higher compared with the sequential group (T=2.27, P=0.043) (Fig. 5A). In the animal model of combination treatment, the NLR was 0.58±0.08 in the concurrent treatment group and 0.26±0.06 in the sequential treatment group (P<0.01) (Fig. 5B).