To the Editor: Lung cancer, one of the most common types of tumor, is also the leading cause of cancer death, accounting for an estimated 1.8 million deaths worldwide. Early recognition of lung cancer is critical. It has been established that nitric oxide and its byproducts play a role in pathophysiological processes of lung cancer, such as tumor immunity, inflammation, and lung tumor progression.[1] Exhaled nitric oxide (eNO) concentrations can be measured by non-invasive devices. The alveolar concentration of nitric oxide (CaNO) has been proposed as a marker of distal airway inflammation, but its value in non-small cell lung cancer (NSCLC) is unknown. The tumor microenvironment has received great attention in recent years, and growing evidence suggests that inducible nitric oxide synthase (iNOS) is a key mediator of immune activation and inflammation.[2] The concentrations of NO, nitrite, and nitrotyrosine were discovered to be elevated in lung cancer patients. Airway epithelial cells, vascular endothelial cells, resident macrophages, and recruited inflammatory cells all play roles in the production of NO in exhaled gas.[3] It is particularly important to clarify the connection between NO and lung cancer. This study was approved by the Ethics & Clinical Research Committee of Nanfang Hospital (No. NFEC-2021-397). Informed consent was obtained from all study participants. Patients with pulmonary nodules and lung-occupying lesions, who visited the pulmonary function examination unit at Nanfang Hospital of Southern Medical University from May 2021 to June 2022, were included in the study, and eNO testing was performed prior to the pulmonary function tests (PFTs). Patients were excluded if they were under the age of 20 years or older than 80 years, had concurrent bronchial asthma, allergy, tuberculosis, or community-acquired pneumonia, or were taking an oral steroid or inhaled corticosteroid (ICS). This study ultimately included 164 patients with pathologically confirmed diagnoses; 123 were diagnosed with NSCLC and 41 with benign lung lesions. Besides, this study recuited 103 healthy people from Nanfang Hospital, who had no pulmonary disease and no inflammation for at least one month before recrutiment, as control group. The eNO measurements were taken using an online approach with a handheld nitric oxide analyser (NANO COULOMB, Sunvou Medical Electronics Co., Ltd, Wuxi, China), and the results are presented in parts per billion (ppb). The fractional exhaled nitric oxide (FeNO) was tested at a flow rate of 50 mL/s (FeNO50), which was eatablished as the standard expiratory flow rate for measuring FeNO by the American Thoracic Society (ATS) and the European Respiratory Society (ERS). In addition to FeNO50, exhalations at a flow rate of 200 mL/s (FeNO200) were measured. CaNO was calculated automatically based on the data obtained from the above measurements through a two-compartment model.[4] In our study, the results of CaNO <1 ppb were recorded as CaNO = 1 ppb. Patients were instructed to refrain from strenuous physical activity and to abstain from eating and drinking for at least 1 hour prior to the assessment. Patients underwent PFTs using a Jaeger instrument (E Jaeger GmbH, Germany). The measurements of eNO and PFTs were carried out on the same day, and PFTs were carried out after the measurement of eNO to reduce the impact of forced breathing. The values of forced vital capacity (FVC), forced expiratory volume in one second (FEV1), forced expiratory volume in one second to forced vital capacity ratio (FEV1/FVC), maximum mid-expiratory flow (MMEF), mean forced expiratory flow at 75%, 50%, and 25% of the FVC (MEF75, MEF50, and MEF25), and the proportion of measured value of above indicators to the predicted value were all recorded. Each participant provided demographic information such as sex, age, and smoking history. Relevant clinical and laboratory data were collected from the electronic medical records system. In addition, the results for serum tumor markers (carcinoembryonic antigen [CEA], cytokeratin 19 fragment [CYFRA21-1], and squamous cell carcinoma associated antigen [SCC]) were recorded. We only included the results of blood samples collected within 48 h before and after the eNO test. The continuous variables were expressed as the mean ± standard deviation or the median (Q1, Q3), and the categorical variables are expressed as numbers (%). Propensity score matching (PSM) was used to match both NSCLC and benign lung lesion groups with healthy controls, respectively. The Mann-Whitney test or independent samples t-test was used to compare continuous variables between groups, the chi-squared test was used to analyze categorical variables, and the Spearman correlation coefficient (ρ) was used to investigate correlations between eNO and PFT parameters. GraphPad Prism Version 9 (San Diego, CA, USA) and IBM SPSS 26.0 (SPSS Inc., Chicago, IL, USA) were used to conduct statistical analyses, produce receiver operator characteristic (ROC) curves, and generate the figures. Statistical significance threshold value was defined as an adjusted P value of 0.05. There were 123 subjects (67 males and 56 females) in the NSCLC group and 41 subjects (19 males and 22 females) in the benign lung lesion group. Age, sex, BMI, and proportion of ever-smokers were not significantly different between the two groups. Among patients in the NSCLC group, adenocarcinoma was confirmed in 104 patients and squamous cell carcinoma was confirmed in the remaining 19 patients. In the NSCLC group, 19 patients had pathologically confirmed stage III/IV advanced NSCLC, and the rest were less than stage III. Based on medical records, the PD-L1 tumor proportion score (TPS) was evaluated in 61 individuals with NSCLC. There were 37 patients with PD-L1 expression, and 24 were negative. Pathological diagnoses in the benign lung lesion group included inflammatory nodules, granulomatous inflammation, inflammatory pseudotumor of the lung, atypical adenomatous hyperplasia, and sclerosing pneumocytoma. There were no significant differences in lung function parameters between NSCLC and benign lung lesions groups [Supplementary Table 1, https://links.lww.com/CM9/B597]. We observed significant differences in FeNO200 and CaNO levels between individuals with NSCLC and those with benign lung lesions (8.0 [6.2, 10.3] ppb vs. 7.3 [6.1, 9.0] ppb, P = 0.005 and 3.7 [2.2, 5.1] ppb vs. 3.5 [2.1, 4.1] ppb, P = 0.011, respectively), but not in FeNO50 levels. We matched the two groups separately to the healthy control population using PSM (matching variables included age, sex, BMI, and smoking history) for reducing bias. The NSCLC group was matched to 65 healthy controls, with a matching tolerance of 0.02, and the benign lung lesion group was matched to 33 individuals. We discovered that both the NSCLC and benign lung lesion groups had significantly higher FeNO50, FeNO200, and CaNO levels than the healthy control group (all P <0.05). Within NSCLC group, the differences in CaNO between the adenocarcinoma and squamous cell carcinoma groups were statistically significant (4.6 [3.4, 4.7] ppb vs. 7.4 [6.4, 10.5] ppb, P = 0.002). We did not find a difference between the two pathological subtypes in FeNO. In terms of pulmonary function parameters, neither CaNO nor FeNO50 demonstrated a statistically significant association with FEV1 and FVC in the NSCLC group. Only CaNO exhibited a significant negative correlation with the small airway function parameters (MEF25, MEF50, and MMEF, P <0.05) in the NSCLC group [Supplementary Table 2, https://links.lww.com/CM9/B597]; in particular, the group of patients with predicted MMEF% (MMEF%pred) less than 65% was linked to the high CaNO levels (patients with MMEF%pred ≥65%: 4.6 [3.3, 6.3] ppb, n = 68; patients with MMEF%pred <65%: 6.2 [3.9, 9.2] ppb, n = 51; P = 0.028). Demographic characteristics, such as age, have been shown to affect eNO. In our study, when comparing eNO grouped by sex, age, and smoking status in NSCLC patients, we discovered that the CaNO level was higher in patients aged >60 years than that in patients aged ≤60 years (6.3 [3.9, 10.0] ppb vs. 4.6 [3.5, 6.7] ppb, Z = -2.074, P = 0.038). We did not observe statistically significant differences in eNO when NSCLC patients were categorized by CEA and CYFRA21-1 thresholds. But, we found that lower CaNO values in PD-L1 expression-negative group (4.1 [3.1, 6.4] ppb vs. 6.2 [3.8, 9.3] ppb, P = 0.047). There were no significant differences in both FeNO50 and CaNO among patients with different tumor stages [Supplementary Table 3, https://links.lww.com/CM9/B597]. The purpose of this study was to explore the clinical value of eNO in NSCLC. Our results demonstrate that CaNO and FeNO200 levels were significantly elevated in NSCLC. One possible explanation is that proinflammatory cytokines and iNOS are preferentially expressed by tumor-associated cells in the tumor microenvironment. CaNO was correlated with clinical parameters, especially with regard to impairment in small airway function. Zeng et al[5] found that CaNO was inversely correlated with small airway parameters in asthmatic subacute cough. Elevated CaNO indicates the presence of small airway inflammation, and the CaNO of NSCLC group had statistically significant correlations with MEF50, MEF25, and MMEF in our study. The results implied that alveolar inflammation may affect small airway function in NSCLC. This study is a single-center clinical study with a relatively small sample size, particularly for patients with benign lung lesions, making it challenging to conduct a more thorough and in-depth analysis of FeNO and CaNO. Subsequently, larger sample size studies are needed to further explore the application of eNO in the diagnosis and treatment of NSCLC. In conclusion, our research showed that people with NSCLC had higher CaNO and FeNO200 values than both health control and patients with benign lung lesion. In NSCLC patients, CaNO in squamous cell carcinoma was higher than that in adenocarcinoma. CaNO was associated with age in NSCLC patients, and high CaNO was also observed in patients who had high PD-L1 expression. In addition, higher CaNO values were observed in NSCLC patients who had small airway dysfunction. This study adds evidence on the use of eNO for NSCLC diagnosis. Funding This work was supported by grants from the National Natural Science Foundation of China (Nos. 82170032 and 81970032). Conflicts of interest None.
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