Preoperative fractional exhaled nitric oxide in differentiating pulmonary nodules and pathological examination of non-small cell lung cancer: a retrospective cohort study

Abstract

Background

Lung cancer remains the leading cause of cancer-related mortality worldwide, and preoperative differentiation of pulmonary nodules remains challenging. We aimed to investigate the clinical value of preoperative fractional exhaled nitric oxide (FENO) in distinguishing benign pulmonary nodules from malignant pulmonary nodules and characterizing its relationship with pathological features of non-small cell lung cancer (NSCLC).

Methods

After exclusions from 1,046 consecutive patients, 550 patients were included and classified into malignant (NSCLC) and benign groups based on postoperative pathological examination. Using the Mann-Whitney U test, FENO levels were compared between the two groups. Receiver operating characteristic (ROC) curve analysis was conducted with the optimal cutoff value determined based on the Youden index. Multivariate binary logistic regression analysis was conducted to assess the independent association of FENO with the malignant behavior of NSCLC. In the malignant group, FENO levels were compared across pathological types, tumor-node-metastasis (TNM) stages, and programmed death-ligand 1 (PD-L1) expression groups using the Mann-Whitney U test and the Kruskal-Wallis H test. Subsequently, multivariate linear regression analysis was conducted to explore the independent association between FENO and PD-L1 expression.

Results

Preoperative levels of FENO were significantly higher in the malignant group than in the benign group {median [interquartile range (IQR)]: 23 [18–29] vs. 16 [12–23] ppb, P<0.001}. The clinical/imaging-based model (including age, sex, smoking, and nodule size) achieved an area under the ROC curve (AUC) of 0.597 [95% confidence interval (CI): 0.537–0.657]. After incorporating FENO into the model, the AUC increased significantly to 0.716 (95% CI: 0.657–0.774), indicating that FENO provided incremental diagnostic value, with an optimal cutoff value of 19.5 ppb (sensitivity =70.9%, specificity =66.4%). After adjusting for confounders, FENO remained independently associated with malignancy [odds ratio (OR) =1.094; 95% CI: 1.060–1.128; P<0.001]. In the malignant group, FENO levels were significantly higher in squamous cell carcinoma than in adenocarcinoma (P<0.01). Furthermore, FENO levels differed significantly across PD-L1 expression groups (P<0.001), but not across TNM stages (P=0.52). Multivariate linear regression showed that PD-L1 high-expression group (≥50%) was independently associated with higher FENO levels compared with the negative group (B=0.211; 95% CI: 0.085–0.336; P=0.001), whereas the low-expression group (1–49%) showed no significant association (B=0.030; 95% CI: −0.028 to 0.231; P=0.11) and squamous histological subtype (B=0.290; 95% CI: 0.141–0.440; P<0.001) were independently associated with FENO levels.

Conclusions

Preoperative levels of FENO demonstrated moderate diagnostic value for malignant nodules. It also exhibited exploratory associations with the squamous histological subtype and PD-L1 expression. Prospective, multicenter validation is needed before clinical adoption.

Highlight box.

Key findings

• Preoperative fractional exhaled nitric oxide (FENO) levels were significantly higher in malignant nodules than in benign nodules [median (interquartile range): 23.0 (18.0–29.0) vs. 16.0 (12.0–23.0) ppb, P<0.001].

• FENO showed moderate diagnostic value for nodule differentiation, with an area under the receiver operating characteristic curve of 0.716 and an optimal cutoff of 19.5 ppb (sensitivity =70.9%, specificity =66.4%).

• FENO was independently associated with malignancy [odds ratio =1.094; 95% confidence interval (CI): 1.060–1.128; P<0.001].

• FENO levels were higher in squamous cell carcinoma than in adenocarcinoma (P<0.001) and independently associated with the programmed death-ligand 1 (PD-L1) high-expression group (≥50%) (B=0.211; 95% CI: 0.085–0.336; P=0.001).

What is known and what is new?

• FENO is a non-invasive biomarker of airway inflammation. Previous studies suggest elevated FENO in lung cancer, but its role in preoperative nodule differentiation and association with non-small cell lung cancer (NSCLC) pathological features and PD-L1 remain unclear.

• This is the first study to systematically evaluate preoperative FENO for both nodule differentiation and prediction of NSCLC pathological features, including PD-L1 expression. We identified a specific cutoff value (19.5 ppb) and demonstrated FENO’s incremental diagnostic value.

What is the implication, and what should change now?

• Preoperative FENO could serve as a simple, non-invasive adjunctive tool for pulmonary nodule risk stratification.

• The FENO-PD-L1 association suggests exploratory potential for immunotherapy guidance.

• Prospective, multicenter studies are warranted to validate these findings and establish standardized cutoff values before clinical application.

Introduction

Worldwide, lung cancer is the most common cancer in terms of incidence and mortality, with approximately 2.48 million new cases and 1.81 million deaths in 2022 (12.4% and 18.7%, respectively) based on the latest Global Cancer Observatory (GLOBOCAN) data from the International Agency for Research on Cancer (IARC) (1). Low-dose computed tomography (CT) screening has increased the pulmonary nodule detection rate, although most nodules are benign. Preoperative differentiation of benign nodules from malignant nodules, while obtaining reliable pathological data on non-small cell lung cancer (NSCLC), remains challenging. Postoperative pathology is the gold standard method for assessing tumor behavior, but this method is invasive, highlighting the need for non-invasive biomarkers for preoperative risk stratification and personalized treatment of NSCLC.

Fractional exhaled nitric oxide (FENO) is a well-established non-invasive biomarker for airway inflammation, which is commonly measured in patients with asthma and chronic cough. Nitric oxide (NO) dose-dependently affects lung cancer development, angiogenesis, and tumor spread. Low concentrations of NO promote tumor growth, while high concentrations of NO are cytotoxic, highlighting the role of FENO as a prognostic biomarker (2). Programmed death-ligand 1 (PD-L1) is the core biomarker guiding immunotherapy in NSCLC. Wang et al. (3) reported a PD-L1 positive rate of 46.9% in resectable NSCLC (low expression 1–49%: 41.8%; high expression ≥50%: 5%). Specifically, PD-L1 positivity was found to be associated with male sex, smoking, high positron emission tomography (PET)-CT maximum standardized uptake value (SUVmax), and elevated levels of carcinoembryonic antigen (CEA). Regarding FENO and immunotherapy, Suzuki et al. (4) found that anti-PD-1 therapy can significantly increase FENO levels and peripheral eosinophil counts in patients with NSCLC and concomitant chronic obstructive pulmonary disease (COPD), but not in those without COPD. These findings suggest a functional link between FENO and the PD-L1 pathway, underscoring the potential of FENO as a prognostic biomarker for treatment response in NSCLC. However, the value of FENO in differentiating benign nodules from malignant nodules, and its associations with the histological subtype of NSCLC, tumor-node-metastasis (TNM) stage, and PD-L1 expression, remain poorly defined. So far, no study has integrated these multidimensional analyses. Therefore, this study evaluated the clinical value of preoperative FENO in differentiating benign pulmonary nodules from malignant pulmonary nodules and characterizing pathological features and PD-L1 expression in NSCLC. We present this article in accordance with the STROBE and STARD reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-2191/rc).

Methods

Patients

Study population and data collection

This single-center retrospective study enrolled 1,046 consecutive patients who underwent surgical resection or biopsy for pulmonary nodules or masses at the Department of Thoracic Surgery, Tianjin Cancer Hospital. The participants were enrolled between April and December 2025 without intentional selection. Patients were classified into malignant and benign groups based on postoperative pathological examination. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Ethics Committee of Tianjin Cancer Hospital. Informed consent was obtained from all individual participants included in the study.

The inclusion criteria were as follows: (I) definitive pathological diagnosis via surgical resection, transbronchial biopsy, or percutaneous core-needle biopsy; (II) FENO measurement within 14 days preoperatively, with complete and traceable data; (III) PD-L1 testing in the malignant group; and (IV) complete clinical data, including age, sex, smoking history, and imaging and pathological examination reports.

The exclusion criteria were as follows: (I) previous or concurrent other malignancies; (II) active respiratory infection within 4 weeks before surgery; (III) receiving systemic or inhaled corticosteroids within 2 weeks; (IV) neoadjuvant therapy (chemotherapy, radiotherapy, targeted therapy, or immunotherapy); (V) incomplete clinical or imaging data; and (VI) comorbid asthma/COPD/allergic rhinitis (whether stable or exacerbated).

FENO measurement

In a quiet, awake state, all patients underwent FENO measurement within 14 days before surgery. Measurements were conducted using the NIOX respiratory analyzer [Scasia (Beijing) Medical Devices Co., Ltd., Beijing, China] strictly following the American Thoracic Society (ATS) guidelines. The participants were seated and exhaled at a constant flow rate of 50 mL/s through a disposable filter for 6–10 seconds. FENO values were automatically recorded and expressed in ppb.

Collection of clinical and pathological data

Clinical data, including age, sex, and smoking history (never-smoker, former smoker, or current smoker), were collected from the hospital electronic medical record system.

Imaging data included the maximum diameter of the nodule (cm) reported based on preoperative chest CT.

Pathological data

Regarding postoperative pathological diagnosis (malignant/benign), the histological subtype (adenocarcinoma/squamous cell carcinoma) was recorded for malignant cases.

TNM stage was classified following the 8^th edition of the American Joint Committee on Cancer (AJCC) staging system and coded as an ordinal categorical variable (e.g., stage 0 =0, stage I =1, stage II =2, stage III =3, and stage IV =4).

PD-L1 expression was assessed based on immunohistochemical (IHC) staining. The tumor proportion score (TPS) was recorded and categorized into three groups: negative (<1%) group, low-expression (1–49%) group, and high-expression (≥50%) group. 22C3 was uniformly used as the antibody clone for detection.

Concerning FENO data, the preoperative FENO value (ppb) was collected.

Statistical analysis

Statistical analyses were conducted using SPSS 26.0. Continuous variables were compared using the independent t-test or Mann-Whitney U test, and categorical variables were compared using the χ² test or Fisher’s exact test. Receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic performance of FENO, with the optimal cutoff determined based on the Youden index. Multivariate binary logistic regression was utilized to identify the independent predictors of the malignant phenotype. Within the malignant group, FENO levels across pathological types, TNM stages, and PD-L1 expression subgroups were compared using the Mann-Whitney U test or Kruskal-Wallis H test. To explore the independent associations between FENO levels and PD-L1 expression, log-transformed FENO was analyzed using multivariate linear regression analysis, which was adjusted for histological subtype, smoking, TNM stage, and nodule size. A two-tailed P<0.05 was deemed statistically significant.

Results

After excluding 496 patients, 550 patients were included in the final analysis (Figure 1). Some patients had more than one exclusion reason. The baseline characteristics of the benign and malignant groups are summarized in Table 1. Preoperative FENO levels were significantly higher in the malignant group than in the benign group (P<0.001, Table 1).

Figure 1.

Flowchart of the patient enrollment process. Some patients had more than one exclusion reason. COPD, chronic obstructive pulmonary disease; PD-L1, programmed death-ligand 1.

Table 1. Baseline characteristics of patients with benign and malignant pulmonary nodules.

Characteristics	Malignant (n=443)	Benign (n=107)	P value
Age (years)	62 [54–68]	60 [53–65]	0.06‡
Sex			0.41†
Male	204 (46.0)	54 (50.5)
Female	239 (54.0)	53 (49.5)
Smoking history			0.048†*
Never	189 (42.7)	57 (53.3)
Ever/current	254 (57.3)	50 (46.7)
Maximum nodule diameter (cm)	1.8 [1.8–2.3]	1.7 [1.1–2.0]	0.006‡**
FENO (ppb)	23 [18–29]	16 [12–23]	<0.001‡***

Data are presented as median [IQR] or n (%). ^†, P value calculated using the χ² test. ^‡, P value calculated using the Mann-Whitney U test. Significance codes: *, P<0.05; **, P<0.01; ***, P<0.001. FENO, fractional exhaled nitric oxide; IQR, interquartile range.

Using pathological diagnosis as the gold standard, ROC curve analysis was conducted to evaluate the diagnostic performance of FENO (Figure 2, Table 2). The clinical/imaging-based model comprising age, sex, smoking, and nodule size achieved an area under the ROC curve (AUC) of 0.597 [95% confidence interval (CI): 0.537–0.657]. After adding FENO to the model, the AUC increased significantly to 0.716 (95% CI: 0.657–0.774), suggesting that FENO significantly increased the diagnostic value. The optimal cutoff value was 19.5 ppb based on the Youden index, yielding a sensitivity of 70.9% and a specificity of 66.4% (P<0.001).

Figure 2.

ROC curves of the clinical/imaging-based model with and without FENO for distinguishing benign pulmonary nodules from malignant pulmonary nodules. FENO, fractional exhaled nitric oxide; ROC, receiver operating characteristic.

Table 2. Comparison of diagnostic performance between models with and without FENO.

Model	AUC	95% CI	P value
Clinical/imaging-based model	0.597	0.537–0.657	0.002**
Clinical/imaging-based model + FENO	0.716	0.657–0.774	<0.001***

Significance codes: **, P<0.01; ***, P<0.001. AUC, area under the ROC curve; CI, confidence interval; FENO, fractional exhaled nitric oxide; ROC, receiver operating characteristic.

Using a cutoff value of FENO ≥19.5 ppb as the threshold for a positive test, sensitivity was 70.9% (95% CI: 66.5–75.1%), specificity was 66.4% (95% CI: 56.8–74.9%), positive predictive value was 89.7% (95% CI: 86.5–92.9%), negative predictive value was 35.5% (95% CI: 28.9–42.1%), positive likelihood ratio was 2.11 (95% CI: 1.59–2.63), negative likelihood ratio was 0.44 (95% CI: 0.36–0.52), and overall diagnostic accuracy was 70.0% (95% CI: 66.2–73.8%).

Using the Enter method, variables with P<0.10 in univariate analysis, including smoking history (P=0.049), maximum nodule diameter (P=0.02), and FENO (P<0.001), were entered into a multivariate binary logistic regression model. After adjusting for smoking history and nodule diameter, FENO was identified as an independent predictor of malignancy [odds ratio (OR) =1.094; 95% CI: 1.060–1.128; P<0.001] (Table 3).

Table 3. Univariate and multivariate logistic regression analysis of factors associated with malignant pulmonary nodules.

Variables	Univariate analysis		Multivariate analysis
	OR (95% CI)	P value	OR (95% CI)	P value
Age (per 1 year)	1.010 (0.992–1.029)	0.28	–†	–
Sex (male vs. female)	0.838 (0.549–1.278)	0.83	–†	–
Smoking history (ever vs. never)	1.532 (1.003–2.341)	0.049	1.379 (0.882–2.154)	0.15
Maximum nodule diameter (per 1 cm)	1.397 (1.048–1.861)	0.02	1.314 (1.262–1.795)	0.046*
FENO (per 1 ppb)	1.097 (1.063–1.131)	<0.001	1.094 (1.060–1.128)	<0.001***

^†, not included in the final multivariate model (P>0.10 in univariate analysis). Significance codes: *, P<0.05; ***, P<0.001. CI, confidence interval; FENO, fractional exhaled nitric oxide; OR, odds ratio.

FENO levels were significantly higher in squamous cell carcinoma than in adenocarcinoma (P<0.01). No significant difference was observed across TNM stages, based on the 359 patients with complete staging data (P=0.52) (Table 4).

Table 4. Association of FENO with pathological features in the malignant group.

Pathological features	N (%)	FENO (ppb), median [IQR]	P value
Pathological type			<0.01 † **
Adenocarcinoma	373 (84.2)	22 [17–29]
Squamous cell carcinoma	70 (15.8)	26 [22–29]
TNM staging			0.52‡
0	3	16 [16–23]
I	293	23 [17–28]
II	40	21 [15.5–29.5]
III	20	20 [15.5–29.5]
IV	3	23 [23–87.5]
PD-L1 expression
Negative (<1%)	196	21 [16–27]	<0.001‡***
Low (1–49%)	159	24 [19–30]
High (≥50%)	88	26 [21–32]

^†, P value was calculated using the Mann-Whitney U test. ^‡, P values were calculated using the Kruskal-Wallis H test. Significance codes: **, P<0.01; ***, P<0.001. FENO, fractional exhaled nitric oxide; IQR, interquartile range; PD-L1, programmed death-ligand 1; TNM, tumor-node-metastasis.

Among patients with NSCLC and available PD-L1 data (n=443), FENO levels differed significantly across PD-L1 expression groups (P<0.001), with the high-expression group showing the highest FENO levels (Table 4). Multivariate linear regression analysis confirmed that, unlike smoking history, TNM stage, and nodule size, PD-L1 expression and squamous histological subtype were independently associated with log-transformed FENO levels (both P<0.001) (Table 5).

Table 5. Multivariate linear regression analysis of the factors associated with log-transformed FENO levels in patients with NSCLC.

Variables	B†	95% CI	P value
Histological subtype (squamous vs. adenocarcinoma)	0.290	0.141 to 0.440	<0.001***
PD-L1 expression group (ref.: negative)‡
Low expression (1–49%)	0.030	−0.028 to 0.231	0.11
High expression (≥50%)	0.211	0.085 to 0.336	0.001***
Smoking history (yes vs. no)	−0.009	−0.099 to 0.081	0.85
TNM (per stage increase)	−0.016	−0.093 to 0.061	0.67
Nodule size (per cm)	−0.007	−0.065 to 0.051	0.80

^†, dependent variable: lnFENO. ^‡, PD-L1 expression was entered as a categorical variable using dummy coding, with the negative group (<1%) as the reference category. Significance codes: ***, P<0.001. CI, confidence interval; FENO, fractional exhaled nitric oxide; lnFENO, log-transformed FENO; NSCLC, non-small cell lung cancer; PD-L1, programmed death-ligand 1; TNM, tumor-node-metastasis.

Discussion

NO is a gaseous free radical produced from L-arginine via the catalytic function of NO synthase (NOS). FENO measurement was first reported in 1991 (5,6). FENO possesses established clinical value in the diagnosis and management of chronic respiratory diseases, including asthma, chronic cough, and COPD. The 2011 ATS guidelines proposed FENO as a biomarker of airway eosinophilic inflammation (6). Studies have shown that through chemical modifications, NO is involved in various pathophysiological processes, including inflammation, tumor immunity, and apoptosis, and is associated with the pathogenesis and progression of lung cancer (2). Therefore, FENO holds promise as a potential predictive biomarker for lung cancer.

Using ultraviolet differential optical absorption spectroscopy, Li et al. (7) measured FENO levels in patients with various pulmonary diseases. They observed that compared with control subjects, FENO levels significantly increased in those with malignant tumors (P<0.01), which decreased postoperatively. This observation points to a direct relationship between FENO levels and tumor progression, rather than simply reflecting the underlying airway inflammation. A more recent prospective cohort study with 150 participants (8) reached a similar conclusion. The study indicated that elevated levels of FENO are independently associated with an increased risk of lung cancer, with male sex, older age, and lower BMI also emerging as positive risk factors. A meta-analysis of 50 studies, including 2,551 patients with lung cancer and 1,691 control subjects, confirmed that FENO levels are significantly elevated in patients with lung cancer compared to controls [standardized mean difference (SMD) =3.01; 95% CI: 1.89–4.13; P<0.00001]. The meta-analysis revealed that FENO may contribute to both diagnosis and treatment monitoring, especially in NSCLC, although no cutoff value was proposed (9). Our study addresses this gap by identifying an optimal cutoff of 19.5 ppb, a threshold that can be readily applied in clinical practice. Liu et al. (10) enrolled 172 healthy controls and 164 patients with confirmed lung cancer. They measured FENO online at a flow rate of 50 mL/s and reported higher FENO levels in the cancer group (33.94±15.6 vs. 16.84±4.2 ppb). In our cohort, we also found significantly elevated FENO levels in the malignant group compared with the benign group {median [interquartile range (IQR)]: 23 [18–29] vs. 16 [12–23] ppb, P<0.001}. Taken together, these findings consistently support the role of FENO in lung cancer risk stratification and auxiliary diagnosis. In our study, the clinical/imaging-based model, including age, sex, smoking, and nodule size, achieved an AUC of 0.597 (95% CI: 0.537–0.657). After adding FENO to the model, the AUC increased significantly to 0.716 (95% CI: 0.657–0.774), indicating that FENO significantly increased the diagnostic value. Although this level of accuracy may not be sufficient for standalone use, the non-invasive and point-of-care nature of FENO makes it a practical adjunctive tool, particularly when CT findings are equivocal. Liu et al. (10) described a markedly higher AUC (0.925), which seems to underscore the diagnostic promise of FENO. The modest performance in the present study could be explained by the relatively large number of inflammatory lesions in our benign group and the predominance of early-stage lung adenocarcinomas. Both of these factors likely affected the observed discriminative capacity of FENO.

In multivariate logistic regression analysis adjusted for smoking history and nodule size, FENO remained an independent predictor of malignant pulmonary nodules (OR =1.094; 95% CI: 1.060–1.128; P<0.001). This finding is in line with previous findings on the predictive role of FENO in lung cancer. For instance, Okamoto et al. (11) reported in a prospective study of 105 patients undergoing lung cancer surgery that preoperative FENO independently predicted postoperative pulmonary complications (OR =1.004; 95% CI: 1.016–1.074; P=0.002). Similarly, analyzing data from 292 patients with early-stage NSCLC, Lin et al. (12) found that preoperative FENO is an independent risk factor for postoperative cough (OR =1.106; 95% CI: 1.076–1.137; P<0.001). Liu et al. (13) (n=183) indicated that elevated postoperative levels of FENO (>25 ppb) and postoperative increase in FENO levels (>4 ppb) can help detect postoperative pneumonia in patients with surgically treated lung cancer. These findings suggest that perioperative FENO can serve as an indicator for postoperative pneumonia. Although the outcome measures of the above- mentioned studies differed from those of the present study, they consistently confirmed the independent predictive value of FENO. Proinflammatory cytokines and inducible NOS (iNOS) activation can promote NO production in the tumor microenvironment (14). Tumor immunity and metabolic processes upregulate iNOS activity, thereby promoting NO production by alveolar macrophages (15). Studies have confirmed elevated levels of NO, nitrite, and nitrotyrosine in lung cancer (16), suggesting that chronic airway inflammation promotes the production of reactive oxygen species and NO through inflammatory cell infiltration and activation. Pianigiani et al. (2) noted that NO exerts bidirectional regulatory effects on tumor angiogenesis and immune evasion, suggesting that FENO may indirectly reflect certain aspects of the tumor microenvironment. The T helper type 2 (Th2)-type airway inflammation reflected by FENO is closely associated with the development of lung cancer. Chronic inflammation can induce DNA damage, promote cell proliferation, and facilitate angiogenesis, establishing conditions conducive to tumorigenesis. Elevated levels of FENO may reflect persistent inflammation of the lungs, which is a key driver of malignant transformation. Furthermore, the Th2-dominant immune environment, reflected by elevated levels of FENO, may synergistically interact with the programmed cell death protein 1 (PD-1)/PD-L1 immune checkpoint pathway. Basic studies have shown that Th2-type cytokines, such as interleukin (IL)-4 and IL-13, can upregulate PD-L1 expression in cancer cells via the PI3K-AKT-p65 pathway, forming an immunosuppressive microenvironment. Consistently, the present study identified an association between FENO and PD-L1 expression. Although the OR value for FENO was not as prominent as nodule size, its incremental value as a continuous variable remained stable. Future studies may explore the potential value of dynamic FENO monitoring in predicting postoperative complications or response to immunotherapy.

In the present study, FENO levels were significantly higher in patients with squamous cell carcinoma than in those with adenocarcinoma {median [IQR]: 22 [17–29] vs. 26 [22–29], P<0.01}. This finding is highly consistent with the results of Okamoto et al., who conducted IHC staining on 101 patients with NSCLC and found that iNOS expression in tumor cells exhibits a characteristic tendency toward squamous cell carcinoma (P<0.01). Specifically, they found that FENO levels were significantly higher in patients with iNOS-positive squamous cell carcinoma than in patients with iNOS-negative squamous cell carcinoma (15). Similarly, Liu et al. (10) compared FENO levels across four pathological types, including squamous cell carcinoma, adenocarcinoma, small cell lung cancer (SCLC), and pulmonary carcinoid. They found higher levels of FENO in squamous cell carcinoma than in adenocarcinoma. From a pathophysiological perspective, iNOS overexpression in squamous cell carcinoma cells may enhance the local production of NO, which is consistent with the results of exhaled breath measurements. Thus, FENO may non-invasively capture these airway changes associated with the tumor. This feature gives FENO a unique advantage in the topographic diagnosis of squamous cell carcinoma and may serve as an adjunctive marker for distinguishing squamous carcinoma from non-squamous carcinoma. However, Li et al. (17) did not observe significant differences in FENO levels among patients with different pathological types of lung cancer. Similarly, the blood levels of NO did not differ significantly among patients with different pathological types of NSCLC (17). These discrepancies across studies may be attributable to heterogeneity in the pathological composition of lung cancer cases.

FENO levels were higher in patients with advanced TNM stage (stage IV), although the differences among stages did not reach the threshold of statistical significance (P=0.52). Of note, this analysis included only 359 of the 443 malignant patients because TNM data were missing for 84 cases (19.0%) due to missing pathological T, N, or M descriptors in the original medical records. The small number of stage 0 cases (n=3) and the marked imbalance in sample sizes across stages (stage I: n=293; stage IV: n=20) further precluded any a reliable assessment of trends and reduced the statistical power to detect modest differences. Therefore, our TNM-related findings should be interpreted with caution, and prospective studies with standardized reporting are warranted to confirm this observation.

The present study found that FENO levels in the PD-L1 high-expression group (≥50%) were significantly higher than those in the PD-L1-negative group. The results also revealed a dose-response trend, suggesting a potential association between FENO and the tumor immune microenvironment. Suzuki et al. reported that anti-PD-1 therapy (nivolumab) can significantly increase FENO levels in patients with NSCLC (4), suggesting that dynamic changes in FENO may reflect the activation status of the PD-1/PD-L1 axis. Interferon-γ (IFN-γ) released by T-cells may be involved in the observed effects, which in turn upregulate iNOS expression in airway epithelium, highlighting a functional link between FENO and the PD-L1 pathway. Giatromanolaki et al. (18) examined 98 NSCLC tissue specimens and found extensive infiltration of iNOS-positive tumor-infiltrating lymphocytes (iNOS⁺ TILs) in approximately 50% of cases. Furthermore, iNOS⁺ TIL infiltration was significantly associated with high PD-L1 expression and better overall survival (P=0.002), indicating that iNOS activation coexists with PD-L1 expression in the tumor microenvironment. Therefore, the increase in FENO levels with PD-L1 overexpression may reflect the activation status of iNOS⁺ TILs, with FENO potentially serving as an indirect and non-invasive indicator of this immune feature. FENO is a biomarker of Th2-type airway inflammation, and Th2-type immune responses can further deepen immunosuppression synergistically with the PD-1/PD-L1 axis. Combining FENO with PD-L1 may enable a more comprehensive assessment of the biological characteristics of lung cancer from the dual dimensions of inflammation and immunity, which represents a promising research direction. However, to date, the direct clinical association between FENO levels and PD-L1 expression in the tumor tissues of patients with NSCLC has not been systematically evaluated. An ongoing clinical trial, the FENOTYPE study (NCT05985330) (19), is investigating whether FENO can serve as a biomarker for predicting response to anti-PD-L1 immunotherapy. The results of this trial will provide prospective evidence for the clinical application of FENO in immunotherapy. In the present study, patients with lung cancer and elevated preoperative levels of FENO had higher expression levels of PD-L1 {median [IQR]: 21 [16–27] vs. 24 [19–30] vs. 26 [21–32], P<0.001}. In the multivariate linear regression analysis, compared with the PD-L1 negative group, the high-expression group (≥50%) was independently associated with significantly higher FENO levels (B=0.211; 95% CI: 0.085–0.336; P=0.001), whereas the low-expression group (1–49%) showed no significant association after adjustment for histological subtype, smoking, TNM stage, and nodule size (B=0.030; 95% CI: −0.028 to 0.231; P=0.11). These findings suggest an independent association between FENO and the PD-L1 pathway, particularly at higher levels of PD-L1 expression. Future studies should investigate the potential of dynamic FENO monitoring for predicting postoperative complications or responses to immunotherapy. Furthermore, mechanistic studies are needed to clarify the specific molecular mechanisms through which the iNOS/NO signaling pathway contributes to the development of squamous cell carcinoma and to investigate its crosstalk with immune checkpoint pathways.

Several limitations of this study should be acknowledged. First, the retrospective, single-center design and the high proportion of malignant cases (80.5%) may have introduced selection and spectrum bias, thereby limiting the generalizability of the findings. Second, the inclusion of inflammatory lesions in the benign group may have reduced the diagnostic performance of FENO. Third, FENO was measured at only a single preoperative time point, preventing the assessment of dynamic changes. Fourth, TNM stage data were missing for 84 of 443 NSCLC patients (19.0%), mainly due to incomplete pathological documentation of nodal status, which may have introduced bias into this exploratory analysis. Therefore, these findings should be considered exploratory and validated in prospective, multicenter studies.

Conclusions

Preoperative FENO offers moderate diagnostic value for malignant nodules and is associated with the squamous histological subtype and PD-L1 expression in NSCLC. These findings are preliminary and require prospective, multicenter validation before clinical application.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Ethics Committee of Tianjin Cancer Hospital. Informed consent was obtained from all individual participants included in the study.

Data Sharing Statement

Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-2191/dss

jtd-18-9-1012-dss.pdf ^{(117.7KB, pdf)}

DOI: 10.21037/jtd-2026-2191