Patients
This study is part of a prospective multicenter study aimed to establish a biobank (“BALOTHEK”) containing BALF and blood serum for the investigation of various lung diseases in patients in whom BAL was indicated as part of their routine clinical evaluation. Enrolled patients were retrospectively grouped according to their final diagnosis as confirmed by histology and finally allocated into four groups describing different lung diseases: lung cancer, sarcoidosis, interstitial lung disease (ILD), and drug-related pneumonitis. A fifth group consisted of patients who underwent bronchoscopy for assessment of chronic cough with a normal chest computed tomography (CT) finding (i.e., absence of consolidation, ground-glass opacity, nodule, mass, or interstitial changes) and without evidence of lung disease during a follow-up time of six months. Thus, the latter group served as healthy control group with structural normal lung parenchyma. Exclusion criteria for enrollment in BALOTHEK were history of lung transplantation, patient vulnerability such as pregnancy or emergency setting, as well as possible sampling and processing failures, such as a low BALF recovery rate resulting in less than 10 ml of BALF for the purpose of the biobank or a BAL-to-processing time exceeding 1 h as described elsewhere [24].
Between January 2018 and June 2019, a total of 401 adult patients were prospectively enrolled in the BALOTHEK. After exclusion of patients with conditions possibly influencing the cytokine levels (infection, systemic immunosuppressants including systemic corticosteroids, and alveolar hemorrhage), uncertain diagnosis or malignancy other than lung cancer, and sampling/processing failures, 119 patients were included for the purpose of this study (Fig. 1).
All patient-related data including demographic and clinical data as well as bronchoscopy, radiology, and pathology reports were drawn from patient record files.
Bronchoscopy and BAL technique
All patients underwent FB using Olympus (Olympus, Tokyo, Japan) bronchoscopes (190 series) under moderate sedation using propofol or general anesthesia according to the clinician’s decision. BAL was performed following the standardized procedure described by Baughman by injecting four portions of 50 ml (200 ml in total) of isotonic normal saline into the wedged segmental bronchus leading to the target lesion with the most prominent radiological finding [25]. BALF was recovered by gentle suction with the same syringe and collected in a graduated plastic cylinder. Approximately 50 ml of BALF was reserved for routine clinical analyses; excess fluid was used for the purpose of BALOTHEK.
Processing of BAL fluid and blood specimens
BALF was collected in plastic tubes and centrifuged at 1′000 rounds per minute for 10 min at room temperature. The supernatant was collected and stored at − 80 °C for later analyses. The aliquots were only thawed once for analysis to prevent falsification of cytokine levels by repeated freezing and thawing. The routinely performed analysis of BALF for cell differentiation was performed by ADVIA 2120i (Siemens Healthcare AG, Zurich, Switzerland) via peroxidase staining. Cell differentiation included cell count, macrophages, lymphocytes, neutrophils, eosinophils, mast cells, and plasma cells. Blood samples were drawn as part of the routinely performed pre-interventional peripheral vein access. Whole blood was collected in a 10-ml BD Vacutainer Clot Activator Tube (CAT, Plus Blood Collection Tubes, Becton Dickinson, Plymouth, UK) and centrifuged at 3′500 rounds per minute for 10 min at room temperature. The subsequent process was analogous to the handling of BALF.
Selection of cytokines
We specifically selected cytokines which were verified in previous studies as factors in TME of lung cancer, serving as rationale for their use in this study. Fractalkine is a prohibiting factor for metastasis and has a particularly high expression in the lungs [26]. Granulocyte–macrophage colony-stimulating factor (GM-CSF) can act pro-inflammatory as well as anti-inflammatory depending on the concentration and its environment [27, 28]. When secreted by tumor cells themselves, GM-CSF may lead to immune evasion for the tumor by promoting the development of myeloid suppressor cells [29]. Interferon gamma (IFN-γ) has been shown to be downregulated in progressive tumor disease as a sign for lower expression by natural killer cells [30]. Tumor necrosis factor alpha (TNF-α) is a marker for alveolar macrophage activity and plays a role in inhibiting carcinogenesis as well as angiogenesis [31]. Notably, it has been shown to be increased in exhaled breath condensate in patients with NSCLC in a previous study [32]. Interleukin (IL) 1b and IL-6 also act as markers for alveolar macrophage activity, and have been shown to be increased in BALF of lung cancer patients, with increase of IL-1b acting as a positive prognostic factor and increase of IL-6 acting as a negative prognostic factor for survival in lung cancer [33,34,35]. IL-2 has a strong effect on activation of natural killer cells [36]. Furthermore, it has been shown to be increased in exhaled breath condensate in patients with NSCLC [32]. IL-4 and IL-13 have been shown to have higher histopathological expressions of NSCLC in a previous study [37]. IL-5 had been shown to have an augmenting role in metastasis in lung cancer in mice models [38]. IL-7 has been shown to have an anti-apoptotic and thus pro-carcinogenic effect on lung cancer cells [39]. IL-8 is a chemoattractant for various immune cells as well as an inducer of angiogenesis [4]. Elevated levels of IL-8 have been shown to predict decreased survival in lung cancer [35]. Furthermore, rapid decrease of IL-8 levels during anti-PD-1 treatment correlated with treatment success [40]. IL-10 has an immunosuppressive effect and has been shown to be expressed by alveolar macrophages in TME of primary lung cancer. Increased levels of IL-10 positively correlated with tumor progression (size, metastasis, and poor histologic differentiation) [41]. IL-12 and in its activated form IL-12p70 are activators of natural killer cells as well as alveolar macrophages [4]. In mice models, knock-out of IL-12 induced spontaneous development of lung carcinomas [42]. Additionally, IL-12 and IFN-γ act as necessary mediators of anti-PD-1 treatment [43]. IL-13 is a marker of natural killer cell activity and been shown to be associated with progression and metastasis of lung cancer [44, 45]. IL-17 is a promotor of angiogenesis and cell proliferation as well as an inhibitor of apoptosis. As such, IL-17 has been shown to have a correlation with tumor progression and metastasis of lung cancer [46]. IL-23 suppresses the activity of B cells, T cells, and natural killer cells and thus promotes tumor progression and metastasis in lung cancer [30, 47].
Cytokine analysis
The cytokine analysis was conducted using a cytokine multiple array on a Luminex 200 platform (Luminex Corporation, Austin, TX, USA) with a high sensitivity Milliplex kit (HSTCMAG-28SK-10, Merck Millipore, Darmstadt, Germany). The array included the following cytokines: fractalkine, granulocyte–macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and several interleukins (IL): IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, and IL-23. The acceptance criteria included the % coefficient of variation of the intra-assay, inter-assay, and replicates of low concentrated BALF, as well as the lower limit of detection, the lower limit of quantification, and the linearity.
Statistical analysis
Levels of each cytokine of the case group (lung cancer) were analyzed against two control groups consisting of healthy individuals and patients with other lung diseases (sarcoidosis and ILD).
All statistical analyses were performed using SPSS Statistics for Windows 22.0 (IBM, Armonk, NY, USA). Normal distribution was assessed by the Kolmogorov–Smirnoff test. As all data were not normally distributed, data are reported as median ± interquartile range (IQR) or as percentages, as appropriate. Test for likeness was conducted by Pearson Correlation. Differences of means between the cohorts were calculated by Mann–Whitney U Test. Variables with p value < 0.1 were entered into a multivariate regression model. Minimum power level was set at 0.8. [48] The confidence interval (CI) was defined as 95%. p values of all outcomes were two-sided; a value less than 0.05 was considered statistically significant.
As the complete array of cytokines was analyzed in every patient, measurements of cytokine concentration with a median of 0.00 do neither imply an analytical error in cytokine measurement nor a reduction of patient samples but rather a cytokine concentration below the measurable detection limit in vivo, which in itself was considered an important finding. Consequently, we included all results in the statistical analysis to show the complete set of data in our study.