The present study is part of a prospective multicenter study, which aims to establish a biobank (“BALOTHEK”) using blood serum and BALF for the research of various lung diseases. The samples were acquired from patients in whom BAL was performed for purpose of routine clinical evaluation (BASEC-ID 2017–02307 and 2018 − 01724). Enrolled patients were retrospectively clustered in five groups according to clinical and radiological presentation, confirmed by histology: lung cancer, sarcoidosis, ILD, drug-related pneumonitis and chronic cough. Patients in the latter group served as healthy controls, when there were no pathological findings in the chest CT during a follow-up time of six months. Patients were excluded in case of precedent lung transplantation, general patient vulnerability such as emergencies or pregnancies and errors in sampling or processing of the samples, e.g. BAL to processing time exceeding 60 minutes .
From January 2018 until June 2019, a total of 401 adult patients were enrolled in BALOTHEK. Simultaneously, 240 patients were treated with ICIs at the Departments of Dermatology and Medical Oncology from University Hospital Zurich because of various malignancies. Of these 240 patients, 16 developed typical symptoms (i.e. cough, fever, dyspnea) and CT findings (i.e. COP, NSIP, HP, AIP) suggestive for ICI-associated pneumonitis. After conducting BAL however, in four patients an alternative diagnosis other than ICI-associated pneumonitis had been made (one patient with Melphalan-induced pulmonary toxicity, two patients with acute bronchitis and one patient with chronic cough of unknown origin). Thus, the remaining 12 patients were eventually included with the diagnosis of ICI-associated pneumonitis confirmed by BAL. From these patients BALF could be harvested for purpose of BALOTHEK and for the present study, respectively. In addition, 12 subjects from the control group of BALOTHEK, matched according to gender and age (range +/- five years) were used as healthy controls.
This parallel cohort study was approved by the local ethic review committee (BASEC-ID 2017–02307 and 2018 − 01724).
Blood specimens and processing
All blood samples were collected by nurses proficient in blood drawing as part of the routinely performed pre-interventional peripheral venous access. For differential blood count and whole blood count 10 ml BD Vacutainer K2E tubes (EDTA, Plus Blood Collection Tubes, Becton Dickinson, Plymouth, UK) were used. To gain serum samples, whole blood was collected in 10 ml BD Vacutainer Clot Activator Tube (CAT, Plus Blood Collection Tubes, Becton Dickinson, Plymouth, UK) and centrifuged at 3500 rounds per minute (rpm) at room temperature. Thereafter, the supernatant was aliquoted and eventually stored at − 80 °C for later analyses, according to Valaperti et al. . Once thawed for analysis, the samples were not frozen again.
Bronchoscopy, BAL and processing of BALF
Bronchoscopy was performed in moderate sedation with propofol using Olympus (Tokyo, Japan) flexible bronchoscopes (190 series). BALF was obtained conforming to official recommendations [23, 24] by instillation of isotonic saline solution in four times 50 ml portions into the wedged pulmonary segment that showed the most prominent finding in the most recent chest CT. Through gentle suction of the same syringe that injected the solution, BALF was yielded and filled in designated tubes, absent any further substances such as anticoagulants and preservatives. The recovered BALF was quantitatively expressed in absolute values (ml) and in percent of the instilled volume. BALF was routinely used for cytological and microbiological analyses, whereas the rest served the purpose of this study. For processing, BALF was centrifuged at 1’000 rpm at room temperature, the supernatant was aliquoted and stored at -80 °C in accordance to Valaperti et al. . Once thawed for analysis, the samples were not frozen again. 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.
A Milliplex MAP kit (human high sensitivity T cell magnetic bead panel) customized by Merk Millipore (Darmstadt, Germany) was used to analyze cytokines applying MAGPIX system (Luminex Corporation, Austin, TX, USA). The array contained the following 11 cytokines: interferone-gamma (IFN-gamma), interleukin (IL)-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-12p70, IL-13, IL-17, and tumor necrosis factor alpha (TNF-alpha). This selection of cytokines based on several publications [25–27, 12, 28] investigating inflammatory biomarkers in drug-induced pneumonitis as well as specifically ICI-associated pneumonitis. The preparation of standards was composed of serial dilution 1:4 of each stock standard to generate seven standard concentrations which were used to create a five-parameter logistic curve-fit standard curve with the xPONENT software (Luminex Corporation, Austin, TX, USA). Before quantifying cytokines, the high sensitivity bead panel was successfully validated and calibrated, showing a correct standard curve for each cytokine. Cytokines were determined in BALF as well as in serum.
Continuous data are reported as median ± interquartile range (IQR) or as mean ± standard deviation (SD), as appropriate. Normal distribution was tested using the Shapiro-Wilk test. To express comparisons between groups, Chi-squared test or Fisher’s exact test was used for categorical variables and the Mann-Whitney U test or independent t-test for continuous variables. The Bonferroni correction was used adjust p-values for multiple comparisons to avoid the risk of a type I error. P values of less than 0.05 were considered to be statistically significant and were based on two-sided hypothesis. All analyses were conducted using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corporation, Armonk, NY, USA) and R Core Team, 2013; R version 4.0.3 (2020-10-10).