Evaluation of cytokines in the tumor microenvironment of lung cancer using bronchoalveolar lavage fluid analysis.

INTRODUCTION: Lung cancer is the leading cause of death by cancer. In recent years, immunotherapy with checkpoint inhibitors (ICI) emerged as a promising new therapeutic approach. However, a deeper understanding of the immunologic responses adjacent to the tumor known as tumor microenvironment (TME) is needed. Our study investigated TME of lung cancer by analyzing cytokines in bronchoalveolar lavage fluid (BALF). MATERIALS AND METHODS: Between January 2018 and June 2019, 119 patients were prospectively enrolled in this study. For each cancer patient, levels of 16 cytokines (fractalkine, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and 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) were measured in BALF and serum and compared to healthy individuals and patients with other lung diseases. RESULTS: There were several significant differences of cytokine levels of patients with lung cancer compared to healthy individuals. However, none of them remained in the multivariate analysis compared to other lung diseases in either BALF or serum. Furthermore, there were no significant differences between the groups in cell differentiation of either BALF or serum. Cytokine levels in BALF were generally near the lower detection limit and showed almost no correlation with their respective levels measured in serum of the same individual. CONCLUSIONS: Cytokines in BALF and serum of lung cancer patients may indicate unspecific inflammation. BAL is not recommendable as a tool to investigate TME of lung cancer. Therefore, cytokines measured in BALF are probably not appropriate as predictors in patients treated with ICIs.

Variability of cytokine concentration in whole blood serum and bronchoalveolar lavage over time.

Measurement of cytokines in peripheral blood and bronchoalveolar lavage fluid (BALF) is a useful method to assess human immune responses in a large range of pulmonary diseases. One of the major pre-analytical challenges of cytokine analysis is the quality and stability of cytokines in the timeframe between sample collection and the separation of supernatant from cells. To evaluate if the method of storage may affect cytokine quantification, whole blood and BALF were collected, aliquoted, and left at room temperature (RT) to be processed at different time points. In addition, sera and BALF were left either at RT or at 4 °C for 24 h after cell separation to test cytokine variations in the absence of cells. Samples were analysed by a multiple array containing ten cytokines. Most of the cytokines analysed (interleukin (IL)-4, IL-5, IL-6, IL-12p70, IL-13, IL-17A, IL-23, interferon (IFN)-γ, and tumour necrosis factor (TNF)-α) did not show significant variations in whole blood and BALF. Levels of IL-8 however, increased after storage of whole blood and BALF for 24 h at RT. Ex vivo IL-8 production seems to correlate with higher numbers of macrophages in collected BALF. These data demonstrate that many cytokines are stable for a brief time after sample collection. For IL-8, freshly collected whole blood and BALF should be quickly processed and frozen to avoid false positive results.

Early Spirometry Following Bronchoscopic Lung Volume Reduction with Endobronchial Valves.

Bronchoscopic lung volume reduction (BLVR) by endobronchial valve (EBV) implantation has been shown to improve dyspnea, pulmonary function, exercise capacity, and quality of life in highly selected patients with severe emphysema and hyperinflation. The most frequent adverse event is a pneumothorax (PTX), occurring in approximately one-fifth of the cases due to intrathoracic volume shifts. The majority of these incidents are observed within 48 h post-procedure. However, the delayed occurrence of PTX after hospital discharge is a matter of concern. There is currently no approved concept for its prevention. Particularly, it is unknown whether and when respiratory manoeuvers such as spirometry post EBV treatment are feasible and safe. As per standard operating procedure at the University Hospital Zurich, early spirometry is scheduled after BLVR and prior to the discharge of the patient in order to monitor treatment success. The aim of our retrospective study was to investigate the feasibility and safety of early spirometry. In addition, we hypothesized that early spirometry could be useful to identify patients at risk for late PTX, which may occur after hospital discharge. All patients who underwent BLVR using EBVs between January 2018 and January 2020 at our hospital were enrolled in this study. After excluding 16 patients diagnosed post-procedure with PTX and four patients for other reasons, early spirometry was performed in 61 cases. There was neither a clinically relevant PTX during or after early spirometry nor a late PTX following hospital discharge. In conclusion, we found early spirometry, conducted not sooner than three days following EBV treatment, to be feasible and safe. Furthermore, early spirometry seems to be a useful predictor for successful BLVR, and it may help to decide whether a patient can be discharged. Given the small sample size and the retrospective design of our study, a prospective study that includes routine chest imaging after early spirometry to definitively exclude PTX is needed to recommend early spirometry as part of the standard protocol following EBV treatment.

Analysis of cytokines in serum and bronchoalveolar lavage fluid in patients with immune-checkpoint inhibitor-associated pneumonitis: a cross-sectional case-control study.

PURPOSE: Immune-checkpoint inhibitors (ICI) present a new treatment for malignancies by boosting the immune system. This has led to a variety of immune-related adverse events, including ICI-associated pneumonitis (ICIaP). Diagnosis thereof is often challenging, and its pathogenesis has not yet been fully understood. The aim of this cross-sectional case-control study was to investigate cytokines in serum and bronchoalveolar lavage fluid (BALF) expressed in patients with ICIaP compared to controls consisting of healthy individuals, patients with lung cancer and patients with interstitial lung diseases (ILD) other than ICIaP. METHODS: From January 2018 until June 2019, 401 adult patients with various lung diseases were prospectively enrolled in a BALF- and serum biobank, called BALOTHEK. Of these, 12 patients were diagnosed with ICIaP (Pembrolizumab, Ipilimumab, or both, and Durvalumab) serving as case group. Subjects with one of three diagnosis groups from BALOTHEK, including lung cancer, ILD other than ICIaP, and healthy individuals, served as matched controls. The following 11 cytokines were simultaneously analyzed in BALF and serum of each study participant: interferon gamma, tumor necrosis factor alpha, interleukin (IL) 1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-12p70, IL-13 and IL-17A. This study was approved by the local ethic review committee (BASEC-ID 2017-02,307 and 2018-01,724). RESULTS: Absolute number and percentage of lymphocytes in BALF of patients with ICIaP were significantly higher compared to control groups. For the investigated cytokines in BALF, a significant increase of IL-6 level was shown for patients with ICIaP compared to control groups (p = 0.031, adjusted for multiple comparisons). CONCLUSION: Cytokine profile assessed in BALF shows promising potential for facilitating diagnosis and understanding of pathophysiology of ICIaP. IL-6 may not only contribute to better understanding of pathophysiology but also herald therapeutic implications for Tocilizumab.

Diagnostic Value of Bronchoalveolar Lavage for Diagnosis of Suspected Peripheral Lung Cancer.

BACKGROUND: There is a paucity of data concerning the benefit of bronchoalveolar lavage (BAL) for the diagnosis of suspected peripheral lung cancer (PLC). The aim of this study was to investigate the diagnostic value of BAL for the diagnosis of suspected PLC. PATIENTS AND METHODS: All flexible bronchoscopies that included BAL among other modalities (brush, forceps, washing) for the diagnosis of a suspected PLC performed between 2009 and 2013 were analyzed in this retrospective study. RESULTS: A total of 260 patients were included. Malignancy was present in 61%. BAL's sensitivity for the diagnosis of malignancy was 29%, and overall diagnostic yield of BAL was 46%. However, only 1% of cancer diagnoses would have been missed in the absence of BAL. In the multivariable analysis, the size of lesion (odds ratio [OR], 1.17; 95% confidence interval [CI], 1.02-1.33; P = .023), the presence of bronchus sign (OR, 4.73; 95% CI, 1.06-21.08; P = .042), and the presence of mediastinal/hilar lymphadenopathy (OR, 3.37; 95% CI, 1.53-7.41; P = .002) were associated with improved BAL true-positive ratio relating to diagnosis of malignancy. However, the effect of lesion size on sensitivity was small (area under the curve, 0.31; 95% CI, 0.23-0.40; P < .001). Ground-glass lesions were not associated with improved BAL diagnostic value. The number needed to test for BAL for the diagnosis of malignancy or pulmonary infection was 37. CONCLUSION: Conventional BAL has a low diagnostic value for the diagnosis of suspected PLC, and the low number needed to test does not qualify BAL as a recommended routine investigation for the diagnosis of suspected PLC for either solid or ground-glass lesions.