Circulating and Local Expression Levels of Survivin and Fibulin-3 in Benign and Malignant Respiratory Diseases

Study design and participants The current prospective cohort study has been conducted with 73 patients of both sexes, with recently diagnosed benign and malignant respiratory diseases recruited from Cardio-Thoracic Surgery and Oncology Departments, Qena University Hospitals, South Valley University, Egypt. The included patients were categorized into 4 groups. Group A included 21 patients with lung cancer, group B included 21 patients with various benign lung diseases, group D included 15 patients with malignant pleural mesothelioma (MPM) and group E included 16 patients having various benign pleural diseases. In addition, 20 age and sex matched unrelated healthy subjects serve as the control group (group C). Patients with renal failure, hepatic failure, severe cardiopulmonary compromise, coagulopathy or hemodynamically unstable were excluded The study was approved by the Ethics committee of the Faculty of Medicine, South Valley University, Qena, Egypt, and it was conducted in accordance with the Declaration of Helsinki. Informed written consent has been obtained from every included subject. The study duration was two years from January 1st, 2017 to December 30th, 2019.

Data collection Full history, thorough clinical examinations were taken for every included patient. Additionally, radiological assessments by plain CXR and CT, and routine laboratory investigations (serum lactate dehydrogenase (LDH), total protein, albumin, liver and kidney functions, ESR and CBC) were performed. Multiple lung biopsies were taken and sent for histopathological examination when indicated. Thoracentesis was done for cases with proved pleural effusion. The standard pleural fluid analysis was done including: pH, biochemical testing of pleura/serum (LDH, glucose, albumin and Adenosine deaminase (ADA), cytology and microbiological testing (Z-N, L-J culture) and differential cell count) and Light's original criteria (ratio of pleural fluid/serum protein >0.5, ratio of pleural/serum LDH >0.6 or pleural fluid LDH more than two-thirds of the upper limit of normal serum value) to discriminate exudative from transudative pleural effusions. Thoracoscope was done for patients with indecisive cytology or pleural fluid analysis, and multiple pleural biopsies were taken for histopathological examination. The malignant pleural effusion was diagnosed if pleural fluid cytology or pleural biopsy findings were positive for malignancy.

Staging of MPM was done using van Meerbeeck et al, while that of lung cancer was assessed according to Lim et al.

Blood samples and pleural effusion fluid collections Five mls of venous blood was withdrawn from every included subject using serum separator gel tubes and was allowed to clot at room temperature for 30 minutes and then centrifuged for 15 minutes at 1000g. The separated sera were stored into aliquots using 1 ml cryotubes at -800C for later biochemical assays of survivin and fibulin-3. Additionally, 10 ml of pleural fluids from the included patients were centrifuged for 15 minutes at 1000g and stored into aliquots using 1 ml cryotubes at -800C for later molecular assays of survivin and fibulin-3 expression levels using western blot analysis.

ELISA assays of IGF-I and survivin Quantitative determinations of serum survivin and fibulin-3 were achieved using commercially available ELISA assay kits supplied by Chongqing Biospes Co., Ltd, China with catalog numbers BYE3519 and BYEK2017 respectively. The assays were performed using microplate ELISA reader (EMR 500, USA), according to manufacture protocol.