Survivin and Fibulin-3 in Benign and Malignant Respiratory Diseases
Condition: Bronchial Neoplasm Benign
MeSH terms: Bronchial Neoplasms; Respiratory Tract Diseases; Respiration Disorders
Study type: Observational

Summary: 
      Purpose: Survivin is a common member of the inhibitors of the apoptosis protein (IAP) family
      with a dual role in promoting cell proliferation and preventing apoptosis. Fibulin-3, a
      matrix glycoprotein, was recently presented as a promising novel biomarker for malignant
      pleural mesothelioma. The aim of this study was to validate the expression levels of survivin
      and fibulin-3 in benign and malignant respiratory diseases.

      Patients and methods: The study included 73 patients, with various benign and malignant
      respiratory diseases. For validation of the data, a control group including 20 healthy
      subjects was chosen. The clinical and radiological assessments of the included individuals
      were done. The serum survivin and fibulin-3 levels were measured using ELISA assays kits,
      while their local expressions in the lung and pleura were assessed using western blot
      analysis.
    

Detailed description: 
      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.

      Western blotting assessments of survivin and fibulin-3 expression levels Lung and pleural
      biopsies were homogenized in ice-cold RIPA lysis buffer(Sigma-Aldrich, Milan, Italy),
      containing 1% protease inhibitor cocktail (Cell Signaling Technology, Inc., MA, USA) using
      Potter-Elvehjem rotor-stator homogenizer (glass/teflon homogenizer), fitted with a Teflon
      pestle and stored frozen at -70 °C for subsequent assessment of tissue nuclear survivin and
      fibulin-3 expressions by Western blotting technique.

      Proteins in each corresponding lung , pleural tissue homogenates or pleural effusion sample
      were denatured at 95 °C for 5 minutes in 2× Laemmli buffer followed by addition of 5%
      2-mercaptoethanol. SDS-PAGE electrophoresis was achieved by loading 50 µg protein per lane at
      75 volts through resolving gel (18% for survivin and 15% for fibulin-3) followed by 125 volts
      during approximately 2 hours and transferred to a PVDF membrane using T-77 ECL semidry
      transfer unit (Amersham BioSciences UK Ltd) for 2 hours. Immunoblotting was performed by
      incubating the PVDF membrane in TBS buffer containing 0.1% Tween and 5% non fat milk for one
      hour at 4°C, followed by overnight incubation at 4°C with rabbit anti-survivin polyclonal
      antibody (Bioss Inc., Massachusetts, USA) and rabbit anti-fibulin-3 polyclonal antibody
      (Novus Biologicals, LLC, Littleton, CO, USA) at a dilution of 1:1500. After being washed
      three times with TBST buffer, each membrane was incubated for 1 hour at room temperature with
      an alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Novus Biologicals,
      LLC, Littleton, CO, USA) at a dilution of 1:5000. After being washed four times in TBST, the
      membrane bound antibody was detected with a commercially available BCIP/NBT substrate
      detection Kit (Genemed Biotechnologies, Inc., CA, USA). Equivalent protein loading for each
      lane was confirmed by stripping and re-blotting each membrane at 4°C against mouse monoclonal
      anti β-actin antibody (Santa Cruz Biotechnology, Inc., CA, USA) at a dilution of 1:5000. The
      analysis was repeated 3 times to assure the reproducibility of results. Quantification was
      performed using ImageJ software and expressed as the band density relative to that of
      β-actin.

      Statistical analysis Data entry and data analysis were done using SPSS version 19
      (Statistical Package for Social Science). Data were presented as a number, percentage, the
      mean and standard deviation for parametric data. Chi-square test and Fisher exact test were
      used to compare qualitative variables. Independent t-test was used to compare quantitative
      variables between two groups. Pearson correlation was done to measure the correlation between
      quantitative variable. Medcalc Program was used to calculate sensitivity, specificity,
      positive and negative predictive values with calculation of the AUC (95% CI). P-value was
      considered statistically significant when <0.05.
    

Intervention name(s): ELISA assays and Westren blot analysis of survivin and fibulin-3 expression
Intervention type(s): Diagnostic Test
Intervention description(s): expression analysis of biomarkers
Intervention MeSH terms: BIRC5 protein, human
Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Probability Sample
Study population: 
        any patients with Benign or Malignant Respiratory Diseases and agree to included in the
        study in addition to healthy controls
      
Keywords: Survivin; Fibulin-3; Lung cancer; MPM