Bronchial Neoplasm Benign Clinical Trial
Official title:
Circulating and Local Expression Levels of Survivin and Fibulin-3 in Benign and Malignant Respiratory Diseases
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.
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.
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