RESULTS OF LIQUID BIOPSY STUDIES BY NEXT
GENERATION SEQUENCING IN PATIENTS WITH
ADVANCED STAGE NON-SMALL CELL LUNG CANCER:
SINGLE CENTER EXPERIENCE FROM TURKEY
Buyuksimsek M1,*, Togun M2, Oguz Kara I1, Bisgin A3,4, Boga I4, Tohumcuoglu M1,
Ogul A1, Evren Yetisir A1, Sahin B1, Erdem Sumbul H5, Mirili C6
*Corresponding Author: Mahmut Buyuksimsek, M.D., Department of Oncology, Çukurova University
Faculty of Medicine, Sarican, Adana, Turkey. Tel: +90-536-862-20-26. Fax: +90-322-338-70-72.
E-mail: mahmutbuyuksimsek@gmail.com
page: 17
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MATERIALS AND METHODS
Patient Selection. A prospective review of 100 patients
in Adana, Turkey with a diagnosis of advanced/
metastatic NSCLC, whose physician requested clinical
ccfDNA based genomic profiling from January 2017 to
January 2019, were performed. All patients included in the
analysis had metastatic or inoperable disease. A second liquid
biopsy was performed in eight patients who developed
progressive disease after the first treatment. All patients
provided their written consent to the genomic profiling.
Targeted Multi-Gene Panel Testing. Customized
targeted multiple gene panel consisting of 19 genes (AKT1,
ALK, BRAF, DDR, ERBB2, ESR1, KIT, KRAS, MAP2K1,
NRAS, NTRK, PDGFRA, PIK3CA, PTEN, ROS1, RICTOR,
EGFR, MET, FGFR1 gene mutations, and RICTOR, EGFR,
MET, FGFR1, ERBB2 gene amplifications) was used for
NGS. The ccfDNA was extracted from whole peripheral
blood collected in 10 mL PaxGene (PreAnalytiX GmbH,
Hombrechtikon, Switzerland) biological sample tubes. The
results of NGS were obtained after the following steps: separation
of plasma from the samples, isolation of ccfDNA from
the plasma, target region enrichment in an appropriate quality
and quantity, library preparation, clonal amplification
and NGS steps. Afterwards, bioinformatic analyses were
performed to determine the quality and variant analysis according
to the clinical information of the patients to interpret
the variants. All the workflow was carried out at Çukurova
University AGEN TEM (Adana Genetic Diseases Diagnosis
and Treatment Center), Adana, Turkey via the GeneReader
NGS system (Qiagen GmbH, Hilden, Germany).
The isolation of ccfDNA was performed using the
circulating cfDNA isolation Kit (QIAamp circulating nucleic
acid kit; Qiagen GmbH) with the help of a vacuum
system (QIAvac 24 Plus; Qiagen GmbH). The ccfDNA
concentrations were determined using fluorometric DNA
quantitation device (Qubit 3.0, Thermo Fisher Scientific,
Waltham, MA, USA). The samples with adequate DNA
concentrations were used for further laboratory workflow.
The target enrichment of the region of interest was amplified
by PCR (polymerase chain reaction). The bar coding
and library preparation step was then performed. Thereafter,
the samples were loaded into flowcells to be sequenced
in the GeneReader NGS system (Qiagen GmbH).
Bioinformatic Analysis and Interpretation. The
most complicated and difficult step is the accurate analysis
of the large data from the sequenced NGS samples by an
experienced medical geneticist and the team. The data with
appropriate quality were selected before the data analysis.
The selected data were compared with the reference
genome data. The Human Genome Mutation Database
(HGMD) (http://www.hgmd.cf.ac.uk/ac/index.php0, Catalogue
of Somatic Mutations in Cancer (COSMIC) (https://
cancer. sanger.ac.uk/cosmic), 1000 Genome Frequency
and Ingenuity Knowledge Base databases (https://www.
internationalgenome.org/1000-genomes-browsers), and
SIFT, BSIFT (https://sift.bii.a-star.edu.sg/), PolyPhen-2
(http:// genetics.bwh.harvard.edu/pph2/dbsearch.shtml)
and Clin Var (https://www.ncbi.nlm.nih.gov/clinvar/) in
silico analysis were used for variant analysis. Then, selected
variants were analyzed using bioinformatics tools
to classify and evaluate them according to their clinical
impacts, for potential influence on the treatment strategies,
and to confirm the clinical diagnosis. The low quality variants
were also assessed for the samples’ status and clinical
status of the patient. All the variants were evaluated in two
steps: the first one was for its quality and possible effects
independent of clinical diagnosis and mostly on the basis
of quality control parameters such as for ward/reverse
read balance and coverage. The second evaluation was
performed for clinical diagnosis and possible effect on the
sensitivity and/or the resistance to the treatment protocols.
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