Page 246 - EJMO-9-1

Eurasian Journal of Medicine and
Oncology
EGFR and PIK3CA mutations in lung cancer patients

designed this study to examine the prevalence of EGFR, PCR reactions were conducted in a final volume of 22 μL,
PIK3CA, and HER2 alterations and their association with containing 2 μL of DNA, 2 μL of 10× Dream Taq Buffer,
clinicopathological features. These insights aim to enhance 2 μL of 10 mM deoxyribonucleotide triphosphates, 0.6 μL
the understanding of mutational profiles in NSCLC within of 10 μM forward and reverse primers (Table 1), and 0.5
the Moroccan context, supporting clinicians in tailoring U of Taq polymerase enzyme (Thermo Fisher Scientific,
targeted treatment strategies for this population. France). Thermal cycling was initiated with incubation
at 96°C for 3 min, followed by 40 cycles of 30 s at 96°C,
2. Materials and methods primer annealing for 60 s at the specific T (Table 1),
m
2.1. Collection of tissue samples and extension for 40 s at 72°C. A final elongation step
was performed for seven minutes at 72°C. For each PCR
From August 2019 to July 2022, we consecutively collected
fresh biopsies from 90 Moroccan LC patients who underwent reaction, a negative control containing all components of
surgery at the Thoracic Surgery Department of the Ibn the master mix except the DNA sample was included. The
PCR amplicons were then analyzed by electrophoresis on a
Rochd University Hospital (Casablanca, Morocco). For each 1.5 – 2% agarose gel and visualized after ethidium bromide
biopsy, one portion was fixed in 10% formaldehyde buffer
for routine pathological diagnosis, while another portion staining (10 mg/mL).
was immediately stored in RNAlater (Ambion, United 2.2.3. Sequencing of target genes
®
States of America) and preserved at −80°C for molecular
analyses. Cancer staging was determined according to the For DNA sequencing, amplicons were purified using
2021 World Health Organization LC classification. 21 the ExoSAP-IT™ system (Applied Biosystems, USA)
according to the manufacturer’s instructions. The mixtures
Patient information, including age, gender, smoking were incubated at 37°C for 15 min to remove primers
history, and disease site, was retrieved from the archives of and nucleotides, followed by 15-min incubation at 80°C
the Thoracic Surgery Department. The study was approved to inactivate the enzyme. Sequencing was carried out
by the Ethics Committee of Ibn Rochd University Hospital, using the BigDye™ Terminator v3.1 Cycle Sequencing Kit
and written consent was obtained from each participant. (Applied Biosystems, United States of America) in a 10 μL
2.2. Mutational profiling reaction containing 2 μL of purified PCR product, 1 μL of
BigDye Terminator, 0.5 μL of 10 μM forward or reverse
®
2.2.1. Deoxyribonucleic acid (DNA) extraction primer, and 6.5 μL of nuclease-free water. The sequencing
Genomic DNA was isolated from fresh lung biopsies using reaction included denaturation at 96°C for one minute,
a standard protocol, which involved overnight digestion followed by 25 cycles of 96°C for 10 s, primer annealing
with proteinase K at 37°C, followed by phenol/chloroform at 50°C for 5 s, and extension at 60°C for 4 min. After
extraction. The quantity and quality of the extracted sequencing, the products were purified using Sephadex
22
DNA were assessed using a NanoDrop 2000 (Thermo G-50 gel-exclusion chromatography (GE Healthcare Life
Fisher Scientific, United States of America). Sciences, USA) to remove the remaining sequencing
products. The purified sequences were then analyzed with
2.2.2. Polymerase chain reaction (PCR) amplification an ABI 3130XL DNA analyzer (Applied Biosystems, USA),
Exons 18 – 21 of EGFR, exon 20 of HER2, and exons 9, and the resulting sequences were compared to the human
and 20 of PIK3CA were individually amplified using PCR. genome reference using the NCBI-BLAST tool.

Table 1. List of primers and their characteristics
Gene Exon Forward primer 5’ – 3’ Reverse primer 5’ – 3’ T (°C) Fragment size (bp)
m
EGFR 18 CAAATGAGCTGGCAAGTGCCGTGTC GAGTTTCCCAAACACTCAGTGAAAC 60 400
19 GCAATATCAGCCTTAGGTGCGGCTC CATAGAAAGTGAACATTTAGGATGTG 58 372
20 CCATGAGTACGTATTTTGAAACTC CATATCCCCATGGCAAACTCTTGC 60 408
21 CTAACGTTCGCCAGCCATAAGTCC GCTGCGAGCTCACCCAGAATGTCTGG 58 415
HER2 20 CTCTCAGCGTACCCTTGTCC CCTAGCCCCTTGTGGACATA 60 273
PIK3CA 9 AATCATCTGTGAATCCAGAGG ATGCTGAGATCAGCCAAAT 55 273
20 CATTTGAGCAAAGACCTGAAGG TGAGCTTTCATTTTCTCAGTTATC 58 370
Abbreviations: EGFR: Epidermal growth factor receptor; HER2: Human epidermal growth factor receptor 2; PIK3CA: Phosphoinositide 3-kinase
catalytic subunit alpha.

Volume 9 Issue 1 (2025) 238 doi: 10.36922/ejmo.7111