Mutational Analysis of PIK3CA Gene from FFPE of Non-Small Cell Lung Cancer in Non-Smokers
Mutational Analysis of PIK3CA Gene from FFPE of Non-Small Cell Lung Cancer in Non-Smokers
Farah Bilal*, Abdulmohsen Alhejaily, Shahida Husnain
Department of Microbiology and Molecular Genetics, Quaid e Azam Campus, University of The Punjab, Lahore, Pakistan.
Abstract | PIK3CA is considered important component of PI3K pathway. Being a regulatory gene for RAS, PI3K and EGFR pathways, any change in PIK3CA gene sequence plays vital role in several cancers including lung cancer. Purpose of this study was to evaluate the prevalence of common nucleotide changes (E545K/E542K and H1047R/H1047L) occur in PIK3CA gene in non smoker patients of non-small cell lung cancer (NSCLC). Somatic mutational analysis was done by QMC-PCR following direct sequencing with sangers’ method. Only one sample detected p.E545K mutation at c.1633G>A. Another most common mutation was observed in PIK3CA exon 20 which leads to change A>G at codon 1047. This transition converts amino acid histidine to arginine. Our study concluded p.H1047R most frequent while p.E545K rarely found mutations in PIK3CA gene in NSCLC population. Present study can be proved as road map in setting PIK3CA mutations as potential therapeutics target for NSCLC in non-smokers.
Received: February 01, 2019
Revised: March 21, 2019
Accepted: June 03, 2019
Published: June 26, 2019
FB did experiments and wrote the manuscript. AA presented the idea of the research and designed the project. SH upervised the study and reviewed the manuscript.
NSCLC: Non-small Cell Lung Cancer, FFPE: Formalin Fixed paraffin Embedded tissue, PIK3CA: Phosphatidylinositol 3-kinase Catalytic Alpha Subunit
Farah Bilal, email@example.com
To cite this article: Bilal, F., Alhejaily, A. and Husnain, S., 2019. Mutational analysis of pik3ca gene from ffpe of non-small cell lung cancer in non-smokers. Punjab Univ. J. Zool., 34(1): 97-100. http://dx.doi.org/10.17582/journal.pujz/2019.34.1.97.100
Single nucleotide changes which occur frequently in several cancers can be reason of tumor initiation and progression. These types of mutations trigger cancer called driver mutations and are vital target in therapeutics treatment of cancers (Pao and Girard, 2011). driver mutations lead to change in expressions of genes work up/down stream of signaling pathway which results in cancer by promoting malignant growth or switching off tumor suppressor genes (Thompson et al., 2016). The signaling pathways promoting cell growth (Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways) involve several significant genes like KRAS, BRAF and PIK3CA. Mutations of these genes can drive cell towards cancer (Vakiani and Solit, 2011). Phosphatidylinositol-3-kinase (PI3K) protein has crucial role in cell proliferation and metabolism. Any abnormality in these kinases can results in cancers. PI3K has its two subunits, regulatory and catalytic subunit (Shull et al., 2012). PIK3CA (Phosphoinositide-3-kinase catalytic alpha polypeptide) gene’s catalytic unit has special importance in cancer study of being mutated in several cancers including lung cancer (Yamamoto et al., 2008). PIK3CA mutations were found most frequently in exon 9 (helicase domain) and exon 20 (catalytic domain) which are called hot spot regions. The mutations E545K/E542K in exon 9 and H1047R/H1047L in exon 20, considered as vital oncogenic targets (Chaft et al., 2012). Purpose of our study was to check the prevalence of most common mutations in PIK3CA exon9 and exon20 in non-small cell lung cancer (NSCLC) of non-smokers. PIK3CA mutations can be potential therapeutic target for treatment of anti-EGFR therapy resistant in never smokers and basic clinical characteristics can be proved best prognostic factors in diagnosis of NSCLC in non-smokers (McCubrey et al., 2012).
Materials and Methods
This was a retrospective study. Histological confirmed NSCLC cases were selected from the department of pathology National Guard Hospital Biobank Riyadh. 50 tissue samples were selected from these confirmed NSCLC patients with no-smoking history. All samples were selected based on availability of formalin-fixed paraffin-embedded tissue samples (FFPE) (from lung cancer patients) and minimum 70% malignant appearance of tumor cells. Patient’s baseline clinical characteristics (age, gender and family history of lung cancer) were noted. Tumor-rich area was micro dissected and DNA extraction was done by tissue by QIAamp DNA Mini Kit (Qiagen, Germany). DNA quantification was done by using Nano Drop ND-1000 spectrophotometer (Nano DropTechnologies, Wilmington, DE, USA). Quick multiplex consensus–polymerase chain reaction (QMC-PCR) was performed for DNA amplification followed by sanger’s sequencing. Targeted outer primers for PIK3CA exon 9 (F 5’-CTGTGAATCCAGAGGGGAAA-3’, R 5’-GCACTTACCTGTGACTCCATAGAA-3’) and PIK3CA exon 20 (PIK20F 5’-TGAGCAAGAGGCTTTGGAGT-3’, PIK20R 5’-CCTATGCAATCGGTCTTTGC-3’) were used in first PCR reaction. final volume of 25 µl reaction mixture was prepared. Each reaction mixture consisted of DNA template (20ng), all outer primers (each concentration of 0.400 mM) and 13HotShot master mix. first PCR was performed using thermal cycle sequence: First cycle at 95oC for 10 minutes, at 95oC for 1 seconds 45 cycles and at 55oC last cycle for 1 second. Amplicon of this PCR was diluted (1:100) with PCR grade water and used as template for final specific diagnostic PCR. Reaction mixture of 10 µl total volume was used including all components with same concentration as in previous PCR. Internal primer (0.25µM) were used for PIK3CA exon9 (F 5’-AAGGGAAAATGACAAAGAACAG-3’, R 5’-CACTTACCTGTGACTCCATAGAAA-3’) and PIK3CA exon 20 (F 5’-GCAAGAGGCTTTGGAGTATTTC-3’, R 5’-TTTTCAGTTCAATGCATGCTG-3’) separately for final diagnostic PCR. Duplicate reaction for each target in specific diagnostic PCR was run on AB 7500 fast PCR thermal cycler. Direct sequencing reaction was done by using purified amplicon of last PCR reaction as template and universal M13 primers (M13F: TGTAAAACGACGGCCAGT, M13R: CAGGAAACAGCTATGACC). Sequencing was done by using commercial prepared kit (Big Dye Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems, Foster City, CA). The sequence data was analyzed by DNA Sequence Analysis Software version 3.1.1 and POP 7 (Applied Biosystems, Life Technologies) on ABI 3700 BioAnalyzer.
Results and Discussion
The (PI3K)/AKT pathway plays vital role in cell survival by regulating several cellular processes like differentiation, migration and proliferation. Any disturbance in this pathway’s mechanisms can lead to cell malignancy and death. Variations in genes involved in (PI3K)/AKT pathway have been reported in many cancers (Steelman et al., 2011). PI3K has two main subunits one is catalytic p110 and other is p85 which is called as regulatory subunit because of its involvement in regulation of cell division. Mutations in these subunits results in tumorigenesis (Chang et al., 2003). The aim of our study was to detect mutations in hot spot region of PIK3CA gene in NSCLC of non-smokers. We used QMC-PCR for amplification of targeted sequence in PIK3CA gene which is relatively cheap, quick and précised technique. Basically it is a nested PCR which ensures the specificity of target amplification in FFPE tissue sample (Ebili et al., 2017). Two common mutations were noted in PIK3CA gene. One mutation E545 (NM_006218.3) was observed in helicase domain shown in Figure 1. Another commonmutation H1047 (NM_ 006218.1) was also seen in kinase domain. Figure 2 showed A>G transition (p.H1047R) at codon 1047 in exon 20, observed in 16 samples (32%) of total 50 NSCLC samples while 34 NSCLC tumor showed wild type for the same position. This mutation (p.H1047R) results in histidine amino acid conversion to arginine amino acid. Second mutation was observed in only one sample (2%) from total NSCLC population. This mutation presented G>A change at codon 1633 in exon 9 of PIK3CA. The transition leads to substitute glutamic acid with lysine amino acid at position E545K. No co-existence of mutations was observed in PIK3CA exon 20 and exon 9 in any sample. both observed mutations in current study were also reported previous in pancreatic cancer and considered hotspot mutations which also effect the activation of Akt signaling (Schönleben et al., 2006). The same mutations of PIK3CA gene were also reported in other cancers like colorectal (32%), gastric (25%) and in lung cancer (4%) (McCubrey et al., 2012). But unfortunately, limited information published on NSCLC of nonsmokers. However, describing prevalence of PIK3CA mutations in non-small lung cancer of nonsmokers Barbareschi et al. (2007) reported high frequency of PIK3CA mutations in exon9 than exon20 in breast cancer, contrasting their data present study confirmed PIK3CA exon 20 mutations were more frequent than exon9 mutation in NSCLC population. We examined only one mutation E545K in helicase domain of one NSCLC tumor which is common mutation reported in many cancers specially in colorectal that was Glu545 to Lys545 (E545K) and thought to interrupt p85 N-terminal SH2 domain inhibitory interaction (Miled et al., 2007). Contrasting our finding another study reported more frequent mutations of PIK3CA exon 9 in NSCLC (Yamamoto et al., 2008). SNPs encoding K545E and H1047R have signiﬁcant role in enhancing migrating and growth activity of epithelial cells (Okudela et al., 2007). These observed factors predict mutations in PIK3CA exon 20 can be a potential targets for treatment of NSCLC in response of resistant NSCLC against anti-EGFR therapy hence can be recommended to identify better response to chemotherapies (Mao et al., 2012). The small number of non-small lung cancer population limited mutational importance of PIK3CA gene. This issue can be resolved by increasing more patient’s data to present study can lead to new drug therapy for NSCLC in future.
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