Sains Malaysiana 53(11)(2024): 3683-3693

http://doi.org/10.17576/jsm-2024-5311-12

Sensitive Detection of PIK3CA Exon 20 H1047R Breast Cancer Based on Low-Cost Intercalary Dye SYBR Green I Real-Time qPCR Assay

(Pengesanan Sensitif PIK3CA Exon 20 H1047R Kanser Payudara Berdasarkan Pewarna Interkalari Kos Rendah SYBR Green I Ujian Asai qPCR Masa Nyata)

DESRIANI¹, AZAMRIS², PRIMARIADEWI RUSTAMADJI³, INHERNI MARTI ABNA⁴, IRSYAD IBADURRAHMAN¹, ASRUL MUHAMMAD FUAD¹, DINI NURDIANI¹, YULIAWATI YULIAWATI¹, NURULIAWATY UTAMI¹, NENG HERAWATI¹, NAJMIATUL FITRIA⁵ & MUHAMMAD ALI WARISMAN¹

¹Research Center for Genetic Engineering, Research Organization for Life Sciences and Environment, National Research and Innovation Agency. 16911 Jl. Raya Km 46 Cibinong, Kabupaten Bogor, Jawa Barat, Indonesia

²Division of Surgical Oncology, Medical School of Andalas University, 25127 Jl. Perintis Kemerdekaan no 94, Padang, Sumatera Barat, Indonesia

³Division of Anatomy Pathology, Faculty of Medicine, Universitas Indonesia, 10430 Jl. Salemba Raya No 6, Kenari, Kec Senen, Jakarta, Indonesia

⁴Pharmacy Study Program, Faculty of Health Science, Esa Unggul University, 11510 Jl. Arjuna Utara No 9, Duri Kepa, Jakarta, Indonesia

⁵Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Andalas University, 25175 Limau Manis, Pauh, Padang, Sumatera Barat, Indonesia

Diserahkan: 21 Mac 2024/Diterima: 4 September 2024

Abstract

Breast cancer is associated with an excessive function of somatic mutation in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), specifically in exon 9 and 20 hotspots. Exon 20, which contains H1047R, has more potential for an oncogenic mutation than exon 9. Detection of PIK3CA/H1047R mutation has also been reported for molecular diagnosis and therapeutic application. Sensitive methods are required because mutants are mostly present at low levels in a mixture with the wild type. Therefore, this study aimed to explore the development of a low-cost and sensitive PIK3CA exon 20 H1047R detection method using intercalary dye real-time qPCR (quantitative polymerase chain reaction), SYBR Green I. The method was primer design, formulation, specificity, limit of detection, reproducibility, repeatability, and genotyping of 15 DNA (deoxyribonucleic acid)-frozen tissue patient samples, which were further confirmed by PCR sequencing for validation. DNA primer proportion formulations were 0.25 µM PIK3CA exon 20 (WT), 0.65 µM PIK3CA exon 20 H1047 (MT), and 0.2 µM reverse primer in 10 μl total volume. The results showed that compared to the gold standard genotyping PCR sequencing (6.6-20%), the developed method had a higher sensitivity of 5%. The coefficients of intra- and inter-variability were between 0.01-0.19%, suggesting the developed method was repeatable and reproducible with a low mean pipetting error. Genotyping of 15 DNA breast cancer patient samples showed a wild-type genotype, which was in 100% agreement with the PCR sequencing result. The developed method showed sensitive, reproducible, and repeatable results,  potentially applicable in prognosis and therapeutic predictions.

Keywords: Breast cancer; Exon 20; H1047R; mutation; PIK3CA

Abstrak

Kanser payudara dikaitkan dengan fungsi berlebihan mutasi somatik dalam subunit pemangkin fosfatidilinositol-4,5-bisfosfat 3-kinase α (PIK3CA), khususnya di exon 9 dan 20 titik panas. Exon 20 yang mengandungi H1047R lebih berpotensi untuk mutasi onkogenik berbanding exon 9. Pengesanan mutasi PIK3CA/H1047R juga telah dilaporkan untuk diagnosis molekul dan aplikasi terapeutik. Kaedah sensitif diperlukan kerana mutan kebanyakannya terdapat pada tahap rendah dalam campuran dengan jenis liar. Oleh itu, kajian ini bertujuan untuk meneroka pembangunan kaedah pengesanan PIK3CA exon 20 H1047R yang kos rendah dan sensitif menggunakan qPCR masa nyata pewarna interkalari (tindak balas rantai polimerase kuantitatif), SYBR Green I. Kaedahnya ialah reka bentuk primer, formulasi, kekhususan, had pengesanan, kebolehasilan, kebolehulangan dan genotaip 15 sampel pesakit tisu DNA (asid deoksiribonukleik)-beku, yang selanjutnya disahkan oleh penjujukan PCR untuk pengesahan. Formulasi perkadaran primer DNA ialah 0.25 µM PIK3CA exon 20 (WT), 0.65 µM PIK3CA exon 20 H1047 (MT) dan 0.2 µM primer songsang dalam 10 μL jumlah isi padu. Keputusan menunjukkan bahawa berbanding dengan penjujukan PCR genotaip piawaian emas (6.6-20%), kaedah yang dibangunkan mempunyai kepekaan yang lebih tinggi sebanyak 5%. Pekali intra-dan inter-kevariabelan adalah antara 0.01-0.19% menunjukkan kaedah yang dibangunkan boleh diulang dan boleh dihasilkan semula dengan ralat pipet min yang rendah. Genotaip 15 sampel pesakit kanser payudara DNA menunjukkan genotaip jenis liar yang 100% bersetuju dengan hasil penjujukan PCR. Kaedah yang dibangunkan menunjukkan keputusan yang sensitif, boleh dihasilkan semula dan boleh diulang, berpotensi digunakan dalam ramalan prognosis dan terapeutik.

Kata kunci: Exon 20; H1047R; kanser payudara; mutasi; PIK3CA

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