Development of a multiplex allele-specific qPCR approach for testing PIK3CA mutations in patients with colorectal cancer

Oscorbin, Igor P.; Beginyazova, Oguljan P.; Khlistun, Inna V.; Shamovskaya, D. V.; Oskina, N. А.; Филипенко, М. Л.

doi:10.1016/j.heliyon.2022.e11804

Heliyon

2022

DOI: 10.1016/j.heliyon.2022.e11804

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Development of a multiplex allele-specific qPCR approach for testing PIK3CA mutations in patients with colorectal cancer

Igor P. Oscorbin

Oguljan P. Beginyazova

Inna V. Khlistun

et al.

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Cited by 2 publications

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A Multiplex Assay for Fast PIK3CA Hotspot Mutation Characterization in a Single Specimen by 3-Color Digital PCR Analysis

Helmijr,

Motta,

Jongbloed

et al. 2024

The Journal of Applied Laboratory Medicine

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Background Activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene have been detected often in solid tumors. Targeted therapy for mutant PIK3CA is now available in the clinic, making molecular diagnostics pivotal. Our aim was to design a multiplex digital PCR (dPCR) assay to evaluate the 4 most common PIK3CA hotspot mutations simultaneously to characterize and quantify these in liquid biopsies. Methods A multiplex assay was developed to detect exon 9 p.E542K and p.E545K mutations, and exon 20 p.H1047L and p.H1047R mutations using the Stilla 3-color dPCR Naica system. The assay was evaluated on stock and pre-amplified DNA from cell lines with the above mutations as single and pooled samples, and on cell-free DNA (cfDNA) from healthy blood donors (HBDs) and breast cancer patients, to determine detection thresholds and diagnostic accuracy. Results The assay distinguished all 4 PIK3CA mutations in (cf)DNA, and also when dual mutations were present. Detection thresholds of stock and pre-amplified cfDNA samples were 0.11 and 0.40 copies/uL (cp/uL) for mutant copies concentration, and 0.003% and 0.68% for variant allele frequencies (VAFs), respectively. The assay confirmed the PIK3CA (mutation) status as defined by targeted next-generation sequencing (NGS) in 82 out of 96 patients that were mutant for PIK3CA, and in 11 out of 12 patients with wild-type PIK3CA. Conclusions Our designed multiplex dPCR assay detected PIK3CA mutations with high accuracy in stock and pre-amplified cfDNA. Furthermore, it is affordable and demands less cfDNA input when compared to available uniplex dPCR assays and NGS analyses.

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A Multiplex Assay for Fast PIK3CA Hotspot Mutation Characterization in a Single Specimen by 3-Color Digital PCR Analysis

Helmijr,

Motta,

Jongbloed

et al. 2024

The Journal of Applied Laboratory Medicine

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Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers

et al. 2023

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Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer’s 3′-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template′s background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

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Development of a multiplex allele-specific qPCR approach for testing PIK3CA mutations in patients with colorectal cancer

Cited by 2 publications

References 46 publications

A Multiplex Assay for Fast PIK3CA Hotspot Mutation Characterization in a Single Specimen by 3-Color Digital PCR Analysis

A Multiplex Assay for Fast PIK3CA Hotspot Mutation Characterization in a Single Specimen by 3-Color Digital PCR Analysis

Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers

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