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Highly Sensitive Detection of PIK3CA Mutations by Looping-Out Probes-Based Melting Curve Analysis

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Abstract

PIK3CA mutations have important therapeutic and prognostic implications in various cancer types. However, highly sensitive detection of PIK3CA hotspot mutations in heterogeneous tumor samples remains a challenge in clinical settings. To establish a rapid PCR assay for highly sensitive detection of multiple PIK3CA hotspot mutations. We described a novel melting curve analysis-based assay using looping-out probes that can enrich target mutations in the background of excess wild-type and concurrently reveal the presence of mutations. The analytical and clinical performance of the assay were evaluated. The developed assay could detect 10 PIK3CA hotspot mutations at a mutant allele fraction of 0.05–0.5% within 2 h in a single step. Analysis of 82 breast cancer tissue samples revealed 43 samples with PIK3CA mutations, 28 of which were confirmed by Sanger sequencing. Further testing of 175 colorectal cancer tissue samples showed that 24 samples contained PIK3CA mutations and 19 samples were confirmed by Sanger sequencing. Droplet digital PCR supported that all mutation-containing samples undetected by sequencing contained mutations with a low allele fraction. The rapidity, ease of use, high sensitivity and accuracy make the new assay a potential screening tool for PIK3CA mutations in clinical laboratories.

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Data Availability

All data generated or analyzed during this study are included in this published article and its additional files.

Abbreviations

PI3Ks:

Phosphatidylinositol-3-kinases

BC:

Breast cancer

CRC:

Colorectal cancer

MAF:

Mutant allele fraction

ARMS:

Amplification refractory mutation system

COLD-PCR:

Co-amplification at lower denaturation temperature-polymerase chain reaction

LNA:

Locked nucleic acid

ddPCR:

Droplet digital PCR

NGS:

Next-generation sequencing

LPO:

Looping-out

gDNA:

Genomic DNA

FFPE:

Formalin-fixed paraffin-embedded

dF/dT:

The negative derivative of fluorescence over temperature

T m :

The melting point

ΔT m :

The Tm difference

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Funding

This study was supported by National Natural Science Foundation of China (Grant No. 81672110).

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Author notes

  1. Boheng Xu, Yanping Lan, Danjiao Luo have equally contributed to this article.

Authors and Affiliations

  1. Department of Molecular Diagnostics, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou, 510060, Guangdong, China

    Boheng Xu

  2. Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, 361102, Fujian, China

    Boheng Xu, Yanping Lan, Danjiao Luo, Yangsi Zheng, Runfang Ni, Qiuying Huang & Qingge Li

  3. The First Affiliated Hospital of Xiamen University, Xiamen, 361003, Fujian, China

    Guoqiang Su

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  1. Boheng Xu
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Contributions

BHX, YPL and DJL design and performed experiments, analyzed data and co-wrote this paper. YSZ, RFN and GQS collected samples and the data. QYH and QGL provided financial support and supervised the study proposal. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Qiuying Huang or Qingge Li.

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical Approval

Approval of the research protocol by an Institutional Reviewer Board: this study was approved by the medical ethics committee of Xiamen University (approval No. XDYX2021017).

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Xu, B., Lan, Y., Luo, D. et al. Highly Sensitive Detection of PIK3CA Mutations by Looping-Out Probes-Based Melting Curve Analysis. Biochem Genet 62, 77–94 (2024). https://doi.org/10.1007/s10528-023-10408-7

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