A novel hotspot specific isothermal amplification method for detection of the common PIK3CA p.H1047R breast cancer mutation

Sci Rep. 2020 Mar 12;10(1):4553. doi: 10.1038/s41598-020-60852-3.

Abstract

Breast cancer (BC) is a common cancer in women worldwide. Despite advances in treatment, up to 30% of women eventually relapse and die of metastatic breast cancer. Liquid biopsy analysis of circulating cell-free DNA fragments in the patients' blood can monitor clonality and evolving mutations as a surrogate for tumour biopsy. Next generation sequencing platforms and digital droplet PCR can be used to profile circulating tumour DNA from liquid biopsies; however, they are expensive and time consuming for clinical use. Here, we report a novel strategy with proof-of-concept data that supports the usage of loop-mediated isothermal amplification (LAMP) to detect PIK3CA c.3140 A > G (H1047R), a prevalent BC missense mutation that is attributed to BC tumour growth. Allele-specific primers were designed and optimized to detect the p.H1047R variant following the USS-sbLAMP method. The assay was developed with synthetic DNA templates and validated with DNA from two breast cancer cell-lines and two patient tumour tissue samples through a qPCR instrument and finally piloted on an ISFET enabled microchip. This work sets a foundation for BC mutational profiling on a Lab-on-Chip device, to help the early detection of patient relapse and to monitor efficacy of systemic therapies for personalised cancer patient management.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / diagnosis*
  • Breast Neoplasms / genetics
  • Cell Line, Tumor
  • Class I Phosphatidylinositol 3-Kinases / genetics*
  • DNA Primers / genetics
  • Early Detection of Cancer
  • Female
  • Humans
  • Lab-On-A-Chip Devices
  • Liquid Biopsy
  • MCF-7 Cells
  • Molecular Diagnostic Techniques / instrumentation*
  • Molecular Diagnostic Techniques / methods
  • Mutation, Missense*
  • Nucleic Acid Amplification Techniques / instrumentation*
  • Nucleic Acid Amplification Techniques / methods
  • Pilot Projects
  • Proof of Concept Study

Substances

  • DNA Primers
  • Class I Phosphatidylinositol 3-Kinases
  • PIK3CA protein, human

Supplementary concepts

  • LAMP assay