A novel hotspot specific isothermal amplification method for detection of the common PIK3CA p.H1047R breast cancer mutation.
Melpomeni KalofonouKenny Malpartida-CardenasGeorge AlexandrouJesus Rodriguez-ManzanoLing-Shan YuNicholas MiscouridesRebecca AllsoppKelly L T GleasonKatie GoddardDaniel Fernandez-GarciaKaren PagePantelis GeorgiouSimak AliR Charles CoombesJacqueline A ShawChristofer ToumazouPublished in: Scientific reports (2020)
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.
Keyphrases
- loop mediated isothermal amplification
- circulating tumor
- nucleic acid
- case report
- metastatic breast cancer
- high throughput
- papillary thyroid
- cell free
- end stage renal disease
- single molecule
- breast cancer risk
- polycystic ovary syndrome
- circulating tumor cells
- ejection fraction
- newly diagnosed
- sensitive detection
- ultrasound guided
- chronic kidney disease
- childhood cancer
- squamous cell
- single cell
- gene expression
- type diabetes
- peritoneal dialysis
- free survival
- pregnancy outcomes
- genome wide
- deep learning
- intellectual disability
- cervical cancer screening
- label free
- autism spectrum disorder
- patient reported