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.
Highlights
Breast cancer (BC) has a lifetime incidence risk of 12%, with an overall survival of more than 70% of reported cases when early detection is possible[1,2]
These properties of Loop-mediated isothermal amplification (LAMP) coupled with its higher amplification yield allows it to be utilised in tandem with standard microchip technology used for chemical sensing such as the Ion-Sensitive Field-Effect Transistors (ISFETs)[16]
Specific amplification of the PIK3CA p.H1047R gene mutation in isothermal conditions was achieved following the unmodified self-stabilising (USS)-SNV-based loop mediated isothermal amplification (sbLAMP) method described by MalpartidaCardenas et al.[15]
Summary
Breast cancer (BC) has a lifetime incidence risk of 12%, with an overall survival of more than 70% of reported cases when early detection is possible[1,2]. These properties of LAMP coupled with its higher amplification yield allows it to be utilised in tandem with standard microchip technology used for chemical sensing such as the Ion-Sensitive Field-Effect Transistors (ISFETs)[16]. Besides lab-based validation, we demonstrate on-chip specific detection of both WT and MT alleles using an ISFET-based Lab-on-Chip platform Using this fully-electronic chemical sensing technology in combination with the molecular methods developed here, we are in a position to create a lab-free, cost-effective sample-to-result system for routine monitoring of a panel of breast cancer mutations through liquid biopsy profiling of ctDNA, to be used as a diagnostic companion to stratify patient treatment.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
