Current statistics provided by the World Health Organization calculate an annual worldwide cancer mortality rate at a level of 10 million. In Europe, this equates to one in every four deaths (26%) with the four most common cancer types identified as lung, breast, colorectal and prostate. In women, breast cancer is the most commonly occurring cancer accounting for 30% of all new diagnoses 1.
The current standard for cancer diagnosis and primary therapy response relies on tumour biopsies and conventional imaging approaches. Whilst providing a valuable snapshot, there is an urgent need to profile and assess tumour evolution over time, guiding predictive, preventive, and personalized medicine to inform treatment decisions in response to changes in tumour biology at all phases of cancer care 2. Liquid biopsy monitoring of plasma derived circulating tumour DNA (ctDNA) holds great promise in achieving this, with proof of principle demonstrated using approaches to target single variants by droplet digital PCR (ddPCR), real-time PCR, plus broader multigene and genome wide next-generation sequencing (NGS) approaches. Progress is greatly dependent on technological advances of highly sensitive techniques offering exquisite selectively over high background levels of wild-type alleles in a sample. Tzanikou and Lianidou 3 comprehensively review a variety of techniques and their clinical utility in breast cancer.
To circumvent the long turnaround times and requirement for expensive thermal-cycling machinery associated with NGS, ddPCR and real-time PCR methods, isothermal amplification reactions have emerged as attractive alternatives. Loop mediated isotheral amplification (LAMP) 4 in particular has received significant attention as a molecular diagnostic tool due to its ability at the point of sample collection to provide highly sensitive and specific results at a significantly lower cost 5–8. If validated, this approach could be ideally suited for liquid biopsy where 1 mm tumour is predicted to be associated with 1 copy of ctDNA in the circulation 9, ctDNA making up as little as 0.01% total cfDNA 10.
Several variations of LAMP have been successfully demonstrated. Itonaga et al., 11 developed a PNA-LNA mediated LAMP approach for mutant KRAS allele detection (helpful for patient stratification with anti-EGFR therapies), reproducibly detecting mutant alleles within samples diluted down to 0.1% mutant to wild type ratio, in contrast to 1% mutant to WT ratio when using a PNA-clamping PCR approach. Kumasaka et al., 12 developed mutation-oriented LAMP (MO-LAMP), a 5-primer reaction (FIP, BIP, F3, B3 plus loop backwards primer) plus a PNA for the sensitive detection of KRAS gene mutations. Arjuna et al., 13 developed allele-specific LAMP (AS-LAMP), a typical 6-primer LAMP reaction, incorporating the T790M EGFR mutation or wild type counterpart in the backward inner primer (BIP). Subsequent application on cfDNA template material (termed CF-LAMP) included a pre-LAMP conventional PCR using LAMP outer primers (F3 and B3) to enrich for copies of target cfDNA prior to CF-LAMP 14. Our previous work describes a universal self-stabilising single base-LAMP method (USS-sbLAMP) 5, a typical 6-primer reaction encoding single base variant selectivity at the 5’ end of both the forward and backward inner primers (FIP and BIP), coupled with unmodified self-stabilising (USS) competitive primers for enhanced prevention of un-specific wild type variant base amplification. Using this method, selective amplification of the PIK3CA c.3140 A > G (H1047R) cancer mutation was validated using DNA from patient breast tumour tissue samples and the assay successfully piloted on an ISFET (ion-sensitive field-effect transistor) based CMOS integrated Lab-on-Chip (LoC) system 15. Furthermore, this approach was applied to assays targeting the estrogen receptor gene, ESR1 p.E380Q and p.Y537S mutations, both reproducibly detecting 1,000 copies of synthetic DNA in under 25 minutes on the LoC platform, with further optimisation ongoing towards reproducible 100 copy sensitivity 16.
Whilst these methods show great promise, the short length and heavily fragmented nature of cfDNA 17 remains a challenge. Sequencing-based approaches identify a prominent peak at 166 bp with several factors shown to influence the recovery and quality of plasma cfDNA 18. Optimal LAMP primer design typically exceeds this target length, requiring a minimum of 200 bp sequence for a 4-6 primer reaction as typified by our own previous 214 bp and 208 bp LAMP reactions 15,16. This study aims to demonstrate a more compact allele specific LAMP reaction, more likely to fit within the cfDNA template span (see Figure 1 schematic). Mini-LAMP selectivity for mutant over wild type is maintained via dual FIP and BIP 3’- mutation recognition plus competitive inhibition of non-selective allele amplification with the use of universal self-stabilising (USS) primers. Termed AS-Mini-LAMP + USS and spanning 155 bp of the ESR1 c.1138G>C E380Q template DNA, this approach selectively amplified synthetic DNA to a sensitivity of 500 mutant copies in under 25 minutes ahead of validation via ddPCR on E380Q confirmed patient cfDNA material.