Simultaneous quantification of multiple single nucleotide variants in PIK3CA ctDNA using mass-tagged LCR probe sets

Circulating tumor DNA (ctDNA) in blood carries genetic variations associated with tumors. There is evidence indicating that the abundance of single nucleotide variant (SNV) in ctDNA is correlated well with cancer progression and metastasis. Thus, accurate and quantitative detection of SNVs in ctDNA may benefit clinical practice. However, most current methods are unsuitable for the quantification of SNV in ctDNA that usually differentiates from wild-type DNA (wtDNA) only by a single base. In this setting, ligase chain reaction (LCR) coupled with mass spectrometry (MS) was developed to simultaneously quantify multiple SNVs using PIK3CA ctDNA as a model. Mass-tagged LCR probe set for each SNV including mass-tagged probe and three DNA probes was firstly designed and prepared. Then, LCR was initiated to discriminate SNVs specifically and amplify the signal of SNVs in ctDNA selectively. Afterward, a biotin-streptavidin reaction system was used to separate the amplified products, and photolysis was initiated to release mass tags. Finally, mass tags were monitored and quantified by MS. After optimizing conditions and verifying performance, this quantitative system was applied for blood samples from breast cancer patients, and risk stratification for breast cancer metastasis was also performed. This study is among the first to quantify multiple SNVs in ctDNA in a signal amplification and conversion manner, and also highlights the potential of SNV in ctDNA as a liquid biopsy marker to monitor cancer progression and metastasis.