107P Ultra-sensitive monitoring mutations in cancer patients using superRCA mutation detection assays

Rare tumor-specific mutations in patient samples serve as excellent markers to monitor the course of malignant disease and responses therapy clinical routine, improved assay techniques are needed for broad adoption. We describe herein - superRCA assays which provides rapid highly specific detection DNA sequence variants present at very low frequencies samples. Using a standard flow cytometer we demonstrate precise, ultra-sensitive single-nucleotide mutant sequences from cells against 100,000-fold excess DNA, follow patients treated acute myeloid leukemia (AML). Sequence interest first enriched by targeted PCR amplification sample converted circles that subjected rolling-circle (RCA). Padlock probes or wild-type then used probe repeated RCA products with exquisite specificity, followed circularized probes. The large clusters result each starting circle referred products. limit high precision consequences selective genotyping target combination numbers may be conveniently analyzed cytometry. As illustrated UPN125, NGS-analysis failed detect remaining IDH2 p.R172K mutation after initial treatment was therefore paused, although later ddPCR analyses both clearly revealed clone, subsequently leading relapse this patient. Even levels leukemic post SCT-setting would prompt action, mainly reducing immunosuppressants boost immunological effect SCT order eradicate clones risk giving rise relapse. procedure is suitable routine use virtue its sensitivity simplicity. 3-hr protocol only requires five additions sample, separated incubations, before reaction using cytometer.