BIOM-26. RAPID IDENTIFICATION OF IDH1-R132 VARIANTS IN GLIOMA WITH LOCKED NUCLEIC ACID LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LNA-LAMP)

Abstract

Abstract Point mutations that impact the catalytic site of isocitrate dehydrogenase 1 (IDH1) are associated with an extended overall survival of approximately 3.8 years for glioma patients compared to 1 year for those with wild-type IDH1. The difference in survival benefit is associated with the maximal surgical resection of IDH1 mutant tumors, making intraoperative knowledge of the mutational status valuable. Unfortunately, current methods used to determine IDH1-R132 mutational status are incompatible with obtaining real-time results. We have developed a panel of assays that use LNA-LAMP to identify commonly encountered IDH1-R132 variants (R132H, R132C, R132G, R132S, R132L) within 40 minutes. These assays require little preanalytical processing, no complex equipment, and yield results which are easily interpreted by the naked eye. Tumor samples are initially subjected to a 5-minute alkaline digest, then added directly to LNA-LAMP reactions containing primers unique to each IDH1-R132 variant. Discrimination between the wild-type and mutant allele is achieved with the integration of locked nucleic acids within primers. A reaction with an IDH1-R132R specific primer serves as the sample positive control, as all tumor lysates are expected to contain wild-type IDH1 DNA. Lysates are incubated for 35 minutes in a hot water bath, followed by visual interpretation using a pH-based colorimetric indicator, where samples positive for the mutation appear a vibrant yellow while those lacking the mutation remain pink. This method has been rigorously tested with patient-derived tumor lysates, yielding reproducible and accurate results, and has an analytical sensitivity of about 20% mutant DNA in a background of wild-type DNA. This sensitivity is comparable to that of Sanger Sequencing while simultaneously offering a more cost-effective and efficient workflow. LNA-LAMP is amenable for use in the intraoperative window to allow patients with an IDH1-R132 mutation the benefit of a maximal surgical resection.