Abstract
Hotspot codon 132 mutations (R132xIDH1m) are frequent in intrahepatic cholangiocarcinoma (ICC), are druggable by anti-IDH1m agents, and could represent a marker of disease progression. Developing an assay to identify R132xIDH1m would provide a useful tool to select patients benefitting from targeted treatments. We tested a quantitative real-time allele-specific polymerase chain reaction (qPCR)-based method to detect the main R132xIDH1m in an Italian ICC series (n = 61) of formalin-fixed paraffin-embedded (FFPE) samples, and on circulating-free DNA samples. The outcomes were compared with nested PCR/Sanger sequencing. Reconstitution experiments of plasmids harboring the different R132xIDH1m mixed with wild-type (WT) DNA demonstrated that qPCR is able to detect at least 2% of all mutated allele. High efficiency was also observed on patient-derived mutated DNA mixed with WT DNA (up to 10% and 0.3 ng of mutated template); qPCR detected 16.4% of mutated samples (one R132G, three R132C and six R132L) while nested PCR/Sanger sequencing only 8.2% (four R132L and one R132G). In a single patient with an R132C-mutated tumor, qPCR was also performed on plasma samples collected at four time-points, observing an increase correlating with disease progression. In conclusion, we developed a qPCR assay which could represent a fast, inexpensive and sensitive tool both for detection of R132xIDH1m in ICC samples and monitoring disease progression from liquid biopsy.
Highlights
Intrahepatic cholangiocarcinoma (ICC) is a malignancy arising from the epithelial cells of the intrahepatic bile duct branches of the second-order or beyond
We set up a quantitative real-time allele-specific polymerase chain reaction assay to detect with high efficiency, comparable with generation sequencing (NGS) data [21], the specific IDH1 mutation (IDH1m) both in tissue and in plasma samples
We developed a quantitative real-time allele-specific polymerase chain reaction (qPCR) assay able to identify the main IDH1R132x mutations (R132C, R132G, R132H, R132L, R132S) in formalin-fixed paraffin-embedded (FFPE) samples and cfDNA
Summary
Intrahepatic cholangiocarcinoma (ICC) is a malignancy arising from the epithelial cells of the intrahepatic bile duct branches of the second-order or beyond. The mutational status of the main ICC cancer related genes was analyzed in different cohorts of patients. A particular subgroup of ICC resulted enriched for IDH1 mutation (IDH1m) and is characterized by DNA hypermethylation, which influenced the gene expression and the cell differentiation [13]. We evaluated the incidence of mutations in the exon 4 of IDH1, focusing on the IDH1 R132x mutation, in an Italian series of 61 primary ICCs. We set up a quantitative real-time allele-specific polymerase chain reaction (qPCR) assay to detect with high efficiency, comparable with generation sequencing (NGS) data [21], the specific IDH1m both in tissue and in plasma samples. We compared the sensitivity of qPCR with Sanger sequencing, the conventional and cheapest method to detect gene mutations, frequently used in routine molecular diagnostics, but characterized by lower sensitivity
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