Automated JAK2V617F quantification using a magnetic filtration system and sequence-specific primer-single molecule fluorescence detection.

We established an automated mutational analysis detection system using magnetic filtration and the sequence-specific primer-single molecule fluorescence detection (SSP-SMFD) assay to identify the janus activating kinase-2 (JAK2)(V617F). DNA was extracted from 100 microL of whole blood automatically by a magnetic filtration system. The JAK2 1849G-->T mutation occurs in chronic myeloproliferative disorder (CMPD), and the detection of this change has diagnostic potential. To detect and semiquantitate this mutation, we used two artificial oligonucleotides (wild-type specific and mutated-type specific) and performed the SSP-SMFD assay using an automated fluorescence cell sorter measuring device. The SSP-SMFD assay can detect the presence of a minimum of 5% of the mutated artificial oligonucleotide, thus indicating that this technique is available in detecting contamination of at least 5% cells with the homozygous JAK2(V617F) mutation. Based on this technique, we analyzed 94 patients with CMPD and compared with the results obtained by the polymerase chain reaction (PCR)-direct sequence. Two homozygous JAK2(V617F) patients were identified as heterozygous JAK2(V617F) by the PCR-direct sequence, and four patients judged as wild-type JAK2 by the PCR-direct sequence were identified as heterozygous JAK2(V617F) by the SSP-SMFD method. Our automated system is simple and suitable for high-throughput analysis in detecting JAK2(V617F) with a threshold detection limit of 5%.

Genomic Sequencing Identifies ELF3 as a Driver of Ampullary Carcinoma.

Ampullary carcinomas are highly malignant neoplasms that can have either intestinal or pancreatobiliary differentiation. To characterize somatic alterations in ampullary carcinomas, we performed whole-exome sequencing and DNA copy-number analysis on 60 ampullary carcinomas resected from clinically well-characterized Japanese and American patients. We next selected 92 genes and performed targeted sequencing to validate significantly mutated genes in an additional 112 cancers. The prevalence of driver gene mutations in carcinomas with the intestinal phenotype is different from those with the pancreatobiliary phenotype. We identified a characteristic significantly mutated driver gene (ELF3) as well as previously known driver genes (TP53, KRAS, APC, and others). Functional studies demonstrated that ELF3 silencing in normal human epithelial cells enhances their motility and invasion.