detect the common Myeloproliferative Neoplasm- associated mutations - PowerPoint PPT Presentation

Development of a NGS approach to simultaneously detect the common Myeloproliferative Neoplasm- associated mutations in JAK2 , CALR , and MPL T. Frawley, C. O’Brien, M. Percy, E. Conneally, E. Vandenberghe, S. Langabeer, & K. Haslam

Myeloproliferative Neoplasms MPN classification Overlapping clinical features of MPN

MPN driver mutations PV ET PMF

Profile of mutations in MPN Molecular Diagnostic Algorithm JAK2 MPL CALR

Next Generation Sequencing Ion Torrent PGM

Materials and Methods NGS of 35 samples Multiplex PCR PCR Multiplex positive for MPN Primer Design Product Optimisation Optimisation associated Equalisation mutations

Results - optimisation NGS GEL JAK2 Ex 12 No of CALR Reads MPL JAK2 Ex 14 Primer Concentration

NGS results Known mutations Mutations sequenced No of Gene Positive Mutation HGVS.c Detected Samples Mutation 1 CALR 52bp del 52 bp del c.1099_1150del 1 52bp del 52 bp del c.1099_1150del L367T c.1132G>C 52bp del c.1099_1150del 1 52bp del 100% 12bp del 13bp del c.1126_1143delinsTG concordance 5bp ins c.1154_1155insTTCTC 3 5bp ins 34bp del c.1099_1132del 1 34bp del 49bp del c.1097_1149del 1 49bp del 2bp ins c.1123_1125delinsTGTTT 1 2bp ins 31bp del c.1111_1141del 1 30bp del 46bp del c.1100_1145del 1 46bp del 19bp del c.1199_1218del 1 19bp del W515L c.1544G>T 5 MPL W515L W515K c.1543_1544TG>AA 3 W515K V617F c.1849G>T 14 JAK2 V617F K539L c.1615_1616delinsTT 1 K539L

Increased Resolution AAA del TGTTT ins CALR 2bp ins c.1123_1125delinsTGTTT

Conclusion • Single assay vs stepwise algorithms • Simultaneous screening for rare and common mutations • 100% specificity • Good Sensitivity • Increased resolution • Sample preparation is labour intensive for high throughput • Automation