Miguel Alcaide1, Stephen Yu1, Kevin Bushell1, Daniel Fornika1, Julie S Nielsen2, Brad H Nelson2, Koren K Mann3, Sarit Assouline3, Nathalie A Johnson3, Ryan D Morin4. 1. Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada; 2. Deeley Research Centre, BC Cancer Agency, Victoria, BC, Canada; 3. Department of Medicine, Jewish General Hospital, Montreal, Quebec, Canada; 4. Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada; Genome Sciences Centre, BC Cancer Agency, Vancouver, BC, Canada. rdmorin@sfu.ca.
Abstract
BACKGROUND: A plethora of options to detect mutations in tumor-derived DNA currently exist but each suffers limitations in analytical sensitivity, cost, or scalability. Droplet digital PCR (ddPCR) is an appealing technology for detecting the presence of specific mutations based on a priori knowledge and can be applied to tumor biopsies, including formalin-fixed paraffin embedded (FFPE) tissues. More recently, ddPCR has gained popularity in its utility in quantifying circulating tumor DNA. METHODS: We have developed a suite of novel ddPCR assays for detecting recurrent mutations that are prevalent in common B-cell non-Hodgkin lymphomas (NHLs), including diffuse large B-cell lymphoma, follicular lymphoma, and lymphoplasmacytic lymphoma. These assays allowed the differentiation and counting of mutant and wild-type molecules using one single hydrolysis probe. We also implemented multiplexing that allowed the simultaneous detection of distinct mutations and an "inverted" ddPCR assay design, based on employing probes matching wild-type alleles, capable of detecting the presence of multiple single nucleotide polymorphisms. RESULTS: The assays successfully detected and quantified somatic mutations commonly affecting enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) (Y641) and signal transducer and activator of transcription 6 (STAT6) (D419) hotspots in fresh tumor, FFPE, and liquid biopsies. The "inverted" ddPCR approach effectively reported any single nucleotide variant affecting either of these 2 hotspots as well. Finally, we could effectively multiplex hydrolysis probes targeting 2 additional lymphoma-related hotspots: myeloid differentiation primary response 88 (MYD88; L265P) and cyclin D3 (CCND3; I290R). CONCLUSIONS: Our suite of ddPCR assays provides sufficient analytical sensitivity and specificity for either the invasive or noninvasive detection of multiple recurrent somatic mutations in B-cell NHLs.
BACKGROUND: A plethora of options to detect mutations in tumor-derived DNA currently exist but each suffers limitations in analytical sensitivity, cost, or scalability. Droplet digital PCR (ddPCR) is an appealing technology for detecting the presence of specific mutations based on a priori knowledge and can be applied to tumor biopsies, including formalin-fixed paraffin embedded (FFPE) tissues. More recently, ddPCR has gained popularity in its utility in quantifying circulating tumor DNA. METHODS: We have developed a suite of novel ddPCR assays for detecting recurrent mutations that are prevalent in common B-cell non-Hodgkin lymphomas (NHLs), including diffuse large B-cell lymphoma, follicular lymphoma, and lymphoplasmacytic lymphoma. These assays allowed the differentiation and counting of mutant and wild-type molecules using one single hydrolysis probe. We also implemented multiplexing that allowed the simultaneous detection of distinct mutations and an "inverted" ddPCR assay design, based on employing probes matching wild-type alleles, capable of detecting the presence of multiple single nucleotide polymorphisms. RESULTS: The assays successfully detected and quantified somatic mutations commonly affecting enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) (Y641) and signal transducer and activator of transcription 6 (STAT6) (D419) hotspots in fresh tumor, FFPE, and liquid biopsies. The "inverted" ddPCR approach effectively reported any single nucleotide variant affecting either of these 2 hotspots as well. Finally, we could effectively multiplex hydrolysis probes targeting 2 additional lymphoma-related hotspots: myeloid differentiation primary response 88 (MYD88; L265P) and cyclin D3 (CCND3; I290R). CONCLUSIONS: Our suite of ddPCR assays provides sufficient analytical sensitivity and specificity for either the invasive or noninvasive detection of multiple recurrent somatic mutations in B-cell NHLs.
Authors: Miguel Alcaide; Stephen Yu; Jordan Davidson; Marco Albuquerque; Kevin Bushell; Daniel Fornika; Sarah Arthur; Bruno M Grande; Suzan McNamara; Mathilde Couetoux du Tertre; Gerald Batist; David G Huntsman; Luca Cavallone; Adriana Aguilar; Mark Basik; Nathalie A Johnson; Rebecca J Deyell; S Rod Rassekh; Ryan D Morin Journal: Sci Rep Date: 2017-09-05 Impact factor: 4.379
Authors: Lori E Lowes; Scott V Bratman; Ryan Dittamore; Susan Done; Shana O Kelley; Sabine Mai; Ryan D Morin; Alexander W Wyatt; Alison L Allan Journal: Int J Mol Sci Date: 2016-09-08 Impact factor: 5.923
Authors: Sarah E Arthur; Aixiang Jiang; Bruno M Grande; Miguel Alcaide; Razvan Cojocaru; Christopher K Rushton; Anja Mottok; Laura K Hilton; Prince Kumar Lat; Eric Y Zhao; Luka Culibrk; Daisuke Ennishi; Selin Jessa; Lauren Chong; Nicole Thomas; Prasath Pararajalingam; Barbara Meissner; Merrill Boyle; Jordan Davidson; Kevin R Bushell; Daniel Lai; Pedro Farinha; Graham W Slack; Gregg B Morin; Sohrab Shah; Dipankar Sen; Steven J M Jones; Andrew J Mungall; Randy D Gascoyne; Timothy E Audas; Peter Unrau; Marco A Marra; Joseph M Connors; Christian Steidl; David W Scott; Ryan D Morin Journal: Nat Commun Date: 2018-10-01 Impact factor: 14.919