Technical ReportsDetection of MYD88 L265P Mutation by Real-Time Allele-Specific Oligonucleotide Polymerase Chain ReactionJiménez, Cristina BSc*,†; Chillón, María del Carmen PhD, BSc*,†; Balanzategui, Ana BSc*,†; Puig, Noemí PhD, MD*,†; Sebastián, Elena MD*,†; Alcoceba, Miguel PhD, BSc*,†; Sarasquete, María E. PhD, BSc*,†; Conde, Isabel P. BSc*,†; Corral, Rocío MD*,†; Marín, Luis A. PhD, BSc*,†; Paiva, Bruno PhD, BSc*,†; Ruano, Montserrat AS*,†; Antón, Alicia AS*,†; Maldonado, Rebeca AS*,†; San Miguel, Jesús F. PhD, MD*,†; González, Marcos PhD, MD*,†; García-Sanz, Ramón PhD, MD*,†Author Information *Department of Hematology, University Hospital of Salamanca †Center of Investigation in Cancer (CIC), Instituto Biosanitario de Salamanca (IBSAL), Salamanca, Spain C.J. and R.G.-S.: the initial designers of the study. C.J., E.S., M.C.C., and A.B.: carried out all molecular studies. M.R., A.A., and R.M.: provided technical assistance. R.G.-S., M.A., and L.A.M.: carried out the analysis. I.P.C., M.E.S., N.P., and R.C.: helped in the molecular analysis and data collection. B.P.: responsible for the immunophenotyping and cytogenetic analysis of the patients included in this series. C.J.: prepared the initial version of the paper. J.F.S.M. and M.G.: main responsible of the global and molecular groups, respectively, and they were the people responsible of the final revision of the draft as well as the ones who gave the final approval of the version to be published. R.G.-S.: reviewed the conception and design of most of the work and rewrote the paper and made the final upload of the paper. Partially supported by the grant from the Spanish ISCIII (PI12/02311), the grant reference HUS412A12-1 from the “Consejería de Educación de la Junta de Castilla y León” and the grant reference RD12/0036/0069 from “Red Temática de Investigación Cooperativa en Cáncer (RTICC)”, Spanish ISCIII. The authors declare no conflict of interest. Reprints: Ramón García-Sanz, PhD, MD, Department of Hematology, University Hospital of Salamanca, Paseo de San Vicente, 58-182, Salamanca 37007, Spain (e-mail: [email protected]). Received September 20, 2013 Accepted October 30, 2013 Applied Immunohistochemistry & Molecular Morphology: November/December 2014 - Volume 22 - Issue 10 - p 768-773 doi: 10.1097/PAI.0000000000000020 Buy Metrics Abstract MYD88 L265P mutation has been reported in ∼90% of Waldenström’s Macroglobulinemia (WM) patients and immunoglobulin M (IgM) monoclonal gammopathies of uncertain significance (MGUS), as well as in some cases of lymphoma and chronic lymphocytic leukemia. The present study aimed to develop a real-time allele-specific oligonucleotide PCR (ASO-RQ-PCR) to detect the MYD88 L265P mutation. We first evaluated the reproducibility and sensitivity of the technique with a diluting experiment of a previously known positive sample. Then, we evaluated the applicability of the methodology by analyzing 30 selected patients (10 asymptomatic WM, 10 symptomatic WM, and 10 IgM MGUS) as well as 10 healthy donors. The quantitative ASO-PCR assay could detect the MYD88 L265P mutation at a dilution of 0.25%, showing an inverse correlation between the tumor cell percentage and the cycle threshold (CT) value, thus allowing for tumor burden quantitation. In addition, mutated cases were distinguished from the unmutated by >10 cycles of difference between CTs. To sum up, ASO-RQ-PCR is an inexpensive, robust, and optimized method for the detection of MYD88 L265P mutation, which could be considered as a useful molecular tool during the diagnostic work-up of B-cell lymphoproliferative disorders. © 2014 Lippincott Williams & Wilkins, Inc.