title: Multiplex sequencing for EZH2, CD79B, and MYD88 mutations using archival cytospin preparations from B-cell non-Hodgkin lymphoma aspirates previously tested for MYC rearrangement and IGH/BCL2 translocation.
abstract: BACKGROUND
  Gene rearrangements and specific translocations define some B-cell non-Hodgkin lymphoma (NHL) subtypes. Genome-wide mutational studies have revealed recurrent point mutations with prognostic implications. The goals of this study were to evaluate the feasibility of applying a multiplex mutation assay to archival cytospin preparations (CPs) and to investigate the rate of EZH2, CD79B, and MYD88 mutations in B-cell NHL samples previously tested for MYC rearrangement and/or IGH/BCL-2 translocation.
  
  
  METHODS
  DNA was extracted from archival CPs of B-cell NHL cases with previous fluorescence in situ hybridization (FISH) assays for MYC rearrangement and/or IGH/BCL-2 translocation. Multiplex sequencing was performed for the detection of EZH2 (Y641), CD79B (Y196), and MYD88 (L265) mutations. Sanger sequencing was applied to samples with positive results and failed assays.
  
  
  RESULTS
  Eighty-eight archival CPs were available from 40 patients. Alterations detected by FISH were: MYC rearrangement (10 cases), IGH/BCL-2 translocations (21 cases), dual translocations (6 cases), and other abnormalities for IGH/BCL-2 (23 cases) and for MYC (16 cases). DNA concentration ranged from 1.88 to 62.85 ng/µL (mean, 9.46 ng/µL). Successful results were obtained in 88.0% of the specimens submitted to multiplex sequencing. With Sanger sequencing, 2 additional mutated cases were found, and all cases with mutations were confirmed. Eight specimens showed mutations: 6 for EZH2, 1 for CD79B, and 1 for MYD88. Among them, 5 cases showed concurrent MYC and/or IGH/BCL-2 translocations and 2 revealed abnormal signals of IGH/BCL-2 and MYC.
  
  
  CONCLUSIONS
  CPs archived for up to 6 years are a reliable source of high-quality genomic material for multiplex sequencing. Almost all B-cell NHL with point mutations showed concurrent chromosomal abnormalities.
authors: Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.|Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada.
authors: Gilda da Cunha
authors: Gda C
authors: Santos
authors: Department of Pathology, Santa Casa Medical School, Sao Paulo, Brazil.|Fleury Laboratories, Sao Paulo, Brazil.
authors: Mauro Ajaj
authors: MA
authors: Saieg
authors: Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.|Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada.
authors: Hyang Mi
authors: HM
authors: Ko
authors: Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.|Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada.
authors: William R
authors: WR
authors: Geddie
authors: Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.|Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada.
authors: Scott L
authors: SL
authors: Boerner
authors: Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada.
authors: Kenneth J
authors: KJ
authors: Craddock
authors: Division of Medical Oncology and Hematology, University Health Network, Toronto, Ontario, Canada.
authors: Michael
authors: M
authors: Crump
authors: Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.|Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada.
authors: Denis
authors: D
authors: Bailey
mesh_terms: D000328:Adult; D000368:Aged; D044963:Biopsy, Fine-Needle; D001707:Biopsy, Needle; D051925:CD79 Antigens / Q000737:chemistry* / Q000235:genetics; D015331:Cohort Studies; D016208:Databases, Factual; D000071221:Enhancer of Zeste Homolog 2 Protein; D005260:Female; D015321:Gene Rearrangement; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D016393:Lymphoma, B-Cell / Q000235:genetics* / Q000473:pathology; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008875:Middle Aged; D053594:Myeloid Differentiation Factor 88 / Q000737:chemistry* / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry; D017354:Point Mutation / Q000235:genetics*; D063151:Polycomb Repressive Complex 2 / Q000737:chemistry* / Q000235:genetics; D011237:Predictive Value of Tests; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D014178:Translocation, Genetic; D000096985:Calponins
chemical_list: D051925:CD79 Antigens; D000071221:Enhancer of Zeste Homolog 2 Protein; D053594:Myeloid Differentiation Factor 88; D010750:Phosphoproteins; D063151:Polycomb Repressive Complex 2; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; C497883:CD79B protein, human; C507779:MYD88 protein, human; C558855:SPECC1L protein, human; D000096985:Calponins; C498409:EZH2 protein, human
keywords: B-cell non-Hodgkin lymphoma; CD79B; DNA mutational analysis; EZH2; IGH/BCL2; MYC; MYD88; MassARRAY spectrometry; cytospin preparation; fine needle biopsy