title: Immunohistochemical loss of 5-hydroxymethylcytosine expression in acute myeloid leukaemia: relationship to somatic gene mutations affecting epigenetic pathways.
abstract: OBJECTIVE
  Genes affecting epigenetic pathways are frequently mutated in myeloid malignancies, including acute myeloid leukaemia (AML). The genes encoding TET2, IDH1 and IDH2 are among the most commonly mutated genes, and cause defective conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), impairing demethylation of DNA, and presumably serving as driver mutations in leukaemogenesis. The aim of this study was to correlate 5hmC immunohistochemical loss with the mutation status of genes involved in epigenetic pathways in AML.
  
  
  RESULTS
  Immunohistochemical staining with an anti-5hmC antibody was performed on 41 decalcified, formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with AML. Archived DNA was subjected to next-generation sequencing for analysis of a panel of genes, including TET2, IDH1, IDH2, WT1 and DNMT3A. TET2, IDH1, IDH2, WT1 and DNMT3A mutations were found in 46% (19/41) of the cases. Ten of 15 cases (67%) with TET2, IDH1, IDH2 or WT1 mutations showed deficient 5hmC staining, whereas nine of 26 cases (35%) without a mutation in these genes showed loss of 5hmC. It is of note that all four cases with TET2 mutations showed deficient 5hmC staining.
  
  
  CONCLUSIONS
  Overall, somatic mutations in TET2, IDH1, IDH2, WT1 and DNMT3A were common in our cohort of AML cases. Immunohistochemical staining for 5hmC was lost in the majority of cases harbouring mutations in these genes, reflecting the proposed relationship between dysfunctional epigenetic pathways and leukaemogenesis.
authors.affiliation: Department of Pathology, University of Massachusetts Memorial Medical Center and University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Minoti
authors.initials: M
authors.lastname: Magotra
authors.affiliation: Department of Pathology, University of Massachusetts Memorial Medical Center and University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Ali
authors.initials: A
authors.lastname: Sakhdari
authors.affiliation: Department of Pathology, University of Massachusetts Memorial Medical Center and University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Lee
authors.affiliation: Department of Pathology, University of Massachusetts Memorial Medical Center and University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Keith
authors.initials: K
authors.lastname: Tomaszewicz
authors.affiliation: Department of Pathology, University of Massachusetts Memorial Medical Center and University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Dresser
authors.affiliation: Department of Pathology, University of Massachusetts Memorial Medical Center and University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Lloyd M
authors.initials: LM
authors.lastname: Hutchinson
authors.affiliation: Department of Pathology, University of Massachusetts Memorial Medical Center and University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Woda
authors.affiliation: Department of Pathology, University of Massachusetts Memorial Medical Center and University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Chen
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis; D004252:DNA Mutational Analysis; D044127:Epigenesis, Genetic; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D049670:History, 17th Century; D006801:Humans; D007150:Immunohistochemistry; D015470:Leukemia, Myeloid, Acute / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation
chemical_list: D014408:Biomarkers, Tumor; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine
keywords: 
  IDH
  ; 5-hydroxymethylcytosine; TET2; acute myeloid leukaemia; epigenetics; immunohistochemistry