title: Pseudonegative BCL2 protein expression in a t(14;18) translocation positive lymphoma cell line: a need for an alternative BCL2 antibody.
abstract: OBJECTIVE
  The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein expression in most follicular lymphomas. However, a small number of cases lack BCL2 expression despite carrying the t(14;18)(q32;q21) translocation. This study aims to explore the mechanism accounting for the lack of BCL2 protein expression when the t(14;18) translocation is present.
  
  
  METHODS
  BCL2 expression in the t(14;18) positive cell lines FL18, Karpas-422, SU-DHL-4 and SU-DHL-6, was analysed by Western blotting and by immunohistochemistry using two different antibodies. FISH analysis was performed to confirm the cytogenetic changes in the cell lines and real time quantitative PCR was used to evaluate the BCL2 mRNA level. Sequence analysis of translocated BCL2 was performed on FL18, Karpas-422, SU-DHL-4 and SU-DHL-6 cell lines.
  
  
  RESULTS
  In FL18, Karpas-422, and SU-DHL-4, the BCL2 mRNA level correlated with the BCL2 protein expression. In contrast, BCL2 protein was not detected in SU-DHL-6 line using standard anti-BCL2 antibody (BCL2/124), despite the presence of the t(14;18) translocation and high level of mRNA. cDNA sequencing of translocated BCL2 showed three mutations in the SU-DHL-6 cell line, one of which resulted in an amino acid substitution (I48F) in the region recognised by the standard BCL2 antibody, whereas the other two were silent mutations at aa71 and aa72. Interestingly, when BCL2 expression was tested with an alternative antibody, E17, the protein was detected in SU-DHL-6, suggesting that the 'negativity' of SU-DHL-6 line for BCL2 using the standard antibody is spurious. Amino acid changes were found in Karpas-422 (G47D, P59L) and SU-DHL-4 (P59T, S117R) but these did not affect BCL2 detection.
  
  
  CONCLUSIONS
  This study suggests that some somatic mutations of the translocated BCL2 gene may prevent epitope recognition by BCL2 antibodies, and hence cause false negative expression using the standard antibody. It is recommended that in practice all BCL2 negative cases should routinely be stained with an alternative antibody to prevent false negativity.
authors.affiliation: *Pathology Department, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
authors.forename: Noraidah
authors.initials: N
authors.lastname: Masir
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Campbell
authors.forename: Margaret
authors.initials: M
authors.lastname: Jones
authors.forename: David Y
authors.initials: DY
authors.lastname: Mason
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002883:Chromosomes, Human, Pair 14 / Q000235:genetics; D002887:Chromosomes, Human, Pair 18 / Q000235:genetics; D018985:Epitopes, B-Lymphocyte / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008224:Lymphoma, Follicular / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000276:immunology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014178:Translocation, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger