title: Detection of clonal antigen receptor gene rearrangement in dogs with lymphoma by real-time polymerase chain reaction and melting curve analysis.
abstract: BACKGROUND
  Molecular techniques that detect canine lymphoma cells by their clonal antigen receptor gene rearrangement play an increasing role for diagnosis as well as for monitoring minimal residual disease during and after cytostatic therapy. However, the methods currently available are time-consuming and/or cost-intensive thus impeding the use in clinical routine. The aim of the present study was to develop and evaluate a real-time polymerase chain reaction (PCR) with subsequent melting curve analysis (MCA) for the detection of clonally rearranged antigen receptor genes in dogs with B and T cell lymphoma on non formalin-fixed and paraffin-embedded lymph node samples.
  
  
  RESULTS
  In lymph node aspirates from 30 dogs with multicentric B cell lymphoma, real-time PCR with MCA detected clonal rearrangement in 100% and conventional PCR with polyacrylamide gel electrophoresis (PAGE) in 93% of samples. Both methods correctly identified clonality in 80% of lymph node aspirates of 10 dogs with T cell lymphoma. None of the two PCR systems detected clonal rearrangement in samples from 9 dogs with lymph node hyperplasia. Using a dilutional series with regular lymphoid desoxyribonucleic acid (DNA), detection limits of lymphoma DNA were as low as 0.8% and 6.25% for B and T cell clonal rearrangement with real-time PCR and MCA and at 3.13% and 12.5% with the conventional system. Median absolute detection limits of lymphoma DNA were shown to be at 0.1 ng and 1 ng for the B and T cell immunophenotype with the real-time PCR system and at 10 ng each with conventional PCR and PAGE.
  
  
  CONCLUSIONS
  Real-time PCR with MCA is a convenient and reliable method with a good analytical sensitivity. Thus, the method may assist the detection of clonal antigen receptor gene rearrangement in canine lymphoma patients in a clinical setting also in the presence of small amounts of neoplastic cells.
authors.affiliation: Small Animal Clinic, University of Veterinary Medicine, Buenteweg 9, 30559 Hannover, Germany. K.Langner@gmx.de.
authors.forename: Kathrin F A
authors.initials: KF
authors.lastname: Langner
authors.forename: Alexa E
authors.initials: AE
authors.lastname: Joetzke
authors.forename: Verena
authors.initials: V
authors.lastname: Nerschbach
authors.forename: Nina
authors.initials: N
authors.lastname: Eberle
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Schuberth
authors.forename: Mirja
authors.initials: M
authors.lastname: Koy
authors.forename: Ingo
authors.initials: I
authors.lastname: Nolte
authors.forename: Daniela
authors.initials: D
authors.lastname: Betz
mesh_terms: D000818:Animals; D004283:Dog Diseases / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000378:metabolism / Q000662:veterinary*; D016399:Lymphoma, T-Cell / Q000235:genetics / Q000378:metabolism / Q000662:veterinary*; D009691:Nucleic Acid Denaturation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011946:Receptors, Antigen / Q000235:genetics / Q000378:metabolism*
chemical_list: D011946:Receptors, Antigen