title: Heteroduplex PCR analysis of rearranged immunoglobulin genes for clonality assessment in multiple myeloma.
abstract: OBJECTIVE
  Molecular analysis by PCR of monoclonally rearranged immunoglobulin (Ig) genes can be used for diagnosis in B-cell lymphoproliferative disorders (LPD), as well as for monitoring minimal residual disease (MRD) after treatment. This technique has the risk of false-positive results due to the "background" amplification of similar rearrangements derived from polyclonal B-cells. This problem can be resolved in advance by additional analyses that discern between polyclonal and monoclonal PCR products, such as the heteroduplex analysis. A second problem is that PCR frequently fails to amplify the junction regions, mainly due to somatic mutations frequently present in mature (post-follicular) B-cell lymphoproliferations. The use of additional targets (e.g. Ig light chain genes) can avoid this problem.
  
  
  METHODS
  We studied the specificity of heteroduplex PCR analysis of several Ig junction regions to detect monoclonal products in samples from 84 MM patients and 24 patients with B cell polyclonal disorders.
  
  
  RESULTS
  Using two distinct VH consensus primers (FR3 and FR2) in combination with one JH primer, 79% of the MM displayed monoclonal products. The percentage of positive cases was increased by amplification of the Vlamda-Jlamda junction regions or kappa(de) rearrangements, using two or five pairs of consensus primers, respectively. After including these targets in the heteroduplex PCR analysis, 93% of MM cases displayed monoclonal products. None of the polyclonal samples analyzed resulted in monoclonal products. Dilution experiments showed that monoclonal rearrangements could be detected with a sensitivity of at least 10(-2) in a background with >30% polyclonal B-cells, the sensitivity increasing up to 10(-3) when the polyclonal background was <1% of polyclonal B-cells.
  
  
  CONCLUSIONS
  Heteroduplex analysis of PCR amplified products is a simple and quick alternative for detecting monoclonally rearranged Ig genes in MM. This can be applied for diagnosis of B cell LPD and as a previous step in MRD strategies.
authors: Hematology Service, University Hospital of Salamanca, Spain.
authors: R
authors: R
authors: García-Sanz
authors: R
authors: R
authors: López-Pérez
authors: A W
authors: AW
authors: Langerak
authors: D
authors: D
authors: González
authors: M C
authors: MC
authors: Chillón
authors: A
authors: A
authors: Balanzategui
authors: M V
authors: MV
authors: Mateos
authors: I
authors: I
authors: Alaejos
authors: M
authors: M
authors: González
authors: J J
authors: JJ
authors: Van Dongen
authors: J F
authors: JF
authors: San Miguel
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology / Q000473:pathology; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D006801:Humans; D009101:Multiple Myeloma / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods