Fluorescent polymerase chain reaction and capillary electrophoresis for IgH rearrangement and minimal residual disease evaluation in multiple myeloma.
OBJECTIVE
Several polymerase chain reaction (PCR)-based techniques for tracking minimal residual disease (MRD) in B-lymphoproliferative disorders have been recently proposed. These procedures show significant variation in sensitivity and specificity. We describe an alternative assay based on fluorescent PCR combined with capillary electrophoresis and GeneScan analysis, to identify the monoclonal immunoglobulin heavy chain (IgH) rearrangement in multiple myeloma (MM) and to provide a semi-quantitative evaluation of MRD by limiting dilutions.


METHODS
Different sets of family specific primers derived from the leader region and from the framework-1 of IgH were used, with a unique reverse fluorescent primer JH. The malignant clone was identified by GeneScan and sequenced. Two tumor primers, mapping in the complementarity determining regions CDRII and CDRIII, were designed for each patient. A comparison between the nested-PCR approach and direct fluorescent PCR was performed for three patients in complete clinical remission after autologous or allogeneic bone marrow transplantation.


RESULTS
Thirty-six consecutive patients with MM were screened and monoclonality was identified in about 70% of the cases. Molecular MRD evaluation was performed in 18 patients using tumor primers. This method allowed identification of 1 neoplastic cell among 10(4)-10(6) normal cells. In three cases, negative by nested-PCR and agarose gel electrophoresis, gene scanning showed persistence of the neoplastic clone, despite the negativity of the immunofixation.


CONCLUSIONS
Capillary electrophoresis of fluorescent fragments with gene scanning provides a simple, rapid and reproducible method to detect IgH rearrangement and to evaluate MRD. Furthermore, the sensitivity reached is up to 1 log higher than that of the conventional approach with nested-PCR, even though two steps of specificity are maintained.
Novella
Elisabetta
E
Department of Cell Therapy and Hematology, San Bortolo Hospital, Vicenza, Italy.
Giaretta
Ilaria
I
Elice
Francesca
F
Madeo
Domenico
D
Piccin
Andrea
A
Castaman
Giancarlo
G
Rodeghiero
Francesco
F
D000328:Adult; D000368:Aged; D017931:DNA Primers / Q000592:standards; D019075:Electrophoresis, Capillary / Q000592:standards; D005453:Fluorescence; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods / Q000592:standards*; D009101:Multiple Myeloma / Q000175:diagnosis*; D018365:Neoplasm, Residual / Q000175:diagnosis; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity
D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains