The evaluation of minimal residual disease in multiple myeloma by fluorescent molecular beacons in real time PCR of IgH gene rearrangements and correlation with flow cytometry.

PURPOSE: Multiple myeloma (MM) patients relapse after a period of time despite longer disease-free survival due to novel treatment options. In this study we aimed to assess the value of real-time polymerase chain reaction (RT-PCR) for detecting the immunoglobulin heavy chain (IgH) gene rearrangement using allele-specific molecular beacons as fluorescence probes to quantify minimal residual disease (MRD) and also to correlate post-treatment flow cytometric detection of plasma cells' (PCs) expression of CD19, CD38, CD45, CD56 and CD138 in MM.

METHODS

After diagnosis of 17 MM patients, the CDR1, CDR2 and CDR3 regions of the IgH gene were analysed and sequenced to identify IgH's clonal nature. Unique sequences of the clonal IgH rearrangement were used to design specific molecular beacon probes for each MM patient. Examined were also the co-expression of CD19, CD38, CD45, CD56, and CD138 molecules in bone marrow aspirates of patients with MM by flow cytometry.

RESULTS

Detection of MRD was positive in 13 (76%) of 17 patients by RT-PCR. The infiltration ratio was significantly correlated with CD138 expression (p=0.009). Significant correlation was also found between RT-PCR detection of MRD and CD138 expression (p=0.006). Nevertheless, no correlation was observed among other surface antigens (CD38, CD45, CD56).

CONCLUSION

Our results indicated that RT-PCR with specific molecular beacons provide a feasible, accurate and reproducible method for the determination of MRD in MM. Flow cytometry detection of CD138 expression may be used as a disease marker in addition to RT-PCR.