Characterization of Oncogene Dysregulation in Multiple Myeloma by Combined FISH and RT-PCR Analyses.

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus and various partner loci frequently are associated with multiple myeloma (MM). We investigated the expression profiles of the FGFR3/MMSET, CCND1, CCND3, MAF genes, which are involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), and t(14;16)(q32;q23), respectively. The analysis was performed by RT-PCR from purified plasma cell populations from 57 MMs and we compared the results with the presence of translocations as assessed by dual-color FISH. A t(4;14) was found in 11MMs, t(11;14) in 9 MMs, t(6;14) in 5 MM, and t(14;16) in 4 MMs. In all cases, the translocations were associated with the spiked expression of target genes. Furthermore, gene expression profiling enabled the identification of putative translocations causing dysregulation of CCND1 (1 MM) and MAF (1 MM) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. IGH-MMSET hybrid transcripts were found in 10 of the 57 (17.7%) MM samples. There was complete concordance between the findings of RT-PCR and FISH analyses of the MM samples, with 19.2% (11/57) t(4; 14) detected by FISH. Samples were separated further into three major groups based on the size of the RT-PCR product. The 1064bp, 438bp, and 275bp of IGH-MMSET were found in 7, 2, and in 1 sample, respectively. We then screened all 57 MM samples for the expression of FGFR3 using RT-PCR, with primers amplifying the 283bp fragments. Specific transcripts were detected in 11 (19.2%) samples that validate the t(4; 14) from cytogenetic studies. In the remaining 46 MM patients without t(4; 14), and 10 normal bone marrow controls, the FGFR3 amplified transcript was barely detectable. Only one patient sample without t(4; 14) revealed detectable levels of FGFR3 expression. Thus, RT-PCR assay for FGFR3 expression can detect all cases with evident or cryptic t(4; 14) translocation (P< 0.01). Using the primers corresponding to 7–10 exon in 11 cases of MM patients with overexpression of FGFR3, we directly sequenced the FGFR3 cDNA fragments amplified by PCR. Polymorphism (GGC>GGT) was detected in nine of the 11 patients. This polymorphism was tightly associated with higher expression of FGFR3. No FGFR3 mutations were found in the remaining 2 MM patients with overexpression of FGFR3. Our data indicate that RT-PCR is a sensitive and reliable method for the detection of FGFR3 and IGH-MMSET. Translocation t(4; 14) in MM detected by FISH can be validated by RT-PCR method. We examined our result by the Chi-Square test and revealed 90% sensitivity and 100% specificity. The Youden Index remains 0.9. This rapid and reliable detection of FGFR3 and IGH-MMSET overexpression may have practical clinical utility in the analysis and monitoring of the disease in MM patients with t(4; 14). Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations in MM.