MOLECULAR PATHOGENESIS OF MULTIPLE MYELOMA

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"Our major focus has been to identify and characterize translocations to the IgH locus (chromosome 14q32.3) in multiple myeloma (MM) cell lines and tumors. Until recently, IgH translocations were detected only infrequently by conventional karyotypic analysis of MM tumors, and the partner chromosome was identified rarely (14q+). To determine the frequency of IgH translocations and the identity of partner chromosomes (and oncogenes) involved in the pathogenesis of MM, we assembled a panel of 30 EBV negative MM cell lines. These lines were analyzed by a combination of conventional karyotypes, FISH using IgH and partner chromosome probes, spectral karyotyping (SKY), and Southern blotting plus cloning to identify molecular translocation breakpoints. Although these studies are not yet fully completed, we are able to conclude that: 1) each line has one or more IgH translocations; 2) most IgH translocations involve IgH switch regions; 3) cloned breakpoints are scattered over a large region, often hundreds of kb from the dysregulated, overexpressed oncogene; 4) at least 7 lines have two independent IgH translocations; 5) we have reported the first example of a translocation that simultaneously dysregulates two potential oncogenes (with t[4;14] translocations, there is dysregulation of FGFR3 and a novel gene called MM.SET that is related to the MLL gene); 6) the same molecular translocation was present in the primary tumor cells of the 4 lines for which we were able to obtain tumor; 7) three chromosomal loci (cyclin D1 at 11q13; FGFR3 tyrosine kinase receptor and MM.SET at 4p16.3; and the c-maf basic zip transcription factor at 16q23) each account for about 20% of IgH translocations in MM, even though the 4;14 and 14;16 translocations are not detected by conventional karyotypes; 8) our results, together with results from other laboratories demonstrate the involvement of 6 additional recurrent chromosomal partners and 9 single incidence partners involved in an Ig translocation in MM. Recently we have developed FISH probes that will enable us to efficiently detect IgKappa and IgLambda translocations. We are continuing additional SKY and FISH analyses with IgH and IgL probes, as well as molecular cloning and expression studies on these lines and primary tumor samples to gain a more complete insight into the role of Ig translocations in MM. Our working hypothesis is that translocations to Ig loci provide one of the initial immortalizing events in the molecular pathogenesis of myeloma, and occur during plasma cell development in germinal centers." "A second focus is to clarify the significance of our finding that there is selective expression of one c-myc allele in 7 informative MM cell lines (confirmed in the corresponding tumor in 2 cases) despite the apparent absence of a translocation, rearrangement, or amplification involving the c-myc locus. From a combination of FISH and SKY analyses, we have evidence for karyotypic abnormalities of the c-myc locus in virtually all MM cell lines that we have examined. Thus it seems clear that the selective expression of one c-myc allele in MM lines is a consequence of a tumor specific structural abnormality that alters the chromosomal context of one of the two parental c-myc alleles. In all informative cases, it is clear that the myc structural abnormality was present in the primary tumor as well as in the cell line. The molecular mechanism and time of occurrence of c-myc structural abnormalities during pathogenesis of MM remain to be determined" "A third focus is to define other kinds of genetic and phenotypic abnormalities in MM. First, we have screened for ras and FGFR3 mutations in a panel of 36 MM lines, and for FGFR3 mutations in 6 of 30 primary MM tumors that have the t(4;14) translocation, with preliminary results consistent with mutation in ras or FGFR3 contributing to tumor progression in about 40% of MM tumors. Second, we are screening for p53 and bax mutations in the cell lines. Third, with Lou Staudt, we are doing a lymphochip analysis of mRNA expression in 28 of our well-characterized MM cell lines."