Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3

Abstract

Dysregulation of oncogenes by translocation to the IgH locus (14q32) is a seminal event in the pathogenesis of B-cell tumours. In multiple myeloma (MM), translocations to the IgH locus have been reported at an incidence of 20-60%. For most translocations, the partner chromosome is unknown (14q+); for the others, a diverse array of chromosomal partners have been identified, with 11q13 (cyclin D1) the only chromosome that is frequently involved. Recently, we developed a Southern-blot assay that detects translocation breakpoint fragments in most MM tumours, including those with no translocation detected by conventional karyotyping. In a continuing analysis of translocation in 21 myeloma cell lines and primary tumours, we show that the novel, karyotypically silent translocation t(4;14)(p16.3;q32.3) is present in five lines and at least three of ten primary tumours. The chromosome-4 breakpoints are clustered in a 70-kb region centromeric to the fibroblast growth factor receptor 3 gene (FGFR3), the apparent dysregulated oncogene. Two lines and one primary tumour with this translocation selectively express an FGFR3 allele containing activating mutations identified previously in thanatophoric dwarfism. We propose that after the t(4;14) translocation, somatic mutation during tumour progression frequently generates in FGFR3 protein that is active in the absence of ligand.

Fig. 1

Translocation breakpoints involving 4p16.3. a, Diagram of der(14) and der(4) breakpoints resulting from a translocation into Sµ. The centromere is to the left, and the figure is not to scale. Structural elements include enhancers (3'E and 5'E), switch region (Sµ) and coding segments (Cµ and JH). Thick horizontal lines depict 3'- and 5'Sµ probes. Vertical lines represent restriction enzyme sites, X = XbaI, H = HindIII, S = SphI. b, Distribution of 4p16.3 breakpoints within the distal 70 kb of a 2-Mb cos-mid contig spanning the Huntington's disease region, centromeric to the FGFR3 gene. The diagram is drawn to scale. Solid arrows indicate the positions of the cloned breakpoints for the OPM2, JIM3, H929 and KMS11 myeloma cell lines and for the tumour sample (PCL-1), while a dashed arrow shows where the breakpoints for UTMC2 have been mapped by Southern-blot analysis. Next to each arrow is indicated the type and orientation of the switch region associated with the breakpoint; the shaded designation indicates breakpoints determined by Southern blotting. The sites of restriction enzymes, indicated by a capital letter (H for HindIII, S for SphI, X for XbaI), as well as the position of the probes, indicated by thick lines, used for cloning the breakpoint or for Southern-blot analysis are depicted in the enlarged sections. The entire sequence (except for three short gaps in L75b9) of the most distal cosmid contig (L190b4, L184d6 and L75b9) in the Hunting-ton's disease region is known. The transcription orientation of FGFR3 gene is from the T7 end to the T3 end of pC385.12 (M.R. Alther, pers. comm.r).

Fig. 2

Identification of chromosomal rearrangements affecting the 4p16.3 locus. Southern blots were performed on 10 µg of DNA from three MM cell lines (KMS11 in a, H929 in b, and UTMC2 in d), from a tumour sample (PCL-1 in c) and from placenta (P). The DNA was digested with the indicated restriction enzyme and hybridized sequentially with the switch and the chromosome 4p16.3 probes listed below each line. Arrows identify the rearranged bands that co-hybridize with the switch and chromosome 4p16.3 probes as a consequence of the translocations t(4;14)(p16;q32). Additional bands not seen in placental DNA or designated by the arrow represent other rearrangements unrelated to the translocation; for instance, the 15-kb band detected by the 3'Sα probe in H929 (b) identifies a productive µ to α switch recombination event. It should be noted that the absence or altered migration of germline bands in some of the samples (for example, UTMC2 probed with 5'Sµ, 2d) is due to multiple kinds of switch recombination events (for example, translocation, productive rearrangement), genetic polymorphisms, or deletion. We refer only to the bands related to the t(4;14) translocation products.

Fig. 3

Expression of FGFR3 in MM. a, RT-PCT assay: a fragment of 598 bp has been amplified from four MM cell lines (UTMC2, KMS11, H929 and OPM2) and four tumour samples (PCL-1, JIM3-T, MM-T1 and MM-T2) by 35 cycles with a primer pair specific for the 3' half of the second immunoglobulin-like domain and the juxtapose-membrane (JM) region of the FGFR3 gene, b, Northern-blot assay: Poly(A)⁺ RNA from 100 µg of total RNA from six MM cell lines was fractionated on an agarose gel, blotted, and then hybridized sequentially with FGFR3 and β-actin probes. After five hours of exposure, the 4.5 kb-band corresponding to FGFR3 mRNA was detected in OPM2, H929 and KMS11, whereas a much longer exposure (63 hours) was needed for detection in UTMC2. c, Immunoblotting detection of FGFR3. Protein lysates from five MM cell lines were immunoprecipitated with a polyclonal rabbit antiserum against FGFR3. The immune complexes were separated on a 7.5% polyacrylamide SDS gel, blotted and detected with the same antiserum. Specific bands of 115 and 135 kD were detected in all four MM cell lines positive both by RT-PCT and northern-blot analysis. d, Selective expression of an FGFR3 variant allele in KMS11 by SSCP analysis. A primer pair flanking the juxta-membrane and transmembrane region of FGFR3 generate a 164-bp fragment by PCR from KMS11 genomic DNA and cDNA, and from placental genomic DNA (P). The products were then labelled with ³²P-dCTP, denatured and fractionated on a native 10% polyacrylamide gel, and detected by autoradiography. The informative region of the gel is shown.

Fig. 4

Two-colour fluorescence in situ hybridization of the UTMC2 MM line with FGFR3 and chromosome-14 probes. The chromosome-14 probe generates a red signal, whereas other chromosomes are stained blue. The FGFR3 cosmid clone, located on chromosome band 4p16, generates green and yellow signals, respectively, on chromosomes that are counterstained blue and red. The FGFR3 cosmid clone hybridized to the telomeric band of two chromosomes 4 (normal map position) and to the telomeric band of two chromosomes 14 [translocation t(4;14)(p16;q32)].The other two chromosomes 14 showed only the hybridization signal of the chromosome 14–specific painting probe.