Dysregulation of oncogenes by translocation to the IgH locus (14q32) is a seminal event in the pathogenesis of B-cell tumours 1 . 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 [2][3][4][5][6] . 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 6 . In a continuing analysis of translocations 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 an FGFR3 protein that is active in the absence of ligand.As described in detail elsewhere 6 , we generated paired probes immediately upstream and downstream of the repetitive sequences in each switch region-for example, 5'Sµ and 3'Sµ probes. By Southern-blot analysis, candidate translocation breakpoint fragments were identified as rearranged fragments that hybridize to only one switch probe. Fig. 1a illustrates a balanced translocation into Sµ, with the 3'Sµ probe detecting the der(14) breakpoint and the 5'Sµ probe detecting the der(4) breakpoint. Using this approach, we cloned one translocation breakpoint fragment involving chromosome 4 from each of five samples: four MM lines (KMS11, H929, OPM2 and JIM3) and one plasma cell leukaemia tumour (PCL-1). In each case, the novel non-Ig sequences at the breakpoint and at one end of the fragment were identical to sequences present in a cosmid contig that is centromeric to the fibroblast growth factor receptor 3 (FGFR3) gene at 4pl6.3 (Fig. 1) probes that were used in Southern-blot analyses to detect and map reciprocal breakpoint fragments for KMS11, H929 and PCL-1 (Fig. 2a-c). For example, as shown in Fig. 2a for the der(4)(5'Sµ) reciprocal breakpoint in KMS11 ( Fig. 1), a 5'Sµ probe detected a rearranged 7.9-kb XbaI fragment (compared to 7.5-kb fragment in placental [P] DNA) and a 4pl6.3 probe (9226 in Fig. 1b) that detects a 13.8-kb germline fragment in the tumour and in placental DNA, co-hybridizes to the 7.9-kb XbaI fragment in KMS11. These results, together with Southern blots using other enzymes, are consistent with the der(4) (5'Sµ) translocation breakpoint occurring near the cloned der(14) (3'Sµ) translocation breakpoint for K...

In multiple myeloma, karyotypic 14q32 translocations have been identified at a variable frequency (10-60% in different studies). In the majority of cases, the partner chromosome has not been identified (14q؉), and in the remaining cases, a diverse array of chromosomal partners has been implicated, with 11q13 being the most common. We developed a comprehensive Southern blot assay to identify and distinguish different kinds of immunoglobulin heavy chain (IgH) switch recombination events. Illegitimate switch recombination fragments (defined as containing sequences from only one switch region) are potential markers of translocation events into IgH switch regions and were identified in 15 of 21 myeloma cell lines, including seven of eight karyotyped lines that have no detectable 14q32 translocation. From all nine lines or tumor samples analyzed further, cloned illegitimate switch recombination fragments were confirmed to be IgH switch translocation breakpoints. In three of these cases, the translocation breakpoint was shown to be present in the primary tumor. These translocation breakpoints involve six chromosomal loci: 4p16.3 (two lines and the one tumor); 6; 8q24.13; 11q13.3 (in three lines); 16q23.1; and 21q22.1. We suggest that translocations into the IgH locus (i) are frequent (karyotypic 14q32 translocations and͞or illegitimate switch recombination fragments are present in primary tumor samples and in 19 of 21 lines that we have analyzed); (ii) occur mainly in switch regions; and (iii) involve a diverse but nonrandom array (i.e., frequently 11q13 or 4p16) of chromosomal partners. This appears to be the most frequent genetic abnormality in multiple myeloma.

Previously we reported that a karyotypically silent t(4;14)(p16.3;q32.3) translocation is present in about 25% of multiple myeloma (MM) tumors, and causes overexpression of FGFR3, which is 50 to 100 kb telomeric to the 4p16 breakpoints. Frequent FGFR3 kinase activating mutations in MM with t(4;14) translocations substantiate an oncogenic role for FGFR3. We now report that the 4p16 breakpoints occur telomeric to and within the 5′ introns of a novel gene,MMSET (Multiple Myeloma SETdomain). In normal tissues, MMSET has a complex pattern of expression with a short form (647 amino acids [aa]) containing an HMG box andhath region, and an alternatively spliced long form (1365 aa) containing the HMG box and hath region plus 4 PHD fingers and a SET domain. Although t(4;14) translocation results in IgH/MMSET hybrid transcripts, overexpression of MMSET also occurs from endogenous promoters on 4p16. Given the homology to HRX/MLL1/ALL1at 11q23 that is dysregulated by translocations in acute leukemia, we hypothesize that dysregulation of MMSET contributes to neoplastic transformation in MM with t(4;14) translocation. This is the first example of an IgH translocation that simultaneously dysregulates two genes with oncogenic potential: FGFR3 on der(14) andMMSET on der(4). © 1998 by The American Society of Hematology.

Preliminary results from a pilot trial on trastuzumab's mechanism of action against operable breast tumors overexpressing Her2 suggested a role for antibody-dependent cell cytotoxicity (ADCC). To examine factors affecting ADCC intensity and variability, we extended this study to the phenotypic and functional analysis of circulating mononuclear cells in 18 patients. ADCC was induced by trastuzumab therapy in 15 of 18 patients (83%). Inability to develop ADCC in three patients did not depend on inadequate levels of trastuzumab because further increase in its concentration in vitro was ineffective. Rather, susceptibility to develop ADCC was fairly predicted by test with trastuzumab before therapy and was correlated to the number of lymphocytes coexpressing CD16 and CD56. Phenotypic analysis at the end of ADCC evaluating down-regulation of CD16, and up-regulation of CD69 and CD107a, confirmed that natural killer (NK) cells and CD56 + T cells were involved in productive engagement of trastuzumab. Also, the killing efficiency of CD16 + lymphocytes was influenced by 158 V/F polymorphism of Fc;RIII (CD16), whereas variations of CD247 on NK cells were consistent with trends between ADCC before and after therapy. Complete pathologic response was observed in one patient showing ADCC of outstanding intensity, whereas four cases of partial response showed intermediate ADCC; none of the three patients unable to mount ADCC had significant tumor regression. These data indicate that quantity and lytic efficiency of CD16 + lymphocytes are major factors for ADCC induction by trastuzumab, and confirm that breast cancer responses to short-term trastuzumab monotherapy may depend on involvement of the ADCC mechanism. [Cancer Res 2007;67(24):11991-9]