ALBERT

Description: Abstract 4045 Multiple Myeloma (MM) is characterized by accumulations of chromosomal abnormalities throughout the course of disease. Recurrent IgH translocations such as t(4;14) or deletion (del)13 occur during early stages of disease or perhaps as originating events. Other abnormalities appear to be acquired late in the disease process, after development of frank MM, including p53 deletion (Ch17) and amplification of CKS-1B (Ch1). We have previously identified drug resistant clonotypic B lymphocytes in MM that have malignant characteristics and can xenograft MM to immunodeficient mice. Using an automatic scanning system we scanned bone marrow (BM) cytospin slides stained with May-Grünwald Giemsa to identify lymphocytes and to determine whether or not they share the same chromosomal abnormalities that are found in autologous plasma cells (PC). For 200 MM patients, we performed interphase FISH using commercial probes, to detect deletion of chromosome 13, any translocations in 14q32, t(4;14)(p16;q32),t(11;14)(q13;q32), deletion of p53 and a custom probe to detect amplification of CKS-1B. For patients harboring a given chromosomal abnormality in their PC, we found the identical abnormalities in BM lymphocytes for 35% of patients having del13, 30% having t(4;14) and 23% having t(11;14). Likewise we detected amplification of locus 4p16, 11q13, 14q32, trisomy 1 and trisomy 17 in 45%, 15%, 30%, 37.5% and 44% respectively in the lymphocytes of patients demonstrating those same abnormalities in the PC. In a more limited series of experiments using immuno-FISH, we observed that BM lymphocytes with chromosomal abnormalities expressed CD20. In contrast, amplification of CKS-1B or p53deletion were undetectable in lymphocytes from patients having these abnormalities in their PC. We are currently analyzing the impact of abnormalities present in BM lymphocytes with regards to the clinical parameters and survival. An update of these results will be given at the meeting. Given the compelling evidence that B cells comprise a clinically important compartment of the MM clone, this work suggests that fundamental genetic events shared by all compartments of the MM clone include recurrent IgH translocations and Ch13 deletions. However, further clonal expansion occurring during disease progression and after relapse may be restricted to the PC population, characterized by acquisition of p53 deletions and CSK1B amplification. Furthermore in overt MM, clonal expansion may simultaneously occur on at least two levels, firstly that of the B cells which persistently give rise to PC, and secondly that of PC themselves as they continue clonal expansion in the absence of contributions from the B cells. Abnormalities such as del13 or IgH translocations characterize early events in the disease while p53del or amplification of CKS-1B may occur as progression events restricted to PC. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2981 Typically MM involves a single monoclonal protein, predicting a single CDR3 peak. We have shown that a sizeable number of patients do not fit this pattern. RT-PCR and genomic PCR (gPCR) were performed using universal primers that bind to FR3/JHc or FR3/CH1 to identify the monoclonal CDR3. In a cohort of 49 MM patients, 10 (20%) exhibited two CDR3 peaks, one of the clinically identified MM clone and another “second” clone. With one exception, a biclonal gammopathy, these second clones were not detected serologically by serum protein electrophoresis or immunofixation. Molecular biclonality is categorized into 3 groups based on the isotype of second clones: group I (1 IgD and 4 IgG MM) where second clone has a pre-switch isotype; group II (3 IgG MM) where the second clone has a post-switch isotype and group III (2 IgA MM) where the second clone exhibits ongoing class switch recombination. All MM clones undergo somatic hypermutation (2.44-15.03%, median = 8.55%). For second clones, 2/10 are unmutated (0-1.05%, below 2% cut off) and 8/10 are mutated (2.44-13.54%, median = 5.90%). The VH3 gene family was utilized in 6/10 of MM clones and 5/10 of second clones. For 10/10 patients, the clinical MM clones are unrelated to second clones as shown by different combinatorial VDJ, different length of CDR3 and different VDJ junction. Biclonal distribution was compared by grading of CDR3 peak heights generated by gPCR. For 9/10 patients, the MM clone is most frequent in BM, 3 of which also predominated in blood. For 6/10 patients, second clones predominated in blood. Q-PCR of mononuclear cells (MNC) showed that second clones ranged from 0.06–24% (median = 1%, n=7) in BM and 0.1–31% (median = 1.45%, n=6) in blood. Chromosomal abnormalities in plasma cells (PC) were seen in 8/8 patients analysed; among these, 4 had t(11;14). Other genetic abnormalities included deletion 13q14, amplification 1q21, trisomy and tetrasomy of chromosome 1. For 4/5 patients where second clones could be distinguished from the MM clone by IgH immuno-FISH, chromosomal abnormalities were detected in the second clone, which in some cases were different from those in the MM clone. Longitudinal analysis was studied in 3 patients. For case 1, a biclonal gammopathy, the IgGλ clone exhibited deletion 13q14 and amplification 1q21, and was dominant in a sternal biopsy (IgGλ/IgGκ = 3.6%/

Description: Abstract 783 Malignant plasma cells from patients with multiple myeloma (MM) display recurrent chromosome translocations involving the immunoglobulin heavy chain locus (IGH) and CCND1 [t(11;14)] or FGFR [t(4;14)]. It is poorly understood why recombination events between chromosomes recur at specific breakpoints in the human genome, but spatial proximity of translocation-prone gene loci (TPGL) is influential. Utilizing 3D fluorescent in situ hybridization and 3D analysis techniques, we have measured the spatial and radial positioning of translocation-prone and control loci in the nuclei of non-malignant cells to determine a propensity to translocate. Purified CD34+ progenitors and CD19+ B cells were analyzed; none had translocations. Utilizing these cell subsets (n=900 cells) from MM patients having recurrent IGH translocations in autologous plasma cells, and comparable subsets (n=300 cells) from healthy donors, we show that spatial genome organization contributes to the formation of recurrent translocations. We show that IGH, CCND1, and FGFR3 are preferentially positioned in close proximity relative to each other in presumptively normal CD34+ progenitors and CD19+ B-cells from these MM patients, and that the clinical frequency of IGH translocations correlates with relative positioning (p=0.002 for CD34+ cells; p=0.017 for CD19+ cells), and with radial positioning (p=0.05 for CD34+ cells; p=0.043 for CD19+ cells). We also show that non-malignant CD19+ B-cells from MM patients display cancer-specific radial positioning of TPGL in the nucleus (p≤0.008), suggesting a predisposition to translocate. In addition to preferred positions within the nucleus, genes also localize to favoured locations within their respective chromosome territory (CT). Genes have been shown to position outside of their respective chromosome territory. Although proximity of potential translocation partners is necessary for translocation events to occur, others have shown that translocation events between adjacent chromosomes take place at CT boundaries, or in the space of intermingling between adjacent CTs. Active transcription ‘factories' are also present within the area of intermingling. We observed that at least one of the two alleles of CCND1 and FGFR3, is positioned outside of its chromosome territory in 59% of CD19+ cells from MM patients, whereas a control locus TGFBR2, is positioned outside its chromosome territory in only 25% of the same cells. We find myeloma-specific positioning of TPGL in a subset of B-cells that lack translocations and are predominantly polyclonal, defining them as non-malignant. It seems likely that the original parent B-cell that gave rise to MM also harboured spatially proximal TPGL prior to the formation of physically translocated loci. Together, these observations support the likelihood that translocations occur only in specific sites within the nucleus. Recent reports indicate that ongoing transcription is necessary for functional activity of IGH recombination enzymes, and that these same enzymes have off-target effects on proximal genes. IGH and its translocation partners FGFR3 and CCND1 may come together briefly to a transcription ‘factory' outside of their respective CTs and be acted upon by IGH recombination enzymes. We speculate that cancer-specific positioning of active TPGL in B-cells from MM patients promotes the formation of clinically important IGH translocations, perhaps through co-localization of TPGL to nuclear transcription ‘factories' outside of CTs. The strong correlation between locus positioning and the clinical frequency of recurrent IGH translocations involving these loci is consistent with the idea that spatial proximity in the nucleus is an important contributor to the high frequency of recurrent translocations. CCND1 and FGFR3 are positioned outside their CT and in close proximity to IGH as compared to TGFBR2 and c-MAF, both of which are more peripheral in the nucleus and position out of their respective CTs at lower frequency. This is consistent with the fact that c-MAF/IGH translocations are relatively infrequent and TGFBR2/IGH translocations have not been reported. Our results suggest that the formation of recurrent IGH translocations in MM is determined not only by spatial proximity of gene loci and radial positioning in the cell nucleus, but also by positioning of loci outside of their chromosomal territories. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3930 Risk associations with genetic changes, especially SNPs, can be most meaningfully interpreted when the functional relevance of the involved gene(s) is known. Further, using targeted sequencing to detect SNPs provides for unequivocal allele “calling” in each patient, as well as identification of any linked low penetrance mutations that might influence risk. HAS1 is overexpressed and aberrantly spliced in malignant cells from multiple myeloma (MM) and Waldenstrom macroglobulimenia (WM), but not in healthy donors (HD); HAS1Vb correlated with reduced survival in a cohort of MM patients1. In transfectants, HAS1 variants are oncogenic in vivo and/or in vitro2. As shown here, a set of three intron 3 SNPs in linkage disequilbrium have significantly different genotype and allele frequencies and robust hazard ratios in people with B lineage malignancies as compared to age matched controls and to a set of unlinked exon 3 HAS1 SNPs. In all patient and control groups, all five SNPs met Hardy-Weinberg equilibrium. We sequenced an 850bp segment of the HAS1 gene (exon 3, intron 3) from PBMC of 307 Caucasian individuals, including 86 MM, 70 monoclonal gammopathy of undetermined significance (MGUS), 25 WM, 40 B chronic lymphocytic leukemia (CLL), 15 affected and 21 unaffected members of a monoclonal gammopathy prone Icelandic kindred, and 60 age-matched HD. Using direct or subclone sequencing, we evaluated the frequencies of NCBI designated minor alleles for the 5 SNPs. Bioinformatics and in vitro mutagenesis experiments confirmed that intron 3 plays a central role in clinically relevant splice site selection and aberrant intronic HAS1 splicing3. The linked intron 3 HAS1 SNPs (rs11084110, rs11084109 and rs11669079) in patient groups have significantly different host genotype and allele frequencies from those of HD. The frequencies of the unlinked exon 3 HAS1 polymorphisms (rs61736495, rs11084111) on the same 850bp amplicon were not significantly different between patients and HD, providing internal controls for sequencing artefacts. Associations between risk of B cell malignancy and HAS1 SNPs were evaluated using the logistic regression model. Compared to age matched controls, the HAS1 intron 3 SNPs were significantly associated with MM and MGUS for genotype frequencies (p.01 to.05) and allele frequencies (p.01 to.0007); the association was even stronger for CLL and WM (genotype frequencies p

Description: Abstract 3931 Clinically important aberrant splicing of the hyaluronan synthase 1 gene (HAS1) occurs in malignant B cells from multiple myeloma (MM) and Waldenstrom macroglobulimenia (WM) patients but is undetectable in B cells from healthy donors (HD)1. HAS1 splice variants are generated by aberrant alternative splicing (AS) within exons 3, 4 and 5, involving exon 4 skipping (Va), partial intron 4 retention (Vc, Vd) or a combination of both (Vb). Aberrant splicing of HAS1Vb has previously shown to correlate with reduced survival in a cohort of MM patients1. In patients, frequent mutations have been identified in introns 3 and 4, suggesting their roles in AS1. To identify genetic regions involved in splice site selection leading to HAS1Vb expression, we performed in vitro sequence manipulation of introns 3 and 4. We established a mammalian expression system to analyse HAS1 splicing by fusing a minigene extending from exon 3 to exon 5 with the upstream cDNA sequence. In an unaltered sequence of the HAS1 minigene, splicing of HAS1 full length predominated, with trace amount of variants, including the novel HAS1Vd, identified for the first time in transfectants. HAS1Vd shared an alternative acceptor site with HAS1Vb (retained 59 bp of downstream intron 4) but unlike HAS1Vb, retained exon 4. Analysis of peripheral blood mononuclear cells (PBMC) revealed that 9% of healthy donors (n=102) and MM patients (n=93) expressed HAS1Vd. About 2% coexpressed HAS1Vb and Vd. In this cohort of patients, HAS1Vb was found in 20% of unfractionated MM PBMC compared to 5% in HD PBMC, supporting our previous finding that HAS1Vb was restricted to sorted MM B cells1and suggesting that in HD PBMC, non-B cells are responsible for this splicing. While MM PBMCs expressed HAS1Vb〉Vd, HD PBMCs and transfectants expressed HAS1Vd〉Vb, indicating that splicing directed by the minigene construct was substantively different from that occurring in MM patients. In transfectants, partial deletion of HAS1 intron 4 increased HAS1Vd expression but did not affect HAS1Vb despite the fact that both variants use the same 3' splice site in intron 4. Proper joining of exons 3, 4 and 5 required a minimum of 172 bp of downstream intron 4 and acceptor site selection may be regulated by sequence between 172 and 84 bp upstream of exon 5. Thus, changes in intron 4 alone were insufficient to promote the splicing pattern observed in patients (i.e., elevated HAS1Vb). We then employed site directed mutagenesis to alter multiple G-rich regions in downstream intron 3, a strategy which enhanced exon 4 skipping as shown by increased HAS1Va expression, but did not lead to HAS1Vb splicing. Minimal manipulation to promote exon 4 skipping included base changes within the 2 tandem G-repeats (8 bp) located 73 bp upstream of exon 4, where splicing enhancer/silencer sequences were also identified. Thus, changes in intron 3 also affected the HAS1 splicing profile but like deletions in intron 4, were insufficient on their own to promote HAS1Vb expression. We next developed expression constructs that combined deletion in intron 4 with mutations in intron 3. We are able to demonstrate that when both introns were co-modified, the splicing pattern shifted towards increased HAS1Vb expression, similar to that observed in malignant cells from MM patients. Our previous work showed that deletions or mutations in the 3' end of intron 4 are frequent in MM; in silico analysis predicts splicing to form HAS1Vb2. To determine whether intron 3 genetic changes occur in patients, we sequenced intron 3 from genomic DNA of 50 MM PBMC. In 22/50 patients, 18 recurrent mutations unique to MM were identified in intron 3; many were also recurrent in our previous study2. Individual mutations recurred in 2–7 patients. Among these, 17/18 recurrent mutations increased the G-C content of intron 3 and 6/18 created or disrupted G runs in intron 3. This supports the idea that in MM patients, cumulative variations in introns 3 and 4 alter splice site selection, operationally resulting in loss of HAS1Vd and enhanced expression of the clinically relevant HAS1Vb variant. We speculate that individuals who accumulate genetic variations in introns 3 and 4 of HAS1 are predisposed to aberrant splicing of HAS1 which may contribute to development of malignancy in MM and WM. Disclosures: Belch: Celgene: Research Funding; Onyx: Research Funding.