ALBERT

Description: Abstract 2842 Poster Board II-818 In addition to full length (FL) transcripts, clinically significant HAS 1 splice variants (Va, Vb and Vc) have been previously identified in multiple myeloma (MM) and Waldenstrom's macroglobulinemia. Increased HAS1Vb expression correlates with poor survival in a cohort of MM patients. Here, we show that directed mutation of HAS1 intron 3 alters HAS1 splicing and generates a pattern of HAS1 variant expression that mimics patterns detected in MM patients. This suggests that hypermutation of HAS1 and consequent expression of HAS1 splice variants may contribute to oncogenesis in MM. HAS1FL comprises of 5 exons (2089 bp); Va skips exon 4 (133 bp); Vb skips exon 4 and partially retains 59 bp of intron upstream of exon 5 (+59); Vc has all 5 exons and partially retains 26 bp of intron downstream of exon 4. In MM, frequent intronic mutations have been observed in introns 3 and 4, suggesting possible contributions to HAS1 alternative splicing. We have utilized a mammalian expression system to analyse HAS1 splicing by fusing a minigene extending from exon 3 to exon 5 (g345) with the upstream cDNA sequences. HAS1 expression is determined by transfection and RT-PCR using appropriated primer sets. This study focuses on identification of intronic mutations that may affect HAS1 splicing. We target mutations on (A/U)GGG motif because of its high abundance in HAS1 intron 3. The (A/U)GGG repeat was also shown to enhance the splicing of alternative intron in chicken β-tropomyosin (Sirand-Pugnet, P, et al, NAR, 1995, 23, 3501) and intronic G runs could work in a combinatorial way to control the selection of the proper 3' splice site in human thrombopoietin (Marcucci, R, et al, NAR, 2006, 35, 132). A 580 bp long human HAS1 intron 3 is GC-rich and comprises of 28 (A/U)GGG motifs (sequentially identified as G1, G2.., G28). HeLa cells transfected with an unmutated intron 3 construct mainly produce FL with a small amount of HAS1Va, a profile that is similar to CD40L/IL-4 activated normal B cells. Site directed mutagenesis of all 28 (A/U)GGG motifs (G1-28) abolished FL expression, but not HAS1Va, suggesting that these sequence alterations are highly unfavorable for constitutive splicing. It may be due to the loss of essential cis-acting element(s) and/or undesirable conformational changes that prevent spliceosome formation. Mutagenesis of G1-18 is shown to eliminate constitutive expression by increasing the usage of multiple alternative donor sites. Mutagenesis of G19-28 produces more HAS1Va than FL, presumably due to increased exon 4 skipping events. An increased Va/FL ratio could also be achieved by mutagenesis of G25-28 or G27-28, suggesting that this subregion is important for pathway selection. Mutagenesis was also studied in del1 construct, a unique derivative of HAS1 minigene that partially deletes intron 4. Similar to g345, del1 produces FL and HAS1Va as well as promotes expression of novel HAS1Vd, an isoform that includes +59 bp (like Vb) and exon 4. Alteration of HAS1 splicing profile caused by mutagenesis shown in g345 series is also observed in del1 series. Additionally, there is a shift from Vd to Vb expression in all constructs analysed (del1/G1-28, del1/G1-18, del1/G19-28, del1/G25-28 and del1/G27-28), a pattern of aberrant splicing that found in MM patients. Thus, in del1, increased exon 4 skipping events promote both Va and Vb expression. Sequencing of HAS1 intron 3 in a cohort of 50 MM patients indicates that recurrent mutations are found in the G repeat regions and that new repeats are generated by recurrent MM-specific HAS1 mutations. This suggests that mutation of the HAS1 construct mimics HAS1 mutation events that occur in MM patients themselves, and contributes to the clinically significant aberrant HAS1 splicing we have reported in MM (Adamia et al. Blood, 2008, 112, 5111; Blood, 2005, 105, 4836). Overall, critical mutations that could alter HAS1 expression and the ratio of HAS1 variants to FL were identified in intron 3. In intron 4, critical mutations that increase the usage of alternative splice site (+59) remain to be studied. We speculate that cumulative mutations within these two intronic sequences could bring the two events together to promote HAS1Vb splicing. While trans-acting elements are likely to regulate RNA splicing and its pathway, our studies clearly suggest that intronic mutations play an important role in the aberrant splicing of human HAS1, with probable contributions to disease progression in MM. Disclosures: No relevant conflicts of interest to declare.

Description: In MM and WM, we identified aberrant HAS1 splice variants that were absent from normal donors (HD) and B-CLL. Here we sequenced multiple subclones from multiple cell subsets to show that aberrant HAS1 splicing results from cryptic splice sites activation. Aberrant splicing defects are the consequences of genetic variations (GVs) detected in the sequence of classical splicing elements as well as within exons and introns. To investigate HAS1 splicing in MM and WM patients, we sequenced the HAS1 gene segments involved in abnormal splicing events. HAS1 from buccal epithelial cells (BEC) represented the host genotype, and hematopoietic progenitors (HP), T, B and plasma cells (PC) as the normal and malignant components of the hematopoietic lineage in MM/WM. 197 GV were found in 16 MM or WM patients, but none in 9B-CLL, MGUS or HD. We found 60 germline (defined as present in BEC and hematopoietic cells) and 137 somatic GV (defined as GV found in HP, T, B and/or PC, but absent from BEC). These somatic GV include 97 tumor-specific GV found in MM and/or WM B and PC and 40 hematopoietic origin GV identified in HP, T, B and PC, but not in BEC. Some GV were recurrent, detected in more than one patient. Recurrent GV (24 in MM and 22 in WM) included both germline and somatic GVs, 6 tumor-specific, 6 hematopoietic and 14 germline origin GV, as well as 20 NCBI-SNPs. The distribution of GV indicated that some of the recurrent germline and somatic GV are restricted to MM, some are restricted to WM and some are shared by both MM and WM. None were found in B-CLL, MGUS or HD. The patterns of germline GV observed in MM and WM suggests that MM and WM patients, but not B-CLL, inherit recurrent germline GV that are necessary but not sufficient for progression to malignancy. Acquisition of recurrent, somatic HAS1 GV in HP further increases the risk of developing MM or WM. Transformation may become inevitable when tumor-specific recurrent GV are acquired. Interestingly, we detected increased homozygosity for the mutated allele of some germline GV in all cell types (PC, B, T, HPs and BECs) from MM and/or WM patients. These GV were detected in 75-90% of subclones analyzed. Mutational analysis of minigene constructs demonstrated a distribution of GV as clusters in the vicinity of HAS1splicing elements, allowing us to classify them as “splicing mutations”. This is supported by an in vitro splicing assay, which confirmed that a combination of germline and somatic GVs leads to aberrant HAS1 splicing (see abstract by Kriangkum et al., ASH 2007). Our study demonstrates that the impact of inherited and acquired GV on HAS1 gene splicing is manifested only in the context of accompanying tumor-specific HAS1 GV, that in combination give rise to the clinically significant aberrant splicing of HAS1. It also indicates that MM and WM are closely related at the genomic level, with the same recurrent somatic mutations independently arising in both diseases. Accumulation of somatically acquired HAS1 mutations in HP, in the context of inherited predispositions to MM and WM may represent a very early stage of pre-malignant development. Similar to leukemias, initiation events that contribute to MM and WM pathogenesis may arise from mutations which first accumulate during the non-malignant or pre-malignant stages of hematopoietic differentiation in progenitor cells.

Description: BACKGROUND: The centrosome is the cellular organelle that nucleates the mitotic spindle. Polo-like kinase 1 (Plk1), a centrosome-associated serine/threonine kinase, serves as a key regulator of multiple steps in mitosis. Plk1 is overexpressed in a broad spectrum of tumor types, and its expression often correlates with poor patient prognosis. We have previously observed centrosome amplification in myeloma, and have validated other components of the myeloma centrosome and mitotic apparatus as therapeutic targets. The present study explored the expression of Plk1 in myeloma and the effect of BI2536, a potent and selective inhibitor of Plk1, on myeloma cells in the pre-clinical setting. METHODS: Plk1 expression was assayed in a panel of myeloma cell lines (MMCL) and AutoMACS-purified CD138+ patient bone marrow plasma cells (BMPC). The anti-myeloma effects of BI2536, alone or in combination with conventional agents, were assayed on myeloma cells with proliferation (MTS) and apoptosis (Annexin V/propidium iodide) assays. The phenotype of treated cells was examined with DNA content analysis and immunofluorescence microscopy. The efficacy of BI2536 monotherapy was evaluated in NOD/SCID mice bearing RPMI8226 xenografts. RESULTS: Plk1 is ubiquitously expressed in myeloma to varying degrees in both MMCL and BMPC. BI2536 inhibited the proliferation of MMCL (RPMI 8226, U266, LP-1 and KMS-11) and patient BMPC at nanomolar concentrations. The addition of BI2536 was able to overcome resistance to dexamethasone. Bortezomib in combination with BI2536 had significantly increased anti-myeloma effects compared to the use of either agent alone. BI2536-treated MMCL accumulated 4N DNA content prior to undergoing apoptosis. The phenotype of BI2536-treated cells is consistent with inhibition of Plk1, showing prometaphase arrest and monopolar mitotic spindles in a dose-dependent fashion. BI2536 induces regression of human myeloma xenografts in NOD/SCID mice. Taken together, BI2536 is a promising new agent for the treatment of multiple myeloma. This work provides further evidence that Plk1 and the amplified myeloma centrosome are targets for therapy.

Description: The treatment of multiple myeloma (MM) with new drugs like Velcade and Revlimid shows promise in the clinic. These two drugs give excellent partial to complete remissions, but the disease invariably returns indicating escape of the malignant clone. The current clinical practices for quantifying minimal residual disease (MRD), such as plasma cell counts, lack needed sensitivity. PCR based assays amplify the clonotypic IgH VDJ gene rearrangement in myeloma and identify all compartments of the malignant clone, providing a unique molecular signature to monitor disease. Every myeloma cell carries one copy of the specific IgH VDJ rearrangement and provides a quantitative measure of the number of malignant cells remaining in each patient. No previous work has quantified MRD after treatment with either Revlimid or Velcade. TaqMan real-time quantitative PCR (rqPCR) assays are able to quantify the level of disease burden but probes are expensive and difficult to design, making this assay unsuitable for routine clinical use. SYBR green, however, is an inexpensive alternative to probes and utilizes the same unique molecular VDJ signature with consistency and reliability (r2=0.9997). PCR product specificity is confirmed using melting curve analysis. This is the first report of the successful use of SYBR green in quantifying the level of malignant disease in MM. The specific clonal rearrangement is identified for each patient. Patient specific primers are designed and verified as being present in the majority of plasma cells using single cell PCR. A cloned IgH VDJ product for each patient provides a standard curve for each patient-specific rqPCR. A cloned B2Microglobulin PCR product provides an independent standard curve to quantify cell number. After correcting for copy number differences, the percentage of malignant cells is calculated by dividing the number of VDJ molecules by the number of B2M molecules. For all tests, the level of residual disease in bone marrow was measured using 150ng of DNA. In patients achieving complete clinical remission during treatment with Revlimid, the SYBR green method detected 0.104% clonal cells with a range of 0.014%–0.26%. In patients with a partial remission, 3.034% clonal cells were detected, with a range of 0.62%–7.79%. A subsequent bone marrow from one Revlimid treated patient revealed no clonal cells this using SYBR Green method. In patients treated with Velcade at the time of relapse, the remaining clonal cells averaged 1.042% with a range of 0.00075%–3.11%. When Velcade was used as a frontline therapy followed by a stem cell transplant, 0.10% clonal cells remained with a range of 0%–0.287%. In one dexamethasone treated patient who achieved a complete remission, 0.018% clonal cells were found. The SYBR green rqPCR assay for patient specific clonal VDJ was shown to reliably quantify residual disease. To date, our results suggest that frontline treatment with Velcade may lead to lower levels of residual disease than treatment at the time of relapse. In Revlimid treated patients, the extent of residual disease correlates with clinical classifications of complete or partial remission. The significance of these low numbers of malignant cells remains to be established, but the persistent occurrence of relapse suggests they are clinically relevant.

Description: In this study, we show that the hyaluronan synthase 1 (HAS1) gene undergoes aberrant intronic splicing in multiple myeloma (MM). In addition to HAS1 full length (HAS1FL), we identify 3 novel splice variants of HAS1, HAS1Va, HAS1Vb, and HAS1Vc, detected in patients with MM or monoclonal gammopathy of undetermined significance (MGUS). HAS1Vb and HAS1Vc undergo intronic splicing with creation of a premature stop codon. MM cells expressing one or more HAS1 variants synthesize extracellular and/or intracellular hyaluronan (HA). Expression of the HAS1Vb splice variant was significantly correlated with reduced survival (P = .001). Together, alternative HAS1 gene splicing, the correlations between HAS1 splicing and HA synthesis, and the correlations between HAS1 splicing and reduced survival of MM patients support the hypothesis that the family of HAS1 protein plays a significant role in disease progression. Further, expression of HAS1Vb, in conjunction with HAS1FL and/or other HAS1 variants, may lead to accumulation of intracellular HA molecules and an impact on receptor for HA-mediated motility (RHAMM)-mediated mitotic abnormalities in MM. This study highlights the potential importance of HAS1 and its alternative splicing in pathophysiology of MGUS and MM. (Blood. 2005;105: 4836-4844)

Description: Interphase fluorescence in situ hybridization (FISH) is widely used as a diagnostic tool for known genetic abnormalities due to its sensitivity for detection of cryptic aberrations, such as t(4;14)(p16;q32) in multiple myeloma (MM). In many cancers, chromosomal abnormalities are prognostic indicators that also predict response to therapy. Tests to determine the type and extent of these abnormalities are increasingly essential for more informed diagnosis and choice of treatment strategies. However the cost and complexity of the current FISH protocols, and the variability arising from differences in technical approach and subjective evaluation of hybridization patterns has compromised its widespread utilization. To create a standardized platform that will be accurate, robust, cost-effective and easy to use in any clinical setting, we have developed a microfluidic platform that enables simultaneous assessment of 10 chromosomal abnormalities or 10 patients, on a single chip. Microfluidic chips are hybrid polymer/glass microsystems with miniaturized networks of wells and channels, incorporating valves, heaters and fluidic control. The 10 channel microfluidic chip used here is the size of a microscope slide. Each channel requires only 1/10 the amount of probe used in conventional FISH, thus substantially reducing the cost per test. All the probes tested gave comparable results to conventional testing. Three cell lines and three ex-vivo PBMC samples from MM patients were tested against four different chromosomal probe sets, to detect translocation (4:14), any 14q32 translocation, deletion of chromosome 13 or deletion of p53. We used a mixture of patient sample and cell line to test the robustness of our technology and were able to successfully distinguish abnormal patterns with percentages that were comparable to FISH on microscope slides. On-chip FISH was highly reliable with consistent results in multiple test runs. To automate the process of reading slide, a computer vision algorithm was developed to provide a quantitative and objective measure of staining patterns, and to eventually eliminate the requirement for human intervention. This strategy uses artificial intelligence to distinguish probe from background staining, to identify and quantify the number of cells with different chromosomal patterns. This visual processing algorithm has been validated against human interpretation and provides a sensitive and unbiased method to distinguish signal and noise within stained cells. Although reliable and reproducible hybridization occurred in as little as four hours, to further reduce the time required for FISH testing, methods to enhance the hybridization were examined. These included chip designs that implemented mechanical or electrokinetic pumping. Both methods improved the hybridization and are currently being optimized. On-chip FISH appears to be versatile, fast and inexpensive, making fully automated FISH testing a possibility. Compared to conventional methods, these first iterations of on-chip FISH provide a 10-fold higher throughput and a 10 fold reduction in the cost of testing. On-chip FISH technology holds promise for sophisticated and cost-effective screening of cancer patients at every clinic visit in any health care setting, thus facilitating the delivery of personalized cancer care targeted to the genetic characteristics of each individual. Funded by CIHR, NSERC and Western Economic Diversification.

Description: In multiple myeloma (MM) and Waldenstrom’s macroglobulinemia (WM), we identified three alternatively and/or aberrantly spliced HAS1 transcripts—HAS1Va, HAS1Vb and HAS1Vc. Statistical analysis of samples taken from 172 untreated MM patients showed that expression of HAS1Vb, an intronic splice variant, strongly correlates with poor survival (P=0.005). We investigated the molecular basis of intron retention during HAS1 splicing in MM and WM patients. We speculated that aberrant HAS1 splicing and the associated reduced survival of MM patients, resulted from an accumulation of mutations in aberrantly spliced regions of HAS1. Exons and introns 3 and 4 of the HAS1 gene were sequenced, because they are hotspots for splicing aberrations. Sequencing of HAS1 was performed for a total 11 patients with WM and MM and 2 healthy donors. HAS1 gene templates for sequencing were isolated from a multiple sorted cell subpopulations, including malignant B and plasma cells (PC), non-malignant T cells and buccal epithelial cells (BECs), as well as hematopoietic progenitor cells (HPCs) from mobilized blood of MM patients. We detected sets of inherited and acquired genetic variations in HAS1 that were recurrent within 5–11 of the MM and WM patients analyzed, but absent from healthy donors. We also identified genetic variations that were unique to individual patients. Those HAS1 mutations found in all cell types tested, including BECs and from the hematopoietic cells (B, PC, T and HPCS) were classified as germline mutations. Those mutations found in hematopoietic cells but absent from BECs were classified as hematopoietic origin which acquired during the lifetime of the individual. Mutations identified only in malignant MM and WM B cells and PCs (absent from T cells, HPCs and BECs) were classified as acquired tumor specific mutations. Recurrent HAS1 mutations were found among both inherited and acquired sets of mutations. Some recurrent HAS1 mutations were common to both MM and WM. The high frequency of inherited HAS1 mutations suggests that they confer predisposition for developing MM or WM. Our sequencing analysis suggests that in MM and WM, sequential accumulation of genetic variations occurs as hematopoietic cells differentiate. Our data also suggest that hematopoietic origin mutations are necessary but by no means sufficient to drive HAS1 gene splicing. Effects of hematopoietic origin mutations on HAS1 splicing are manifested in malignant MM cells in context of additional tumor specific mutations, which are acquired by circulating B cells and passed to their plasma cell progeny. This suggests that mutations which lead to aberrant splicing of HAS1 pre-mRNA undergo mutational selection events, and leave a mutational “trace” throughout the hematopoietic cell lineage, including tumor cells. Existence of same mutational events detected in HAS1 gene from MM and WM supports the speculation that the precursors of both diseases may undergo a series of shared genetic events, diverging only when tumor specific mutations accumulate in distinct subsets of B lineage cells. In silico comparison of splicesomal assembly between wild type and mutated HAS1 gene gave a pattern that precisely predicts partial retention of intron and aberrant splicing of the HAS1 gene.

Description: Abstract 2841 Poster Board II-817 Introduction: Monoclonal gammopathy of undetermined significance (MGUS) is the most common plasma cell disorder. A small proportion of MGUS transform to multiple myeloma (MM) or other B lineage malignancies. Hyaluronan synthase I (HAS1) is overexpressed in many human cancers, including MM. Here we show that intron 3, a region involved in aberrant splicing of HAS1, is heavily mutated in 34 patients with MGUS. Aberrant splicing of HAS1 correlated with poor outcome in a cohort of MM patients. HAS1 is GC-rich and as such is likely to be subject to mutation events. We recently reported a series of nearly 200 HAS1 mutations in 17 MM and Waldenstrom's macroglobulinemia (WM) patients, that were absent from healthy donors (Adamia et al, Blood, 2008, 112:5111). Forty-nine of these HAS1 mutations were recurrent, defined as being present in more than one patient. None of them were present in sequenced regions of HAS1 from a small series of MGUS patients. Mutated HAS1 DNA directs aberrant splicing of HAS1 and in vitro mutagenesis of HAS1 intron 3 alters HAS1 splicing patterns (see abstract by Kriangkum et al.), mimicking the aberrant splicing seen in MM patients. To sequence HAS1 from a larger cohort, we developed a streamlined sequencing strategy that allowed rapid sequencing of exon 3 and intron 3, regions of the HAS1 gene rich in mutations. Our objective was to identify HAS1 mutations in MGUS as compared to a larger cohort of MM and healthy donors. Many reports have shown familial clusters of MGUS and MM and of other B cell malignancies involving MGUS, supporting the idea that genetic factors, and possibly genetic predispositions, contribute to the development of disease. Patients and Methods: We sequenced HAS1 regions from 34 MGUS patients, 50 MM patients, and 45 healthy donors. In all cases, we sequenced an 850 bp region covering exon 3 and intron 3 of the HAS1 gene. For each patient, we sequenced eight HAS1 region subclones, in both directions. To predict impact of recurrent HAS1 mutations on the splicing process, we performed in silico analysis using online bioinformatic tools. Results: To date we have identified, 117 HAS1 mutations in HAS1 gene in MGUS patients including 5 frequently detected NCBI single nucleotide polymorphisms (SNPs). Although the same NCBI SNPs were also identified in healthy donors, increased homozygosity was detected in MGUS patients compared to healthy donors. Overall, among the novel HAS1 mutations, 32 (27%) were shared between MGUS and MM but absent in healthy donors, defining them as recurrent. Seven of the 32 mutations (23%) shared between MGUS and MM were recurrent in 2-3 individual MGUS patients; three of the recurrent MGUS mutations were missense mutations present in the coding region of exon 3, changing the protein sequence. Our initial bioinformatic analysis showed that the two of the shared recurrent mutations were found in the SR protein binding sites of the pre-mRNA and may contribute to aberrant splicing of the primary HAS1 transcript. We predict that our ongoing analysis HAS1 mutations in MGUS patients will identify a subpopulation(s) at high risk of aberrant splicing and thus at increased risk of transformation to overt MM. Conclusion: HAS1 appears to be hypermutated, perhaps due to its GC-rich nature. HAS1 mutations were identified in patients with a clinical diagnosis of MGUS (range of 1-14 mutations per MGUS). Thirty-two HAS1 mutations were recurrent in MGUS and MM but were absent from healthy donors. Some of these mutations are predicted to have functional significance for protein sequence and pre-mRNA splicing, possibly leading to increased risk for or predisposition to disease. Although genetic background and environmental context are likely to be important factors, our report of novel mutations in HAS1 of MM and WM patients, confirmed now in MGUS, suggests that mutations in HAS1 may play a key role in oncogenesis. Further genetic analysis of patient populations and the biochemical characterization of the mutated proteins will provide considerable insight into the function of HAS1 during transformation from MGUS to overt MM. Disclosures: No relevant conflicts of interest to declare.

Description: Multiple Myeloma (MM) is clinically characterized by accumulations of plasma cells in the bone marrow (BM). We have identified drug resistant clonotypic B lymphocytes in the peripheral blood of MM patients that have malignant characteristics and the ability to xenograft MM to immunodeficient mice. Using an automated scanning system (Bioview Duet), for blood samples from 72 MM patients, we scanned cytospin slides stained with May-Grunwald Giemsa to identify lymphocytes and determine whether they have the same chromosomal abnormalities that characterize autologous plasma cells. The location on the slide of each morphologically identified cell is recorded during an initial scan, followed by FISH, and examination of the same cells for genetic abnormalities. For this study, we chose probes to detect 1) deletion of chromosome 13 using D13S319 (Vysis), and 2) the t(4;14)(p16;q32) translocation using a dual fusion probe (Vysis). Both abnormalities correlate with adverse prognosis. Lymphocytes from PBMC of healthy donors show 2.8+/−1% of lymphocytes with Ch13 deletion, and less than 1% with t(4;14). An MM PBMC sample was considered to have lymphocyte abnormalities if it scored above a cut off value of 10% for Ch13 deletions and 2% for t(4;14), this is likely to underestimate the extent of chromosomal abnormalities in MM B cells. The number of abnormal lymphocytes in MM PBMC is sufficiently large, and the number of abnormal cells in comparable populations from healthy donors is sufficiently small, that we are readily able to detect significantly increased numbers of abnormal lymphocytes in MM PBMC. We found that peripheral lymphocytes from 19/60 (32%) MM patients have Ch13 deletion (27%) and/or t(4;14) (26%); for those patients with an available BM sample, these same abnormalities were found in their BM plasma cells. Interestingly, intraclonal heterogeneity is apparent in malignant cells from a t(4;14) patient; these plasma cells include a minority population with apparent Ch14 monosomy (23%) and a major population having two copies of Ch14 (77%). This was internally controlled in that for both populations, the plasma cells have two copies of the fused t(4;14) chromosome, and the polymorphonuclear cells on the same slide were normal. One MM patient had Ch13 deletion in 60% of lymphocytes from PBMC, consistent with the 63% of BM plasma cells from this same patient showing Ch13 deletion. For another MM patient known to be t(4;14)+, sorted sIgM+ B cells were analyzed and found to include a small subset with t(4;14) translocations. For 2/6 MM PBMC, abnormalities in Ch19 were detectable in 25–37% of lymphocytes. For mobilized blood autografts, preliminary data indicates the presence of lymphocytic cells with detectable Ch13 deletion. By performing FISH after immunostaining with anti-CD20 of PBMC from MM patients whose bone marrow plasma cells had Ch13 deletion/monosomy, we detected chromosomal abnormalities in CD20+ B cells from MM blood. No abnormalities were detected for CD20+ B cells from healthy donors. In the context of our previous work, this analysis demonstrates that two molecular signatures of MM, clonotypic IgH gene rearrangements and, as reported here, prognostically important chromosomal abnormalities are found among circulating CD20+ B cells from the blood of patients with MM. This work confirms that the MM clone includes circulating B lymphocytes harbouring chromosomal deletions or IgH switch region translocations known to be clinically significant in MM, further implicating them as a source of relapse.

Description: Multiple myeloma (MM) is a B-lineage malignancy characterized by diverse genetic subtypes and clinical outcomes. The recurrent immunoglobulin heavy chain (IgH) switch translocation, t(4;14)(p16;q32), is associated with poor outcome, though the mechanism is unclear. Quantitative reverse-transcription–polymerase chain reaction (RT-PCR) for proposed target genes on a panel of myeloma cell lines and purified plasma cells showed that only transcripts originating from the WHSC1/MMSET/NSD2 gene are uniformly dysregulated in all t(4;14)POS patients. The different transcripts detected, multiple myeloma SET domain containing protein (MMSET I), MMSET II, Exon 4a/MMSET III, and response element II binding protein (RE-IIBP), are produced by alternative splicing and alternative transcription initiation events. Translation of the various transcripts, including those from major breakpoint region 4-2 (MB4-2) and MB4-3 breakpoint variants, was confirmed by transient transfection and immunoblotting. Green fluorescent protein (GFP)–tagged MMSET I and II, corresponding to proteins expressed in MB4-1 patients, localized to the nucleus but not nucleoli, whereas the MB4-2 and MB4-3 proteins concentrate in nucleoli. Cloning and localization of the Exon 4a/MMSET III splice variant, which contains the protein segment lost in the MB4-2 variant, identified a novel protein domain that prevents nucleolar localization. Kinetic studies using photobleaching suggest that the breakpoint variants are functionally distinct from wild-type proteins. In contrast, RE-IIBP is universally dysregulated and also potentially functional in all t(4;14)POS patients irrespective of fibroblast growth factor receptor 3 (FGFR3) expression or breakpoint type.