Wier, Scott A. Van

Abstract Background: Clonal cytogenetic aberrations define multiple myeloma (MM) into unique subsets of the disease each one with unique clinical and biologic features, associations with laboratory and pathologic markers and presumed differential influence on disease progression. While it is widely accepted that translocations represent primordial events in the pathogenesis of MM, it is controversial whether all translocations can be associated with disease stability and non-malignant behavior (i.e. in MGUS/SMM). We have found the translocations in cases of MGUS using dual fusion, cIg enhanced, interphase FISH, while others have found this translocation at lower prevalence using single fusion interphase FISH or the RT-PCR for IgH/MMSET. We thus sought to clarify by expanding the study of the t(4;14) among patients with MGUS and SMM. Materials and methods: We studied 210 patients who had the diagnosis of MGUS and SMM and entered into the Mayo Clinic Dysproteinemia database. Patients were excluded if they had active MM, extramedullary disease or plasmacytoma. Patients with isolated lytic bone lesions could be included if they had no overt evidence of progression. All studied patients had provided written informed consent for the collection of a research bone marrow aliquot that was collected at the time of routine bone marrow procurement. All patients had cytospin slides that were studied for a t(4;14) using cIg-FISH as previously described by us and using a double fusion FISH strategy. The importance of this is that the strategy will detect unbalanced t(4;14) with loss of der(14). Results: Twenty patients (9.5%) had evidence of the chromosome translocation. Detailed demographic data was available in 19; 14 were women (74%), 13 of 19 (68%) had SMM and there was no bias for heavy chain IgA (26%). Patients with t(4;14) were slightly younger than those without the abnormality (52 vs. 61 years), had similar hemoglobin concentrations, had slightly higher concentration of the serum M spike (2.0 vs 1.75 g/dl) and had overall slightly higher bone marrow plasmacytosis (median 23% vs 10%). Patients were followed since diagnosis for a median of 33 months (range 2–162 months). Three patients progressed to active MM at 12, 12 and 26 months after original diagnosis (all three originally having indolent/SMM). Conclusion: The t(4;14) can occur in patients with MGUS and SMM and without evidence of progression to MM. The prevalence is similar to that observed in MM, albeit slightly lower (9.5% versus 14%). In most “benign” plasma cell neoplasms the translocation is associated with greater plasmacytosis and thus enriched for in the smoldering MM category. It is also likely that the t(4;14) confers a higher risk of progression to MM given; a) its enrichment in SMM, b) the slightly higher prevalence of the translocation in MM as opposed to MGUS/SMM, and c) the well documented aggressive clinical course of MM with t(4;14).

Abstract Chromosome 13 abnormalities (Δ13), monosomy or deletion, are seen in greater than 50% in patients with Multiple Myeloma (MM) and are associated with poor survival and reduced response to therapy. Δ13 is suggestive of a tumor suppressor model. In this study, we use a comprehensive, high-throughput approach involving array-based technologies to identify genes on chromosome 13 that are important in the pathogenesis and/or progression of MM. Array-based Comparative Genomic Hybridization (aCGH) is being conducted to identify a minimum region of 13q loss in a series of MM cell lines and patient samples. DNA is isolated, labeled and hybridized with a differentially labeled normal DNA reference to determine gene/genomic copy number changes. Arrays are analyzed to search for the minimum region of loss based upon single copy loss for a series of nearby mapping transcripts in each cell line. Data will be cross-referenced to determine the true minimum region of 13q loss in MM. Also, to discover genes harboring potential inactivating mutations, we are using inhibition of nonsense-mediated RNA decay (NMD) coupled with cDNA microarray. This modification of the Gene Identification by NMD Inhibition (GINI) method, first introduced by Noensie and Dietz (2001), is used to identify genes on chromosome 13 that may be inactivated due to premature truncating codons (PTCs). This method is based upon a cell’s natural ability to survey for and degrade RNA species that contain PTCs by activation of the nonsense mediated RNA decay (NMD) pathway. Treating cells with the drug Emetine can inhibit NMD and increase the expression of genes likely harboring truncating mutations. Preliminary data have revealed several candidate genes, with increased ratios after Emetine treatment, which map to chromosome 13. Upon defining a minimum region of loss based upon aCGH data, we will prioritize genes from our NMD study for mutational analysis. It our hope to also perform an array-based CpG island screen to simultaneously analyze all CpG islands on chromosome 13 to detect genes possibly inactivated by hypermethylation. This comprehensive, organized approach incorporating high-throughput techniques for the detection of genomic loss, mutation, and methylation is the first of its kind to our knowledge and may maximize the potential for the identification of a 13q tumor suppressor in MM.

Abstract Introduction: FISH is able to recognize chromosomal deletions and translocations with a greater sensitivity than conventional cytogenetics. Specific abnormalities have been associated with prognosis. Initial observations suggest a poor outcome for patients with -17p13.1, chromosome 13 abnormalities (Δ13) and t(4;14)(p16.3;q32). In contrast a good outcome has been shown in some series for patients with t(11;14)(q13;q32). We analyzed the value of FISH in patients receiving high dose therapy . Patients and Methods: We studied by cIg-FISH 226 patients undergoing high dose therapy at Mayo Clinic between 1/1990 and 9/2001. All patients had a pretransplant cIg-FISH done on cytospin slides from marrow aspirates for t(11;14)(q13;q32), t(4;14)(p16.3;q32), and -17p13.1(p53). Information was available regarding Δ13 for all patients (+ in 52%). Results: The prevalence of the abnormalities were: t(11;14)(q13;q32) 17% (n=197), t(4;14)(p16.3;q32) 13% (n=153), and -17p13.1 11% (n=168). The overall survival (OS)was significantly shortened in patients with t(4;14)(p16.3;q32) (18.2 vs. 43.3 mo, p=0.001) (figure) and patients with -17p13.1 (14.7 vs. 38.6 mo, p=.01). OS was not different for patients with the t(11;14)(q13;q32) (36.2 vs. 34.8 mo, p=ns). Likewise time to progression (TTP) was shortened in patients with t(4;14)(p16.3;q32) (8.5 vs 17.7 mo, p=.001) and -17p13.1 (8.3 vs. 16.2 mo, p=0.005). TTP was also not affected significantly by the t(11;14)(q13;q32) (20.7 vs. 14.9 mo, p=NS). To dissect the specific contribution of t(4;14)(p16.3;q32) we did a subset analysis of patients who also had Δ13, since 85% of patients with t(4;14)(p16.3;q32) are expected to have Δ13. Of 84 studied for both abnormalities 22 had both Δ13 and t(4;14)(p16.3;q32). The OS was significantly shorter in patients with both abnormalities versus those with Δ13 alone (26.6 vs. 18.2 months, p=0.001). When a multivariable analysis of the impact of Δ13 and t(4;14)(p16.3;q32) were placed into a Cox model the hazard function for t(4;14)(p16.3;q32) was greater than Δ13 (2.6 versus 1.6). Δ13 had only borderline significance in this model (p=0.06). Conclusion: We have been unable to corroborate the improved outcome after transplant for patients with t(11;14)(q13;q32). As has been reported in patients with conventional and high dose therapy -17p13(p53) and t(4;14)(p16.3;q32) have clinical importance for estimation of OS and TTP. In this patient group the t(4;14)(p16.3;q32) carried a greater adverse impact than did Δ13, and identifies a subset of patients whose time to progression is 8.5 months. These patients likely do not benefit from autologous transplant and are candidates for novel therapeutic approaches. Outcome Successful determination Patients with translocation or deletion Survival (months) with/without abnormality Time to Progression (months) with/without abnormality * p<0.01;**p<0.001 t(11;14)(q13;q32) 197 34(17%) 36.2/34.8 20.7/14.9 p53 168 18(11%) 14.7/38.6 * 8.3/16.2 * t(4;14)(p16.3;q32) 153 26(17%) 18.2/43.3 **figure 8.5/17.7 ** t(4;14)(p16.3;q32)/Δ13+ patients 84 22(26%) 18.2/26.6 8.2/12.8 * Figure Figure

Abstract Waldenström macroglobulinemia (WM) is a lymphoid neoplasm associated with accumulation of lymphoplasmacytic cells in the bone marrow and an elevated monoclonal IgM in the serum. Although the genetic and molecular mechanisms behind the pathogenesis of WM are not well understood, losses of the long arm of chromosome 6 have been found as the only recurrent genomic aberration. These deletions, mostly starting at 6q21, are observed in upwards of 50% of patients and involve the majority of the clonal cells. To better characterize pathologic gene expression signatures in different types of WM at the molecular level (6q deleted versus not), we examined gene expression level changes of more than 20000 human gene probes in bone marrow tissues of clinically proven WM patients. We used Affymetrix HG-U133A v2 chips (n = 24). Clonal cells were purified using a combination of anti-CD138 and anti-CD19 Miltenyi™ magnetic microbeads. Based on cIgM-FISH results, we classified samples into two groups - 6q normal (ND) group and 6q deletion (D) group. We then proceeded to identify genes that showed differential expression between the two groups. We selected 165 differentially expressed genes (DEG) using SAM, and underwent further investigation of biological annotations of them using Gene Ontology™ (GO) terms. Among165 DEG, only 32 showed high level of expression in ND group compared to D group while 133 showed high level of expression in D group. As expected, all genes that reside in 6q region showed under-expression in D group, constituting 53% (17 genes) of genes under-expressed in D group (32 genes). According to GO analysis, the 6q-deletion-induced under-expression affects many biological processes, and is associated with a transcriptional upregulation of downstream genetic cascades. This likely indicates release of regulatory control by a tumor suppressor gene. Genes involved in cell growth and maintenance, cell communication and morphogenesis are also over-expressed in D group. Interestingly, a gene that is involved in innate immune response and inflammatory response shows under-expression in D group but is not accounted for by other mechanisms. In similar vein, a gene that is involved in negative regulation of cytokine biosynthesis showed over-expression in D group. We think these transcriptional characteristics can provide basic framework for differentiating subtypes of WM and search for genes associated with WM pathogenesis

Abstract Introduction Lenalidomide is an immunomodulatory drug (IMID) used to treat Multiple Myeloma (MM). Although a role for cereblon (CRBN)-mediated degradation of Ikaros proteins (IKZF1 and IKZF3) has been shown, the complete molecular and biochemical mechanisms responsible for lenalidomide-mediated anti-MM activity and/or resistance are undiscovered. Therefore, we aimed to analyze whether IMIDs (thalidomide, lenalidomide, and pomalidomide) are inducing oxidative stress in MM and what determines these drugs varying sensitivity and/or resistance. Methodology Amplex Red Assay has been performed to analyze IMIDs-mediated inhibition of H2O2 decomposition in both, in-vitro and in-vivo assays. Lentiviruses were prepared in 293T cells for CRBN, IgL-λ & IgL-k, and Bim knockdown experiment. Quantification of MM cellular anti-oxidative capacity for determining IMID sensitivity was standardized with H2O2-mediated oxidation of FADH2 and NAD(P)H. To measure apoptosis and gene expression analysis 106 cells were incubated with lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. Results We discovered that IMIDs inhibit peroxidase-mediated decomposition of H2O2 in both, in vitro horseradish peroxidase (HRP) assays and in human MM cell lines (HMCLs). Of the IMIDs analyzed, pomalidomide was the more potent inhibitor. H2O2 treatment effectively degraded IKZF1 and IKZF3 in HMCLs. To confirm the central role of CRBN in IKZF1 and IKZF3 degradation by H2O2-induced oxidative stress, we used CRBN knockdown OPM2 isogeneic cells and the CRBN-overexpressing OCIMY-5 cell line. We treated both sets of isogenic cell lines with lenalidomide and H2O2 for 3 hours, and we showed that H2O2 similarly mediates IKZF1 and IKZF3 degradation in a CRBN-dependent fashion. Next, we tested viability of CRBN present and absent cell lines with increasing concentrations of lenalidomide and H2O2 for 3 days. Lenalidomide-induced cytotoxicity was CRBN dependent, but H2O2 was not after 3 days, as shown by MTT assays. The capacity of MM cells to decompose H2O2 was measured via a biochemical test that quantitatively measured cellular anti-oxidative capacity. IMID sensitivity was well correlated with cellular anti-oxidative capacity, likely, cells more efficiently decompose H2O2was resistant and cells were not sensitive to IMID. This result shows that antioxidant capacity determines lenalidomide sensitivity among HMCLs with similar CRBN protein expression. We discovered that lenalidomide-mediated cytotoxicity in MM was attributable to oxidative damage of intracellular immunoglobulin proteins. By using several sets of isogenic cells lines with and without CRBN expression, we confirmed that lenalidomide treatment caused accumulation of IgL dimers only in CRBN-positive cells. Lenalidomide-induced IgL dimerization lead to decreased secretion and consequent intracellular accumulation of IgL, as evidenced by unchanged IgL mRNA expression, increased total intracellular IgL protein, and decreased secretion of IgL. After 72 hours of lenalidomide treatment we found decreased XBP-1u, increased XBP-1s, and over-expressed GRP78/BiP endoplasmic reticulum stress (ERS) marker proteins in CRBN positive cells but not in CRBN knock-down cells. We observed Bim requirement, especially BimEL, after lenalidomide treatment in CRBN-positive lenalidomide-sensitive cells. Our data reveals that lenalidomide-mediated; progressive ERS can positively enhance bortezomib-induced apoptosis in an in-vitro MM model. We pretreated MM cells with lenalidomide and then treated them with bortezomib. OPM2 cells pretreated with lenalidomide for 2 days clearly showed increased sensitivity to bortezomib-induced apoptosis compared with cells that were not pretreated. Conclusion IMIDs inhibit H2O2 decomposition. Ikaros protein degradation is a consequence of H2O2 mediated oxidative stress. Therefore, cells producing high H2O2 and with less antioxidative capacity are more sensitive to IMIDs. On the basis of this discovery, we would be able to predict which patients will benefit from IMIDs-mediated therapy and develop new drugs other than IMIDs that can inhibit intracellular H2O2 decomposition in MM. At present, CRBN may be required for IMIDs to effectively inhibit H2O2decomposition. Disclosures Chirackal: Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity. Fonseca:Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity.

Key Points IMiDs inhibit TrxR-mediated intracellular decomposition of H2O2 and caused oxidative stress in MM cells. MM cells with lower antioxidative capacity were more vulnerable to lenalidomide-induced H2O2 accumulation and its associated cytotoxicity.

Abstract The plasma cell malignancy Multiple Myeloma (MM) is a rare but deadly form of cancer with 5 year survival rates of ∼30%. Epidemiological studies have suggested that MM exacts an especially heavy burden on African American (AA) patients. We examined a multi-ethnic cohort of MM tumors to assess potential differences in the frequency of molecular events in tumors derived from either African American (AA) or European American (EA) patients. The frequency of 14q32 translocation breakpoints at the IgH locus was compared among a series of data from 115 AA MM patients from three studies and EA MM from the Eastern Cooperative Oncology Group (ECOG). In addition, we analyzed matching genome-wide copy number and transcriptional data among 45 AA MM tumors and 196 EA MM tumors to determine differences in the frequency of molecular events in tumors from these populations. No differences were found for specific translocation subtypes; however, we detected a statistically significant difference in the overall frequency of IgH translocations between the two groups, which occurred more frequently in EA patients (52% vs. 40%; p = 0.032). When assessing genomic copy number events previously shown to be associated with poor outcome in MM, frequencies of alterations were not significant after correcting for multiple testing. Furthermore, there was not a significant difference in the association of high-risk disease using expression profiling. Our study represents the first comprehensive assessment of the frequency and distribution of molecular alterations in MM tumors from both AA and EA patients and could help shed light on possible biological features associated with population differences in incidence and outcome in MM. Citation Format: Angela S. Baker, Esteban Braggio, Susana Jacobus, Sungwon Jung, Dirk Larson, Terry Therneau, Angela Dispenzieri, Scott A. Van Wier, Gregory Ahmann, Joan Levy, Louise Perkins, Seungchan Kim, Kim Henderson, David Vesole, S. Vincent Rajkumar, Dianne F. Jelinek, John Carpten, Rafael Fonseca. Characterization of myeloma tumors from a multi-ethnic cohort using cytogenetics and genomic analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2021. doi:10.1158/1538-7445.AM2013-2021

Key PointsIn this study, African American MM patients have a significantly lower frequency of IgH translocations than European American patients.

Abstract Detection of specific chromosomal abnormalities by FISH and metaphase cytogenetics allows risk stratification in multiple myeloma; however, gene expression profiling (GEP) based signatures may enable more specific risk categorization. We examined the utility of 2 GEP-based risk stratification systems among patients undergoing initial therapy with lenalidomide in the context of a phase 3 trial. Among 45 patients studied at baseline, 7 (16%) and 10 (22%), respectively, were high-risk using the GEP70 and GEP15 signatures. The median overall survival for the GEP70 high-risk group was 19 months versus not reached for the rest (hazard ratio = 14.1). Although the medians were not reached, the GEP15 also predicted a poor outcome among the high-risk patients. The C-statistic for the GEP70, GEP15, and FISH based risk stratification systems was 0.74, 0.7, and 0.7, respectively. Here we demonstrate the prognostic value for GEP risk stratification in a group of patients primarily treated with novel agents. This trial was registered at www.clinicaltrials.gov as #NCT00098475.

Abstract Nonrandom recurrent chromosomal abnormalities are ubiquitous in multiple myeloma (MM) and include, among others, translocations of the immunoglobulin heavy chain locus (IgH). IgH translocations in MM result in the up-regulation of oncogenes, and include more commonly t(11;14)(q13;q32), t(4;14)(p16;q32), and t(14;16)(q32;q23). Based on the recurrent nature of these translocations and their finding since the early stages of the plasma cell (PC) disorders, we hypothesized that they would confer biologic and clinical variability. In addition, deletions of 13q14 and 17p13 have also been associated with a shortened survival. We used cytoplasmic Ig—enhanced interphase fluorescent in situ hybridization to detect deletions (13q14 and 17p13.1), and translocations involving IgH in 351 patients treated with conventional chemotherapy entered into the Eastern Cooperative Oncology Group clinical trial E9486/9487. Translocations were frequently unbalanced with loss of one of the derivative chromosomes. The presence of t(4; 14)(p16;q32) (n = 42; 26 vs 45 months, P &lt; .001), t(14;16)(q32;q23) (n = 15; 16 vs 41 months, P = .003), – 17p13 (n = 37; 23 vs 44 months, P = .005), and – 13q14 (n = 176; 35 vs 51 months, P = .028) were associated with shorter survival. A stratification of patients into 3 distinct categories allowed for prognostication: poor prognosis group (t(4;14)(p16;q32), t(14; 16)(q32;q23), and – 17p13), intermediate prognosis (– 13q14), and good prognosis group (all others), with median survivals of 24.7, 42.3, and 50.5 months, respectively (P &lt; .001). This molecular cytogenetic classification identifies patients into poor, intermediate, and good risk categories. More importantly it provides further compelling evidence that MM is composed of subgroups of patients categorized according to their underlying genomic aberrations.