Hyperdiploid Multiple Myeloma Research Articles

Patterns of Change in Multiple Myeloma (MM) Clone Size with Autologous Hematopoietic Stem Cell Transplantation (ASCT) Assessed By Next Generation Sequencing (NGS) in Patients (pts) Receiving Modern Therapy

Background Measurable residual disease (MRD) is a quantitative, dynamic prognostic marker of progression free and overall survival in MM. With modern triplet and quadruplet induction strategies deep responses are the norm and the incremental effect of ASCT impact may vary substantially among pts and be influenced by disease and treatment features. However, the accurate characterization of the impact of ASCT is diluted by measuring a disease surrogate (paraprotein) instead of clone size. We examine the quantitative impact of ASCT on MM clone size by serial NGS testing before and after ASCT and evaluate disease and induction treatment features that modulate this impact. Methods We included pts with newly diagnosed MM from five US centers, who received modern triplet and quadruplet induction followed by single melphalan ASCT between 3/2018 and 5/2023 with MRD testing (ClonoSEQ®) pre and post ASCT. ClonoSEQ® platform utilizes NGS to quantify unique immunoglobulin genes rearrangements associated with MM clone, therefore directly capturing clone size. We excluded pts with clone size <10 -5 prior to ASCT. We explored patient-, disease-, and treatment features influencing clone size reduction with AHCT by 1 log 10. We used binary logistic regression to identify factors associated with ≥1 log 10 clone reduction (60-100 days post ASCT). Results One hundred and forty-nine pts with NDMM had pre and post ASCT results and pre ASCT MRD ≥10 -5 and met the inclusion criteria. The median age was 63 years (35-79), 37 pts (25%) ≥70 years, 62% male, 27% Black, 23% ISS3 and 16% RISS3. Seventy-three pts (49%) had high risk chromosomal abnormalities [HRCA, gain/amp(1q), t(4;14), t(14;16), t(14;20) or del(17p)]); 72 pts (48%) had 0 HRCA, 50 (34%) had 1 HRCA, and 23 (15%) had 2+ HRCA. AntiCD38 monoclonal antibody (mAb) was part of induction in 125 (84%) pts [68 pts quadruplet containing carfilzomib; 53 pts quadruplet containing bortezomib], 28 pts received triplets. Eighty-seven pts (58%) had >1log 10 reduction in MRD burden with ASCT. The median log reduction in MRD burden was 1.15 (IQR 0.65-1.75). For pts with 0, 1 and 2+ HRCA, 47% (34/72), 68% (34/50), 74% (17/23) had a >1 log 10 reduction in clone size and the median reduction was 0.96 log 10 (IQR 0.47-1.66), 1.24 log 10 (IQR 0.89-1.91) and 1.5 log 10 (IQR 0.89-2.20), respectively (Figure). Among pts without HRCA, those with hyperdiploid MM had a trend towards less frequent ≥1 log 10 clone size reduction with ASCT (38% vs. 57%, p=0.18). Overall, we saw reduction ≥1 log 10 in clone size in 61% of pts receiving anti-CD38 mAb in induction (N=125) vs. 46% among those who did not (N=24, p= 0.18). Among pts treated with melphalan 200 mg/m 2 (N=113) compared to those who received 140 mg/m 2 (N=36), ≥1 log 10 reduction in clone size was noted in 61% vs. 50% (p=0.25), respectively. In multivariable analysis that included stage, antiCD38 mAb in induction, use of carfilzomib in induction, HRCA and melphalan dose, the presence of HRCA was the only factor associated with greater than 1 log 10 reduction in MRD burden with ASCT [1 HRCA, OR 2.34 (1.07-5.14); ≥2 HRCA, OR 2.71 (0.91-8.03); p=0.049]. Conclusions The impact of high dose melphalan and AHCT on MM clone size is heterogeneous, of greater magnitude in pts with one or more high-risk chromosome abnormalities, and less pronounced in pts with standard risk disease, particularly hyperdiploid MM. The use of antiCD38 mAb in induction regimens or dose of melphalan does not impact the effect of ASCT on MM clone size, likely owing to the distinct mechanism of action of melphalan. This dataset provides context to explore risk- and response-adapted use of ASCT and use of new cellular and non-cellular immunotherapies as post-induction consolidative strategies.

P-346 Internal tandem duplications of the BCMA transmembrane domain are common in hyperdiploid multiple myeloma and associated with constitutive NF-kB signaling

P-346 Internal tandem duplications of the BCMA transmembrane domain are common in hyperdiploid multiple myeloma and associated with constitutive NF-kB signaling

Vκ*MYC Mouse Model Recapitulates Key Genomic Defining Events in Multiple Myeloma

Background: The Vk*MYC transgenic model of multiple myeloma (MM) is based on the sporadic AID-induced activation of MYC in a single germinal center (GC) B-cell, in a mouse strain (C57Bl6) that spontaneously develops monoclonal gammopathy. With age, Vk*MYC mice universally develop a progressive expansion of class-switched, somatically hypermutated, monoclonal plasma cells. Myeloma defining events occur only after long latency, suggesting that additional mutations are required to induce malignancy. Here, we interrogated the genomic landscape of Vk*MYC MM to capture somatic mutations selected during progression. Methods: We interrogated whole genome (WGS) and exome sequencing (WES) from 55 purified plasma cell (PC) from Vκ*MYC transgenic mice tumors. Additional 27 and 12 Vκ*MYC mice were investigated with array comparative genomic hybridization (aCGH) and mate-pair low coverage WGS, respectively. Our analysis included 34 individual de novo tumors from aged Vk*MYC mice, 37 individual tumors from recipient mice transplanted with tumor cell lines established from de novo donors, and 13 tumors selected to propagate in vitro. Results: Tumor mutational burden was lowest in de novo samples (median 2.3 mutations/Mb, range 0.3-4.3 mutations/Mb) and highest in in vitro samples (median 5.8 mutations/Mb, range 1.2-12.7 mutations/Mb), similar to that of human MGUS and MM, respectively. To identify genes affected by nonsynonymous mutations more frequently than expected by chance (i.e., driver genes under positive selection), we ran dndscv. A total of 11 significant driver genes were detected, 7 of which are known driver genes in human MM: Dusp2, Trp53, Nfkbia, Pim1, Tent5c (Fam46c), Hist1h1c, Hist1h1e. Driver genes not previously described in MM were: Apobec3, Pten, Cd74, Cd79b. Additional nonsynonymous mutations were detected in driver genes frequently altered in MM including Kras, Nras, Dis3, as well as histone modifiers and NF-kB regulators. Differential mutation analysis revealed bi-allelic inactivation of Trp53 and Pten were more frequent in in vitro samples compared to de novo and transplanted tumor, in line with the almost universal p53 inactivation reported in human MM cell lines. As in human hyperdiploid MM, GISTIC and molecular time analyses revealed evidence of simultaneous multi-gain events of chromosomes 1, 9, 11, and 15. As reported previously, we observed frequent deletion of chromosome 5. There were numerous recurrent focal CNAs involving oncodrivers. These included focal deletion of tumor suppressors genes Cdkn2a, Cdkn2b, Cdh11, Ncor1, Rb1, Trp53, Kdm6a and Pten, as well as amplification of oncogenes Mdm4, Mcl1, and Kras. We detected several structural variants and complex events throughout the Vκ*MYC genome, similar to human MM. Importantly, we found chromothripsis in five (14%) cases. Additionally, in three Vκ*MYC mice we detected chromosomal translocations involving the immunoglobulin heavy or light chain loci that partnered and induced overexpression of MM associated genes such as Irf4, Map3k14 and Pou2af1. Overall, mutations activating the NF-kB and STAT3 pathways were the most common events, each present in more than half MM. Overall, the genomic landscape of the Vκ*MYC model resembles hyperdiploid MM in humans, with MYC translocations and enrichment of events affecting the NF-κB pathway. Finally, analysis of mutational signatures uncovered similar signatures present in human MM, including aging, APOBEC, and poly-eta in the GC, among others. In particular, APOBEC mutational activity was detected in more than half of Vκ*MYC MM, further supporting the similarities with human MM where APOBEC mutational activity is detectable in 80-90% of human MM and progressive precursor, but it is undetectable in the stable precursor conditions. Conclusions: Overall, these data reveal a progressive accumulation of genomic events from de novo, to transplant, to in vitro tumors that mimic the natural progression from newly diagnosed, to relapsed disease, to cell lines noted in human MM. Thus, our results highlight how the transformation pathways to malignant PC are conserved between mice and men. Given the long lead time between precursor diagnosis and malignant transformation to MM that limit the ability to investigate the earliest phases of tumorigenesis in human MM, the Vκ*MYC transgenic mouse model of human MM represents a unique opportunity to overcome this temporal limitation.

Suppression of Multiple Myeloma Via Inhibition of EFNB2 Reverse Signaling

Multiple myeloma (MM) cells display a reciprocal relationship with neighboring niche cells in the bone marrow (BM), particularly vascular endothelial cells (BM ECs). This relationship drives disease initiation, progression, and therapy resistance, but the mechanisms underlying this interaction remain incompletely understood. Here, we show that silencing ephrin B2 (EFNB2) blocked MM cell proliferation and survival in vitro and abrogated MM engraftment and repopulation in vivo. EFNB2 expression is also increased in MM patients with adverse-risk cytogenetics and decreased survival, suggesting an important role for EFNB2 in MM disease progression. To identify novel regulators of MM pathogenesis, we performed transcriptome analyses of ECs from multiple human tissues and analyzed their capacity to promote MM growth. The EPH receptor, EPHB1, was overexpressed in human ECs that support MM expansion in culture. Silencing EPHB1 expression in ECs using siRNA or a lentiviral shRNA abrogated MM cell expansion compared to EC co-cultures treated with non-targeting shRNA and blocked the expansion of MM cells capable of in vivo repopulation in immune deficient mice. Additionally, transgenic Vk*MYC mice, which develop indolent murine MM through activation-induced deaminase-dependent MYC activation in germinal B cells, displayed increased expression of EPHB1 and EPHB4 in BM ECs compared to wild type BM ECs. We found that expression of EFNB2, an EPHB1/4 binding partner, is upregulated in 3 human MM cell lines and in primary MM cell populations from patients, whereas other EPHB1/4 binding partners were either not detected or expressed at a low level. shRNA-mediated inhibition of EFNB2 in MM cells blocked MM cell cycling, leading to a near 70-fold reduction in MM cell growth at day 12 of culture, and blunted MM cell repopulation when transplanted into NSG mice. In order to determine the therapeutic potential of targeting EFNB2 in MM in vivo, we transplanted irradiated NSG mice with MM cells and subsequently administered a single chain variable fragment (scFv) targeting EFNB2 on days +4, +6, +10 and +12. Mice treated with the EFNB2 scFv displayed significantly decreased engraftment of MM cells in vivo compared to control mice. MM reconstitution in vivo. Silencing EFNB2 expression in MM cells downregulated several pathways including those involved in cellular movement, cell death and survival, cellular assembly, cell cycle and DNA repair. Several STAT5 target genes were also downregulated in MM cells treated with shRNA-EFNB2, supporting the notion that STAT5 signaling is activated by ephrin B proteins (Bong et al. Proc Natl Acad Sci. 2007;104(44):17305-17310). Consistent with these findings, EFNB2 overexpression via dCas9-VPR increased MM cell survival, proliferation and STAT5 phosphorylation. Conversely, silencing EFNB2 decreased STAT5 activation, increased MM apoptosis, and decreased MM proliferation. Importantly, STAT5 inhibition in MM cells overexpressing EFNB2 blocked EFNB2-mediated MM cell proliferation, suggesting that EFNB2-mediated MM growth is dependent on STAT5. Overexpressing a mutant form of human EFNB2 with GFP replacing the intracellular cytoplasmic PDZ signaling domain did not result in cell growth comparable to overexpressing the full-length EFNB2. Silencing EPHB4, the main EFNB2 binding partner expressed on MM cells, resulted in significant impairment of growth in wild-type MM cells but not those with full-length EFNB2 overexpression. Updated downstream signaling and in vivo data of these will be presented at the meeting. Interrogation of MM clinical databases, including the Total Therapy studies (Zhan F. et al. Blood. 2006;108(6):2020-2028) and the CoMMpass data (Multiple Myeloma Research Foundation, https://research.themmrf.org/, accessed on 20 October 2021, n = 773 patients), revealed that EFNB2 expression is increased in MM patients with adverse risk cytogenetics, including t(4;14) and gain of chromosome 1q, while lower EFNB2 expression is associated with the more favorable, "standard risk" disease category, hyperdiploid MM. Increased EFNB2 expression is also associated with advanced stage disease and decreased survival in MM patients. Our analyses suggest that EFNB2 signaling represents an important new mechanistic target in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Study of DNA ploidy in newly diagnosed multiple myeloma by flow cytometry and its correlation with disease prognosis and outcome

Objectives: Multiparameter flow cytometry is increasingly being used in determining DNA content in several hematological malignancies where abnormal DNA ploidy is a useful prognostic marker. This study was done to analyze the DNA ploidy of plasma cells in multiple myeloma (MM) by flow cytometry and explore its role as a prognostic factor. Material and Methods: Propidium-iodide staining technique and gating based on light scatter properties were used. DNA index (DI) of the tumor sample was calculated as the ratio of the DNA content of the G0-G1 population of the myeloma cells with the normal control cells (lymphocytes) present in the same sample. Based on DI, ploidy was categorized as diploidy, hypodiploidy, and hyperdiploidy and the results were correlated with the clinical outcome. Results: Among 32 patients, none had hypodiploid DNA content, 8 (25%) patients had hyperdiploid DNA, and 24 (75%) patients had diploid DNA. There was no significant association between DI and international staging system staging (P = 0.68), Eastern Cooperative Oncology Group Performance Status (P = 0.59), and post-induction remission status (P = 0.10). The median overall survival (OS) in the study patients was 20 (CI 11.4–28.8) months and the median progression-free survival was not reached. There was no difference in the OS among patients with diploid MM and hyperdiploid MM (P = 0.84). Conclusion: Although hyperdiploid MM has been reported to have a better prognosis than diploid MM, we did not find any significant difference possibly due to the small sample size. Nevertheless, flow cytometry is a useful tool in DNA ploidy analysis and its role as a prognostic factor in various hematologic malignancies including MM can be further explored.

16p Deletion Involving BCMA Locus Is Frequent and Predominantly Observed with del17p

16p Deletion Involving BCMA Locus Is Frequent and Predominantly Observed with del17p

False-Negative Centromere 15 Probe Results in Association with African Ancestry in Plasma Cell Dyscrasias

Purpose:Multiple myeloma (MM) is a plasma cell (PC) malignancy with an increasing incidence in the United States. Fluorescence in situ hybridization (FISH) is currently the gold-standard diagnostic assay to detect recurrent genomic abnormalities of prognostic and therapeutic significance in MM. Hyperdiploid MM, a standard risk abnormality, is characterized by gains of odd-numbered chromosomes including chromosome 15. While gains of chromosome 15 have been reported in approximately 53% of standard risk MM patients (Chretien, Blood, 2015), losses of chromosome 15 are rarely reported. Enumeration of chromosome 15 centromere is detected using the alpha satellite FISH probe. We previously discovered ~9% of cases had false-negative chromosome 15 FISH results when compared to Mate Pair Sequencing (MPseq) (Smadbeck, BCJ, 2019). To our knowledge, no previous studies have reported a reduced performance of the alpha satellite D15Z4 FISH probe in PC FISH studies. A false-negative result for D15Z4 could result in an underreporting of trisomy 15 and overreporting of monosomy 15. Here, we investigated the incidence of false-negative D15Z4 FISH results by examining the presence of monosomy 15 from patients with a monoclonal gammopathy.Methods:We identified 1231 samples from patients with a monoclonal gammopathy who had undergone uniform testing to identify MM-specific cytogenetic abnormalities. DNA from bone marrow samples was genotyped on the Precision Medicine Research Array and biogeographical ancestry was assessed using the Geographic Population Structure Origins tool (Elhaik, Nat Commun, 2014; Home DNA 2016). FISH of immunoglobulin (cIg)-stained PCs was performed as described (Baughn, BCJ, 2018). Chromosome 15 centromere probe (Vysis CEP 15 D15Z4, Abbott Molecular, Abbott Park, IL) was used for chromosome 15 enumeration. Samples were divided into two groups: Cases with or without evidence of monosomy 15 by FISH. Mann-Whitney U test was applied to compare difference between groups for data deviating from normal distribution. A Chi-squared test evaluating the differences across these two groups was used.Results:We identified monosomy 15 in 31 cases (2.5% of cohort). Monosomy 15 was observed in nearly 100% of both the PC (cIg+) and non-PC (cIg-) populations, by contrast to the other recurrent PC-specific genomic abnormalities which were restricted to only the PC population. All samples with a banded analysis revealed most metaphases had two normal chromosome 15s demonstrating that the D15Z4 FISH probe failed to correctly enumerate the number of chromosome 15 centromere signals. Of the samples with a monosomy 15 result, the median African ancestry was 76.2% (95% percentiles: 77.5%-89.0%), while the median African ancestry of the non-monosomy 15 cohort was 2.3% (95% percentiles: 0%-87.7%) (). Although not all patients with >50% African ancestry had evidence of monosomy 15, 26/31 (83.9%) of patients with monosomy 15 had >50% African ancestry. Accordingly, patients with African ancestry (similarity ≥ 0.70) had 8.02-fold (95% confidence interval: 3.73-17.25, P = 9.92 X10 -8) increased possibility to have monosomy 15. Of cases with monosomy 15, 22 (71.0%) had an IGH rearrangement with the most prevalent IGH partner being CCND1. The incidence of t(11;14) was greater in the monosomy 15 group (13/31:41.9%), in contrast to the non-monosomy 15 group (330/1200=27.5%) (P = 0.12).Conclusions:We report a polymorphic variant of the chromosome 15 centromere resulting in a false-negative FISH result using the D15Z4 FISH probe. Surprisingly, the false-negative FISH result was not uniformly distributed among patients and was highly enriched in individuals of African ancestry. This may be due to a variation of the chromosome 15 alpha satellite in some individuals that prevents the D15Z4 FISH probe from sufficiently binding (O'Keefe, Genome Research, 2000). Although this study focused on identification of monosomy 15, the incidence of a false-negative result is likely greater than we have reported in this study since we did not evaluate cases that had a true trisomy 15 that was reported as normal chromosome 15 enumeration by FISH. This study highlights a critical need to ensure diagnostic reagents have equal performance for all patients independent of their ancestry. Evaluation of genomic events using unbiased approaches such as whole genome sequencing may circumvent some of these limitations. DisclosuresElhaik: DDC: Consultancy. Baird: DDC: Current Employment. Kumar: Antengene: Consultancy, Honoraria; Bluebird Bio: Consultancy; Beigene: Consultancy; BMS: Consultancy, Research Funding; Tenebio: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Novartis: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Carsgen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

Racial and Age-Related Differences in Impacts of High-Risk Cytogenetic Abnormalities on Survival in Multiple Myeloma in a Nationwide Electronic Health Record-Derived Database

Background: The incidence of multiple myeloma (MM) and enrichment of cytogenetic abnormalities differ significantly between racial/ethnic groups in the US, and their significance in determining myeloma progression and survival is not well understood. Whole genome sequencing has identified unique mutational signatures in MM, including an age-related process common in hyperdiploid myeloma. Our purpose was to describe racial and age-related differences in the impact of high-risk cytogenetic abnormalities (HRCAs) on survival in MM.Methods: We conducted a retrospective cohort study of adult MM patients starting first-line therapy between January 2011 and May 2021 using the nationwide Flatiron Health electronic health record-derived de-identified database. Patient-level demographic and clinical characteristics were ascertained using structured and unstructured data, curated via technology-enabled abstraction. Patients who had documented fluorescence in situ hybridization testing within 30 days prior to or 90 days following the start of first-line treatment were included. HRCAs, including gain or amplification 1q21, deletion 17p, t(4;14), t(14;16) and t(14;20), were identified and categorized as 0, 1, or 2+ HRCAs. Our outcomes of interest were real world progression free survival (rwPFS) and overall survival (rwOS). Cox proportional hazards models were used to calculate adjusted hazard ratios (HR) and 95% confidence intervals (CI), adjusted for demographic and clinical characteristics and treatment including time-dependent receipt of autologous stem cell transplantation.Results: From a cohort of 4889 MM patients, there were 790 (16%) Black and 2995 (61%) White patients with median ages at diagnosis of 68 and 70 years, respectively. Compared to White patients, a higher proportion of Black patients had IgG M-protein (61% vs 55%) and a lower proportion had 1+ HRCAs identified (31% vs 34%). Among all racial groups, compared to patients aged <65 years (N=1771), a higher proportion of patients aged 65+ years (N=3118) had IgA M-protein (21% vs 17%) and 1+ HRCAs identified (35% vs 33%).Multivariable models showed evidence of significant statistical interaction between age and prevalence of HRCA for rwPFS (P-int: 0.02). Among White patients, having 2+ HRCAs (“double-hit MM”) compared to no HRCAs was associated with worse rwPFS in both younger and older patients (<65 years: HR 2.88, 95% CI 1.93-4.32, P<0.01; 65+ years: HR 1.51, 95% CI 1.18-1.94, P<0.01). Among Black patients, associations between double-hit MM and rwPFS were attenuated and not statistically significant regardless of age (<65 years: HR 1.81, 95% CI 0.69-4.74, P=0.23; 65+ years: HR 1.61, 95% CI 0.92-2.81, P=0.09).Similarly, we also found evidence of statistical interaction between age and prevalence of HRCA for rwOS (P-int: 0.02). Among White patients, double-hit MM was significantly associated with worse rwOS but the magnitude of increased risk differed for younger (HR 3.39, 95% CI 2.24-5.14, P<0.01) and older (HR 1.61, 95% CI 1.27-2.05, P<0.01) patients. Double-hit MM was significantly associated with worse rwOS among older Black patients (HR 1.78, 95% CI 1.03-3.06, P=0.04), but not younger Black patients (HR 1.60, 95% CI 0.58-4.40, P=0.36).Conclusions: In this cohort of newly diagnosed MM patients treated in routine practice, having double-hit MM was differentially predictive of poor survival across age groups. Double-hit MM was associated with worse rwPFS and rwOS among White patients, but these trends were less consistent among Black patients. Our current understanding of cytogenetic risk stratification of MM requires further study and additional data for identifying low- and high-risk subsets of patients across different ages and racial groups.Figure. Kaplan-Meier survivor functions for rwPFS in White (Panel A) and Black (Panel B) patients by age group and number of HRCAs [Display omitted] DisclosuresCalip: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company; Pfizer: Research Funding. Ascha: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company. Wang: Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment. Pierre: Flatiron Health, Inc: Current Employment; Roche: Current holder of stock options in a privately-held company. Maignan: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company. Wadé: Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment. Leng: Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment. Seymour: Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Current equity holder in publicly-traded company; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Flatiron Health Inc: Current Employment. Patel: Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Neparidze: Eidos Therapeutics: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Research Funding; Janssen: Research Funding.

Clonality Analysis Reveals the Order of Acquisition of Copy Number Alterations in Multiple Myeloma

Multiple myeloma (MM) is characterized by a wide heterogeneity at both clinical and genomic level. Two major subgroups of MM have been identified: hyperdiploid MM (HMM) characterized by the trisomy of eight odd number chromosomes (3, 5, 7, 9, 11, 15, 19, and 21); and the non-hyperdiploid (NHMM) often associated with hemizygous deletion of chromosome 13, translocations involving the immunoglobulin heavy chain gene and copy number alterations (CNA) in other chromosome regions such as 1q, 6q, 8p, and 16q. Although, aneuploidy is a hallmark of MM, the sequence of occurrence of these complex genomic rearrangements during the MM development is unknown. Here we have studied, utilizing large datasets, when and in what sequence does each of these genomic events occurs, in both MM subgroup.We performed copy number profiling on 336 CD138+ MM cells from newly diagnosed patients and validated the results on another set of 103 samples. All patients were uniformly treated and have clinical data with median 6 years of follow up. We have used both cytoscan array as well as whole genome sequencing, as needed for the analysis. We have analyzed the data using dual quantile normalization, residual variability removal and segmentation using HG19. For each sample than we have extracted kernel smooth over a window of genomically contagious log2 ratio as smoothed signal. Clonality was then assessed by using deviation from two copies. We have also calculated clonality occurrence index which considers number of patients with clonal change for specific chromosome or cytoband.Our initial results showed that even though copy number gains were usually clonal and early events, while deletions, except 13q, were subclonal and also late events. Focused analysis on HMM samples showed that chromosome 15 is likely the first event with detected clonality in 82% of the cases, followed by occurrence of trisomies involving chromosomes 9 and 19 with clonality observed in 70 and 68% of the cases, respectively. For trisomies involving these three chromosomes, the clonality index was 42, 35 and 35 respectively, suggesting an early hyperdiploid events. Trisomies involving chromosomes 3, 5 and 11 occur subsequently and have clonality index of 25. Finally, chromosomes 7 and 21 trisomies are late events with clonality index of 15. This information is confirmed in an independent data set.In NHMM deletion 13 is a frequent and early clonal event while the other chromosomal aberrations such as del 1p, del 6q and del 17p are usually subclonal and hence late events. Del 13 is also observed in more than 25% of HMM patients where the clonality for del 13 was higher in the HMM chromosomes (chr 9, 15 and 19) compared to del13 (89% vs 65%, respectively) indicating that HMM occurs before del13 in these patients. Monosomy 14 is observed in 10% of patients and usually occurs at the same time with deletion 13, differently from other deletions such as 16q which is likely to happen after del13. Our analysis also showed that clonal or subclonal events on different regions across the genome actually carries much more information than just the global copy number data, e.g . clonality of chromosome 13 and chromosome 17 carry poor prognosis (p< 0.006, p <0.00026, respectively) while subclonality does not.Our decomposition analysis shows the potential order of occurrence of aneuploidy in MM and provides a new understand of the development of disease. This information now can be utilized to consider various genes resident within the early and late event chromosomes for their potential role in evolution of the disease. DisclosuresRichardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Moreau:Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Consultancy, Honoraria; Amgen: Honoraria; Millennium: Consultancy, Honoraria; Onyx Pharmaceutical: Consultancy, Honoraria; Celgene, Janssen, Takeda, Novartis, Amgen, Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Consultancy, Honoraria. Anderson:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; MedImmune: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Attal:Sanofi: Consultancy; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; JANSSEN: Consultancy, Research Funding.

CST6 Is a Small Autocrine Molecule That Targets Myeloma Growth and Bone Destruction

Bone destruction is a major complication of multiple myeloma (MM). Healthy bone is constantly remodeled through bone resorption by osteoclasts and bone formation by osteoblasts. New bone formation in MM is virtually non-existent, because differentiation of osteoblasts is inhibited by DKK1, a Wnt-β-catenin signaling inhibitor secreted by MM cells, reported by our group in NEJM, 2003. MM in its early stages is totally dependent on its microenvironment and for the hyperdiploid type MM this dependence is perpetual. Based on concordant gene expression signatures, predominantly driven by recurrent translocations and hyperdiploidy, we have classified MM into 7 distinct molecular entities. One subgroup, with significantly less bone disease and superior event-free and overall survival following high-dose therapy and stem cell transplantation than the other subgroups, defined as the Low Bone (LB) disease subgroup. Consistent with the LB phenotype, we have observed a strong inverse correlation between DKK1 and CST6 expression and by analyzing gene expression profiling (GEP) and RNA-sequencing data of more than 1,000 myeloma patients, we identified CST6 as the most upregulated gene in the LB subgroup. The aim of the present study was to determine the role of Cystatin E/M (CST6) in MM biology and to apply this knowledge to prevent both bone disease and MM cell growth. CST6, a 14-17 kD secretory protein, is a lysosomal protease inhibitor and suggested tumor suppressor gene. We showed that overexpression of CST6 in human MM cell lines prevents MM cell growth in vitro and in vivo. Also, purified CST6 protein from conditioned media of CST6-overexpressing MM cells significantly inhibits MM cell growth (p&amp;lt;0.01) and RANKL-induced osteoclast differentiation (p&amp;lt;0.01), decreases MM cell-induced bone destruction (p&amp;lt;0.05), and extends MM mouse survival (p&amp;lt;0.01). Mechanistic studies indicate that CST6 abrogates the alternative NF-kB signaling pathway as evidenced by a decrease in nuclear p52 protein in CST6-treated osteoclast precursors. Cathepsin K (CTSK), an osteoclast specific cysteine protease involved in bone resorption, was inhibited by CST6. GEP studies of whole bone marrow biopsies (WBMBx) across a spectrum of samples show higher expression of CTSK in WBMBx relative to purified plasma cells, while levels in MM remission WBMBx were higher than seen in healthy adult donors, MGUS/SMM, and newly diagnosed MM. Importantly, CTSK levels where not significantly different between remission and relapsed MM WBBx. These data show that Cathepsin K levels, and therefore osteoclasts, are elevated in the bone marrow of MM in remission and that these levels are similar to that seen in relapsed MM. Based on GEP data and experimental confirmation, we conclude that CST6, secreted by MM cells could be used clinically to target MM cells and prevent bone damage in MM. Inhibiting CTSK by CST6 in MM remission may aid in the prevention of MM relapse. Disclosures van Rhee: CDCN: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotech: Consultancy; EUSA: Consultancy.

Genome-Wide Somatic Alterations in Multiple Myeloma Reveal a Superior Outcome Group.

PURPOSEMultiple myeloma (MM) is accompanied by heterogeneous somatic alterations. The overall goal of this study was to describe the genomic landscape of myeloma using deep whole-genome sequencing (WGS) and develop a model that identifies patients with long survival.METHODSWe analyzed deep WGS data from 183 newly diagnosed patients with MM treated with lenalidomide, bortezomib, and dexamethasone (RVD) alone or RVD + autologous stem cell transplant (ASCT) in the IFM/DFCI 2009 study (ClinicalTrials.gov identifier: NCT01191060). We integrated genomic markers with clinical data.RESULTSWe report significant variability in mutational load and processes within MM subgroups. The timeline of observed activation of mutational processes provides the basis for 2 distinct models of acquisition of mutational changes detected at the time of diagnosis of myeloma. Virtually all MM subgroups have activated DNA repair–associated signature as a prominent late mutational process, whereas APOBEC signature targeting C>G is activated in the intermediate phase of disease progression in high-risk MM. Importantly, we identify a genomically defined MM subgroup (17% of newly diagnosed patients) with low DNA damage (low genomic scar score with chromosome 9 gain) and a superior outcome (100% overall survival at 69 months), which was validated in a large independent cohort. This subgroup allowed us to distinguish patients with low- and high-risk hyperdiploid MM and identify patients with prolongation of progression-free survival. Genomic characteristics of this subgroup included lower mutational load with significant contribution from age-related mutations as well as frequent NRAS mutation. Surprisingly, their overall survival was independent of International Staging System and minimal residual disease status.CONCLUSIONThis is a comprehensive study identifying genomic markers of a good-risk group with prolonged survival. Identification of this patient subgroup will affect future therapeutic algorithms and research planning.

The Landscape of Genome Wide Somatic Alterations Identifies a Good-Risk Group in Newly Diagnosed Multiple Myeloma

Multiple Myeloma (MM) is a plasma cells malignancy with number of recent therapeutic options that has improved outcomes with median survival now stretching beyond 8 years. There has been an intense search to identify genomic and laboratory correlates of outcome for high risk patients. However, a subgroup of patients have a long survival but genomic segmentation of this important group which can be potentially cured has not been identified. We here described first such attempt at identifying a subset of patients with long survival. We have analyzed data from 205 newly-diagnosed uniformly-treated MM patients using both deep whole genome (WGS) (80x) and whole transcriptome sequencing (RNAseq). Median number of SNVs, small insertion and deletion per megabase were 2.18 [0.49 - 14.52], 0.077 [0.024 - 0.17] and 0.12 [0.03 - 0.26] respectively. MM subgroups defined by FISH or copy number alterations (CNAs) had significantly different mutational load (Kruskal-Wallis p-value = 0.003). In general, Hyperdiploid MM (HMM) patients had lower SNV load compared to other subgroups and t(14;16) had the highest mutational load per megabase (Dunn test FDR &lt; 0.25). These subgroups also showed significant differences for mutational process utilization. Mutational processes associated spontaneous deamination of 5-methylcytosine to thymine was significantly high in HMM, APOBEC activity was very strong in t(14;16) MM and DNA repair was significantly different in t(6;14), del17p and t(11;14). Importantly, the mutations associated with clonal cell population and thus in early phases of MM cell progression were driven by spontaneous or enzymatic deamination (Signature 1), somatic hypermutation in lymphoid cells (Signature 9) and Signature 17 (ANOVA p value &lt; 1e-16) and surprisingly APOBEC activity was constant among the clones (ANOVA p value &gt; 0.05). However, mutations associated with homologous recombination (HR) and nucleotide excision repair (NER) activity were significantly enriched in the subclonal mutations (ANOVA p value &lt; 1e-16) suggesting their role in later stages of MM progression. Based on this information, we next calculated the homologous recombination repair defects using copy number alterations from WGS data. We identified that HR score generated using WGS copy number information predicted outcome in newly diagnosed myeloma. Further analysis of this data identified that newly diagnosed MM patients with hyperdiploid MM (HMM) and no HR- repair deficiency have superior outcome with 6 year overall survival (OS) probability for this of 100% in IFM/DFCI 2009 cohort (Logrank test p value = 0.0012). We next validated this finding using MMRF CoMMpass dataset and we confirmed 6-yr OS probability of 95% (Logrank test p value = 0.0016) in this independent dataset (Figure 1). We further investigated RNAseq data between these genomically defined groups and identified that chromosome and telomere maintenance pathways (FDR &lt; 0.01) and low bone disease associated genes described by Zhan et al. (FDR = 0.004) were up regulated in low-risk group. Finally, patients who were able to maintain HR, had lower mutation rate for TP53 (0% vs 9%, FDR &lt; 0.05), CSMD1 (0% vs 6%, FDR &lt; 0.05), FAT3/FAT4 mutations (1.7% vs 15%, FDR &lt; 0.05), however surprisingly higher mutation rate for NRAS (42% vs 14%, p value &lt; 0.001). In addition, DNA damage associated processes predicted by SNVs trinucleotides were significantly lower in the low-risk group (12% vs. 20%, p value &lt; 1e-05) providing further support to this data. In conclusion, we report a detailed genomic profile using deep DNA and RNA sequencing and identify a genomically-defined sub group that is predicted to have a long survival. This study also identifies the role of HR in myeloma with potential for its translational application in both prognosis as well as therapy. Figure 1 Disclosures Richardson: Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Moreau:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Thakurta:Celgene: Employment, Equity Ownership. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Avet-Loiseau:celgene: Consultancy, Other: travel fees, lecture fees, Research Funding; takeda: Consultancy, Other: travel fees, lecture fees, Research Funding. Munshi:Takeda: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy.

Super-Enhancer Profiling Identifies Novel Oncogenes and Therapeutic Targets in Multiple Myeloma

Background: Multiple myeloma (MM) is an aggressive neoplastic plasma cell cancer characterized by diversely cytogenetic abnormalities. MM can be divided into subtypes with immunoglobulin heavy chain (IGH) gene translocations involving CCND1-3, FGFR3/MMSET, MAFs and hyperdiploid myeloma containing trisomies of several odd numbered chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. Although several new drugs have been introduced into clinic, treatment for MM patients remains challenge and refractory/resistant to therapy is often seen. Thus, a better understanding of the molecular pathogenesis of MM can lead to generate new prognostic classification and identify new therapeutic targets. Super-enhancers (SEs) are defined as large clusters of cis-acting enhancers, marked by high level bindings of acetylation of histone H3 lysine 27 (H3K27ac) and mediator complex. SEs have been shown to control genes for maintaining cellular identity and also key tumor drivers in various malignancies. Methods: H3K27Ac ChIP-seq and RNA-seq were performed on primary MM patient samples, MM cell lines. Normal plasma cells and lymphoma cell lines were served as controls. We systematically compared SEs and their associated genes of normal and cancerous tissue. THZ1, a CDK7 inhibitor, was used to efficiently down-regulate SE-associated genes. Combinatory analysis of THZ1-sensitive and SE-associated gene revealed a number of promising MM oncogenes. CRISPR/Cas9 technology and ectopic expression experiments in conjunction with cellular functional assays were performed to determine the effects of candidate SE-genes on MM cells. Circularized chromatin conformation capture followed by sequencing (4C-seq) was applied to explore the direct contact of SE and promoter. Results: SE analysis uncovered some cell lineage-specific transcription factors (TFs) and known oncogenes in MM. Several key TFs, including IRF4, PRDM1, MYC and XBP1, were identified in most MM samples, confirming the origin of MM cells. These data reinforce the concept that SE establishment is a key component of MM biology. The acquisition of SEs around oncogene drivers is widely observed during tumorigenesis. ST3GAL6 and ADM were two known oncogenic drivers in myeloma cells, which were associated with super-enhancers in all MM samples but not in normal plasma cell and lymphoma cells. We also found SE constituents for multiple subtype-specific key oncogenes such as CCND1 in t(11;14) cells, C-MAF in t(14;16) cells, and NSD2 and FGFR3 in t(4;14) cells. Furthermore, THZ1 showed prominent anti-neoplastic effect against MM cells. SE-associated genes were more sensitive to THZ1 compared with those genes associated with typical enhancers (TEs). By overlapping THZ1-sensitve gene with SE-associated genes, we identified a number of novel MM oncogenes, including MAGI2, EDEM3, HJURP, LAMP5, MBD1 and UCK2 as a potential druggable kinase. The expression level of MAGI2 and HJURP confers poor prognosis in several MM datasets. MAGI2 silencing in MM cells decreased cell proliferation and induced apoptosis. qRT-PCR and Western blot analysis confirmed the overexpression of HJURP in t(4;14) cells relative to non-t(4;14) MM cells. Furthermore, 4C-seq analysis revealed the physical interaction between HJURP-SE and promoter and THZ1 treatment diminished this interaction. Motif search at SE constituents revealed a highly significant enrichment of NSD2 recognition. Significant reduction of NSD2 binding at HJURP-SE region was observed in KMS11 infected with NSD2-specific shRNAs. Interestingly, blocking SE sites by CRISPR/Cas9i or silencing HJURP by shRNA led to decreased HJURP expression and cell apoptosis, whereas overexpression of this gene promoted cell growth. Taken together, our data demonstrated that HJURP is a novel SE-associated oncogene in t(4;14) MM. Conclusions: Our integrative approaches by combing H3K27Ac ChIP-seq, RNA-seq and THZ1-sensitive transcript defined the landscape of SE and identified SE-associated novel oncogenes, as well as lineage-specific TFs in MM. Furthermore, we also revealed subtype-specific SE-driving oncogenic program in MM. Taken together, these results not provide novel insight into the MM pathology, but also offer novel, potential therapeutic targets, such as MAGI2, and HJURP for the treatment of MM patients. Disclosures No relevant conflicts of interest to declare.

Oncogenic MAF in Co-Operation with IRF4 Confers Extensive Chromatin Re-Arrangement in Plasma Cells and Generates ‘Neo-Enhancers’ That Regulate Genes Critical for Myeloma Biology

Overexpression of the transcription factor MAF, as a result of its juxtaposition to the IgH enhancer [MAF-translocated t(14;16)], is a myeloma-initiating event in 3-5% of patients with multiple myeloma (MM) and confers a poor prognosis. MAF is also overexpressed in another 40% of cases, often in co-operation with the oncogene MMSET. The mechanisms by which MAF overexpression impacts on the regulatory genome to generate the MAF-driven oncogenic transcriptome and its direct targets are not known. To address this, we employed a multi-layer -omics approach using primary myeloma plasma cells (PC) as well as myeloma cell lines (MMCL). First, we determined the chromatin accessibility and transcriptome profiles of MAF-translocated myeloma by performing ATAC-seq and RNA-seq, respectively, in purified bone marrow CD138+ PC from two patients with t(14;16) and three healthy donors. We identified 6,640 differentially accessible regions, 87% of which displayed enhanced chromatin accessibility in MAF samples compared to normal PC. Secondary analysis comparing this with ATAC-seq data from a set of 28 other MM samples, including hyperdiploid, MMSET and CCND1-translocated MM, revealed 33% of those regions to be MAF subgroup specific (1,949 regions), with the rest shared between MAF and other cytogenetic groups. Gene annotation and pathway enrichment analysis using GREAT confirmed overrepresentation of the MF myeloma patient signature, as previously identified in microarray datasets. RNA-seq detected significant upregulation of approximately 900 genes in MAF samples compared to normal counterparts, including MAF itself (top 4th hit) as well as its presumed targets (CCND2, ITGB7 and NUAK1). Next, we obtained the MAF cistrome using ChIP-seq in the MAF-translocated MMCL MM1.S and integrated it with the primary PC ATAC-seq data. This revealed that 31% (618/1,949) of the differentially accessible regions in MAF-translocated MM PC are also MAF-bound. Additional overlay with ENCODE ChromHMM epigenome map showed that 47% of MAF binding sites are on active enhancers and 42% on active promoters signifying potential direct regulation of the corresponding genes. Next, we superimposed the accessible and MAF-bound loci on the epigenomic landscapes of normal PC and other B-cell types using their corresponding ChromHMM maps (Blueprint consortium data). Interestingly, 56% (345/618) of the MAF-specific regions were not active in any stage of B cell development. This suggests that aberrant MAF overexpression and chromatin binding in PC is associated with de novo activation of these chromatin regions, over half of which (200/345; 58%) are enhancers; we termed these 'neo-enhancers'. Upon de novo motif analysis of MAF ChIP-seq in MAF-translocated JJN3 and MM1.S MMCL, we confirmed MAF as the first and, interestingly, IRF4 as the second top hit, suggesting a possible MAF-IRF4 functional interaction in myelomagenesis. Indeed, overlay of the accessible MAF-bound loci with IRF4 ChIP-seq data in MM1.S revealed 63% co-occupancy (including 62% of "neo-enhancers"), proposing a novel and extensive co-operative chromatin-based network between the two transcription factors. Final integration of the accessible MAF-bound regions with the paired transcriptomes of primary myeloma PC revealed a set 206 candidate enhancer-gene pairs. Strikingly, we identified two IRF4-cobound "neo-enhancers" linked to overexpression of TLR4 and CCR1, two genes known for their roles in myeloma cell proliferation and migration. We confirmed significant downregulation of both genes upon shRNA-mediated knockdown of MAF in the two MAF-translocated MMCL, MM1.S and JJN3, as well as the lethality of MAF depletion. Further, MAF overexpression in MAF-negative myeloma backgrounds led to transcriptional upregulation of these genes, further validating them as MAF targets. While CRISPR/Cas9i experiments targeting TLR4 are ongoing, preliminary results validated the functional role of the "neo-enhancer" in CCR1 gene expression. In conclusion, we demonstrate for the first time an extensive re-organisation of the PC chromatin conferred by oncogenic MAF in MM; we reveal its extensive co-operation with IRF4 in this process; we validate the directly MAF-regulated genes and functionally characterise neo-enhancers of key MAF-dependent genes that in addition to MAF itself are also critical for myeloma biology. Disclosures Hatjiharissi: Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

Abstract 750: Gene Signatures to Distinguish Amyloid Cardiomyopathy Risk in Multiple Myeloma Patients

Amyloid light chain (AL) amyloidosis results from tissue deposition of clonal light chains, most commonly produced by clonal plasma cells. AL amyloidosis is closely associated with multiple myeloma (MM), another disease which arises from clonal plasma cell proliferation. Here we aim to identify gene signatures to distinguish AL cardiomyopathy (AL-CM) risk in MM patients. We utilized publicly available data sets and applied Graph Cluster Perturbation approach to cluster the co-expression networks based on pathways in MM and AL-CM utilizing 225 samples from four datasets: GSE42955 study (12 dilated CM, 12 ischemic CM and 5 control LV human heart tissues), GSE95077 study (16 amiloride drug treated/untreated myeloma cell line samples), GSE24128 study (16 newly diagnosed AL with monoclonal plasma cell samples over- or under-expressing cyclin D1), and GSE6477 study (76 primary bone marrow samples from hyper-diploid myeloma patients and 80 non-hyperdiploid MM patients). From these data sets, the networks for extracellular matrix organization, immune system, innate immune system, metabolism, and neutrophil degranulation pathways for MM and amyloid CM were extracted. Summary: Ranking of the perturbed genes based on pathways similarity index in MM and AL resulted in a panel of genes: CD44, NRAS, GRAP2, CTLA4, GSN, CCND1, NFKB1, and IRF1 (p= &lt;0.01). As shown in Figure , the high hazard ratio of this gene cluster in MM with AL-CM (3.76; p=0.021) compared to MM without CM (0.96; p=0.032) suggests the potential of this gene panel to distinguish high or low risk groups in MM based on survival.

PS1431 IDENTIFICATION OF THE TRUE HYPERDIPLOID MULTIPLE MYELOMA SUBSET BY COMBINING CONVENTIONAL KARYOTYPING AND FISH ANALYSIS

Background:Prognostic evaluation of Multiple Myeloma (MM) by FISH is focused on high risk (HR) cytogenetic aberrations, namely t(4;14), t(14;16) and deletion of 17p13. Hyperdiploid MM (HRD‐MM), instead, that accounts for about 50% of newly diagnosed MM, is not routinely investigated because multiple probes are required to test over the conventional markers. However, in the era of novel agents, the identification of hyperdiploidy may be helpful in MM prognostication since trisomic MM is responsive to lenalidomide treatment and retains a favourable outcome.Aims:The aim of our study was to elaborate a flow chart of FISH prognostic assessment of MM that, beside the analysis of conventional high risk abnormalities, allows to identify those cases with favourable prognosis within hyperdiploid MM.Methods:Bone marrow of 292 patients affected by newly diagnosed MM was studied by conventional karyotype (CK) and on separated plasmacells by FISH for HR cytogenetic aberrations including t(4;14), t(14;16), 17p13 deletion and 1q21 gain. Patient's characteristics including ISS stage, symptoms at diagnosis, type of treatment and OS were collected.Results:Among our entire cohort, CK was productive in 76 patients and based on the number of chromosome of clone (47–57 chromosomes) 50 patients (66%) were classified as hyperdiploid. All patients received treatment with novel agents, including bortezomib (99%), lenalidomide (70%) or both (70%).Of note, cytogenetic classification of HRD‐MM, beyond the definition of ploidy, includes a heterogeneous group characterized not only by numerical abnormalities, but also by several structural aberrations. Therefore, even the ploidy level was not enough to characterize hyperdiploid MM. As a matter of fact, within HRD‐MM patients, high risk chromosomal changes were detected in 26/50 (52%) cases, including 21 (42%) with gain 1q, one with t(4;14) and 8 (16%) with 17p deletion. Finally, 20 (40%) patients displayed an IgH translocation. Major features associated to decreased OS were trisomy 19 (32 vs 62 months, p = 0.0194), gain 1q (30 vs 56 months, p = 0.0371), and IgH translocations (33 vs 57 months, p = 0.0285) (Figure 1A to C).Provided that definition of HRD‐MM is based on trisomies of odd chromosomes, we further classified our patients according to the number of trisomies in cases with &gt; 5 trisomies (trisomic HRD‐MM, T‐HRD‐MM) and cases with &lt;5 trisomies (non trisomic HRD‐MM, N‐HRD‐MM). The group with &gt; 5 trisomies (n = 26) was characterized by lower rates of overall FISH alterations (&lt;2, 92% vs 31%, p = 0.0001). More in detail, lower frequencies of IgH translocations (23% vs 75%, p = 0.0014) and HR FISH changes (5% vs 50%, p = 0.0023) were detected. Even monosomy 13 was less represented in this subset of patients (15% vs 69%, p = 0.008) as compared to N‐HRD‐MM. Interestingly, concomitant trisomy of 9, 11 and 15 chromosomes was described in 53% of T‐HRD‐MM patients but it was completely absent in N‐HRD MM group (p = 0.0002), indicating that in our cohort these three numerical changes represent the most powerful signature to identify HRD MM. Finally, T‐HRD MM subset displayed better outcome towards N‐HRD MM (57 vs 32 months, p = 0.001, Figure 1D).Summary/Conclusion:In our cohort of patients the combination of banding analysis and FISH for HR aberrations identify some genetic features that correlate with the presence of true hyperdiploid clone. In fact, less than two FISH aberrations, absence of HR aberrations and IgH translocation and the concomitant presence of trisomies of chromosome 9, 11 and 15 identify the hyperdiploid MM subset characterized by a favourable outcome.image

Deciphering the chronology of copy number alterations in Multiple Myeloma

Multiple myeloma (MM) and its precursor condition MGUS are characterized by chromosomal aberrations. Here, we comprehensively characterize the order of occurrence of these complex genomic events underlying MM development using 500 MGUS, and MM samples. We identify hyperdiploid MM (HMM) and non-HMM as genomically distinct entities with different evolution of the copy number alterations. In HMM, gains of 9,15 or 19 are the first and clonal events observed as clonal even at MGUS stage. These events are thus early and may underlie initial transformation of normal plasma cells to MGUS cells. However, CNAs may not be adequate for progression to MM except in 15% of the patients in whom the complex subclonal deletion events are observed in MM but not MGUS. In NHMM, besides the driver translocations, clonal deletion of 13 and 1q gain are early events also observed in MGUS. We combined this information to propose a timeline for copy number alteration.

Cereblon-binding proteins expression levels correlate with hyperdiploidy in newly diagnosed multiple myeloma patients

Immunomodulatory drugs (IMIDs) are very effective in the treatment of multiple myeloma (MM). The description of their cereblon-mediated mechanism of action was a hallmark in MM research. Although the importance of IMID-induced degradation of cereblon-binding proteins is well described in vitro, the prognostic value of their expression levels in MM cells is less clear. Based on recently published data showing somewhat conflicting RNA levels, we analyzed the association between the levels of the Ikaros family zinc finger protein 1 (IKZF1), IKZF3, and karyopherin subunit alpha 2 (KPNA2) proteins measured by flow cytometry and prognostic parameters in 214 newly diagnosed MM patients who were randomized in the GMMG HD6 trial. No statistically significant associations between the expression levels and age, gender, light chain type, International Staging System (ISS) stage or cytogenetic high- and normal risk groups could be identified. Hyperdiploid MM cells expressed significantly higher levels of IKZF1, IKZF3 and KPNA2 than nonhyperdiploid cells. In contrast, translocation t(11;14) was associated with significantly lower expression levels. In conclusion, the observed overexpression of cereblon-binding proteins in MM cells with gain of chromosomes 5, 9, 11, 15, and 19 is consistent with the previously proposed positive regulation of MYC by IKZF1 and IKZF3, as well as MYC activation in hyperdiploid MM cells.

Poor overall survival in hyperhaploid multiple myeloma is defined by double-hit bi-allelic inactivation of TP53.

Hyperhaploid multiple myeloma is a rare numerical aberration group defined by a range of 24-34 chromosomes, which is associated with a poor prognosis with a 5-year survival rate of 23%. Hyperhaploid patient samples (n=8) were sequenced and copy number and mutations identified. Samples had a median of 13 monosomies (range 12-14), which in general were those not associated with trisomies in hyperdiploid samples. The chromosomes traditionally trisomic in hyperdiploid myeloma were disomic in hyperhaploid myeloma with retention of heterodisomy. We examined the hyperhaploid samples for frequently mutated genes and found that 8/8 (100%) hyperhaploid samples had a mutation in TP53, exceeding the overall rate of mutation in newly diagnosed patients (5.5%), indicating an oncogenic dependency in this group. All samples with TP53 mutation also had monosomy of chromosome 17, indicating bi-allelic inactivation of TP53. As such, this high risk group is part of double-hit myeloma.

Mieloma multipel: aspek patogenesis molekuler

Multiple myeloma (MM) is a neoplastic plasma disorder that is characterized by clonal proliferation of malignant plasma cells in the bone marrow, monoclonal protein in the blood or urine and associated organ dysfunction. It is preceded by a premalignant tumor which is share genetic abnormalities, monoclonal gammopathy of undetermined significance (MGUS). Although remarkable progress has been achieved, but pathogenesis of MM is still very complex. Multiple myeloma appears to arise from the malignant transformation of germinal-center B-lymphocyte. The first oncogenic events in MM appear to occur in the germinal center due to error in isotype class switching and somatic hypermutation. MM is divided into two distinct genetic subtypes: (1) hyperdiploid myeloma is characterized by multiple trisomies of chromosome 3, 5, 7, 9, 11, 15, 19 and 21; (2) non-hyperdiploid in contrast is characterized by recurrence translocations t(4;14), t(14;16), t (14;20); t(6;14) and t(11;14). A unifying event in the pathogenesis of MM is the dysregulated expression of cyclin D gene. Genetic aberrations occur in MM and also in premalignant state (MGUS), suggesting that genetic mutations alone are necessary, but not sufficient for myeloma transformation. A “ random second hit model” was proposed. Hypothetical second hits are: additional genetic changes ( RAS mutation, p16 methylation, p53 mutation), proliferation due to cell cycle dysregulation, evasion of programmed cell death and changes in bone marrow microenvironment. A complex interaction with the BM microenvironment , characterized by activation of osteoclast and supression of osteoblast , leads to lytic bone lesions.