DIS3 Mutations Research Articles

The genomic profiling of high-risk smoldering myeloma patients treated with an intensive strategy unveils potential markers of resistance and progression

Smoldering multiple myeloma (SMM) precedes multiple myeloma (MM). The risk of progression of SMM patients is not uniform, thus different progression-risk models have been developed, although they are mainly based on clinical parameters. Recently, genomic predictors of progression have been defined for untreated SMM. However, the usefulness of such markers in the context of clinical trials evaluating upfront treatment in high-risk SMM (HR SMM) has not been explored yet, precluding the identification of baseline genomic alterations leading to drug resistance. For this reason, we carried out next-generation sequencing and fluorescent in-situ hybridization studies on 57 HR and ultra-high risk (UHR) SMM patients treated in the phase II GEM-CESAR clinical trial (NCT02415413). DIS3, FAM46C, and FGFR3 mutations, as well as t(4;14) and 1q alterations, were enriched in HR SMM. TRAF3 mutations were specifically associated with UHR SMM but identified cases with improved outcomes. Importantly, novel potential predictors of treatment resistance were identified: NRAS mutations and the co-occurrence of t(4;14) plus FGFR3 mutations were associated with an increased risk of biological progression. In conclusion, we have carried out for the first time a molecular characterization of HR SMM patients treated with an intensive regimen, identifying genomic predictors of poor outcomes in this setting.

Kinetics and Biology of Circulating Tumor Cells (CTCs) and Measurable Residual Disease (MRD): Two Dynamic High-Risk Clones in Multiple Myeloma (MM)

BACKGROUND CTCs and MRD are two small clones associated with a high risk of progression in smoldering MM (SMM) and post treatment relapse in active MM. Despite growing knowledge about their clinical significance and biological features, these questions remain unanswered: 1) What is the prognostic value of CTC and MRD kinetics? 2) Is MM dissemination and on-treatment resistance driven by specific genomic alterations? 3) Are CTCs and MRD genetically related? AIM Analyze the kinetics and molecular features of CTCs and MRD in SMM and MM patients. METHODS CTC kinetics were investigated using NGF in 164 SMM patients with ≥3 assessments collected every 6 months in the iMMunocell study. MRD kinetics were investigated using NGF in 277 MM patients with ≥4 assessments in BM aspirates after induction, transplant, consolidation, and yearly during maintenance (GEM2012MENOS65-GEM2014MAIN trials). Unsupervised clustering based on CTC and MRD kinetics was performed using CONNECTOR. Molecular features of BM tumor cells at diagnosis were compared to paired CTCs in 94 SMM and MM patients and to paired MRD in 96 MM patients. In 10 cases, genomic data from tripartite BM tumor cells, CTCs, and MRD was available. Tumor samples were isolated based on patient-specific aberrant phenotypes using FACS. Whole exome and RNA-seq were performed in 343 and 217 samples, respectively. RESULTS Based on the assessment of CTCs in 754 PB samples, SMM patients clustered into 3 groups: sustained undetectable (n = 20), stable (n = 71) and evolving (n = 73) CTC levels. With a median follow-up of 2 years, the respective 2y progression-free rates were 100%, 94% and 81% ( P =.003). Risk stratification based on CTC kinetics (C-index 0.68) outperformed a single baseline assessment (C-index 0.61) and, along with the IMWG 20/2/20 model, had independent prognostic value (HR 3.4; P = .01). Patients with high risk cytogenetics showed more frequently evolving CTC levels. Studying genetic drivers of tumor egress, we found CTCs to closely resemble BM tumor cells (88% concordance at copy number [CNA] and somatic mutational [SNV] level). No recurrent cell-specific SNV or CNA was identified. CTC's transcriptional profile revealed 268 DEGs enriched in interferon alpha, gamma response and cell-adhesion pathways. Next, the prognostic value of MRD dynamics was analyzed according to 1,759 assessments, identifying 3 patient clusters: sustained undetectable (n = 100), stable (n = 104), and evolving (n = 73) MRD levels. Respective 6-year rates of PFS after consolidation were 90%, 75% and 5% ( P <.001), and of OS were 94%, 95% and 66% ( P<.001). Risk stratification based on MRD kinetics (C-index 0.82) outperformed a single MRD assessment before maintenance (C-index 0.62), the R-ISS (C-index 0.57) and R2-ISS (C-index 0.61) at diagnosis. MRD cells showed heterogeneous genomic evolution (60% median concordance) diverging from BM tumor cells at diagnosis due to de novo CNA and SNV that emerged at a subclonal level, particularly after transplant. After induction, MM driver mutations were detected or remained clonal in 13/19 (68%) MRD, whereas after transplant, this was true for only 5/13 (38%) MRD. No recurrent cell-specific SNV or CNA was identified. The transcriptional signature of MRD was treatment dependent, with common molecular features of on-treatment resistance found in all 96 patient-paired samples analyzed after various treatment stages. The 334 DEGs were commonly enriched in KRAS signaling hallmarks. Genomic profiles of tripartite BM tumor cells, CTCs and MRD from 10 MM patients confirmed a patient-specific mutational landscape with genomic heterogeneity in MRD. For instance, one patient exhibited BRAF & DIS3 mutations and del(13q) detected at baseline in BM tumor cells and CTCs, which persisted in MRD. After induction, de novo clonal SNVs, including a NEK11 mutation, emerged, followed by the appearance of 3 subclonal CNA after transplant, possibly contributing to resistance. CONCLUSIONS This is the largest dataset analyzing the kinetics and molecular features of CTCs and MRD in SMM and active MM. We showed superior prognostic value of CTC and MRD kinetics over single assessments. Tumor dissemination seems to be driven by transcriptional priming rather than acquired secondary genetic events, while on-treatment resistance is linked to genomic and transcriptional evolution, without recurrent genetic alterations and with common molecular signatures of resistance.

Late B Cell-Specific Dis3-Knockout Mice Do Not Develop Plasma Cell Neoplasm

Recent advances in next-generation sequencing have unveiled genetic abnormalities associated with multiple myeloma (MM). Of note, DIS3 mutations have been observed in ~10% of MM patients, and 13q deletion including the DIS3 gene locus are present in ~40% of MM patients. Furthermore, DIS3 mutations/deletions have been shown to be associated with poorer prognosis in MM. Regardless of high incidence of DIS3 mutations/deletions and their relation to adverse outcome, the roles of DIS3 in hematopoiesis and myelomagenesis remains incompletely understood. Here we show that Dis3 is required for normal hematopoiesis and Dis3 deficiency is not sufficient for the development of plasma cell neoplasm in mice. We first explored DIS3 functions in hematopoiesis by generating Dis3 conditional knockout (KO) mice (Dis3 fl/fl;Mx-Cre). To avoid possible effects of Dis3 deficiency on nonhematopoietic cells, we transplanted bone marrow (BM) cells from Dis3 fl/fl;Mx-Cre mice into lethally irradiated CD45.1 + recipient mice. Four weeks after transplantation, we knocked out Dis3 by inducing Cre recombinase via an intraperitoneal injection of Poly(I:C). The deletion of Dis3 in BM cells was confirmed by genotyping and quantitative real time PCR. Importantly, loss of Dis3 in hematopoietic cells resulted in severe pancytopenia at 2 weeks after Dis3 deletion. Consistent with this observation, Dis3 KO mice showed reduced BM counts with relative preservation of mature lymphocytes. A flow cytometric analysis of the BM exhibited lower frequencies of Lin -Sca-2 +c-Kit + (LSK), hematopoietic stem, and multipotent progenitor cells in Dis3 KO mice compared with wild-type (WT) mice. Dis3 KO mice also exhibited lower frequencies of common myeloid progenitors, granulocyte/macrophage progenitors, and megakaryocyte/erythroid progenitors compared with WT mice. A frequency of common lymphoid progenitors (CLPs) was comparable between WT and Dis3 KO mice, whereas absolute numbers of CLPs were significantly reduced in Dis3 KO mice than in WT mice. These results indicate that Dis3 is indispensable for normal hematopoiesis, and LSK and myeloid progenitor cells are more dependent on Dis3 than lymphoid progenitor cells. We next examined whether loss-of-function of Dis3 drives plasma cell neoplasm. To do so, we crossed Dis3 fl/wt or Dis3 fl/fl mice with Cgamma1-Cre mice and generated late B cell-specific Dis3 KO mice by activating Cre recombinase via immunizing with NP-CGG in these compound mice. However, these mice did not develop any B cell malignancies and shorten the survival compared with WT mice. In the samples of MM patients, the frequency of DIS3 mutations is significantly higher in t(14;16) samples than in those without t(14;16) (Walker et al. Blood 2018). We therefore utilized t(14;16) model mice (c-Maf TG) (Morito et al. Cancer Res 2011) and generated c-Maf TG; Dis3 fl/wt;Cgamma1-Cre and c-Maf TG; Dis3 flfl;Cgamma1-Cre mice. As reported (Morito et al. Cancer Res 2011), a small fraction of mice with c-Maf TG developed B cell lymphoma after a long latency. However, the addition of Dis3 KO did not increase the frequency of B cell lymphoma and shorten the survival compared with c-MAF TG mice. These results indicate that only Dis3 deficiency and the combination of c-Maf overexpression and Dis3 deficiency do not develop plasma cell neoplasm and suggest that additional oncogenic events are necessary for myelomagenesis. Further investigations are required for elucidating the mechanisms of how loss-of-function of DIS3 is involved in the development of MM.

OR19-02 DIS3 Variants Are Associated with Primary Ovarian Insufficiency

Abstract Disclosure: E.B. Johnstone: None. B. Gorsi: None. E. Coelho: None. B. Moore: Consulting Fee; Self; Fabric Genomics. C. Chow: None. M. Yandell: Consulting Fee; Self; Fabric Genomics. Stock Owner; Self; Fabric Genomics. C.K. Welt: None. Context: A genetic etiology accounts for the majority of unexplained primary ovarian insufficiency (POI). Objective: We hypothesized a genetic cause of POI for a sister pair with primary amenorrhea.Design: The study was an observational study.Setting: Subjects were recruited at an academic institution.Subjects: Subjects were sisters with primary amenorrhea caused by POI, and their parents. Additional subjects included women with POI analyzed previously (n=291). Controls were recruited for health in old age or were from the 1000 Genomes Project (total n=233). Intervention: We performed whole exome sequencing (WES) and data were analyzed using the Pedigree Variant Annotation, Analysis and Search Tool (pVAAST), which identifies genes harboring pathogenic variants in families. We performed functional studies in a D. melanogaster model.Main Outcome: Genes with rare pathogenic variants were identified. Results: The sisters carried compound heterozygous variants in DIS3. The sisters did not carry additional rare variants that were absent in publicly available datasets. DIS3 knockdown in the ovary of D. melanogaster resulted in lack of oocyte production and complete infertility.Conclusions: Compound heterozygous variants in highly conserved amino acids in DIS3 and failure of oocyte production in a functional model suggest that mutations in DIS3 cause POI. DIS3 is a 3’ to 5’ exoribonuclease that is the catalytic subunit of the exosome involved in RNA degradation and metabolism in the nucleus. The findings provide further evidence that mutations in genes important for transcription and translation are associated with POI. Presentation Date: Saturday, June 17, 2023

DIS3 depletion in multiple myeloma causes extensive perturbation in cell cycle progression and centrosome amplification.

DIS3 gene mutations occur in approximately 10% of patients with multiple myeloma (MM); furthermore, DIS3 expression can be affected by monosomy 13 and del(13q), found in roughly 40% of MM cases. Despite the high incidence of DIS3 mutations and deletions, the biological significance of DIS3 and its contribution to MM pathogenesis remain poorly understood. In this study we investigated the functional role of DIS3 in MM, by exploiting a loss-of-function approach in human MM cell lines. We found that DIS3 knockdown inhibits proliferation in MM cell lines and largely affects cell cycle progression of MM plasma cells, ultimately inducing a significant increase in the percentage of cells in the G0/G1 phase and a decrease in the S and G2/M phases. DIS3 plays an important role not only in the control of the MM plasma cell cycle, but also in the centrosome duplication cycle, which are strictly co-regulated in physiological conditions in the G1 phase. Indeed, DIS3 silencing leads to the formation of supernumerary centrosomes accompanied by the assembly of multipolar spindles during mitosis. In MM, centrosome amplification is present in about a third of patients and may represent a mechanism leading to genomic instability. These findings strongly prompt further studies investigating the relevance of DIS3 in the centrosome duplication process. Indeed, a combination of DIS3 defects and deficient spindle-assembly checkpoint can allow cells to progress through the cell cycle without proper chromosome segregation, generating aneuploid cells which ultimately lead to the development of MM.

DIS3: The Enigmatic Gene in Multiple Myeloma.

Recent studies have revealed the genetic aberrations involved in the initiation and progression of various cancers, including multiple myeloma (MM), via next-generation sequencing analysis. Notably, DIS3 mutations have been identified in approximately 10% of patients with MM. Moreover, deletions of the long arm of chromosome 13, that includes DIS3, are present in approximately 40% of patients with MM. Regardless of the high incidence of DIS3 mutations and deletions, their contribution to the pathogenesis of MM has not yet been determined. Herein, we summarize the molecular and physiological functions of DIS3, focusing on hematopoiesis, and discuss the characteristics and potential roles of DIS3 mutations in MM. Recent findings highlight the essential roles of DIS3 in RNA homeostasis and normal hematopoiesis and suggest that the reduced activity of DIS3 may be involved in myelomagenesis by increasing genome instability.

Genomic Landscape of t(11;14) in Multiple Myeloma

Background Multiple myeloma (MM) is a clonal plasma cell malignancy characterized by multistep genetic alterations, including translocations, copy number aberrations (CNA), and mutations. Translocations involving the immunoglobulin heavy chain (IGH) locus contribute to plasma cell immortalization and consequent commitment to the MM disease pathway, however other genetic events largely underlie the complex genomic landscape of MM with molecular and clinical heterogeneity. The t(11;14) translocation is the most common IGH translocation in MM and has been identified as a predictive biomarker of response to venetoclax in relapsed/refractory MM. The objective of this study was to confirm that t(11;14) is stable across the course of disease and across lines of therapy, and to assess the unique genomic landscape of t(11;14) MM. Methods This was a retrospective, single-center, non-interventional study. To confirm t(11;14) stability, longitudinal assessment of 272 patients for t(11;14) status by plasma cell-enriched FISH using the Vysis IGH/CCND1 XT DF FISH probe was performed on bone marrow aspirates (BMAs) collected at diagnosis and relapse. To assess the genomic landscape, 139 patient BMAs were collected from an approved biobank with informed consent and were analyzed by a targeted NGS panel. NGS was performed as described in Bolli et al. (Bolli et al. 2016) using an Agilent capture panel for sequencing on the Illumina NextSeq platform. Raw sequencing data was processed by an in-house bioinformatics pipeline to detect mutations, copy number aberrations and structural variants (SVs). Protein coding mutations with variant allele frequency >10% and minimum sequencing depth >=20X were selected. CNAs were defined as log ratio above 0.3 (copy number gain) or below -0.4 (copy number loss). SVs with 5 or more supporting reads were examined to detect translocations within the IGH locus. Results Among the 272 patients assessed by longitudinal t(11;14) FISH, 118 were t(11;14)+ and 154 were t(11;14)-. All t(11;14)+ patients remained positive from their initial assessment to first and subsequent longitudinal assessments (Figure 1). The t(11;14) FISH fusion (F) patterns were also consistent across longitudinal assessments with 90% of patients possessing and maintaining either a 1F (18%), 2F (69%), or ≥3F (3%) translocation pattern. No t(11;14)- patients acquired the translocation at relapse. Among the 139 samples analyzed for t(11;14), 130 samples had results from both FISH and NGS with a 100% concordance rate; 66 were t(11;14)+ and 64 were t(11;14)-. The mutational landscape in highly recurrent genes was heterogeneous (Figure 2). MAPK pathway mutations were the most common (47/130 pts, 36.2%), with recurrent mutations in BRAF gene more prevalent in t(11;14)- vs t(11;14)+ pts (18.8% vs 6.1%, p<0.05). Conversely, mutations in DIS3 gene were more common in t(11;14)+ vs t(11;14)- pts (21.2% vs 4.7%, p<0.05). DIS3 and BRAF mutations have been associated with poor prognosis in MM. High CNAs are associated with increased genomic instability, resistance to therapy, and decreased survival in MM. Increased CNAs were observed in t(11;14)- vs t(11;14)+ MM (p<0.001). Conclusion This study confirms in a large, longitudinal cohort of patients with MM that t(11;14) is stable from diagnosis to relapse. These analyses also begin to delineate the differentiated genetic architecture of t(11;14) MM, including fewer CNAs associated with increased genomic stability, but increased rates of DIS3 mutations that confer poor prognosis in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeted Next-Generation Sequencing Identifies Additional Mutations Other Than BCR-ABL in Chronic Myeloid Leukemia Patients: A Chinese Monocentric Retrospective Study

The discovery of tyrosine kinase inhibitors (TKIs) has drastically changed the treatment of chronic myeloid leukemia (CML) patients. However, a proportion of patients still show resistance or intolerance to TKI therapy. These patients often carry somatic variants related to myeloid malignancies, indicating that additional mutations other than BCR-ABL1 may lead to TKI treatment failure or disease progression. We retrospectively evaluated 151 CML patients receiving TKI therapy and performed next-generation sequencing (NGS) analysis of 22 CML patients to explore the mutation spectrum other than BCR-ABL1 affecting the achievement of molecular responses. We also performed a comprehensive evaluation of clinical characteristics, distinct TKI options and scoring systems in the correlation with the molecular response of TKI. In total, 161 variants were detected by NGS, most of which were nonsynonymous SNVs (Figure A). The coexistence pattern among high-frequency variants was quite intricate(Figure B). Among 25 genes with allele mutation frequency (VAF) ≥5%, the most frequently mutated gene was ASXL1 (40.9%), followed by KMT2C (27.3%), DIS3 (18.2%), ATM (13.6%), DNMT3A (13.2%) and NOTCH3 (13.6%) (Figure C). NOTCH3 and RELN mutations were only carried by subjects failing to achieve a major molecular response (MMR) at 12 months, suggesting CML patients with NOTCH3 and RELN mutations might have poor long-term treatment effects (Figure D, Figure E). The distribution frequency of ASXL1 mutations was higher in the group that did not achieve MR4.0 at 36 months (P=0.023), indicating that ASXL1 mutation was an adverse factor for the achievement of MR4.0. Furthermore, subjects in this group possessed more mutations related to activation signaling pathway genes (P=0.003) and chromatin modification genes (P=0.029) (Figure F). In the group that failed to achieve MR4.5 at 12 months, there were more subjects carrying >2 gene mutations (9.1% vs. 63.6%, P=0.024), implying that it is less likely to achieve MR4.5 with the increase in the number of mutated genes and that the existence of more than two mutations is a poor prognostic factor for achieving deep molecular response (DMR) (Figure G, Figure H). However, no statistical significance was found in the effects of mutations in ASXL1 (P=0.371), KMT2C (P=0.079), DIS3 (P=0.467), ATM (P=0.280), DNMT3A (P=0.479) and NOTCH3 (P=0.479) on PFS. In the analysis of clinical characteristics, multivariate analysis identified hemoglobin concentration (HB) (relative risk, [RR], 1.023; P=0.08) and BCR-ABLIS level at 12 months (RR, 2.485; P＜0.001) as independent predictive covariates for MR4.5(Figure I). Subjects who received initial second-generation TKI (2GTKI) were more likely to achieve early molecular response (EMR) at 3 months (P=0.012), BCR-ABLIS ≤1% at 6 months (P=0.012) and MMR at 12 months (P=0.018) than initial first-generation TKI (1GTKI). And there did not show statistical significance in achieving BCR-ABLIS≤1% at 6 months, MMR at 12 months and MR4.5 between original 1GTKI (Group A1, Glevic, Novartis, Switzerland) and generic 1GTKI (Group A2, Genike, Chiatai Tianqing Pharmaceutical Group Co., Ltd.). As for scoring system, ELTS score was sensitive in differentiating whether patients could achieve EMR (P=0.001) and MMR (P=0.004). EUTOS scoring system had a significant difference in achieving MMR at 12 months (P=0.049). However, there was no significant difference in subjects stratified by the Hasford and Sokal scoring systems. In conclusion, ASXL1 mutation, activation signal pathway genes, chromatin modification genes and the presence of >2 mutations were adverse factors in achieving molecular response with TKI treatment. HB and MMR were independent factors for DMR, and initial 2GTKI therapy was better than 1GTKI in the achievement of molecular response. For the scoring system, we found that the ELTS score was the best in predicting the efficacy of TKI therapy. Acknowledgement: The work was supported by the Key R&D Program of Zhejiang (No. 2022C03137.), Public Technology Application Research Program of Zhejiang, China (No. LGF21H080003.), the Key Project of Jinhua Science and Technology Plan, China (No. 2020-3-011.), the 2019-2022 Key Medical Discipline (Haematology) Fund of Jinhua, China. Correspondence to: Dr Jian Huang, The First Affiliated Hospital of Zhejiang University School of Medicine. No. 79 Qingchun Road. Hangzhou, Zhejiang, PR China. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNA:RNA hybrid metabolism

The ribonuclease DIS3 is one of the most frequently mutated genes in the hematological cancer multiple myeloma, yet the basis of its tumor suppressor function in this disease remains unclear. Herein, exploiting the TCGA dataset, we found that DIS3 plays a prominent role in the DNA damage response. DIS3 inactivation causes genomic instability by increasing mutational load, and a pervasive accumulation of DNA:RNA hybrids that induces genomic DNA double‐strand breaks (DSBs). DNA:RNA hybrid accumulation also prevents binding of the homologous recombination (HR) machinery to double‐strand breaks, hampering DSB repair. DIS3‐inactivated cells become sensitive to PARP inhibitors, suggestive of a defect in homologous recombination repair. Accordingly, multiple myeloma patient cells mutated for DIS3 harbor an increased mutational burden and a pervasive overexpression of pro‐inflammatory interferon, correlating with the accumulation of DNA:RNA hybrids. We propose DIS3 loss in myeloma to be a driving force for tumorigenesis via DNA:RNA hybrid‐dependent enhanced genome instability and increased mutational rate. At the same time, DIS3 loss represents a liability that might be therapeutically exploited in patients whose cancer cells harbor DIS3 mutations.

The Alternative Splicing Landscape in Multiple Myeloma Is Determined By IGH Translocations and Mutations of RNA Processing Genes

Introduction: Alternative Splicing (AS) plays a key role in regulating numerous cellular processes in both normal and malignant cells. Previous studies have revealed mutations in the spliceosome complex, such as SF3B1, can cause increased AS frequencies in multiple myeloma (MM) patients, and patients with increased levels of AS are associated with a poor prognosis. Other frequently mutated genes involved in RNA processing include DIS3 and FAM46C, thus, systematically investigating other causes of AS abnormalities and pathologies in MM patients is highly necessary.Materials and Methods: RNA-seq data from 598 newly diagnosed MM patients from the MMRF CoMMpass study were utilized to generate AS comparisons. They were previously annotated for cytogenetic, copy number, and mutation data (version IA16). RNA-seq data were aligned to HG38 using STAR and Salmon. SUPPA2 was used for calling AS differences. For each identified AS event, the splicing level was defined by Percentage of Spliced-In (PSI) while the mean difference of splicing levels between two groups was measured by ΔPSI (dPSI) and by the P-value from independent T-tests against PSIs in the two groups. Filtering thresholds were determined to find high-quality differentially spliced events and were filtered for those also present in normal PCs (GSE110486). Geneset enrichment analysis was performed to identify dysregulated pathways caused by differential splicing and differential expression. Survival analysis was performed on clinical annotations of 598 NDMM patients while the Logrank test and Cox regression were used to evaluate the risk of AS and other genomic factors. Kaplan-Meier curves were plotted for various subgroups.Results: We compared 16 major cytogenetic subgroups, including Ig translocations (t(4;14), t(14;16), t(11;14)), hyperdiploidy, mutations in KRAS, NRAS, BRAF, FAM46C, SF3B1, DIS3 and TP53, combined events (t(4;14) plus DIS3 mutation), as well as those with biallelic abnormalities (DIS3, FAM46C, and TP53). Samples with SF3B1 hotspot mutations identified the greatest number of AS events (n=862), and samples with any SF3B1 mutation had approximately half as many. IGH translocations had an equivalent number of AS events to those with SF3B1 mutations, with t(14;16) having the most (n=587) followed by t(11;14) (n=366), and t(4;14) (n=256). We observed an increased number of significant AS events in bi-allelic DIS3 and FAM46C groups (n=404 and 171) compared to their mono-allelic abnormalities (n=114 and 35). As DIS3 mutations are enriched in the t(4;14) subgroup we also examined that interaction and found significantly more AS events (n=481; p<0.01) in the combination compared to either event alone. As expected, KRAS, NRAS and BRAF mutations did not have enrichment for AS events (n=2, 15, 23, respectively).The majority of AS events were unique to each subgroup, exemplifying the AS heterogeneity in these subgroups. Among overlapped events, an alternative first (AF) exon in ACACA was consistently more spliced in t(14;16), t(11;14) and t(4;14) groups (dPSI=0.18, 0.10, 0.12, P=2x10 -5, 2x10 -9, 5x10 -5). ACACA encodes an enzyme that significantly affects MM cell growth and viability, suggesting that similar regulations exist in the three translocation groups. Unique events were also detected including an AF event in MIB2 (E3 Ubiquitin Protein Ligase 2) in the t(11;14) group (dPSI=0.17, P=7x10 -14), and a skipped exon in UBXN4 (related to ER stress) in t(14;16) group (dPSI=0.1, P=3x10 -4). AS heterogeneity also leads to functional heterogeneity in the three groups. Besides commonly downregulated RNA catabolic processes, cell adhesion, migration and mobility related pathways are enriched pathways in t(14;16); cell growth related pathways in t(11;14); and ERK related pathways in t(4;14). High-risk events were identified through survival analysis and included a retained intron in RPS16 in the t(14;16) (Hazard Ratio (HR)=18.81, p=0.004). Similarly, high risk was associated with an AF event in DDX39B in t(11;14) (HR=2.62, p=0.001) and an AF event in COPA in t(4;14) (HR=6.29, p=0.001).Conclusion: AS is defined by multiple genomic events, including primary translocations and mutations in RNA processing genes, DIS3 and FAM46C, and interactions between genomic markers can increase AS. AS events contribute to outcome and some high risk AS events may serve as prognostic marker or potentially novel targets. DisclosuresWalker: Sanofi: Speakers Bureau; Bristol Myers Squibb: Research Funding.

Somatic reversion impacts myelodysplastic syndromes and acute myeloid leukemia evolution in the short telomere disorders

BACKGROUNDGermline mutations in telomerase and other telomere maintenance genes manifest in the premature aging short telomere syndromes. Myelodysplastic syndromes and acute myeloid leukemia (MDS/AML) account for 75% of associated malignancies, but how these cancers overcome the inherited telomere defect is unknown.METHODSWe used ultra-deep targeted sequencing to detect somatic reversion mutations in 17 candidate telomere lengthening genes among controls and patients with short telomere syndromes with and without MDS/AML, and we tested the functional significance of these mutations.RESULTSWhile no controls carried somatic mutations in telomere maintenance genes, 29% (16 of 56) of adults with germline telomere maintenance defects carried at least 1 (P < 0.001), and 13% (7 of 56) had 2 or more. In addition to TERT promoter mutations, which were present in 19%, another 13% of patients carried a mutation in POT1 or TERF2IP. POT1 mutations impaired telomere binding in vitro and some mutations were identical to ones seen in familial melanoma associated with longer telomere length. Exclusively in patients with germline defects in telomerase RNA (TR), we identified somatic mutations in nuclear RNA exosome genes RBM7, SKIV2L2, and DIS3, where loss-of-function upregulates mature TR levels. Somatic reversion events in 6 telomere-related genes were more prevalent in patients who were MDS/AML-free (P = 0.02, RR 4.4, 95% CI 1.2-16.7), and no patient with MDS/AML had more than 1 reversion mutation.CONCLUSIONOur data indicate that diverse adaptive somatic mutations arise in the short telomere syndromes. Their presence may alleviate the telomere crisis that promotes transformation to MDS/AML.FUNDINGThis work was supported by the NIH, the Commonwealth Foundation, the S&R Foundation Kuno Award, the Williams Foundation, the Vera and Joseph Dresner Foundation, the MacMillan Pathway to Independence Award, the American Society of Hematology Scholar Award, the Johns Hopkins Research Program for Medical Students, and the Turock Scholars Fund.

Genomic Discovery and Clonal Tracking in Multiple Myeloma By Cell Free DNA Sequencing

Summary: Development of drug resistance and clonal evolution is a major problem in multiple myeloma (MM) and may be due to genetic heterogeneity and clonal evolution but its longitudinal genetic characterization has been impractical, thus far. We have developed a genomic discovery method to monitor genetic evolution of the disease through whole exome sequencing and low-pass whole genome sequencing of myeloma-derived circulating cell-free DNA (cfDNA). Low-pass whole genome sequencing of 110 cfDNA samples from 75 randomly selected MM patients was used to develop an approach to predict the utility of further whole exome sequencing based on cfDNA concentration as a quick and cost-effective marker. Whole exome sequencing of cfDNA, matched normal blood cells, and bone marrow MM cells obtained from five myeloma patients at the same time point demonstrated that identification of clonal somatic mutations was robust and highly concordant between cfDNA and bone marrow, and provided sufficient resolution to define the subclonal composition by cfDNA. cfDNA allowed longitudinal tracking of disease burden and captured clonal evolution of MM. We provide a framework for effectively utilizing comprehensive discovery-oriented cfDNA sequencing in MM.Methods and Results: In order to estimate the subclonal composition of MM from “blood biopsy”,we collected 110 plasma samples from 75 randomly selected patients with multiple myeloma, who were undergoing treatment, and isolated cfDNA. As a control, we also isolated cfDNA from the plasma of 12 healthy blood donors. We prepared next-generation sequencing libraries and performed low-pass whole genome sequencing to a sequencing depth of 0.1-1 fold coverage across the entire genome for all of these samples. We deployed a computational approach that enables assessment of the fraction of myeloma-derived DNA in cfDNA by utilizing the relative ratio of somatic copy number alterations (SCNAs) from low pass whole genome sequencing data, after correcting for GC bias. Using a detection threshold cutoff of ~5%, we detected myeloma-derived cfDNA in 22% of total MM samples. In two patients from whom multiple serial samples were available, we found strong concordance between the myeloma DNA amount and serum free light chains, suggesting that low pass whole genome sequencing of cfDNA allows tracking of progression of MM longitudinally in patients.We then asked if whole exome sequencing (WES) and robust calling of somatic mutations is possible from cfDNA in myeloma patients. We performed deep whole-exome sequencing of cfDNA from five patients with multiple myeloma in whom we detected the presence of myeloma-derived circulating DNA by low-pass whole genome sequencing of cfDNA. We also performed whole exome sequencing of CD138+ cells that were flow-sorted from the bone marrow of the same patients, as well as CD45- cells as a matched normal sequencing control. cfDNA was obtained on the same day as the bone marrow aspirate, to rule out ongoing clonal evolution between the time of blood and bone marrow harvest. We first determined if canonical mutations that are typically seen in MM can be detected in cfDNA. We identified mutations in NRAS, KRAS, DIS3, TP53, and RB1, all known to be recurrently mutated in MM. These somatic mutations were identified in both the cfDNA as well as in the CD138+ sorted MM cells from the bone marrow aspirate.We then determined that somatic mutations in the BM are well represented in the cfDNA in general, and that the allelic fractions of somatic mutations correlated well between cfDNA and matched bone marrow biopsies (Pearson's r=0.704; 5 patients with a tumor fraction >16%). We found overall that 87% of 840 mutations in the bone marrow were detected in cfDNA and 92% of clonal mutations were also identified in cfDNA. Some distinct subclones could be identified either only in the cfDNA or only in the bone marrow. In one case, extensive clonal evolution occurred during treatment and individual subclones likely accounting for the development of drug resistant disease could be distinguished enabling the reconstruction of evolutionary trees.These results show that low-pass whole genome as well as whole-exome sequencing of cfDNA is feasible in patients with MM, can recapitulate the genetic evolution of MM in the bone marrow and reveals biologically and therapeutically relevant data when detectable. DisclosuresNo relevant conflicts of interest to declare.

Whole Exome Sequencing Reveals High Genetic Diversity in Relapsed Multiple Myeloma

Background: Treatments like lenalidomide and proteasome inhibitors alone or in combination with chemotherapy have dramatically improved the outcome in multiple myeloma in the past years. Although the majority of patients achieves a remission, almost all patients eventually relapse. Previous studies have found recurrent mutations in TP53 and FAM46C (Weinhold et al., Blood 2016) in relapsed multiple myeloma as well as CRBN mutations (Kortüm et al., Blood 2016) specifically in lenalidomide-treated patients.Methods: In order to identify additional genetic aberrations that are associated with therapy resistance, we analyzed CD138-selected multiple myeloma cells in bone marrow samples obtained pretreatment (n=12) and at different time points at progression during treatment (n=17) by fluorescence in situ hybridization (FISH) and whole exome sequencing (WES). Peripheral blood was used as a germline control. The median number of therapy lines between pretreatment and progression samples was 1 (range, 1 - 4). Progression samples were obtained after treatment with lenalidomide (n=16), bortezomib (n=14), or high-dose melphalan/autologous stem cell transplantation (n=9) alone or in combination or as sequential treatment. WES was performed by paired-end sequencing (read length 100 bp) on an Illumina HiSeq platform. The mean coverage was 117x. A variant allele frequency (VAF) of 5% was set as a cut-off for mutation calling.Results: In the pretreatment samples, the median number of cytogenetic aberrations was 2 (range, 1 - 4) and the median number of somatic variants was 39 (range, 24 - 63) per sample. There was no significant change in these numbers in posttreatment samples: median number of cytogenetic aberrations 2 (range, 0 - 4), median number of somatic variants 52 (range, 11 - 107).The most recurrent cytogenetic alterations at diagnosis were +1q21 (n=6), +9q34.2 (n=4), and del13q14 (n=3). At relapse, 13 of the 18 cytogenetic abnormalities detected at diagnosis were still present, 5 were lost and 5 were newly acquired. The most frequently lost aberration was +9q34.2 (n=2) and the most commonly acquired aberrations were +1q21 (n=2) and del13q14 (n=2). In pretreatment samples we found recurrent mutations in NRAS (n=4), KRAS (n=3), DIS3 (n=3), and IGLL5 (n=3). The median stability of mutations between pre- and posttreatment samples was 62% (range, 13 - 81%). NRAS mutations were lost in 2 progression samples and DIS3 in 1 progression sample while KRAS mutations remained stable. We found 430 relapse-specific mutations in the 17 posttreatment samples. Mutations in TP53 (n=4) were the most frequently newly acquired genetic events in progression samples, which is consistent with previous studies. Of the newly acquired gene mutations, only eight genes were affected in more than one progression sample, all other gene mutations were non-recurrent. Among genes involved in the mechanism of lenalidomide we found a DDB1 mutation in one progression sample after lenalidomide treatment. Another patient had an IKZF3 mutation at relapse. However, the mutation was located outside the lenalidomide degron sequence and the VAF was only 5%, suggesting that it was not responsible for lenalidomide resistance in the major clone. No relapse-specific mutations were found in other genes encoding proteins involved in lenalidomide or proteasome inhibitor activity like IKZF1, IRF4, CRBN, CUL4A, CUL4B or proteasome subunits. Pathway analyses revealed that mutations in genes involved in WNT/beta catenin and KRAS signaling were enriched in the progression samples.Conclusions: In conclusion, we found a high degree of genetic heterogeneity in relapsed multiple myeloma. There were no obvious mutations in genes involved in lenalidomide or proteasome activity that might drive drug resistance. TP53 mutations and +1q21 were the most recurrent gene aberrations identified at relapse, however, these events may cause a general robustness of multiple myeloma cells to multiple treatment modalities and might not be treatment specific. These results suggest that the genetic basis for drug resistance is heterogeneous and warrants genetic characterization of further patients by DNA and RNA sequencing as well as functional analyses of candidate genes and pathways. DisclosuresKronke:Celgene: Honoraria, Other: Travel Support; Takeda: Consultancy, Other: Travel Support.

Identifying High-Risk Phenotypes Using Three-Way Feature Interactions in Multiple Myeloma: An Analysis of Publically Available Mmrf Commpass Study

The introduction of next generation sequencing has exponentially increased the number of potential disease targets from a limited panel of probes to markers across the entire genome. Previous studies of NGS data in multiple myeloma have identified key features associated with outcome in additive models, typically showing involvement of TP53, gain of 1q, MYC translocations, and others.We modified the multi-factor dimensionality reduction method (MDR) method, a computationally efficient method for detecting non-linear patterns of gene-gene interactions in genome-wide association studies (GWAS) first proposed by Richie et al, to identify three-way interactions significantly associated with outcome in MM. Specifically, we altered the ranking strategy of the survival adapted Cox-MDR method of Lee et al from balanced accuracy (average of sensitivity and specificity) to the absolute sum of deviance residuals. Due to the high level of censoring (94 events in 559 cases), balanced accuracy ranking favored interactions that identified few high risk cases and failed to validate well on external data.The publically available MMRF CoMMpass trial database (release version IA10) was utilized as primary training set for our MDR method. We included in our comparisons 164 RNA-SEQ genes with significant association with overall survival, the 20 most abundant non-synonymous, non-IG variants, and 24 estimated whole chromosome arm copy number gains or loss calls from the long insert WGS data of 559 baseline cases with outcome and clinical data (median follow-up 1.8 years). We searched for all three-way interactions across 210 total features resulting in the ranking of over 1.5 million unique three-way interactions, adjusted for age, ISS, creatinine level, and treatment by modified MDR. The top three-way RNA-expression interactions from our discovery phase were validated on 540 cases with GEP microarray and clinical data from UAMS Total Therapy 2 and 3 with median follow-up of 4.66 years (publically available data from GSE2658, GSE31161, and GSE24080).Of the top 100 three-way gene expression interactions discovered that we could validate on Affymetrix GEP data, 84 were significant in the UAMS TT2 and TT3 data set (log rank p-value < 0.05). HMGB3, a proposed inhibitor of myeloid differentiation, and DDX51, known to be differentially methylated in ALL, were the most commonly involved genes in the top significant interactions across both data sets. Pathway analysis of the 150 most frequently observed genes across the top 5,000 interactions in CoMMpass revealed an enrichment of DNA replication and cell cycle associated genes: RFC5, MCM2, MCM3, MCM4, MCM6, CCNA2, CDK2, etc.Within the top interactions that included whole arm copy number variants, gain of 1q and loss of 13q were the most frequently observed. We note that the top three-way interactions with gain of 1q paired exclusively with ALYREF, a transcriptional promoter, and one additional gene. Of the top interactions that included a SNV, mutations of DIS3 and DNAH5 were the most frequently observed. Many of the top interactions including DIS3 mutations paired with HMGB3 or TRIB3, a negative regulator of NF-kB and sensitizer to apoptosis, and one additional gene.MDR models are capable of identifying complex interactions of features that are not strictly additive. We observed a variety of interaction models, and present below an example where presence of 2 or more features resulted in overall negative outcome while presence of 1 or fewer did not. This situation was observed for the interaction of high expression of HMGB3, DDX51, and PSMB2, a bortezomib sensitizer in MM cell lines, where high expression in 2 or more of these genes imparted a negative outcome in CoMMpass and TT2/TT3 data sets. In the TT2/TT3 data set, cases identified as high risk from this interaction were enriched for the PR subtype and GEP70 HR.This modified MDR method is an effective tool at mining large genomic data sets for all k -way interactions associated with outcome among binary or multi-level features, adjusted for clinical covariates. Our modifications to MDR method allow us to identify more relevant interactions and increase reliability in validation data. This method could potentially further knowledge of the complex relationships between key features in genomic landscape of MM by discovering novel interactions that identify high-risk phenotypes. [Display omitted] DisclosuresBergsagel:Phosplatin Therapeutics: Research Funding.

DIS3 mutations in multiple myeloma impact the transcriptional signature and clinical outcome

DIS3 gene mutations occur in roughly 10% of patients with multiple myeloma (MM); furthermore, DIS3 expression can be affected by monosomy 13 and del(13q), which occur in approximately 40% of MM cases. Despite several reports on the prevalence of DIS3 mutations, their contribution to the pathobiology of MM remains largely unknown. We took advantage of the large public CoMMpass dataset to investigate the spectrum of DIS3 mutations in MM and its impact on the transcriptome and clinical outcome. We found that the clinical relevance of DIS3 mutations strictly depended on the co-occurrence of del(13q). In particular, bi-allelic DIS3 lesions significantly affected progression-free survival, independently of other predictors of poor clinical outcome, while mono-allelic events mostly affected overall survival. As expected, DIS3 mutations affect the MM transcriptome involving cellular processes and signaling pathways associated with RNA metabolism, and the deregulation of a large number of long non-coding RNA, among which we identified five distinct transcripts as independent predictors of poorer overall survival and nine of worse progression-free survival, with two (AC015982.2 and AL445228.3) predicting both unfavorable outcomes. These findings strongly prompt further studies investigating the relevance of these long non-coding RNA in MM.

Understanding the impact of DIS3 cancer-associated mutations by in silico structure modeling

DIS3 gene is reported as a driver gene specifically mutated in a few cancer types including multiple myeloma. Unraveling the molecular effects of DIS3 mutations on protein structure may provide insights into the functional effect of different substitutions. Hence, the aim of this study is to analyze the structural and functional consequences of hotspot mutations in DIS3 by using various sequence and structure-based bioinformatics methods. In addition to sequence-based programs, the effect of hotspot mutations on protein stability and the overall pathogenicity was investigated using SDM and Missense3D. Moreover, mutations were modeled on protein structure using UCSF Chimera. Changes in residue interactions after mutations were investigated using RINalyzer. In total 6 positions in DIS3 are reported as hotspot positions. Our analysis showed all hotspots were located in highly conserved positions and their substitution resulted in clashes or disruption in interaction with the Mg2+ or nucleic acid. Our findings confirmed analysis of the residue interaction network provides a better insight into the functional and structural impact of the DIS3 mutations. We expect our analysis will be used for designing future experimental evaluation of the mutations in DIS3 gene and it will also help evaluate other cancer driver genes' mutations.

BRAF and DIS3 Mutations Associate with Adverse Outcome in a Long-term Follow-up of Patients with Multiple Myeloma.

Copy-number changes and translocations have been studied extensively in many datasets with long-term follow-up. The impact of mutations remains debated given the short time to follow-up of most datasets. We performed targeted panel sequencing covering 125 myeloma-specific genes and the loci involved in translocations in 223 newly diagnosed myeloma samples recruited into one of the total therapy trials. As expected, the most commonly mutated genes were NRAS, KRAS, and BRAF, making up 44% of patients. Double-Hit and BRAF and DIS3 mutations had an impact on outcome alongside classical risk factors in the context of an intensive treatment approach. We were able to identify both V600E and non-V600E BRAF mutations, 58% of which were predicted to be hypoactive or kinase dead. Interestingly, 44% of the hypoactive/kinase dead BRAF-mutated patients showed co-occurring alterations in KRAS, NRAS, or activating BRAF mutations, suggesting that they play a role in the oncogenesis of multiple myeloma by facilitating MAPK activation and may lead to chemoresistance. Overall, these data highlight the importance of mutational screening to better understand newly diagnosed multiple myeloma and may lead to patient-specific mutation-driven treatment approaches.

Monosomic loss of MIR15A/MIR16-1 is a driver of multiple myeloma proliferation and disease progression.

The most common genetic abnormality in multiple myeloma (MM) is the deletion of chromosome 13, seen in almost half of newly diagnosed patients. Unlike chronic lymphocytic leukemia, where a recurrent minimally deleted region including MIR15A/MIR16-1 has been mapped, the deletions in MM predominantly involve the entire chromosome and no specific driver gene has been identified. Additional candidate loci include RB1 and DIS3, but while biallelic deletion of RB1 is associated with disease progression, DIS3 is a common essential gene and complete inactivation is not observed. The Vk*MYC transgenic mouse model of MM spontaneously acquires del(14), syntenic to human chromosome 13, and Rb1 complete inactivation, but not Dis3 mutations. Taking advantage of this model, we explored the role in MM initiation and progression of two candidate loci on chromosome 13: RB1 and MIR15A/MIR16-1. Monoallelic deletion of Mir15a/Mir16-1 but not Rb1 was sufficient to accelerate the development of monoclonal gammopathy in wildtype mice, and the progression of MM in Vk*MYC mice, resulting in increased expression of Mir15a/Mir16-1 target genes and plasma cell proliferation, which was similarly observed in patients with MM.

DIS3 Mutation in RUNX1-Mutated AML1 Confers a Highly Dismal Prognosis in AML By Repressing Sister Chromatid Cohesion

Defective in Chromatid Joining (DIS3) is a highly conserved exoribonuclease, involved in the 3'-5' degradation of RNAs, which has a major role as a post-transcriptional checkpoint in eukaryotic cells. The controlled degradation of unnecessary RNA transcripts from the cell by the DIS3 family enzymes has been shown to be essential for cell division and mitosis. Exome- sequencing analyses have demonstrated the presence of DIS3 mutations in several cancers with high incidence in patients with multiple myeloma but its role in acute myeloid leukemia (AML) has not been studied. AML is a malignancy characterized by clonal expansion and differentiation arrest of myeloid progenitors in the bone marrow and peripheral blood. M0 and M1 FAB subtypes are both poorly differentiated AML and RUNX1 mutations are associated with poorer outcome. In this work we asked whether we can confirm in the TCGA AML cohort, that RUNX1 mutations were associated with a prognostic significance and whether other co-existing mutations can influence the overall survival (OS). In this cohort of 193 patients, we have found that the presence of RUNX1 mutation is present in patients with worst OS (log rank p-value=0.036, median OS 227 versus 335 months). We then asked if the association with other mutations has an impact on clinical point of view. With a minimum 3 mutations per gene present in the cohort (Fig.1A), RUNX1 was found to be significantly co-associated with mutations present in 4 other genes: PHF6, TTN, ASXL1 and DIS3. RUNX1 associated mutations affecting ASXL1, TTN and PHF6 did not appear to modify prognosis. However, patients presenting both RUNX1 and DIS3 mutations had a dramatically reduced overall survival (log rank p-value=6E-4, median OS 61 versus 334 months) (Fig.1B) suggesting a dramatic co-association of these two mutations independently of TTN, PHF6, and ASXL1. The two patients presenting a co-association of RUNX1 and DIS3 mutations were found to have a leukemia of M1 subtype. One patient with AML-M3 presented an isolated DIS3 mutation without co-association with a RUNX1 mutation (Fig.1C). DIS3 missense mutations found in the cohort affected VacB RNB protein domain, which is implicated in ribonuclease activity and RNA binding of the molecule (Fig.1D). Removing M3 subtype from the TCGA cohort, RUNX1 fusions were found to be associated with M2 subtype, RUNX1 mutations with M0 &amp; M4 subtypes, and double mutant RUNX1/DIS3 in essentially in M1subgroup (p-value = 0.006976). Without M3, patients with double RUNX1/DIS3 mutations were found to exhibit the worst prognosis respectively as compared to patients presenting mutations or fusion of RUNX1 alone (log rank p-value=0.0042). We then compared the transcriptome of M1 patients with double DIS3-RUNX1 mutations as compared to M1 patients with simple RUNX1 mutations. As can be seen in Fig.1F, the first group harbored an important down-regulation of genes involved in the cohesin complex such as SMC1A, RAD21, STAG2, suggesting a major reduction of sister chromatid cohesion functionality (Normalized enrichment score = -2.017, FDR q-value=0.004). There was also a down-regulation of molecules implicated in DNA double strand repair such as MRE11A, RAD21, as well as that of MAD2L1 (a mitosis checkpoint) and centromere proteins such as CENPF. These results suggest that DIS3 mutations are associated with an important loss of control for the entry in mitosis. Overall, we show here for the first time to our knowledge, that the occurrence of DIS3 mutation in AML-M1 patients presenting a RUNX1 mutation confers a dismal prognosis in terms of overall survival. The transcriptome of these patients harbors a loss of functionality for sister chromatid cohesion. The search for DIS3 mutations is therefore warranted in all AML at diagnosis and could be of decisional value for therapeutic strategies. Disclosures Turhan: Incyte: Consultancy, Honoraria; novartis: Honoraria, Research Funding.

&lt;p&gt;Progress in the identification of gene mutations involved in multiple myeloma&lt;/p&gt;

Sequencing studies have been used to determine a spectrum of multiple myeloma (MM) mutations. Mutation of certain genes, including KRAS, NRAS, TP53, FAM46C, DIS3 and BRAF, have a high recurrence rate and may play important roles in the pathogenesis, progression and prognosis of MM. Mutations in DIS3, which encodes a highly conserved RNA exonuclease, lead to loss of function. The expression of FAM46C is highly correlated with the expression of ribosomal protein, but the exact function of FAM46C mutation is unclear. There are mutants of IRF4, which is considered an MM survival factor. Mutations in the gene coding for the DNA damage-binding protein (DDB1) may affect interactions with CUL4A, which is part of the cereblon (CRBN) ubiquitin ligase complex. IRF4is part of the complex, which binds to DNA. These findings might explain the resistance to immunomodulatory. TP53 deletion or mutation is often present in B-cell malignancies and is associated with low response rates. Myeloma pathogenic mutations in ATM have been found in adult lymphatic tumors. XBP1 and PSMB5 mutations may be related to bortezomib resistance. Multiple gene mutations (KRAS, NRAS and BRAF) involved in the same pathway were found a single patient. Identification of driver gene mutations has brought great hope to the field of individualized, targeted medicine for MM.