In multiple myeloma malignant plasma cells expand within the bone marrow. Since this site is well-perfused, a rapid dissemination of “fitter” clones may be anticipated. However, an imbalanced distribution of multiple myeloma is frequently observed in medical imaging. Here, we perform multi-region sequencing, including iliac crest and radiology-guided focal lesion specimens from 51 patients to gain insight into the spatial clonal architecture. We demonstrate spatial genomic heterogeneity in more than 75% of patients, including inactivation of CDKN2C and TP53, and mutations affecting mitogen-activated protein kinase genes. We show that the extent of spatial heterogeneity is positively associated with the size of biopsied focal lesions consistent with regional outgrowth of advanced clones. The results support a model for multiple myeloma progression with clonal sweeps in the early phase and regional evolution in advanced disease. We suggest that multi-region investigations are critical to understanding intra-patient heterogeneity and the evolutionary processes in multiple myeloma.

• Hits in driver genes and bi-allelic events affecting tumor suppressors increase apoptosis resistance and proliferation rate-driving relapse.• Excessive biallelic inactivation of tumor suppressors in highrisk cases highlights the need for TP53-independent therapeutic approaches.To elucidate the mechanisms underlying relapse from chemotherapy in multiple myeloma, we performed a longitudinal study of 33 patients entered into Total Therapy protocols investigating them using gene expression profiling, high-resolution copy number arrays, and whole-exome sequencing. The study illustrates the mechanistic importance of acquired mutations in known myeloma driver genes and the critical nature of biallelic inactivation events affecting tumor suppressor genes, especially TP53, the end result being resistance to apoptosis and increased proliferation rates, which drive relapse by Darwinian-type clonal evolution. The number of copy number aberration changes and biallelic inactivation of tumor suppressor genes was increased in GEP70 high risk, consistent with genomic instability being a key feature of high risk. In conclusion, the study highlights the impact of acquired genetic events, which enhance the evolutionary fitness level of myeloma-propagating cells to survive multiagent chemotherapy and to result in relapse. (Blood. 2016;128(13):1735-1744

Key Points PET false-negativity was seen in 11% of MM patients. PET false-negativity was associated with low hexokinase-2 expression.

Multiple myeloma (MM) is a plasma cell malignancy preceded by a premalignant stage, named monoclonal gammopathy of undetermined significance (MGUS), and often a smoldering phase (SMM). 1, 2 Primary events, which include recurrent translocations of the IgH locus and hyperdiploidy, occur early in pathogenesis, and are followed by the acquisition of secondary genetic events such as MYC structural variants (SV), mutations that activate the RAS or NFkB pathways, mutations of DIS3 or FAM46C that drive precursor stages of disease toward MM. [3][4][5][6] Whole exome sequencing (WES) studies comparing serial MGUS/SMM and MM samples indicate clonal stability, and no significant increase in mutational load in patients that progress rapidly to MM. 7 In contrast, in 33 unselected MGUS patients single-nucleotide variants (SNVs) were less frequent, and no MYC translocations identified. 8 To study the role of MYC in myeloma we performed an integrated genomic analysis of 612 newly diagnosed myeloma (NDMM) patients enrolled in the CoMMpass study, as well as Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms * shared first co-authors Authorship Contributions PLB, WMK and KM originated concept and design of investigation, KMK EB developed custom capture panel, SVW FISH analysis, GA primary samples, YA GD WMK PLB developed additional bioinformatics methods, NK KM CKS PLB WMK performed analyses, and NK KM CKS WMK PLB composed manuscript. We thank JK and MMRF research network for their work on CoMMpass. All authors read and approved of final manuscript. Conflict of Interest DisclosuresRafael Fonseca works as a consultant for AMGEN, BMS, Celgene, Takeda, Bayer, Jansen, Pharmacyclics, Merck, Sanofi, Kite and Juno. He is on the board of Scientific Advisory Board: Adaptive Biotechnologies. Mayo Clinic and Rafael Fonseca hold a patent for prognosticating myeloma using FISH.

Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies.