Abstract: Introduction: Treatment strategies incorporating proteasome inhibitors, immunomodulators, and autologous transplantation induce durable remissions in most newly diagnosed multiple myeloma (NDMM) patients. However, for 20% of patients even the most intensive therapies have not resulted in satisfactory outcomes. Currently available risk scores do not fully appreciate the complex biology of MM and have limited sensitivity and/or specificity for identification of high risk (HR) disease. We therefore aimed to characterize the mutational landscape of transplant-eligible NDMM patients who relapsed within 2 years after treatment initiation, thereby defining true clinical HRMM. To elucidate the clonal structure and evolution in these patients, we performed deep whole genome sequencing (WGS, ~80x) and RNAseq of samples collected at baseline and first relapse. Methods: We included 34 transplanted NDMM patients who experienced early relapse during maintenance within 2 years after treatment initiation. Tumor samples were collected from 20 and 31 patients at baseline and first relapse, respectively. Paired samples taken at both time points were available from 17 patients. WGS and RNAseq data were pre-processed using in-house pipelines. Single nucleotide variants (SNVs), indels, translocations, and copy number variants (CNVs) were called using Platypus, SOPHIA and ACESeq. Subclones were identified using SciClone. RNAseq data was aligned using STAR. Fusion genes were called by Arriba. Differential gene expression was assessed using DESeq2. Results: At baseline, only 12/20 patients would have been classified as HR according to conventional markers, including presence of t(4;14), t(14;16), amp(1q), clonal del(17p) or ISS3. In 5 patients del(17p) was solely observed in a minor sublone, which was selected during treatment and became dominant at relapse in 3 of them. Selection of amp(1q)-positive subclones was seen in 2 patients, illustrating that subclonal amp(1q) or del(17p) are frequent events in HR patients, and - in contrast to recent results - could contribute to early relapse. Translocations involving MYC have also been reported to be of prognostic impact. At baseline 9 of 20 patients were positive for this event, with BMP6 and the lambda locus being the translocation partner in 2 patients each. At relapse we found an additional MYC-lambda, and two MYC-kappa translocations, supporting recent observations that MYC-light chain translocations are associated with aggressive disease. We identified a median of 40 (range: 17-233) nonsilent somatic SNVs per patient at baseline and 61 (range: 14-322) at relapse. Yet, comparing paired samples there was no significant increase in SNVs. In our HRMM set, 21 of 64 recently identified driver genes were mutated at baseline with KRAS (n = 6), TP53 (n = 6), NRAS (n = 2), and DIS3 (n = 2) being the most frequently affected genes. 6 of them - ACTG1, DIS3, FAM46C, NFKB2, RB1, and TRAF3 - were involved in fusion genes. At relapse the number of mutated driver genes increased to 29, and 10 of 31 patients presented with a clonal TP53 mutation. All patients with a TP53 mutation also showed deletion of the second allele or LOH. Including other tumor suppressor genes, such as RB1, CDKN2C, or TRAF3, 12/20 NDMM and 20/31 relapsed patients had at least one bi-allelic aberration, doubling the number of HR patients with such events compared to considering TP53 alone. Longitudinally, we observed all patterns of clonal evolution that were recently described for unselected patients. Stable evolution was primarily seen in patients achieving partial remission, supporting a model where some tumor cells survive in a protective microenvironment (ME). In deep responders, however, branching evolution was the dominant patterns. This observation rather supports strong cell-intrinsic mechanisms and rapid selection of aggressive minor subclones in clinically defined HRMM. Conclusions: Understanding the mutational landscape in HRMM and drivers of early relapse is crucial in order to improve treatment options. Our study highlights the importance of bi-allelic events in HR and suggests that focusing on TP53 is not sufficient, if all HR cases are to be identified. Of note, 3 patients in the entire cohort would have been classified as low risk by conventional risk scores and even at relapse did not carry any bi-allelic event, indicating the existence of unknown somatic HR aberrations or a protective ME. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Goldschmidt:MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Honoraria, Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Molecular Partners: Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding.

Abstract: Introduction: Treatments incorporating autologous transplantation (ASCT), proteasome inhibitors (PIs), and immunomodulatory drugs (IMIDs) induce deep and durable remissions in most multiple myeloma (MM) patients, resulting in prolonged survival. Yet, patients who suffer from early relapse within 2 years of treatment initiation or become refractory to PIs and IMIDs still have a dismal outcome. The mutational landscape in early relapsed MM (ERMM) and relapsed/refractory MM (RRMM) has been comprehensively described. Several aberrations are associated with these two types of high-risk disease but little is known about the biological difference between them. To this end, we have comparatively and sequentially analyzed whole genome and RNA sequencing data from ERMM and RRMM patients. Methods: We included 32 patients who had relapsed within 2 years of first-line therapy (ERMM group, 30 after ASCT). Samples were collected at first relapse. Paired baseline samples were available from 17 patients. For the RRMM group, we included 43 patients with a median of 5 prior lines of therapy (range 2 - 13; 88% with ASCT), who had relapsed after PIs and IMiDs. For 22 of them consecutive tumor samples were available. Sequencing data were pre-processed using in-house pipelines. Mutations, indels, translocations, and copy number variants were called using Platypus, SOPHIA and ACESeq. Mutational signatures were identified with MMSig and subclonal structures with SciClone. Differential gene expression was assessed using DESeq, gene set enrichment analysis was performed with hypeR, and gene fusions were detected with Arriba. Results: Nonsynonymous mutations occurred more frequently in RRMM (median=180) compared to ERMM at first relapse (median=62, p & lt;0.001). While TP53 mutations were more often seen in ERMM (31% of cases vs. 21% in RRMM), NRAS mutations were enriched in RRMM (37% vs. 22%). Bi-allelic inactivation of TP53, RB1, or CDKN2C was more frequent in ERMM (44% vs. 30% in RRMM). In 11/14 ERMM patients these events were already present at baseline. Genes associated with sensitivity to PARP inhibition, homologous recombination deficiency, HECT, Pi3K and NOTCH signaling were more often mutated in RRMM (p & lt;0.05). While mutations associated with PI-resistance were equally common in both groups (~20%), IMID-resistance mutations were more common in RRMM (23% vs 9%). We observed a median number of 60 and 48 fusion genes in ERMM and RRMM, respectively. Fusions involving B2M, TXNDC5, PVT1 and MYC were more frequent in ERMM, while SPINK family and MAGEC1 fusions were more common in RRMM. Analysis of mutational signatures revealed a major impact of signature MM1 (associated with melphalan-exposure), in 66% of RRMM patients. In contrast, only 22% of ERMM samples showed this signature (p & lt;0.001). Signature 3 (defective homologous recombination-based DNA damage repair) was rarely detectable in ERMM (4/32) but one of the major signatures in RRMM (16/43). Analyzing expression profiles, we found upregulated genes in ERMM that were enriched for epithelial-mesenchymal transition, hypoxia, glycolysis and KRAS/IL6-JAK-STAT3 signaling. For RRMM we found no significantly enriched gene set. Yet, 50 upregulated genes were ribosomal protein pseudogenes. Longitudinally, we mainly observed branching evolution in ERMM and RRMM. Major changes in the clonal substructure with new dominant clones were seen in 65% and 55% of ERMM and RRMM, respectively. No changes ("stable" evolution) were rare in both ERMM (3/17), and RRMM (4/22). Conclusions: According to our results ERMM and RRMM are biologically distinct entities of MM. While ERMM is characterized by inactivation of tumor suppressors and upregulation of gene sets associated with hypoxia and glycolysis, RRMM shows mutations in multiple gene networks, upregulation of ribosomal protein pseudogenes with unknown function and a signature linked to defective DNA repair, suggesting multifactorial mechanisms that lead from first relapse to end-stage relapsed refractory disease. Comparing paired samples, we did not observe major difference in evolution patterns between ERMM and RRMM. Yet, the low prevalence of the melphalan MM1 signature in ERMM suggests selection of pre-existing clones in this entity. In contrast, single tumor cells exposed to melphalan are often the precursors of clones dominating at the RRMM stage, indicating that first-line ASCT has a long-term effect on MM evolution. Disclosures John: Proteona: Research Funding. Mueller-Tidow:Janssen-Cilag Gmbh: Membership on an entity's Board of Directors or advisory committees; Deutsche Forschungsgemeinschaft: Research Funding; Deutsche Krebshilfe: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; BiolineRx: Research Funding; Bayer AG: Research Funding; Jose-Carreras-Siftung: Research Funding; Wilhelm-Sander-Stiftung: Research Funding; BMBF: Research Funding. Goldschmidt:Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Incyte: Research Funding; Molecular Partners: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Novartis: Honoraria, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma GmbH: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; Merck Sharp and Dohme (MSD): Research Funding. Raab:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: 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; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Abstract: Virtually all patients with multiple myeloma become unresponsive to treatment over time. Relapsed/refractory multiple myeloma (RRMM) is accompanied by the clonal evolution of myeloma cells with heterogeneous genomic aberrations and profound changes of the bone marrow microenvironment (BME). However, the molecular mechanisms that drive drug resistance remain elusive. Here, we analyze the heterogeneous tumor cell population and its complex interaction network with the BME of 20 RRMM patients by single cell RNA-sequencing before/after treatment. Subclones with chromosome 1q-gain express a specific transcriptomic signature and frequently expand during treatment. Furthermore, RRMM cells shape an immune suppressive BME by upregulation of inflammatory cytokines and close interaction with the myeloid compartment. It is characterized by the accumulation of PD1 + γδ T-cells and tumor-associated macrophages as well as the depletion of hematopoietic progenitors. Thus, our study resolves transcriptional features of subclones in RRMM and mechanisms of microenvironmental reprogramming with implications for clinical decision-making.

Abstract: Infections are a leading cause of morbidity and mortality in patients with multiple myeloma (MM). Methods To examine the effects of modern second-generation novel agent therapy on immune cell subsets, in particular CD4+-T-cells, and infectious complications in patients with relapsed/refractory MM (RRMM), we conducted a prospective cohort study in 112 RRMM patients. Results Substantially decreased CD4+-T-cells & lt;200/µl before initiation of relapse therapy were detected in 27.7% of patients and were associated with a higher number of previous lines of therapy. Relapse therapy with carfilzomib or pomalidomide showed a significant further decrease of CD4+-T-cells. All novel agents led to a significant decrease of B-cell counts. Overall, infections were frequent with 21.3% of patients requiring antibacterial therapy within the first 3 months of relapse therapy, 5.6% requiring hospitalization. However, in the setting of standard antimicrobial prophylaxis in RRMM patients with very low CD4+-T-cells, no significant association of CD4+T-cell count and an increased risk of infection could be detected. Discussion Our findings imply that reduced CD4+-T-cell numbers and infections are common in patients with RRMM. We also demonstrate an association with the number of previous therapies and certain substances suggesting an increased need for personalized prophylaxis strategies for opportunistic infections in this patient cohort.

Abstract: In multiple myeloma spatial differences in the subclonal architecture, molecular signatures and composition of the microenvironment remain poorly characterized. To address this shortcoming, we perform multi-region sequencing on paired random bone marrow and focal lesion samples from 17 newly diagnosed patients. Using single-cell RNA- and ATAC-seq we find a median of 6 tumor subclones per patient and unique subclones in focal lesions. Genetically identical subclones display different levels of spatial transcriptional plasticity, including nearly identical profiles and pronounced heterogeneity at different sites, which can include differential expression of immunotherapy targets, such as CD20 and CD38. Macrophages are significantly depleted in the microenvironment of focal lesions. We observe proportional changes in the T-cell repertoire but no site-specific expansion of T-cell clones in intramedullary lesions. In conclusion, our results demonstrate the relevance of considering spatial heterogeneity in multiple myeloma with potential implications for models of cell-cell interactions and disease progression.

Abstract: Introduction: Despite improvements in the prognosis of multiple myeloma (MM), most patients ultimately relapse and undergo multiple lines of therapy. Due to the immunocompromising effects of virtually all anti-myeloma agents as well as the disease itself, infections are a frequent complication during therapy and the most important cause of mortality in patients with MM. Establishment of clear predictors of infectious complications, especially under therapy with novel agents, is therefore of major clinical importance to identify patients at risk and to guide anti-infective prophylaxis. Methods: In this prospective, observational cohort study we examined the development of CD4+ T-cell numbers during anti-myeloma therapies which were based on the novel agents daratumumab, carfilzomib, elotuzumab, or pomalidomide and their impact on infectious complications in 96 patients with relapsed/refractory MM (median prior lines of therapy: 2 [1-13], median age: 70 years of age [42-90] ). Data on infectious events including CTC-AE severity grading, antimicrobial prophylaxis strategies and vaccination status was collected before start of therapy, after 3 months and after 6 months of therapy. Flow cytometry was used to identify T-cell subsets at all three timepoints. Results: Before start of therapy, 25 patients (26%) had CD4+ cell counts & lt; 200/µl, 75 patients (78%) had CD4+ cell counts & lt; 500/µl. In a multivariate linear regression model the number of previous lines of therapy had a significant negative impact on CD4+-cell numbers at start of relapse therapy (p=0.03), whereas age and active therapy within in the last 6 months did not. With regard to relapse therapy, both pomalidomide and carfilzomib led to a significant reduction in CD4+ cell count after 3 months of therapy (p=0.03 and p= 0.04, resp.) in a multivariate linear regression model. This effect was not noticeable in treatments based on daratumumab. In a multivariate logistic regression analysis with regard to the occurrence of infections ≥ CTC II° within the first 3 cycles of therapy, CD4+ cell count at start of relapse therapy was the only predictor with borderline statistical significance (p=0.06). Conclusions: A significant proportion of patients with relapsed refractory MM show a severe reduction of CD4+ T-cells already at start of relapse therapy, especially after multiple lines of therapy. CD4+ cell count at start of relapse therapy might indicate an increased risk of infectious complications. Additional studies with a larger number of patients are warranted to further elucidate the impact of CD4+ cell count at start of relapse therapy as a predictor of infectious complications in MM and whether it might serve to better identify patients at risk of infectious complications and steer antimicrobial prophylaxis strategies. Disclosures John: Proteona: Research Funding. Mueller-Tidow:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Jose-Carreras-Siftung: Research Funding; Bayer AG: Research Funding; BiolineRx: Research Funding; Wilhelm-Sander-Stiftung: Research Funding; BMBF: Research Funding; Deutsche Krebshilfe: Research Funding; Janssen-Cilag Gmbh: Membership on an entity's Board of Directors or advisory committees; Deutsche Forschungsgemeinschaft: Research Funding. Jordan:priME Oncology: Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees; G1 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Voluntis: Membership on an entity's Board of Directors or advisory committees; Pomme-med: Speakers Bureau; Hexal: Speakers Bureau; Merck: Membership on an entity's Board of Directors or advisory committees; Merck Sharp & Dome: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; medupdate: Speakers Bureau; Helsinn: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Tesaro: Membership on an entity's Board of Directors or advisory committees; ClinSolResearch: Membership on an entity's Board of Directors or advisory committees; Riemser: Research Funding, Speakers Bureau; Amgen: Speakers Bureau; Kreussler: Membership on an entity's Board of Directors or advisory committees; art-tempi: Speakers Bureau. Goldschmidt:Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Incyte: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Novartis: Honoraria, Research Funding; Mundipharma GmbH: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Molecular Partners: Research Funding; Merck Sharp and Dohme (MSD): Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding. Raab:Heidelberg Pharma: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: 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; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: 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.