Abstract: The semiconductor tracker (SCT) is one of the tracking systems for charged particles in the ATLAS detector. It consists of 4088 silicon strip sensor modules. During Run 2 (2015–2018) the Large Hadron Collider delivered an integrated luminosity of 156 fb -1 to the ATLAS experiment at a centre-of-mass proton-proton collision energy of 13 TeV. The instantaneous luminosity and pile-up conditions were far in excess of those assumed in the original design of the SCT detector. Due to improvements to the data acquisition system, the SCT operated stably throughout Run 2. It was available for 99.9% of the integrated luminosity and achieved a data-quality efficiency of 99.85%. Detailed studies have been made of the leakage current in SCT modules and the evolution of the full depletion voltage, which are used to study the impact of radiation damage to the modules.

Abstract: Introduction Malignant plasma cells (PC) in multiple myeloma (MM) are not homogeneously distributed in the bone marrow and spatial genomic heterogeneity is an evolving concept in MM. PC for histopathology, cytogenetic and molecular analyses and minimal residual disease (MRD) assessment are usually obtained from the iliac crest. This might introduce bias into diagnostic procedures since focal lesions (FL) occur in more than 80% of patients. Methods In April 2019, we initiated a prospective trial to compare findings from imaging-guided biopsies of FL detected by PET/CT and paired standard sampling from the posterior iliac crests (IC) in patients with newly diagnosed (NDMM) and relapsed/refractory MM (RRMM). Bone marrow aspirates from FL and paired IC samples were analyzed with next-generation flow (NGF) for phenotypic and MRD assessment (sensitivity level 10 -5) post therapy where applicable. PC from both locations were isolated using anti-CD138 magnetic beads and subjected to whole-exome sequencing (WES). Last year, we reported on longitudinal assessment of spatiotemporal immunogenomic heterogeneity in this cohort (https://doi.org/10.1182/blood-2020-142622). With a longer follow-up, we report clinical significance of the respective findings. Results In total, 18 patients (10 NDMM, 8 RRMM) underwent bone marrow aspirates from the IC and imaging-guided biopsies of paired FL. Para-medullary disease (PMD) was observed in 4 patients (1 NDMM, 3 RRMM). Unshared mutations from WES between both locations were identified in every patient. In 12 out of 18 patients (8 NDMM and 4 RRMM), less than 20% of mutations were unshared between IC and FL. Patients with & gt;20% unshared mutations had RRMM and/or a history of PMD. The prognostic significance of unshared mutations was emphasized by lesion-specific detection of high-risk mutations (e.g. TP53) and copy number variations (e.g. gains/losses of Chr 1 and del(17p)). To investigate whether unshared mutations might be accessible for therapeutic interventions, the Drug Gene Interaction Database was queried. More interactions were found in FL (n=886) compared to IC (n=505). Furthermore, we showed spatially divergent existence of PC that contribute to resistance, e.g. against proteasome inhibition through mutations in proteasome subunit PSMC3. Next, we investigated whether MRD assessment from IC and FL provides congruent results. In three patients with MRD-negative IC bone marrow aspirates, we obtained clinically relevant divergent results from FL biopsies. In a patient in complete response (CR) after 5 cycles RVD (Lenalidomide, Bortezomib, Dexamethasone), autologous stem cell transplant (ASCT) and 5 years of lenalidomide maintenance, follow-up PET/CT showed a single new FL in the caudal right ilium. While NGF showed persistent MRD-negativity in the ICt, NGF from the FL revealed monocloncal PC. Since there were no other signs of progressive disease, the patient was followed with close surveillance. Repeat PET/CT after 5 months showed progression of the FL. Serological evaluation confirmed relapse from CR and systemic treatment was initiated. In another patient with MRD-negative IC bone marrow aspirate after 4 cycles RVD, imaging-guided biopsy of a T10 residual FL showed monoclonal PC. Mass spectrometry of supernatants from IC aspirates showed persistence of monoclonal protein in each patient. Second primary malignancies (SPM) were detected by imaging-guided biopsies: Multiple PET-positive FL occurred in a MRD-negative patient after 4 cycles of RD, ASCT and 3 years lenalidomide maintenance. NGF confirmed MRD-negativity in IC and FL aspirates. NGF detected no malignant cells in the peripheral blood but B-lymphoblasts were detected in one of the PET-positive FL. Allogeneic stem cell transplant was performed after induction therapy and there is no detectable MM or B-acute lymphoblastic leukemia. Conclusions We demonstrate clinical significance of spatial heterogeneity in NDMM and RRMM through site-specific detection of mutations and chromosomal aberrations associated with adverse outcome and drug susceptibility. Our findings showing divergent results from MRD assessments in bone marrow and FL underline the critical role of whole-body imaging for follow-up of patients in remission. Sampling plasma cells solely from the IC limits the ability for complete clinical decision-making. Disclosures Merz: Sanofi: Honoraria; Janssen: Honoraria; BMS: Honoraria; Hexal: Honoraria; GSK: Honoraria. Barnidge: The Binding Site: Current Employment. McCarthy: Janssen: Honoraria, 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; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird: Honoraria, Membership on an entity's Board of Directors or advisory committees; Juno: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hillengass: Beijing Life Oasis Public Service Center: Speakers Bureau; Beijing Medical Award Foundation: Speakers Bureau; Skyline: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Curio Science: Speakers Bureau; GlaxoSmithKline: 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; Axxess Network: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees.

Abstract: Introduction: Therapy and immune mediated processes are associated with clonal evolution in multiple myeloma (MM). In this study, we performed whole-exome sequencing (WES) and single cell RNA sequencing (scRNA-seq) on plasma cells (PC) from bone marrow aspirates of the iliac crest (BM) and corresponding osteolytic lesions (OL) to investigate spatial heterogeneity in patients with newly diagnosed (NDMM) and relapsed/refractory MM (RRMM). Next generation flow (NGF) and T-cell receptor sequencing (TCRseq) were performed to investigate the immunogenomic landscape surrounding malignant PC. Methods: In a prospective trial, 18 patients (NDMM: n=10; RRMM: n=8) consented to an imaging-guided biopsy of an OL in addition to the regular BM sampling. At inclusion, 37 different locations were biopsied. Follow-up samples were obtained from 5 patients in remission after therapy. After CD138+ selection, PC were subjected to WES and scRNA-seq (Chromium, 10x genomics). TCRseq was performed using multiplex PCR (ImmunoSEQ, Adaptive biotechnologies) on the CD138- fraction. For scRNA-seq data analyses, Cell Ranger (v3.1.0) and the Seurat R toolkit (v3.1) were used. TCRseq data were analyzed with immunoSEQ ANALYZER (v3.0) and the immunarch R toolkit (v0.6.6.). NGF was performed to study subsets of T-, B-, NK- and dendritic cells (DC). Results: Median PC infiltration was higher in OL compared to random BM (50.0% vs 12.5%, p=0.041). WES revealed more mutations in RRMM compared to NDMM (median; range: 189;120-523 vs 71;23-136, p & lt;0.001). Based on mutational profiles from WES, 4 of 18 patients showed a branching evolution in PC isolated from OL. Three of the 4 patients had RRMM and one patient with NDMM had a prior history of solitary plasmacytoma. PC were obtained from OL with adjacent extramedullary disease (EMD) in 3 of 4 patients with branching evolution. Among site-specific mutations, we found in one patient two distinct BRAF mutations: V600E in the BM and G469R in the OL. An additional NRAS mutation (G12D) was found in the OL. BRAF G469R and NRAS G12D cause resistance to BRAF inhibitors, although this patient was naïve to BRAF-inhibitors. Clonal evolution was also reflected by chromosomal aberrations, including site-specific chromothripsis of chromosome 1 in a patient with RRMM. Even in patients without spatially divergent clones as detected by WES, scRNA-seq of more than 150,000 PC from 10 patients and 21 different locations revealed multiple clones. Distinct PC clones were identified by differential expression of genes associated with homing to the BM (CXCR4), malignant transformation (Jun/Fos, CD27, CD79a), apoptosis (BCL-2) bone disease (DKK1) and LAMP-5. In a patient with NDMM in remission after induction therapy, scRNA-seq demonstrated the emergence of a PC clone characterized by the overexpression of Interferon-induced genes (ISG15, IFI27, IFI44L) compared to the initially predominant PC clones. Next, we investigated spatiotemporal differences of immune cells. Estimation of median TCR richness using an abundance-based estimator (Chao1) revealed significantly lower values in patients with RRMM (120444; 57706-212744) compared to NDMM (389341; 50318-525082, p & lt;0.001) and nine healthy individuals (460278; 138326-696419, p & lt;0.001). No significant differences were found for TCR clonality as indicated by Simpson's D. While longitudinal tracking of TCR clones at primary diagnosis showed no clonal expansion after treatment, induction therapy restored sample richness in patients with NDMM to levels of healthy individuals (p=0.61). Overlap analysis showed a high concordance of TCR repertoires from OL and random BM with Morisita indices ranging in 90% of patients from 0.80 to 0.95. Nevertheless, significant site-specific expansion of TCR clones was detected. In accordance with TCRseq, NGF showed in the BM of patients with RRMM more regulatory T-cells (p=0.048) and less myeloid DC (p=0.024), Th9 cells and CD8 effector memory T-cells compared to NDMM. Conclusion: We report the first prospective clinical trial to investigate spatiotemporal immunogenomic heterogeneity in multiple myeloma as assessed by WES and scRNA-seq of PC and NGF and TCRseq of the non-PC compartment. We demonstrate spatial evolution and reduced TCR diversity especially in patients with RRMM and/or EMD. ScRNA-seq adds another layer of complexity compared to WES and helps identifying how PC create an immune suppressive BM niche. Disclosures Merz: Amgen, BMS, Celgene, Takeda: Honoraria. Block:GlaxoSmithKline LLC: Current Employment. McCarthy:Karyopharm: Consultancy, Honoraria; Magenta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Juno Therapeutics, a Bristol-Myers Squibb Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board , Research Funding is to Roswell Park, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Starton: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Genentech: Consultancy, Honoraria. Hillengass:Adaptive, Amgen, BMS, Celgene, GSK, Janssen, Oncotracker, Takeda: Honoraria.

Abstract: Spatial heterogeneity is a common phenomenon in metastatic solid tumors and an evolving concept in multiple myeloma (MM). The interplay between malignant plasma cells (PCs) and the microenvironment has not yet been analyzed in MM. For this purpose, we performed bone marrow aspirates and imaging-guided biopsies of corresponding lesions in newly diagnosed MM (NDMM) and relapsed/refractory MM (RRMM) patients. PCs were isolated and subjected to whole-exome sequencing (WES). Non-PCs were studied with next-generation flow (NGF) and T-cell receptor sequencing (TCRseq) to analyze the connection between malignant and nonmalignant cells in the bone marrow and in lesions. Although we observed a strong overlap from WES, NGF, and TCRseq in patients with intramedullary disease, WES revealed significant spatial heterogeneity in patients with extramedullary disease. NGF showed significant immunosuppression in RRMM compared with NDMM as indicated by fewer myeloid dendritic cells, unswitched memory B cells, Th9 cells, and CD8 effector memory T cells but more natural killer and regulatory T cells. Additionally, fewer T-cell receptor (TCR) sequences were detected in RRMM compared with NDMM and healthy individuals. After induction therapy, TCR repertoire richness increased to levels of healthy individuals, and NGF showed more regulatory T cells and myeloid-derived suppressor cells, regardless of depth of response. Clinical significance of imaging-guided biopsies of lesions was demonstrated by detection of monoclonal PCs in patients without measurable residual disease (MRD) in aspirates from the iliac crest as well as identification of secondary primary malignancies in MRD− patients. Furthermore, site-specific clones with different drug susceptibilities and genetically defined high-risk features were detected by our workflow.

Abstract: Patients with myelodysplastic syndromes (MDS) are at risk of early death from cardiovascular complications due to the link between clonal hematopoiesis and endothelial dysfunction. EASIX (Endothelial Activation and Stress Index) has been established to predict endothelial complications after allogeneic transplantation. We investigated the impact of EASIX measured at first diagnosis on survival of patients with lower- and higher-risk MDS (no allogeneic transplantation) in two independent institutions: n  = 192 (training cohort) and n  = 333 (validation cohort). Serum markers of endothelial cell distress were measured and correlated to EASIX. While no effects of EASIX on survival were observed in higher-risk patients, EASIX was associated with shorter survival in patients with lower-risk MDS in both cohorts (univariate: Cohort I: hazard ratio (HR): 1.46; 95% confidence interval (CI) 1.24–1.71; p -value  〈  0.001/Cohort II: HR 1.31 [1.17–1.48]; p -value  〈  0.001). Multivariate Cox regression analysis and prediction error analyses confirmed that EASIX remained a significant predictor of survival after adjustment for age, sex, cytogenetic abnormalities and bone marrow blasts in lower-risk patients. The model of the training cohort could be validated. Serum levels of Angiopioetin-2 correlated significantly with EASIX. We introduce EASIX as an easily accessible and independent predictor for survival in patients with lower-risk MDS.

Abstract: Introduction Patients with myelodysplastic syndromes (MDS) are at risk of early death from cardiovascular complications due to the link between clonal hematopoiesis and endothelial dysfunction. EASIX (Endothelial Activation and Stress Index) has been established to predict endothelial complications after allogeneic stem cell transplantation (alloSCT). EASIX is an endothelial dysfunction related biomarker that can be easily obtained with routine laboratory markers (creatinine× LDH/thrombocytes). We analyzed whether EASIX can predict mortality in higher-risk and in lower-risk MDS patients. Since alloSCT can alter the natural history of MDS and may be an independent contributor to endothelial complications, this analysis was restricted to patients who did not undergo alloSCT during the course of their disease. Patients and Methods We investigated the impact of EASIX measured at first diagnosis on survival of patients with lower- and higher-risk MDS (no allogeneic transplantation) in two independent institutions: n=192 (training cohort) and n=333 (validation cohort). For the purposes of this study, patients with low and intermediate-1 according to IPSS and very low and low-risk according to IPSS-R, respectively, were considered to have lower-risk MDS, while patients with intermediate-2 and high-risk according to IPSS and intermediate, high and very-high according to IPSS-R, respectively, were considered to have higher-risk MDS. Serum markers of endothelial cell distress were measured and correlated to EASIX. Results While no effects of EASIX on survival were observed in higher-risk patients, EASIX was associated with shorter survival in patients with lower-risk MDS in both cohorts (univariate: training cohort: hazard ratio (HR): 1.46; 95% confidence interval (CI) 1.24-1.71; p-value 〈 0.001 / validation cohort: HR 1.31 [1.17-1.48]; p-value 〈 0.001). We visualized this continuous effect of EASIX in lower-risk disease by grouping patients into quartiles according to EASIX (Figure 1 A and B). In both cohorts, patients in the highest quartile had a shorter survival compared to patients in lower quartiles. Confining our analysis to lower-risk patients who did not develop AML within the observation period, EASIX similarly predicted OS in both cohorts (training: n=78, no. of events n=17, HR per log2 increase 1.43 [1.05-1.94]; p=0.02; validation: n=267, no. of events n=93, HR per log2 increase 1.33 [1.21-1.47] ; p 〈 0.00). Multivariate Cox regression analysis and prediction error analyses confirmed that EASIX remained a significant predictor of survival after adjustment for age, sex, cytogenetic abnormalities and bone marrow blasts in lower-risk patients. The model of the training cohort could be validated. No effect of EASIX on survival was found in higher-risk patients from both cohorts in univariate and multivariate analysis. Serum levels of Angiopioetin-2 correlated significantly with EASIX whereas S100A9 or IL-1b did not. However, we observed a strong intra-pathway correlation of S100A9 with IL1b, IL18, IL37, and HMGB1. Conclusion We introduce EASIX as an easily accessible and independent predictor for survival in patients with lower-risk MDS. The strong correlation of EASIX and ANG2 underlines the endothelial nature of this biomarker. Disclosures Germing: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria. Kobbe:Neovii: Honoraria, Other: Travel support; Pfizer: Honoraria, Other: Travel support; Medac: Honoraria, Other: Travel support; Abbvie: Honoraria, Other: Travel support; Biotest: Honoraria, Other: Travel support; Jazz: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support, Research Funding; Roche: Honoraria, Other: Travel support; Amgen: Honoraria, Other: Travel support, Research Funding; MSD: Honoraria, Other: Travel support; Novartis: Honoraria, Other: Travel support; Takeda: Honoraria, Other: Travel support. Kaivers:Jazz Pharmaceuticals: Other: Travel Support. Merz:Janssen: Other: Travel grants; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Abbvie: Other: Travel grants; Celgene: Other: Travel grants; Takeda Vertrieb GmbH: Other: Travel grants, Research Funding. Dreger:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Neovii, Riemser: Research Funding; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy.

Abstract: Osteolytic lesions (OL) characterize symptomatic multiple myeloma. The mechanisms of how malignant plasma cells (PC) cause OL in one region while others show no signs of bone destruction despite subtotal infiltration remain unknown. We report on a single-cell RNA sequencing (scRNA-seq) study of PC obtained prospectively from random bone marrow aspirates (BM) and paired imaging-guided biopsies of OL. We analyze 148,630 PC from 24 different locations in 10 patients and observe vast inter- and intra-patient heterogeneity based on scRNA-seq analyses. Beyond the limited evidence for spatial heterogeneity from whole-exome sequencing, we find an additional layer of complexity by integrated analysis of anchored scRNA-seq datasets from the BM and OL. PC from OL are characterized by differentially expressed genes compared to PC from BM, including upregulation of genes associated with myeloma bone disease like DKK1 , HGF and TIMP-1 as well as recurrent downregulation of JUN/FOS , DUSP1 and HBB . Assessment of PC from longitudinally collected samples reveals transcriptional changes after induction therapy. Our study contributes to the understanding of destructive myeloma bone disease.