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Abstract: Mass spectrometry (MS) can detect multiple myeloma-derived monoclonal proteins in the peripheral blood (PB) with high sensitivity, potentially serving as a PB assay for measurable residual disease (MRD). This study evaluated the significance of PB MS MRD negativity during posttransplant therapy in patients with newly diagnosed multiple myeloma. Serum samples from 138 patients treated in the phase 3 ATLAS trial of posttransplant maintenance with either carfilzomib, lenalidomide, and dexamethasone, or with lenalidomide alone were analyzed using EXENT MS methodology. We established feasibility of measuring MRD by MS in the PB in the posttransplant setting, despite unavailability of pretreatment calibration samples. There was high agreement between MRD by MS in the PB and paired bone marrow (BM) MRD results at the 10-5 threshold, assessed by either next-generation sequencing (NGS) or multiparameter flow cytometry (MFC) (70% and 67%, respectively). Agreement between PB MS and both BM MRD methods was lowest early after transplant and increased with time. MS negativity was associated with improved progression-free survival (PFS), which, in landmark analysis, reached statistical significance after 18 cycles after transplant. Combined PB/BM MRD negativity by MFC or NGS was associated with superior PFS compared with MRD negativity by only 1 modality. Sustained MS negativity carried similar prognostic performance to sustained BM MRD negativity at the 10-5 threshold. Overall, posttransplant MS assessment was feasible and provided additional prognostic information to BM MRD negativity. Further studies are needed to confirm the role and optimal timing of MS in disease evaluation algorithms. The ATLAS trial is registered at www.clinicaltrials.gov as #NCT02659293., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL., (© 2021 by The American Society of Hematology.)

Purpose: Daratumumab is a promising new agent for relapsed/refractory multiple myeloma (RRMM). However, there are limited data on its clinical activity and tolerability in the real-world patients. The purpose of this study is to determine the efficacy and toxicity profile of daratumumab monotherapy in the real-life setting., Patients and Methods: Thirty RRMM patients treated with daratumumab who had previously received at least three treatment lines including a proteasome inhibitor and an immunomodulatory drug or had been double refractory (DRMM) were included to the Polish Myeloma Group observational study., Results: The objective response rate to daratumumab was 42.8%. Median progression-free survival (PFS) and overall survival reached 9.5 and 13.8 months, respectively. Importantly, patients with DR-MM had a significantly shorter PFS than other patients (median PFS of 4.1 vs. 12.1 months). Daratumumab was generally well tolerated, however two patients had their therapy interrupted due to adverse events., Conclusion: Daratumumab monotherapy has significant activity and good tolerance in heavily pretreated RRMM patients., (Copyright © 2019 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.)

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene ( MS4A1 ) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors., (© 2017 by The American Society of Hematology.)

The receptor for hyaluronic acid-mediated motility (RHAMM) is an antigen eliciting both humoral and cellular immune responses in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and multiple myeloma (MM). We initiated a phase 1 clinical trial vaccinating 10 patients with R3 (ILSLELMKL), a highly immunogenic CD8(+) T-cell epitope peptide derived from RHAMM. In 7 of 10 patients, we detected an increase of CD8(+)/HLA-A2/RHAMM R3 tetramer(+)/CD45RA(+)/CCR7(-)/CD27(-)/CD28(-) effector T cells in accordance with an increase of R3-specific CD8(+) T cells in enzyme linked immunospot (ELISpot) assays. In chromium release assays, a specific lysis of RHAMM-positive leukemic blasts was shown. Three of 6 patients with myeloid disorders (1/3 AML, 2/3 MDS) achieved clinical responses: one patient with AML and one with MDS showed a significant reduction of blasts in the bone marrow after the last vaccination. One patient with MDS no longer needed erythrocyte transfusions after 4 vaccinations. Two of 4 patients with MM showed a reduction of free light chain serum levels. Taken together, RHAMM-R3 peptide vaccination induced both immunologic and clinical responses, and therefore RHAMM constitutes a promising target for further immunotherapeutic approaches. This study is registered at http://ISRCTN.org as ISRCTN32763606 and is registered with EudraCT as 2005-001706-37.

The expression of tumor-associated antigens (TAAs) might play a critical role in the control of minimal residual disease (MRD) in acute myeloid leukemia (AML), and therefore might be associated with clinical outcome in AML. In a DNA microarray analysis of 116 AML samples, we found a significant correlation between high mRNA levels of G250/CA9 and longer overall survival (P = .022), a similar trend with high mRNA levels of PRAME (P = .103), and a hint for RHAMM/HMMR. In contrast, for other TAAs like WT1, TERT, PRTN3, BCL2, and LAMR1, we found no correlation with clinical outcome. High expression of at least 1 of the 3 TAAs, RHAMM/HMMR, PRAME, or G250/CA9, provided the strongest favorable prognostic effect (P = .005). Specific T-cell responses were detected in 8 (47%) of 17 patients with AML in complete remission for RHAMM/HMMR-R3 peptide, in 7 (70%) of 10 for PRAME-P3 peptide, and in 6 (60%) of 10 for newly characterized G250/CA9-G2 peptide, a significant increased immune response compared with patients with AML patients who had refractory disease (P < .001). Furthermore, we could demonstrate specific lysis of T2 cells presenting these epitope peptides. In conclusion, expression of the TAAs RHAMM/HMMR, PRAME, and G250/CA9 can induce strong antileukemic immune responses, possibly enabling MRD control. Thus, these TAAs represent interesting targets for polyvalent immunotherapeutic approaches in AML.

The receptor for hyaluronic acid-mediated motility (RHAMM/CD168) has been described as a leukemia-associated antigen. To define T-cell epitopes of RHAMM/CD168 toward specific immunotherapies for acute myeloid leukemia (AML), 10 potential HLA-A2-binding RHAMM/CD168 peptides (R1 to R10) were synthesized based on computer algorithms and screened by enzyme-linked immunospot (ELISPOT) analysis using CD8+ T cells isolated from peripheral blood (PB) of patients with AML and healthy donors. We found that CD8+ cells from 7 of 13 (54%) patients with AML presensitized with peptides R3 (ILSLELMKL) or R5 (SLEENIVIL) specifically recognized T2 cells pulsed with R3 (39%) or R5 (15%) peptide. In contrast, only 4 of 21 (19%) healthy volunteers had CD8+ cells reactive with R3- or R5-pulsed T2 cells after presensitization. The presence of R3 peptide-specific effector T cells in the peripheral blood of patients with AML could be confirmed by staining as HLA-A2/R3 peptide tetramer+ CCR7-CD45RA+ cells. In chromium-51 release assays, peptide-primed CD8+ T cells from patients with AML were able to lyse RHAMM/CD168 peptide-pulsed T2 cells, AML blasts, and dendritic cells generated thereof (AML DCs). Transfection of COS7 cells with RHAMM/CD168 cDNA revealed that peptides R3 and R5 are naturally processed epitopes of RHAMM/CD168 that are presented in an HLA-A2-restricted manner. In summary, RHAMM/CD168 is a promising target for immunotherapies in patients with AML, and we have therefore initiated a clinical vaccination trial with R3 peptide. Because RHAMM/CD168 is also expressed in various other hematologic malignancies and solid tumors, vaccines targeting this antigen may have even wider application.

We have generated transgenic mice expressing the leech anticoagulant hirudin and human tissue factor pathway inhibitor tethered to the cell surface by fusion with fragments of human CD4 and P-selectin. Expression of the transgenes is under the control of the CD31 (platelet endothelial cell adhesion molecule [PECAM]) promoter, limiting expression to endothelial cells, monocytes, and platelets. In addition, the P-selectin sequence directs expression to secretory granules. Functional cell surface expression only occurs when the cells are activated. In a mouse model of systemic lipopolysaccharide (LPS)-induced endotoxemia, we show that expression of either anticoagulant on activated endothelium inhibits the widespread intravascular thrombosis, thrombocytopenia, and consumptive coagulopathy associated with endotoxemia. Importantly, non- LPS-treated transgenic mice had normal baseline bleeding times. We speculate that targeted delivery of anticoagulants to the endothelium may be a strategy worth pursuing in clinical sepsis to improve efficacy of systemic anticoagulation while minimizing potential hemorrhagic side effects.