Higher Bone Marrow Research Articles

Healthy tissue metabolism assessed by [18F]FDG PET/CT as a marker of prognosis and adverse events in advanced Hodgkin lymphoma patients

The aim of the study was to assess healthy tissue metabolism (HTM) using 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography/computed tomography (PET/CT) during chemotherapy in Hodgkin lymphoma (HL) and the association of HTM with baseline metabolic tumour volume (MTV), haematological parameters, adverse events (AEs), early response and progression-free survival (PFS). We retrospectively identified 200 patients with advanced HL from the RATHL trial with [18F]FDG-PET/CT before (PET0) and following 2 cycles of chemotherapy (PET2). [18F]FDG-uptake was measured in bone marrow (BM), spleen, liver and mediastinal blood pool (MBP). Deauville score (DS) 1–3 was used to classify responders and DS 4–5, non-responders. [18F]FDG-uptake decreased significantly in BM and spleen and increased in liver and MBP at PET2 (all p < 0.0001), but was not associated with MTV. Higher BM uptake at PET0 was associated with lower baseline haemoglobin and higher absolute neutrophil counts, platelets, and white blood cells. High BM, spleen, and liver uptake at PET0 was associated with neutropenia after cycles 1–2. BM uptake at PET0 was associated with treatment failure at PET2 and non-responders with higher BM uptake at PET2 had significantly inferior PFS (p = 0.023; hazard ratio = 2.31). Based on these results, we concluded that the change in HTM during chemotherapy was most likely a direct impact of chemotherapy rather than a change in MTV. BM uptake has prognostic value in HL.

Associations of granulocyte colony-stimulating factor with toxicities and efficacy of chimeric antigen receptor T-cell therapy in relapsed or refractory B-cell acute lymphoblastic leukemia

Few studies have reported the associations of granulocyte colony-stimulating factor (G-CSF) with cytokine release syndrome (CRS), neurotoxic events (NEs) and efficacy after chimeric antigen receptor (CAR) T-cell therapy for relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We present a retrospective study of 67 patients with R/R B-ALL who received anti-CD19 CAR T-cell therapy, 41 (61.2%) patients received G-CSF (G-CSF group), while 26 (38.8%) did not (non-G-CSF group). Patients had similar duration of grade 3–4 neutropenia between the two groups. The incidences of CRS and NEs were higher in G-CSF group, while no differences in severity were found. Further stratified analysis showed that the incidence and severity of CRS were not associated with G-CSF administration in patients with low bone marrow (BM) tumor burden. None of the patients with low BM tumor burden developed NEs. However, there was a significant increase in the incidence of CRS after G-CSF administration in patients with high BM tumor burden. The duration of CRS in patients who used G-CSF was longer. There were no significant differences in response rates at 1 and 3 months after CAR T-cell infusion, as well as overall survival (OS) between the two groups. In conclusion, our results showed that G-CSF administration was not associated with the incidence or severity of CRS in patients with low BM tumor burden, but the incidence of CRS was higher after G-CSF administration in patients with high BM tumor burden. The duration of CRS was prolonged in G-CSF group. G-CSF administration was not associated with the efficacy of CAR T-cell therapy.

Varnimcabtagene Autoleucel (ARI-0001) for Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL) and Richter Transformation (RT)

Background: The prognosis of CLL patients that do not respond to targeted therapy is unfavourable, especially in the case of adverse genomic aberrations (e.g. TP53 alterations or complex karyotype) or after transformation to aggressive B lymphoma (Richter transformation, RT). In patients with refractory CLL, anti-CD19 CAR-T (CART19) therapy has achieved complete response (CR) rates of 19-45%, with RT usually excluded from pivotal trials. However, long-term remission in CLL patients still remains a challenge with CART19 therapy. Methods: We present the outcomes of all patients with relapsed or refractory (R/R) CLL with or without RT treated with the academic CART19 varnimcabtagene autoleucel (var-cel) from November/2017 to May/2023, including patients from the CART19-BE-01 trial and subsequent compassionate use programme. Patients received lymphodepletion with fludarabine (90 mg/m2) and cyclophosphamide (900 mg/m2) followed by 0.1-1 (CLL) and 0.5-5 (RT) x10e6 CART19 cells/kg in single or fractionated administration (10%, 30% and 60%) in a single institution. Results: A total of 20 patients underwent leukapheresis and var-cel production. At screening patients had a median age of 58 years (44-74), 47% were females, with a median of 4 prior lines of therapy (2-9) including ibrutinib (89%), venetoclax (53%), R-CHOP (58%) and allo-HCT (21%). All patients had high-risk features, including unmutated IGHV (90%), TP53 lesions (89%), complex karyotype (43%) and RT (63%), as well as elevated LDH (74%), extramedullary disease (84%), and CSF infiltration (16%). Median bone marrow infiltration at screening was 18% (3-85), and median tumour metabolic volume (TMV) was 55 ml (0-1217). Var-cel manufacturing was performed in a median of 8 days (7-9). One patient required 2 apheresis and 1 RT patient died due to progressive disease without receiving var-cel. All patients received bridging therapy (BT), based on BTKi (53%), venetoclax (26%), chemotherapy (10%), steroids (10%) and radiotherapy (5%). Despite BT, all patients received var-cel either with stable disease (44%) or progressive disease (56%), in a median of 29 days (18-81) from apheresis to infusion. Fifty-eight percent of patients received 100% of the planned dose. The remainder received 10-30% due to early cytokine release syndrome (CRS), with a final median infused dose of 1 (0.4-5) x10e6 CART19 cells/kg. CRS was observed in 89% of patients, being severe in 11%. Only mild ICANS was developed in 11%. Tocilizumab and corticosteroids were administered in 44% and 17% of patients, respectively. Median duration of CRS and ICANS was 4.5 days (1-17) and 3 days (2-4), respectively. Absolute B-cell aplasia (0 B-cells/mL) was achieved in 94% of patients in a median of 14 days (7-34), with median duration not reached for CLL and RT. Tmax peak CART19 concentration was observed at day +24 (median, 7-148). With a median follow-up of 8,4 months (0.3-66.7), the overall response rate was 83%, with 83% achieving an MRD-negative CR (flow) in bone marrow (BM) and peripheral blood, and 67% achieving CR on extramedullary lesions (PET/CT). Median PFS and DOR was not achieved for either CLL or RT (figure 1). Disease progression was observed in 7 (39%) patients in a median of 63 days (7-237), 43% of which occurred due to CD19-negative antigen escape despite active CART19 persistence. No relapses have been observed after CR was achieved. By univariate analysis there was no significant differences for PFS regarding presence of RT, SD vs PD at infusion, high vs low LDH, BM infiltration (&amp;gt; or &amp;lt; 20%), or TMV (&amp;gt; or &amp;lt; 25 ml). However, PFS was shorter among patients with both, high BM infiltration plus high TMV, in comparison to patients with either low burden at BM and/or TMV (median PFS 2 months vs not reached, p = 0.0293) (figure 2). Conclusions: Administration of the CART19 var-cel (ARI-0001) was feasible in 95% of CLL/RT patients, with robust and durable var-cel engraftment in 89% of treated patients, and durable complete responses observed in 61% of patients. These initial outcomes or var-cel in this population contrasts with the overall findings of CART19 therapy in CLL/RT and encourages to further develop var-cel on this high-risk population.

Multiomic Characterization of Myelodysplastic Neoplasms (MDS) with Micromegakaryocytes Highlights the Role of EZH2-RUNX1 deregulation in Disease Physiopathology and Response to Targeted Therapies

Micromegakaryocytes (micro-MKs), an unequivocal myeloid dysplastic feature in adults, are present in 15-20% of myelodysplastic neoplasms (MDS). We recently associated the detection of these small megakaryocytes with a mono- or bi-lobated nucleus in bone marrow (BM) smears of MDS patients with an unfavorable prognosis. The aim of the present study was to elucidate the molecular bases of micro-MK formation and its impact on MDS response to targeted therapies. We selected retrospectively a cohort of 40 MDS patient samples subjected to NGS myeloid panel and RNA sequencing and carried out a morphological re-evaluation that included micro-MKs recounting. Cases with isolated del(5q) or SF3B1 mutation were excluded. MDS cases were selected according to the following morphological findings: no dismegakaryopoiesis, dismegakaryopoiesis without micro-MK, low rate of micro-MK (&amp;lt;10%; micro-MK low) and high rate of micro-MK (&amp;gt;40%; micro-MK high). No patient harbored an intermediate (10-40%) rate of micro-MK. A comparative characterization of 8 representative patients was carried out subjecting diagnosis BM samples to Tapestri and Chromium single cell proteogenomic and transcriptomic platforms, gene set enrichment analysis, and deep cytogenetic characterization by optical genome mapping. Functional validation studies were performed using the megakaryoblastic cell line MEG01 differentiated to a mature MK phenotype by exposure to phorbol myristate acetate (PMA), as well as using the CRISPR-Cas9 tool for gene editing. Finally, activity of investigational drugs including 5-azacytidine, lenalidomide, venetoclax, rigosertib and pevonedistat, was evaluated in MEG01-activated cells and in vivo using a chicken embryo chorioallantoic membrane (CAM) model of MDS patient-derived xenograft (PDX). The most frequently mutated genes across all cohorts were ASXL1 (58%), SRSF2 (38%), RUNX1 (30%), TET2 (30%), STAG2 (20%), EZH2 (18%), DDX41 (10%), U2AF1 (10%), and ZRSR2 (10%) . Interestingly, EZH2 and RUNX1 mutations were enriched in the micro-MK high group ( EZH2: 0%, 10%, 14.3% vs. 66.7%, p 0.006; RUNX1: 0%, 20%, 35.7% vs. 83.3%, p 0.003), and both mutations co-occurred in 85.7% (6/7) of the EZH2 mutated cases. Single cell analyses revealed an enrichment of RUNX1 alterations in three populations within micro-MK high BM samples, including hematopoietic stem and progenitor cells, non-classical monocytes and immature erythroid cells. These samples were also characterized by a 5-fold increase in MK progenitor abundance and by the upregulation of a set of 10 EZH2-repressed genes in neutrophil-like cells and myeloid progenitors, with 7q loss presumably affecting the EZH2 locus in 50% of the cases with micro-MK. Using the in vitro MK differentiation model, we further demonstrated that EZH2 downregulation hampered the acquisition of MK surface markers CD41 and CD61, and that venetoclax was the sole agent retaining notable cytotoxic activity in differentiated cells when compared to undifferentiated megakaryoblasts. Accordingly, in a first-in-kind in vivo CAM-MDS transplant model, venetoclax was able to impair BM infiltration by human cells in a PDX model of micro-MK+ MDS. In summary, supporting the notion that EZH2 and RUNX1 exert a crucial role in the regulation of megakaryopoiesis, our present findings suggest that deregulation of EZH2-RUNX1 activity by loss-of-function mutation and/or genetic deletion affecting myeloid precursors, impairs a proper megakaryocytic differentiation process in MDS. Thus, this phenomenon may be considered as a characteristic feature of MDS cases with an elevated proportion of micro-MK. Of note, preliminary in vitro and in vivo results support the use of venetoclax for the treatment of this subgroup of patients with dismal outcome.

High BM plasma S100A8/A9 is associated with a perturbed microenvironmentand poor prognosis in myelodysplastic syndromes.

S100A8/A9 is a proinflammatory protein and plays an essential role in the pathogenesis of myelodysplastic syndromes (MDS) via the S100A8/A9-Toll-like receptors axis. While S100A8/A9 levels have been used as biomarkers in many inflammatory diseases, their clinical relevance has not been conclusively resolved in MDS. To address this, we used an enzyme-linked immunosorbent assay to quantify S100A8/A9 heterodimers in bone marrow (BM) plasma from 215 MDS patients and compared S100A8/A9 levels across patients with various disease risks and genotypes. S100A8/A9 levels correlated with ASXL1 variant allele frequencies significantly. Moreover, mutant ASXL1 with concurrent RUNX1, STAG2, ZRSR2, or EZH2 mutations was associated with higher S100A8/A9 levels. We further showed that higher S100A8/A9 independently predicted inferior leukemia-free survival and overall survival in MDS patients, irrespective of age, Revised International Prognostic Scoring System subgroups, and known detrimental mutations. Lastly, through deep-sequenced transcriptomic analysis, we demonstrated that higher S100A8/A9 in the BM intimated a perturbed microenvironment with enhanced myeloid-derived suppressor cell-mediated tumor immune escape signal, altered metabolism, and impairment in the functions and quantities of CD8+ T cells and NK cells. S100A8/A9 in the BM microenvironment may be a potential biomarker in the prognostication of MDS and target for novel therapy.

Mast cell deficiency prevents BCR::ABL1 induced splenomegaly and cytokine elevation in a CML mouse model

The persistence of leukemic stem cells (LSCs) represents a problem in the therapy of chronic myeloid leukemia (CML). Hence, it is of utmost importance to explore the underlying mechanisms to develop new therapeutic approaches to cure CML. Using the genetically engineered ScltTA/TRE-BCR::ABL1 mouse model for chronic phase CML, we previously demonstrated that the loss of the docking protein GAB2 counteracts the infiltration of mast cells (MCs) in the bone marrow (BM) of BCR::ABL1 positive mice. Here, we show for the first time that BCR::ABL1 drives the cytokine independent expansion of BM derived MCs and sensitizes them for FcεRI triggered degranulation. Importantly, we demonstrate that genetic mast cell deficiency conferred by the Cpa3Cre allele prevents BCR::ABL1 induced splenomegaly and impairs the production of pro-inflammatory cytokines. Furthermore, we show in CML patients that splenomegaly is associated with high BM MC counts and that upregulation of pro-inflammatory cytokines in patient serum samples correlates with tryptase levels. Finally, MC-associated transcripts were elevated in human CML BM samples. Thus, our study identifies MCs as essential contributors to disease progression and suggests considering them as an additional target in CML therapy.Mast cells play a key role in the pro-inflammatory tumor microenvironment of the bone marrow. Shown is a cartoon summarizing our results from the mouse model. BCR::ABL1 transformed MCs, as part of the malignant clone, are essential for the elevation of pro-inflammatory cytokines, known to be important in disease initiation and progression.

The N-Terminal Amino Acid Mutation Site Is a Potential Biological Marker for Precise Risk Stratification in Pediatric Patients with CEBPA-Dm AML

CCAAT/enhancer-binding protein alpha (CEBPA) mutations are a favorable prognostic factor for acute myeloid leukemia (AML) patients in adult and pediatric. However, several previous studies have examined relapse rate may be as high as 30%. To explore some features that predict relapse, we found that 33/345 de novo AML patients (9.5%) harbored a mutation in CEBPA, of which 33 patients had 25 (75.8%) harbored CEBPA-dm and 8 (24.2%) harbored CEBPA-sm. Five (15.2%) relapsed patients were all classical CEBPA-dm children. There were no statistically significant differences in clinical characteristics and survival outcomes between CEBPA-sm and CEBPA-dm patients. The classical CEBPA-dm patients with N-terminal mutation sites among amino acid (AA)1-59 were less likely to relapse[n=0(0%) vs. n=5(45.5%), P=0.011] and had superior RFS and EFS than patients with N-terminal mutation sites concentrated in the AA range 60-120 (RFS: 100.0% vs. 41.6±19.7%, P=0.005; EFS: 92.3±7.4% vs. 41.6±19.7%, P=0.03). Gene expression profiling in 9 classical CEBPA-dm patients (including 3 patients in N- terminal AA mutation sites among 1-59 and 6 patients among 60-120) showed that over-expression of PDLIM1 may be an indicator of poor prognosis [RSF of PDLIM1 high vs. low PDLIM1 expression: 33.3±27.2% vs. 100.0%, P=0.053]. When we applied the "adjusted risk stratification", namely N-terminal AA mutation sites used as one of the stratification indicators based on NCCN guidelines, we observed a more pronounced difference in RFS among adjusted risk groups, especially for the 24 cases classical CEBPA-dm patients (P=0.358 vs. P=0.027 for classical CEBPA-dm patients; P=0.028 vs. P=0.022 for classical CEBPA-dm patients who chemotherapy-only). Multivariate analysis identified that higher bone marrow (BM) blast(≥75%) was an independent risk factors for EFS [hazard ratio (HR): 1.51, 95%CI: 1.25-1.84, P=0.032] and normal karyotype was an independent favorable prognostic factor for RFS (HR: 0.31, 95%CI: 0.12-0.59, P=0.049) in classical CEBPA-dm cohort. N-terminal AA sites range 1-59 appeared to be a protective factor for RFS (HR=0.82, 95%CI: 0.61-1.38, P=0.056). In classical CEBPA-dm cohort receiving chemotherapy-only, higher BM blast(≥75%) at diagnosis was independent prognostic factors for RFS and EFS (HR=0.82, 95%CI: 0.61-1.38, P=0.056). In conclusion, our clinical data suggested that N- terminal AA mutation sites concentrated among 1-59 appear to be a protective factor for RFS in classical CEBPA-dm patients. The "adjusted risk stratification" may be useful in re-assessing the prognosis of classical CEBPA-dm AML particularly in patients belonging to the intermediate-risk subgroup. Over-expression of PDLIM1 may be an indicator of poor prognosis and need to be validated in our future large samples.

CXCR4 (CD184) Expression in Pediatric AML Is Associated with Bone Marrow Retention, Specific Disease Characteristics, and Worse Outcomes: A Report of 1004 Patients from the Children's Oncology Group AAML1031 Protocol

Introduction: CXCR4 (CD184) is a G protein-coupled chemokine receptor specific for CXCL12 that is associated with leukocyte chemotaxis. It is expressed on normal hematopoietic cells as well as hematopoietic and epithelial cancer cells. CXCR4 and CXCL12 have been associated with bone marrow retention of AML and ALL blasts and blocking this interaction via small molecule inhibitors has been investigated as a therapeutic target. CXCR4 has also been implicated in other aspects of tumor progression including metastasis, angiogenesis, and survival. As part of Children's Oncology Group protocol AAML1031 for de novo pediatric AML we prospectively evaluated CXCR4 expression by difference from normal (ΔN) flow cytometry and correlated expression with clinical characteristics and outcome. Given the impact of the bone marrow niche on the administration of chemotherapy we hypothesized that high levels of CXCR4 expression would be associated with unfavorable outcomes. Methods: In the AAML1031 phase 3 trial pediatric patients with de novo AML were randomized to receive standard chemotherapy (Arm A) or standard chemotherapy with bortezomib (Arm B), excepting patients with high allelic ratio FLT3-ITD who received standard chemotherapy plus sorafenib (Arm C). Patients were stratified as low-risk (LR) or high-risk (HR) based on cytogenetic and molecular abnormalities, and end of induction (EOI) minimal residual disease (MRD) by flow cytometry. LR patients received four cycles of the standard chemotherapy backbone while HR patients received three followed by best hematopoietic stem cell transplant. Patients who: (1) submitted a bone marrow aspirate for ΔN at diagnosis and (2) provided consent for correlative biology studies were included in this analysis. All diagnostic specimens were centrally and prospectively evaluated for the expression of CXCR4 by ΔN. Results: Surface CXCR4 expression was available for 1004 patients and expression on AML cells varied across the cohort (range: 54-17,793 molecules/cell). For analysis, the study population was divided into quartiles (n=257-258 patients each) based on expression levels. Quartiles showed no association with sex and were distributed as expected between treatment arms. Correlation with clinical characteristics showed that patients in the top quartile of CXCR4 expression were younger (5.9 vs. 11.4 years, P<0.001) and had higher bone marrow (BM) blast counts but lower peripheral blood (PB) blast counts (76% vs. 66%, p<0.001 and 33% vs. 43%, p=0.01, respectively). These patients were highly enriched for HR KMT2A fusions (43% of total fusion positive patients), as well as t(9;11) and NPM1 mutations; while they had a lower frequency of t(8;21), inv(16), and CEBPA mutations (p<0.002 for all). There was no association between CXCR4 expression and protocol risk group assignment or complete remission rates, but patients in the high expressing CXCR4 quartile were more commonly MRD negative at EOI (p=0.03). Flow cytometry also revealed a striking increase in RAM phenotype (p<0.001), with 76% of all RAM patients allocated to the CXCR4 high expressing quartile. Patients in the top quartile of CXCR4 expression had lower 5-year OS (58% vs. 66%, p=0.036) and EFS (38% vs. 47%, p=0.009) compared to other patients. CXCR4 was strongly associated with increased 5-year relapse risk (RR) (54% vs. 39%, p<0.001). Among patients with HR KMT2A fusions high expression of CXCR4 was associated with an increased 5-year RR (85% vs. 57%, p=0.04). Multivariable analysis for 5-year RR including MRD status and prognostic genetic factors indicates CXCR4 expression is independently associated with relapse, with a hazard ratio of 1.33 (95% CI 1.04-1.7; p=0.026). Conclusions: These data demonstrate that CXCR4 expression is associated with individual HR markers (RAM phenotype and HR KMT2A fusions), but not risk status across the entire study. Despite similar risk status, patients in the top quartile of CXCR4 expression had inferior outcomes. These patients had higher BM blast counts but lower PB blast counts, which has been observed in RAM phenotype, likely due to the impact of CXCR4 on bone marrow retention of leukemia cells. This increased retention may also play a role in treatment resistance, suggesting that the addition of small molecule inhibitors of CXCR4/CXCL12 to chemotherapy may help these high expressing patients, especially in subtypes such as HR KMT2A and RAM phenotype patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Reappraisal of Mast Cell Leukemia Based on a Single Institution Review of 16 Cases: Role of Mast Cell Morphology in Determining Clinical Outcome

Background: Mast cell leukemia (MCL) is the leukemic variant of systemic mastocytosis (SM); in addition to meeting conventional diagnostic criteria for SM, it is defined by presence of ≥20% mast cells (MC) in the bone marrow (BM) aspirate smear. Presence of circulating MC is not mandatory; MCL is subclassified into leukemic and aleukemic variants based on whether MC comprise ≥10% or <10% of circulating leukocytes, respectively. Given the rarity of MCL, important disease aspects remain incompletely defined, including frequency of evolution from antecedent SM, frequency of associated hematological neoplasm (AHN), prevalence of KITD816V/other mutations, and the prognostic relevance/reproducibility of MC morphology. Furthermore, MCL (as currently defined) has a variable clinical course, with acute and chronic disease variants being recognized; one pitfall is that the latter group may be clinically indistinguishable from other SM subtypes with high BM MC burden. The aim of the current project is to identify clinical, laboratory and pathology features that determine prognosis/survival outcome in MCL patients. Methods: The Mayo Clinic clinical and hematopathology databases were interrogated to identify MCL cases. Patients were retrospectively recruited after confirming research authorization and institutional review board approval. The diagnosis of MCL was per conventional diagnostic criteria. Clinical and laboratory data were collected at the time of MCL diagnosis. BM slides were reviewed by 3 hematopathologists (KKR, KK, and DC), with each slide set being independently reviewed by at least 2 pathologists, using prespecified criteria. MC were classified as either mature (atypical MC type I) or immature (atypical MC type II [promastocytes] and metachromatic blasts) based on the predominant MC population identified. (Valent et al., Ann Oncol. 2014 Sep;25(9):1691-1700). Patient follow up was updated in July 2022; data were censored at last follow up or death. Results: A total of 16 MCL patients were identified (8 females, median age 65 years, range 41-75). Six patients (37%) had de novo MCL; the remaining had antecedent mast cell disease (smoldering SM=4, aggressive SM=3, SM+AHN=2, cutaneous mastocytosis=1). The AHN in 2 patients was CMML and MDS/MPN-unclassifiable, respectively. Four patients (25%) had biopsy confirmed cutaneous involvement. Ten (63%) and 9 (64%) patients had hepatomegaly and splenomegaly, respectively. Median (range) hemoglobin was 9.7 g/dL (7.2-11.9), leukocytes 8.7 x 109/L (0.7-43.9), platelets 123 x 109/L (14-633), serum tryptase 614 ng/mL (69-1684), and serum albumin 3.7 g/dL (2.6-4.7). B- and C-findings were present in 15 (94%) and 12 (75%) patients, respectively. Seven (44%) and 5 (31%) patients had circulating MC and blood eosinophil count >500/µL, respectively. Nine of 13 (69%) and 3 of 14 (19%) evaluable patients harbored KITD816V and abnormal karyotype, respectively. Seven (44%) patients displayed a predominantly 'immature’ MC morphology. After a median follow up of 3.9 months, 12 deaths were recorded. The median OS was 15 months; 2 patients underwent allogeneic stem cell transplant (ASCT). Factors associated with significantly inferior overall survival (OS) on univariate analysis were: immature MC morphology (median OS 0.5 vs 20 months; p<0.01), peripheral blood eosinophilia (p<0.01), and absence of KITD816V (p=0.03). No survival difference was seen in 4 patients treated with midostaurin and/or avapritinib. On multivariable analysis, immature MC morphology retained significance for OS (Figure); among 7 patients with immature MC morphology, 5 had died within a month of their diagnosis, while 2 remain alive at 0.2- and 1-months follow-up. By contrast, among 9 patients with mature MC morphology, 7 had died after a follow-up period of 2.5-59 months, including one receiving ASCT and died at 16 months follow-up, while 2 remain alive at 5.6- and 164.2-months follow-up, the latter post-ASCT. Treatment details and NGS data (7 patients) will be presented at the meeting. Conclusions: The current study identifies immature MC morphology as the most important predictor of inferior survival in MCL. Our observations suggest the need to refine current diagnostic criteria for MCL, in order to distinguish a biologically distinct patient population with a uniformly poor clinical outcome (i.e., 'true’ MCL) from those with more favorable outcome, despite high MC burden. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Three Gene Leukemic Stem Cell Signature Score Is Robustly Prognostic in Chronic Myelomonocytic Leukemia

Introduction Leukemic stem cells (LSCs) possess biological properties shared with normal hematopoietic stem cells. They confer resistance to chemotherapy and mediate relapse in acute myeloid leukaemia (AML) as well as in myelodysplastic syndromes (Ho et al., Blood 2016; Wang et al., Blood Advances 2020). A 17-gene stemness score ("LSC17") was predictive of treatment response and overall survival (OS) in AML (Ng et al., Nature 2016). Whilst chronic myelomonocytic leukaemia (CMML) is regarded as a myeloid stem cell neoplasm, the biological impact and prognostic implications of LSCs in CMML have not been explored. To this end, we evaluated the prognostic potential of these 17 genes in CMML patients. Methods We performed mRNA sequencing on bone marrow (BM) samples from 44 CMML patients at presentation, each with extensive clinical and laboratory correlates and longitudinal outcome data. CMML diagnosis was based on The International Consensus Classification (Arber et al., Blood 2022). The expression of each LSC17 gene was evaluated and integrated with clinical and outcome metadata. Results We identified three genes (MMRN1, ADGRG1, and DNMT3B) whose expression correlated significantly with overall survival (OS). An LSC-3 score was constructed based on the weighted sums derived from Cox regression analysis. Patients were divided into high and low LSC-3 score groups, with cut point determined by maximally selected rank statistics. Higher LSC-3 scores were associated with higher age (p=0.016), lower hemoglobin levels (p=0.011), and higher incidence of IDH1/2 mutations (p=0.009). High-score patients had significantly shorter leukaemia-free survival (LFS; median 7.4 months vs 30.5 months, p<0.001) and OS (10.5 months vs 33.9 months, p<0.001, Figure 1A). In subgroup analyses, LSC-3 score retained strong LFS and OS significance in both CMML-1 and CMML-2 stages as well as across dysplastic and proliferative CMML subgroups. Mutations in ASXL1 have been consistently associated with poor prognosis in CMML and incorporated into several existing prognostic systems (Patnaik, Haematologica2022). In patients with unmutated ASXL1, high LSC-3 score robustly correlated with inferior LFS and OS (p=0.001 and p=0.001, respectively). In multivariable analysis, higher LSC-3 score remained an independent adverse risk factor for LFS and OS, irrespective of CPSS and CPSS-Molecular risk status. Time-dependent ROC curve analysis suggested potential for LSC-3 score to improve current risk stratification systems, increasing area under curve when incorporated (Figure 1B). We validated the prognostic performance of the LSC-3 score in three independent CMML cohorts. Remarkably, LSC-3 score retained strong discriminatory performance even in a CMML monocyte RNA-seq dataset, suggesting potential application on peripheral blood samples. GSEA revealed enriched oxidative stress, altered metabolism, and aberrant immune and T-cell functions in LSC-3 high BM samples. CIBERSORTx analysis (Newman et al., Nature Biotechnology 2019) also indicated significantly lower fractions of activated NK-cells (p=0.026) and borderline significant fewer CD8 T-cells (p=0.06) in the BM from high-score CMML patients. Finally, single-sample GSEA highlighted differences in drug sensitivity profiles, including resistance signatures for hypomethylating agents and venetoclax in LSC-3-high patients, which might partially explain the dismal prognosis in this population. Discussion We describe a simple yet powerful three gene expression signature ("CMML-LSC3") as an independent and robust prognostic factor for LFS and OS in CMML patients. We validated this signature in three independent CMML cohorts with linked transcriptomic data, comprising both bone marrow mononuclear and sorted peripheral blood monocytes. We propose that LSC3 could be incorporated to complement existing scoring systems to improve prognostic performance and aid management decisions. Biologically LSC3 stratifies CMML patients by apparent differences in metabolism, immune microenvironment composition and drug resistance, raising the prospect of personalized therapeutic approaches mandating further study. The LSC3 score is being validated prospectively in a UK multicentre phase 2 trial (AMMO trial EudraCT 2021-004585-35). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Glutamate Metabolism Shapes Osteoclast Differentiation: New Potential Therapeutic Target to Block Osteoclastic Bone Resorption in Multiple Myeloma Patients

Multiple Myeloma (MM) cells are characterized by an impaired Glutamine (Gln) metabolism and Gln-addiction feature. As a result of this, MM patients have lower levels of Gln and higher levels of its main metabolite Glutamate (Glu) in the bone marrow (BM) microenvironment as compared with Smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS) patients. Moreover, we have recently shown that MM-dependent Gln-exhausted microenvironment impairs osteoblast differentiation. Based on this evidence, we have hypothesized that alterations of Gln/Glu axis may have a significant impact on the activation of osteoclasts (OCLs) and in the development of osteolytic lesions. We purified BM CD14+ monocytes from a cohort of 30 patients with monoclonal gammopathies (5 MGUS, 12 SMM and 13 newly diagnosed MM (ND-MM)). Cells have been incubated in osteoclastogenic medium in the presence or absence of Glu. The samples were subsequently analyzed by real-time PCR, Western blot and TRAP staining after 3 or 14 days. Amino acid uptake and amino acid content have been assessed under the same conditions. Glu uptake inhibitors have been tested throughout osteoclastogenesis. Our data showed that the presence of Glu significantly increases the number of OCLs generated from MGUS/SMM CD14+ compared with cells differentiated in the absence of the amino acid (p<0.01). In these patients, Glu increases the expression of RANK as well as NF-κB and NFATc1 protein level pointing to a stimulatory effect on osteoclastogenic signaling pathway. On the contrary, CD14+ obtained from MM patients have a lower response to Glu probably as a consequence of the high Glu microenvironment imposed by MM cells. Consistently, MM CD14+ cells are prone to generate more OCLs also in the absence of Glu compared with CD14+ cells obtained from premalignant samples (median number of OCLs: MGUS/SMM 33, MM 123, p=0.02). The analysis of intracellular amino acids (Gln, Glu, and α-ketoglutarate (2-OG)) indicated that Glu content significantly increases after 8 days of differentiation while decreasing after 14 days pointing to a fast accumulation of Glu upon differentiation. The content of both Gln and 2-OG do not vary appreciably throughout the differentiation. These data suggest that OCLs differentiation is associated with an increased uptake of extracellular Glu. Consistently, the activity of the sodium-dependent Glu transporters EAATs, increases already after 3 days of incubation in differentiating medium without Glu, with a further increase in the presence of the amino acid. Expression analysis revealed that, among EAATs, EAAT1 (encoded by SLC1A3 gene) is responsible for the uptake of extracellular Glu upon differentiation both in the presence and in the absence of the amino acid. The expression of other Glu transporters do not change during differentiation. Selective inhibitors of EAAT1, UCPH101 and TFB-TBOA completely block the formation of multinucleated OCLs in the presence of Glu (p<0.01). In parallel to EAAT1 induction, Glu treatment increases the expression of Calcineurin, the serine/threonine phosphatase activated by increased intracellular calcium levels indicating the possible modulation of calcium signaling by Glu. In conclusion, our results indicate that high BM Glu level sustains OCLs formation by stimulating of NF-κB/NFATc1/calcineurin pathway. EAAT1 transporter is responsible for the uptake of extracellular Glu during osteoclastogenesis while its inhibition markedly blocks OCL formation. Therefore, targeting glutamate metabolism could be a therapeutic strategy to block OCL formation in MM patients.

Prognostic impact of MYD88 and CXCR4 mutations assessed by droplet digital polymerase chain reaction in IgM monoclonal gammopathy of undetermined significance and smouldering Waldenström macroglobulinaemia.

Waldenström macroglobulinaemia (WM) is characterized by recurrent somatic mutations in MYD88 and CXCR4 genes. However, limitations arise when analysing these mutations in IgM monoclonal gammopathy of undetermined significance (MGUS) or smouldering WM (SWM) given the lower tumour load. Here, we used droplet digital polymerase chain reaction (ddPCR) to analyse MYD88 L265P and CXCR4 S338* mutations (C1013G and C1013A) in unsorted bone marrow (BM) or cell-free DNA (cfDNA) samples from 101 IgM MGUS and 69 SWM patients. ddPCR was more sensitive to assess MYD88 L265P compared to allele-specific PCR, especially in IgM MGUS (64% vs 39%). MYD88 mutation burden correlated with other laboratory biomarkers, particularly BM infiltration (r=0.8; p < 0.001). CXCR4 C1013G was analysed in MYD88-mutated samples with available genomic DNA and was detected in 19/54 (35%) and 18/42 (43%) IgM MGUS and SWM cases respectively, also showing correlation with BM involvement (r=0.9; p < 0.001). ddPCR also detected 8 (38%) and 10 (63%) MYD88-mutated cfDNA samples in IgM MGUS and SWM respectively. Moreover, high BM mutation burden (≥8% MYD88 and ≥2% CXCR4) was associated with an increased risk of progression to symptomatic WM. We show the clinical applicability of ddPCR to assess MYD88 and CXCR4 in IgM MGUS and SWM and provide a molecular-based risk classification.

MDS-431 Outcomes of Transformation to Acute Myeloid Leukemia after Hypomethylating Agent Therapy in Patients With Myelodysplastic Syndromes

Most patients with myelodysplastic syndrome (MDS) have progression of disease after hypomethylating agent (HMA) failure, including transformation to acute myeloid leukemia (AML). We aimed to understand the clinical characteristics and outcomes of AML transformation after HMA failure in MDS. We conducted a retrospective review of 147 patients with newly-diagnosed MDS from 2017-2021 who later developed HMA failure, focusing on AML transformation. With a median follow-up time of 19.5 months, 71 patients (48%) developed AML after HMA failure. Patients with eventual transformation were more likely to be younger, have higher bone marrow or peripheral blasts, and lower platelet counts at the time of MDS diagnosis. Moreover, these transformed patients had higher-risk MDS by IPSS-R, more adverse cytogenetics (CG), and more frequent TP53 mutations and less frequent SF3B1 mutations at MDS diagnosis. Patients with AML transformation had a shorter time from diagnosis to HMA failure (7.5 vs. 15.0 months, p<0.001) and overall survival (OS) from HMA failure (4.2 vs. 8.4 months, p=0.003). Age <65 years old, higher-risk IPSS-R, and HMA response was associated with AML transformation by multivariate analysis. In patients with AML transformation, the median overall survival (OS) after AML diagnosis was 4.0 months. After transformation, the median number of treatment lines was 1 with 17% not receiving AML-directed therapy. 52% patients continued HMA-based therapy, and 51% received venetoclax. Out of 44 patients with evaluable responses, 6 achieved complete remission (CR) and 17 attained CR with incomplete hematologic recovery, with an overall response rate of 52%. However, most patients died of refractory disease (73%) and/or complications from pancytopenia (19%). The 60-day and 180-day mortality were high at 33% and 69%, respectively. The type or intensity of treatment was not associated with survival. Therapy-related disease, ELN adverse risk, higher risk CG, and TP53 and KRAS mutations were associated with worse survival. Four of 5 patients who underwent allogeneic stem cell transplantation achieved survival over 1 year (17.7-39.6 months). The outcomes of AML transformation after HMA failure MDS remain extremely poor. Further understanding of the biology and disease characteristics of this patient population is warranted for better treatment approaches.

P932: ON-DEMAND PLERIXAFOR WITH CYCLOPHOSPHAMIDE AND G-CSF FOR HEMATOPOIETIC STEM-CELL MOBILIZATION IN MULTIPLE MYELOMA PATIENTS: FINAL RESULTS OF THE MOZOBL06877 STUDY

Background: High-dose melphalan and autologous stem-cell transplant (ASCT) are pillars for the upfront treatment of ASCT-eligible newly diagnosed multiple myeloma (ND)MM patients (pts) or as salvage strategy at relapse. Failure to collect an adequate number of hematopoietic stem cells (HSC) to receive ASCT (<2×106 CD34+ cells/kg) occurs in 5%-15% of MM pts undergoing HSC mobilization with granulocyte colony-stimulating factor (G-CSF) or G-CSF+cyclophosphamide (G-CSF/CY). Aims: We report the final results of the observational MOZOBL06877 study (NCT03406091; partially supported by Sanofi investigation funds) to prospectively assess the performance of HSC mobilization with G-CSF/CY plus on-demand CXCR4 inhibitor plerixafor (PLX) in NDMM pts treated with novel agents. Methods: NDMM pts undergoing HSC mobilization with CY (2-4 g/m2) and G-CSF (5-10 mcg/kg/day) were enrolled and observed up to 30 days after mobilization. According to its label, “on-demand” PLX was administered in pts with <20 CD34+ cells/ul after ≥4 days of G-CSF or in case <1×106 CD34+ cells/kg were collected on the first apheresis day. Pts gave written informed consent. The primary endpoint was the poor mobilizer rate, defined as the rate of pts collecting <2×106 CD34+ cells/kg or requiring PLX. Secondary endpoints were the identification of predictive factors for PLX use and safety during mobilization. Results: 301 NDMM pts were enrolled and analyzed; 72% received induction with bortezomib-thalidomide-dexamethasone, 9% a lenalidomide (Len)-based and 3% a daratumumab (Dara)-based regimen. Overall, 48 pts (16%) were poor mobilizers: 14 (5%) failed to yield ≥2×106/Kg CD34+, while 34 (11%) required rescue with PLX. Among pts yielding <2×106/Kg CD34+ cells, 4 received PLX, while 10 did not. Among pts who successfully collected ≥2×106/Kg CD34+ (n=287, 95%), 34 (12%) required PLX: 23 (68%) due to a CD34+/uL count ≤20 after ≥4 days of G-CSF and 11 (32%) to a collection <1×106/Kg after the first apheresis day. In pts mobilized with PLX, median number of CD34+×106/L cells increased from 17.5 (IQR 10.8-25.6) before PLX to 58.3 (IQR 34.2-100.2) after PLX. Median number of CD34+/Kg collected in pts who did not require PLX was 10.2×106 (IQR 8.3-13.2). In pts who required PLX, a median of 6.5×106 CD34+/Kg (IQR 4.6-9.6) was collected. An adequate HSC yield for ≥2 ASCTs (≥4×106/Kg) was obtained in 96% and 85% of pts not receiving or receiving PLX, respectively. Median number of apheresis days was 1 (IQR 1-2) in pts not requiring PLX and 2 (IQR 1-2) in pts requiring PLX. In a multivariate analysis (Table), factors predicting PLX use were advanced disease stage (R-ISS 3 vs 1-2, OR 5.5, P=0.01), bone marrow (BM) plasma cell (PC) infiltration at diagnosis (PC>60% vs ≤60%, OR 4.2, P<0.001), pre-mobilization absolute neutrophil count (ANC, < vs >2500/mmc, OR 2.8, P=0.01), and the use of Len-based (Yes vs No, OR 3.4, P=0.02) or Dara-based induction regimens (Yes vs No, OR 5.3; P=0.04). Grade 3 infections occurred in 3 pts (1%), with no grade 4-5 reported. Image:Summary/Conclusion: Among NDMM pts treated with novel-agent-based induction and undergoing HSC mobilization with CY/GCSF, “on-demand” PLX in case of low CD34+ cell count or insufficient HSC yield was a safe and effective rescue strategy, reducing mobilization failure from 16% to 5%. Predictive factors for PLX use were advanced disease stage, high BM plasmacytosis, low ANC values before mobilization and the use of Len/Dara during induction. Preemptive PLX in pts with ≥1 risk factors should be investigated to further reduce the risk of mobilization failure.

Decreased serum apolipoprotein A1 level predicts poor prognosis of patients with de novo myelodysplastic syndromes

BackgroundMyelodysplastic syndromes (MDS) is a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells. It has been demonstrated that apolipoproteins A1(ApoA1) are associated with disease risk in many cancer types. However, there still lacks evidence regarding the link between ApoA1 and MDS. This study was designed to investigate the prognostic value of pretreatment ApoA1 levels in MDS patients.MethodsWe retrospectively analyzed a cohort of 228 MDS patients to explore the prognostic value of the serum ApoA1 levels at diagnosis. Patients were divided into the high ApoA1 group and the low ApoA1 group. The prognostic significance was determined by univariate and multivariate Cox hazard models.ResultsMDS patients with low ApoA1 levels had significantly shorter overall survival (OS, P < 0.0001) along with a higher frequency of TP53 mutation (P = 0.002). Based on univariate analysis, age (≥ 60 years), gender (male), lower levels of hemoglobin (< 10 g/dl), HDL (≤0.91 mmol/L), higher bone marrow blast percentage (> 5%), higher IPSS-R scores and poorer karyotype were significantly associated with decreased OS. However, low ApoA1 level did not influence leukemia-free survival (LFS, P = 0.367). Multivariate Cox proportional hazards regression analysis indicated that low ApoA1 level (≤ 1.02 g/L) was also an independent adverse prognostic factor for OS in MDS (P = 0.034).ConclusionsDecreased ApoA1 level predicts a poor prognosis of MDS patients and thus provides a novel evaluation factor for them that is independent of the IPSS-R system.

Abstract 14015: Extracellular Vesicle-Associated microRNAs From the Plasma of HIV-Positive Individuals Enhance Atherosclerosis Burden

Introduction: People living with HIV (PLHIV) have a higher risk of cardiovascular diseases (CVD) compared to HIV negative (HIV - ) individuals. The molecular mechanisms underlying the increased CVD risk remain elusive. Extracellular vesicles (EVs) mediate cell-to-cell communication through cargo transfer, including microRNAs (EV-miRs). Hypothesis: HIV infection results in modification of plasma EVs cargo and these HIV positive EVs (HIV posEVs ), specifically EV-miRs, recapitulate the effects of HIV infection on CVD development. Methods: Atherosclerosis burden was assessed by carotid ultrasound in 74 PLHIV treated with antiretroviral therapy (ART) with viral loads &lt;50 c/ml and 23 HIV - subjects. Circulating endothelial progenitor cell (EPC) levels were measured by colony forming unit (CFU) assay. PLHIV plasma was fractionated into plasma EVs (HIV posEVs ) and plasma-depleted of EVs (HIV plasmadepEVs ). These fractions, EVs from HIV - subjects (HIV negEVs ) and PBS were injected weekly into apoE -/- mice via tail vein for three months. Atherosclerosis burden (H&amp;E and Oil Red O staining), bone marrow (BM) EPC senescence (β-galactosidase staining), proliferation (MTT assay) and migration (“scratch” assay) were examined. RNASeq was used to identify EV-miRs that mediated the effects of HIV posEVs . Tailored EVs (TEVs) loaded with antagomirs for top EV-miRs were generated to determine their capacity to reverse the adverse effects of HIV posEVs in mouse EPCs in vitro . Results: PLHIV had higher atherosclerosis burden and lower circulating EPC numbers. Mice treated with HIV posEVs showed increased atherosclerotic burden, higher BM EPC senescence and lower functionality. HIV posEVs also negatively affected mouse BM EPC senescence in vitro . RNAseq identified four top candidate EV-miRs upregulated in HIV posEVs : let-7b-5p, miR-16-5p, miR-423-5p, and miR-103a-2. TEVs loaded with the antagomirs for let-7b and miR103a-2 counteracted the effects of HIV posEVs on the accelerated senescence of mouse BM EPCs. Conclusion: 1) Plasma EVs from PLHIV reconstitute the effects of HIV infection on accelerated atherosclerosis via negatively affects EPC functionality; and 2) TEVs, targeting candidate EV-miRs counteracts the effects of HIV infection on EPCs.

High-Risk Gene Expression Subtype Provides Molecular Basis for Different Clinical Presentation of Primary and Secondary Plasma Cell Leukemia

Plasma cell leukemia (PCL) is rare but represents an aggressive, advanced form of multiple myeloma where neoplastic plasma cells (PCs) lose dependence on the bone marrow (BM) and circulate in the peripheral blood (PB). PCL is clinically defined by diagnosis of myeloma with ≥20% circulating plasma cells (CPCs), however, several groups have proposed a ≥5% CPC cutoff. PCL is classified as primary (pPCL) if it presents at myeloma diagnosis or secondary (sPCL) if it arises at a later progression event. These presentations of PCL are clinically distinct, with sPCL patients responding poorly to novel therapies and having inferior outcomes compared to pPCL patients. Despite recent advances in myeloma therapy, PCL prognosis remains poor, and the molecular drivers of PCL remain poorly understood.The MMRF CoMMpass study (NCT01454297) is a longitudinal, observational clinical study of 1171 newly-diagnosed myeloma patients. Tumors were characterized using whole genome (WGS), exome (WES), and RNA (RNAseq) sequencing at diagnosis and each progression event. PCs were isolated from BM-derived tumors and when >5% CPCs were detected, PCs were also isolated from the PB, creating a subcohort of patients with sequencing data from both the BM and PB compartments, with some patients assayed longitudinally.The percent CPCs determined using flow cytometry was reported for 982 patients at myeloma diagnosis and 194 patients at progression. Patients with 5-20% CPCs (median = 19 months) at diagnosis had poor overall survival (OS) outcomes compared to those with less than 5% CPCs (median = 95 months, p<0.001). No outcome difference was observed between patients with 5-20% and >20% CPCs (median = 41 months), confirming the findings of previous independent studies. A ≥5% CPC cutoff identified 947 myeloma, 29 pPCL, and 6 sPCL patients in the CoMMpass cohort. Compared to myeloma, pPCL and sPCL patients had poor OS (p<0.001), and after sPCL detection patients had a median OS of only 53 days (range = 0-169 days). For 10 pPCL patients, the percent CPCs was reported at diagnosis and at least one progression event, and patients with persistent CPCs (n = 5, median = 16 months, p<0.01) had poor OS compared to patients with no detectable CPCs at progression (n = 5, median not met, median follow up = 64 months). This underscores the benefit of early eradication of CPCs and repeated CPC measurements in pPCL.The proliferative (PR) gene expression subtype of myeloma has been previously described and defines a high-risk group of patients with diverse genetic backgrounds and inferior outcomes. For PCL patients, we determined the subtype of all BM and PB tumor samples characterized using RNAseq. There was high subtype concordance between paired BM and PB tumor samples (12/13, 92.3%). Overall, 6/23 (26.1%) pPCL patients were in the PR subtype, and PR pPCL patients had poor OS outcomes (median = 10 months, p<0.001) compared to non-PR pPCL patients (median = 55 months). PR emerged as a robust predictor of risk in pPCL, outperforming other molecular and clinical variables including high BM or PB PCs, plasmacytomas, renal failure, high LDH, high B2M, low platelets, t(11;14), del(1p), amp(1q), del(13q), and del(17p), suggesting that RNA subtyping CPCs may represent a non-invasive tool to predict risk in pPCL.At myeloma diagnosis, all sPCL patients with RNAseq data were classified in non-PR subtypes. However, at sPCL, 5/6 (83.3%) patients were in the PR subtype, indicating that sPCL is associated with transition to PR. Two sPCL patients that transitioned to PR acquired biallelic deletion of CDKN2C, and a third acquired biallelic deletion of RB1. Overall, a subset of pPCL (26.1%) but the majority of sPCL (83.3%) patients were in the PR subtype at PCL diagnosis, providing a molecular basis for the different clinical presentations observed between these two groups, including the highly-aggressive nature of sPCL. In summary, this study supports using a lower percent CPC cutoff to clinically define PCL and highlights the importance of repeated CPC measurements in prognosticating pPCL patients. Further, PR RNA subtype emerged as a predictor of risk in pPCL and, given that the majority of sPCL patients were in the PR subtype, provides a molecular basis for the different clinical features observed between pPCL and sPCL patients. DisclosuresMikhael: Amgen: Consultancy; Takeda: Consultancy; BMS: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Sanofi: Consultancy; GSK: Consultancy; Oncopeptides: Consultancy.

The Natural History of Lower Risk MDS: Factors Predicting Progression to High-Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia in Patients with Very Low and Low Risk MDS According to the R-IPSS Criteria

The Natural History of Lower Risk MDS: Factors Predicting Progression to High-Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia in Patients with Very Low and Low Risk MDS According to the R-IPSS Criteria

Single-Cell Transcriptomic Analysis Reveals Loss of Activated Bone Marrow NK Cells in Multiple Myeloma Patients Which Associates with Disease Progression in Mice

IntroductionMultiple Myeloma (MM) disease progression and therapy response are the net result of tumor cell-intrinsic features and tumor cell-extrinsic cues from the bone marrow (BM) microenvironment. Natural killer (NK) cells are mediators of the cytotoxic immune response against MM and are important effector cells in antibody-based immune therapies, especially anti-CD38 monoclonal antibodies such as Daratumumab. Classically, NK cells are divided into a cytotoxic CD56 dim subset, important for antibody-dependent cellular cytotoxicity, and a cytokine-producing CD56 bright subset releasing inflammatory mediators such as IFNγ, TNFα and GM-CSF. However, accumulating evidence suggests greater heterogeneity in the NK cell compartment and modulation of these NK cell subsets could impact disease progression and response to NK cell-driven immunotherapies. Here, we combined the 5TGM1 murine model of MM with single-cell RNA sequencing of bone marrow (BM) NK cells of newly diagnosed MM patients to map NK cell heterogeneity and to investigate their role in MM progression.ResultsTo gain insight in NK cell heterogeneity in MM disease we performed single-cell RNA sequencing on immune cells of viably frozen BM aspirates from 19 newly diagnosed MM patients and 5 non-cancer control patients. NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 resulting in a single-cell transcriptomic dataset of 30,373 NK cells from MM patients and 8,865 NK cells from control patients. Conventional CD56 bright and CD56 dim NK-cells were identified by increased transcription of GZMK or GZMB, respectively. The GZMK +CD56 bright NK cells contained clusters of naïve and activated NK cells. The GZMB+CD56 dim NK cells consisted of 5 subclusters. To identify MM-induced alterations in NK cell subsets, we compared GZMK +CD56 bright vs GZMB+CD56 dim cluster composition and distribution between controls and MM patients. Control BM was dominated by GZMB-transcribing cytotoxic CD56 dim NK cells, resulting in a low ratio of cytokine-producing GZMK +CD56 bright vs cytotoxic GZMB+CD56 dim NK cells. In contrast, MM bone marrow was characterized by heterogeneity of this ratio with a subset of patients presenting with complete reversal of this ratio . In this subset of patients, the altered composition was due to a loss of cytotoxic GZMB +CD56 dim NK cells, and more specifically a loss of NK cells with a transcriptome suggesting recent activation.To better examine the significance of cytotoxic NK cells in MM disease course we utilized the well-established 5TGM1 mouse model. C57Bl/6 and KaLwRij mice both received 10 6 5TGM1-GFP cells intravenously. Three weeks after tumor injection all KaLwRij mice (18/18) developed MM, defined by >5% tumor cells in BM (“unrestrained tumor”) and serum M-protein >2mg/ml. Interestingly, while 39% (7/18) of C57Bl/6 mice had no tumor, 44% (8/18) had low but detectable levels of MM cells (0.1-5% of BM cells, “restrained tumor”) and 17% (3/18) presented with an unrestrained MM with BM tumor load similar to that seen in KaLwRij mice. With time the percentage of mice with unrestrained tumor increased (5/12, 42%) at the expense of restrained tumor (2/12, 16%). We hypothesized that C57Bl/6 mice with low tumor load could represent a model of immune-mediated tumor control. Detailed analysis of the NK cell compartment revealed an expansion of activated mature (CD69 + CD11b +CD27 +) NK cells in C57Bl/6 mice with restrained BM MM (p=0.0031). In contrast, high BM tumor burden in both genotypes was associated with a sharp decline in absolute numbers of activated NK cells.Conclusion:Through a combination of single-cell transcriptomic analyses of the BM immune microenvironment in MM patients and experimental mouse models we found a loss of activated NK cells in a subset of patients and mice. Our data suggests that loss of these activated NK cells is associated with MM progression in vivo. A subset of MM patients presented with a loss of activated cytotoxic GZMB +CD56 dim NK cells in the BM, suggestive of reduced cytotoxic anti-tumor responses. Meanwhile, in vivo, high disease burden only occurred in mice with an absence of activated NK cells. Current analyses are focused on differences in human disease progression and efficacy of Daratumumab-based therapies in patients with various NK cell phenotypes. DisclosuresBroijl: Janssen, Amgen, Sanofi, Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld: Janssen: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

Loss of Spleen Visualization on Whole-Body Diffusion-Weighted Imaging in Patients with Multiple Myeloma Is Associated with Poor Prognosis and High Tumor Burden

IntroductionMultiple myeloma (MM) is caused by the proliferation of monoclonal malignant plasma cells in the bone marrow (BM). Imaging has played a major role in visualizing myeloma lesions, assessing tumor volume, and predicting the prognosis. Recently, we reported that the total diffusion volume (tDV), assessed using a pretreatment whole-body diffusion-weighted imaging (WB-DWI), was associated with a high BM plasma cells (BMPCs) and a poor prognosis in patients with MM (Terao. et al., Eur Radiol 2021). During that study, we unexpectedly found the frequent absence of a spleen signal in patients with MM and its reappearance after treatment. Therefore, this study aimed to investigate the association between spleen visualization changes on WB-DWI and myeloma tumor load and prognosis in patients with MM.MethodsThe data of 96 consecutive patients with symptomatic newly-diagnosed MM (NDMM) at Kameda Medical Center from January 2016 to December 2020, 15 consecutive patients with smoldering MM (sMM), and two autopsied spleens of patients with PC dysplasia were retrospectively reviewed. All patients underwent at least one WB-DWI prior to treatment. The detail of WB-DWI was previously reported (Terao. et al., Eur Radiol 2021).“Loss of spleen visualization” (LSV) was defined as a visual loss of the spleen in maximum intensity projection on the WB-DWI (Fig1). The spleen-to-spinal cord (SC) ratio (SSR) was used in each regions-of-interest (ROI) to compare the signal intensity. The spleen ROIs were defined as non-overlapping ROIs of 30-50 pixels. The SC ROI was the largest ROI without overhanging in the image depicting the maximum size of the spleen. This study was approved by the institutional review board and conducted in accordance with the Declaration of Helsinki. All participants provided informed consent.ResultsThe median patient age was 75.5 years and 81 patients (84.4%) were 65 years or older. Almost all patients (n=91) received proteasome inhibitors (PIs) as remission induction therapy and 33 patients received autologous stem-cell transplantation (ASCT).LSV was observed on the WB-DWI of 56/96 (58.3%) patients with NDMM and in one patient with sMM (1/15, 6.7%). Patients with NDMM and LSV had a higher median BMPC infiltration as assessed by CD138-immunohistochemistry (80.0% vs. 50.0%, p<0.001), a higher median tDV (540.2 mL vs. 137.0 mL, p=0.003), higher rate of ISS stage III (p<0.01), a lower SSR (0.36 vs. 0.96; p<0.001), and lower tDV (540.2 mL vs. 137.0 mL; p=0.003) than those without LSV. The three-year PFS (p=0.27) and three-year OS (p=0.021) were lower in patients with NDMM with LSV (PFS: 51.2% and OS: 72.5%) than in patients without LSV (PFS: 63.4% and OS: 100%).Next, we investigated the spleen signal change of patients who underwent WB-DWI twice or more during treatment (n=74). Of 42 out of the 74 patients with LSV at diagnosis, the spleen during treatment became visible on 31/42 (73.8%) patients. Representative patients with various spleen signal changes during treatment are shown in Figure 3 as group A (n=32; patients without LSV at diagnosis and during treatment), group B (n=31; patients who had LSV at diagnosis but the spleen reappeared after treatment), and group C (n=11; patients who had LSV at diagnosis, and despite treatment response, did not regain the spleen signal). Patients in group C showed significantly worse three-year PFS and OS (not available due to early events) than those in group A and B, even after excluding patients who did not achieve partial response or worse (n=11) (Fig1). In the multivariate analysis, the group C retained its prognostic significance for both PFS (hazard ratio [HR], 1.98, 95% confidence interval [CI] 1.00-3.90, p = 0.049) and OS (HR, 5.16, 95% CI 1.27-21.0, p = 0.022) even after adjustment for age over 70 years and the revised-ISS stage III.At last, to investigate the pathological cause of LSV, we reviewed two patients who underwent autopsies, who had both received WB-DWI within 3 months before their deaths (Fig1). One patient showed diffuse myeloma cell infiltration in the spleen and the other showed the amyloid deposition without myeloma cell infiltration.ConclusionThis study showed that LSV and a low SSR on pretreatment WB-DWI are correlated with a high tumor volume and poor prognosis. As patients with LSV during treatment had very poor prognosis, the relationships between LSV and other variables should be investigated. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.