Steady-state versus chemotherapy-based hematopoietic cell mobilization after anti-CD38-based induction therapy in newly diagnosed multiple myeloma.

BACKGROUND: The incorporation of anti-CD38 monoclonal antibodies (mAb) in induction regimens of newly diagnosed transplant-eligible multiple myeloma (MM) patients has been established as a new standard. However, the optimal strategy of stem cell mobilization in this context is not yet clear. STUDY DESIGN AND METHODS: From May 2020 till September 2022, we retrospectively reviewed patients receiving anti-CD38 mAb-based induction therapy followed by stem cell mobilization either in a steady-state protocol (SSM) using 10 µg/kg granulocyte colony-stimulating factor (G-CSF) for 5 days or in a chemotherapy-based protocol (CM) using 1-4 g/m2 cyclophosphamide and G-CSF. RESULTS: Overall, 85 patients (median age 61 years) were included in the analysis. In total, 90 mobilization attempts were performed, 42 with SSM and 48 with CM. There was no significant difference in the median concentration of CD34+ cells in peripheral blood (PB) prior to apheresis between SSM and CM (61/µL vs. 55.4/µL; p = .60). Cumulative CD34+ yields did not differ between the groups with median of 6.68 and 6.75 × 106 /kg body weight, respectively (p = .35). The target yield (≥4 × 106 CD34+ cells/kg body weight) was reached in 88% (CM) and 86% (SSM), with a high proportion even after a single apheresis session (76% vs. 75%). Plerixafor was found to be more frequently used in SSM (52%) than in CM (23%; p < .01). A total of 83 patients underwent autologous transplantation and all were engrafted. CONCLUSIONS: Stem cell collection in patients undergoing anti-CD38-based induction therapy is feasible with either CM or SSM, although SSM more frequently requires plerixafor.

Perturbations of mesenchymal stromal cells after allogeneic hematopoietic cell transplantation predispose for bone marrow graft-versus-host-disease.

Functional impairment of the bone marrow (BM) niche has been suggested as a major reason for prolonged cytopenia and secondary graft failure after allogeneic hematopoietic cell transplantation (alloHCT). Because mesenchymal stromal cells (MSCs) serve as multipotent progenitors for several niche components in the BM, they might play a key role in this process. We used collagenase digested trephine biopsies to directly quantify MSCs in 73 patients before (n = 18) and/or after alloHCT (n = 65). For the first time, we demonstrate that acute graft-versus-host disease (aGvHD, n = 39) is associated with a significant decrease in MSC numbers. MSC reduction can be observed even before the clinical onset of aGvHD (n = 10). Assessing MSCs instantly after biopsy collection revealed phenotypic and functional differences depending on the occurrence of aGvHD. These differences vanished during ex vivo expansion. The MSC endotypes observed revealed an enhanced population of donor-derived classical dendritic cells type 1 and alloreactive T cells as the causing agent for compartmental inflammation and MSC damage before clinical onset of aGvHD was ascertained. In conclusion, MSCs endotypes may constitute a predisposing conductor of alloreactivity after alloHCT preceding the clinical diagnosis of aGvHD.

Impact of IDH1 and IDH2 mutational subgroups in AML patients after allogeneic stem cell transplantation.

BACKGROUND: The role of allogeneic hematopoietic cell transplantation (alloHCT) in acute myeloid leukemia (AML) with mutated IDH1/2 has not been defined. Therefore, we analyzed a large cohort of 3234 AML patients in first complete remission (CR1) undergoing alloHCT or conventional chemo-consolidation and investigated outcome in respect to IDH1/2 mutational subgroups (IDH1 R132C, R132H and IDH2 R140Q, R172K). METHODS: Genomic DNA was extracted from bone marrow or peripheral blood samples at diagnosis and analyzed for IDH mutations with denaturing high-performance liquid chromatography, Sanger sequencing and targeted myeloid panel next-generation sequencing, respectively. Statistical as-treated analyses were performed using R and standard statistical methods (Kruskal-Wallis test for continuous variables, Chi-square test for categorical variables, Cox regression for univariate and multivariable models), incorporating alloHCT as a time-dependent covariate. RESULTS: Among 3234 patients achieving CR1, 7.8% harbored IDH1 mutations (36% R132C and 47% R132H) and 10.9% carried IDH2 mutations (77% R140Q and 19% R172K). 852 patients underwent alloHCT in CR1. Within the alloHCT group, 6.2% had an IDH1 mutation (43.4% R132C and 41.4% R132H) and 10% were characterized by an IDH2 mutation (71.8% R140Q and 24.7% R172K). Variants IDH1 R132C and IDH2 R172K showed a significant benefit from alloHCT for OS (p = .017 and p = .049) and RFS (HR = 0.42, p = .048 and p = .009) compared with chemotherapy only. AlloHCT in IDH2 R140Q mutated AML resulted in longer RFS (HR = 0.4, p = .002). CONCLUSION: In this large as-treated analysis, we showed that alloHCT is able to overcome the negative prognostic impact of certain IDH mutational subclasses in first-line consolidation treatment and could pending prognostic validation, provide prognostic value for AML risk stratification and therapeutic decision making.

Prevalence and variation of CHIP in patients with aggressive lymphomas undergoing CD19-directed CAR T-cell treatment.

Inflammation plays an important role in chimeric antigen receptor (CAR) T-cell therapy, especially in the pathophysiology of cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Clonal hematopoiesis of indetermined potential (CHIP) has also been associated with chronic inflammation. The relevance of CHIP in the context of CAR T-cell treatment is widely unknown. We evaluated the prevalence of CHIP, using a targeted deep sequencing approach, in a cohort of patients with relapsed/refractory (r/r) B-cell non-Hodgkin lymphoma before and after CAR T-cell treatment. The aim was to define the prevalence and variation of CHIP over time and to assess the influence on clinical inflammation syndromes (CRS/ICANS), cytopenia, and outcome. Overall, 32 patients were included. CHIP was found in 11 of 32 patients (34%) before CAR T-cell therapy. CHIP progression was commonly detected in the later course. Patients with CHIP showed a comparable response rate to CAR T-cell treatment but had an improved overall survival (not reached vs 265 days, P = .003). No significant difference was observed in terms of the occurrence and severity of CRS/ICANS, therapeutic use of tocilizumab and glucocorticosteroids, paraclinical markers of inflammation (with the exception of ferritin), or dynamics of hematopoietic recovery. CHIP is commonly observed in patients undergoing CD19-directed CAR T-cell therapy and is not associated with an inferior outcome.

Salvage treatment with plerixafor in poor mobilizing allogeneic stem cell donors: results of a prospective phase II-trial.

We conducted a prospective clinical trial to investigate the safety and efficacy of plerixafor (P) in allogeneic peripheral blood stem cells (PBSC) donors with poor mobilization response to standard-dose granulocyte colony-stimulating factor (G-CSF), defined by <2 × 106 CD34 + cells/kg recipient body-weight (CD34+/kg RBW) after 1st apheresis. A single dose of 240 µg/kg P was injected subcutaneously at 10 p.m. on the day of the 1st apheresis. Thirty-seven allogeneic PBSC donors underwent study treatment. The median CD34+ count in peripheral blood was 15/µl on Day 1 after G-CSF alone, versus 44/µl on Day 2 after G-CSF plus P (p < 0.001). The median yield of CD34+ cells was 1.1 × 108 on Day 1 and 2.8 × 108 on Day 2. In contrast to a median yield of only 1.31 × 106 CD CD34+/kg RBW on Day 1, triggering study inclusion, a median of 3.74 × 106 CD CD34+/kg RBW were collected with G-CSF plus P on Day 2. Of 37 donors, 21 reached the target cell count of >4.5 × 106 CD34+/kg RBW (57%, 95%CI 40-73%). No donor experienced a severe adverse event requiring treatment. In conclusion, P might be considered on a case-by-case basis for healthy allogeneic donors with very poor stem cell mobilization success after G-CSF.

Clostridium Difficile infections in patients with AML or MDS undergoing allogeneic hematopoietic stem cell transplantation identify high risk for adverse outcome.

Clostridium difficile (CD) infection is the main cause of nosocomial enterocolitis in western countries and in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHCT). Recipients of alloHCT are at high risk for CD infection but large studies in this population are rare and conflicting results have been reported. We analyzed 727 patients with AML or MDS undergoing alloHCT in our center from 2004 to 2015. Ninety-six patients (13%) had CD infection and 103 patients (14%) were identified as asymptomatic carriers by screening at admission and once a week during aplasia. Patients with CD infection had a shorter median overall survival of 8 months (95% CI, 6-36 months) compared with 25 months (95% CI, 17-35 months) for patients without CD infection, (HR 1.4, p = 0.04). CD positive patients were less likely to develop acute graft-versus-host disease (aGvHD; HR 0.6, p = 0.004) compared with CD-negative patients, but did not show differences in gastrointestinal aGvHD (HR 0.9, p = 0.5). Symptomatic patients developed gastrointestinal aGvHD (HR 2.5, p = 0.02) more often compared with asymptomatic CD positive patients. This analysis demonstrates a high prevalence for CD infection in patients undergoing alloHCT. A significant lower overall survival for patients with CD infection could be demonstrated.

Reliable isolation of human mesenchymal stromal cells from bone marrow biopsy specimens in patients after allogeneic hematopoietic cell transplantation.

Isolation of mesenchymal stromal cells (MSCs) from pretreated, hematologic patients is challenging. Especially after allogeneic hematopoietic cell transplantation (HCT), standard protocols using bone marrow aspirates fail to reliably recover sufficient cell numbers. Because MSCs are considered to contribute to processes that mainly affect the outcome after transplantation, such as an efficient lymphohematopoietic recovery, extent of graft-versus-host disease as well as the occurrence of leukemic relapse, it is of great clinical relevance to investigate MSC function in this context. Previous studies showed that MSCs can be isolated by collagenase digestion of large bone fragments of hematologically healthy patients undergoing hip replacement or knee surgeries. We have now further developed this procedure for the isolation of MSCs from hematologic patients after allogeneic HCT by using trephine biopsy specimens obtained during routine examinations. Comparison of aspirates and trephine biopsy specimens from patients after allogeneic HCT revealed a significantly higher frequency of clonogenic MSCs (colony-forming unit-fibroblast [CFU-F]) in trephine biopsy specimens (mean, 289.8 ± standard deviation 322.5 CFU-F colonies/1 × 106 total nucleated cells versus 4.2 ± 9.9; P < 0.0001). Subsequent expansion of functional MSCs isolated from trephine biopsy specimen was more robust and led to a significantly higher yield compared with control samples expanded from aspirates (median, 1.6 × 106; range, 0-2.3 × 107 P0 MSCs versus 5.4 × 104; range, 0-8.9 × 106; P < 0.0001). Using trephine biopsy specimens as MSC source facilitates the investigation of various clinical questions.

Differences in Cellular Composition of Peripheral Blood Stem Cell Grafts from Healthy Stem Cell Donors Mobilized with Either Granulocyte Colony-Stimulating Factor (G-CSF) Alone or G-CSF and Plerixafor.

This study was conducted to characterize and compare peripheral blood stem cell grafts from healthy donors who underwent granulocyte colony-stimulating factor (G-CSF) mobilization and subsequently received 1 dose of plerixafor after insufficient stem cell yields were achieved at the first apheresis. Aliquots from 35 donors were collected from the first apheresis after mobilization with G-CSF alone and from the second apheresis after additional plerixafor administration. Samples were freshly analyzed for cellular subsets by 8-color flow cytometry. Leukapheresis samples mobilized with additional plerixafor showed a significant increase of total nucleated cells, including B cells, CD4+ and CD8+ T cells, and CD34+ hematopoietic stem and progenitor cells. Absolute numbers of plasmacytoid dendritic cells were also significantly increased, whereas no changes were detected for myeloid dendritic cells. Furthermore, absolute numbers of regulatory T cells increased, with naive CD45RA+ regulatory T cells showing the highest rise. Finally, strikingly higher numbers of myeloid-derived suppressor cells were detected in the plerixafor and G-CSF-mobilized graft. The mobilization of peripheral stem cells in healthy donors with G-CSF and plerixafor led to a significant difference in cellular graft composition compared with G-CSF alone. The clinical impact of the different cell composition for the graft recipient warrants further clinical investigation.

Pilot Study on Mass Spectrometry-Based Analysis of the Proteome of CD34⁺CD123⁺ Progenitor Cells for the Identification of Potential Targets for Immunotherapy in Acute Myeloid Leukemia.

Targeting of leukemic stem cells with specific immunotherapy would be an ideal approach for the treatment of myeloid malignancies, but suitable epitopes are unknown. The comparative proteome-level characterization of hematopoietic stem and progenitor cells from healthy stem cell donors and patients with acute myeloid leukemia has the potential to reveal differentially expressed proteins which can be used as surface-markers or as proxies for affected molecular pathways. We employed mass spectrometry methods to analyze the proteome of the cytosolic and the membrane fraction of CD34 and CD123 co-expressing FACS-sorted leukemic progenitors from five patients with acute myeloid leukemia. As a reference, CD34⁺CD123⁺ normal hematopoietic progenitor cells from five healthy, granulocyte-colony stimulating factor (G-CSF) mobilized stem cell donors were analyzed. In this Tandem Mass Tag (TMT) 10-plex labelling-based approach, 2070 proteins were identified with 171 proteins differentially abundant in one or both cellular compartments. This proof-of-principle-study demonstrates the potential of mass spectrometry to detect differentially expressed proteins in two compartment fractions of the entire proteome of leukemic stem cells, compared to their non-malignant counterparts. This may contribute to future immunotherapeutic target discoveries and individualized AML patient characterization.

Arabidopsis EDS1 connects pathogen effector recognition to cell compartment-specific immune responses.

Pathogen effectors are intercepted by plant intracellular nucleotide binding-leucine-rich repeat (NB-LRR) receptors. However, processes linking receptor activation to downstream defenses remain obscure. Nucleo-cytoplasmic basal resistance regulator EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1) is indispensible for immunity mediated by TIR (Toll-interleukin-1 receptor)-NB-LRR receptors. We show that Arabidopsis EDS1 molecularly connects TIR-NB-LRR disease resistance protein RPS4 recognition of bacterial effector AvrRps4 to defense pathways. RPS4-EDS1 and AvrRps4-EDS1 complexes are detected inside nuclei of living tobacco cells after transient coexpression and in Arabidopsis soluble leaf extracts after resistance activation. Forced AvrRps4 localization to the host cytoplasm or nucleus reveals cell compartment-specific RPS4-EDS1 defense branches. Although nuclear processes restrict bacterial growth, programmed cell death and transcriptional resistance reinforcement require nucleo-cytoplasmic coordination. Thus, EDS1 behaves as an effector target and activated TIR-NB-LRR signal transducer for defenses across cell compartments.

SUMO modification of PCNA is controlled by DNA.

Post-translational modification by the ubiquitin-like protein SUMO is often regulated by cellular signals that restrict the modification to appropriate situations. Nevertheless, many SUMO-specific ligases do not exhibit much target specificity, and--compared with the diversity of sumoylation substrates--their number is limited. This raises the question of how SUMO conjugation is controlled in vivo. We report here an unexpected mechanism by which sumoylation of the replication clamp protein, PCNA, from budding yeast is effectively coupled to S phase. We find that loading of PCNA onto DNA is a prerequisite for sumoylation in vivo and greatly stimulates modification in vitro. To our surprise, however, DNA binding by the ligase Siz1, responsible for PCNA sumoylation, is not strictly required. Instead, the stimulatory effect of DNA on conjugation is mainly attributable to DNA binding of PCNA itself. These findings imply a change in the properties of PCNA upon loading that enhances its capacity to be sumoylated.