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Gemtuzumab ozogamicin (GO), a CD33-targeting antibody drug conjugate, previously showed longer relapse-free survival when combined with induction chemotherapy in patients with favorable-risk acute myeloid leukemia (AML). In this patient population, characterized by lower relapse risk as compared to other ELN risk groups, autologous stem cell transplantation (ASCT) can be used as consolidation strategy. However, there are limited data on the impact of GO on the peripheral blood stem cell (PBSC) mobilization potential. We therefore retrospectively analyzed data from 54 AML patients with favorable-risk AML treated with ( n = 17) or without ( n = 37) GO during induction treatment. We observed no significant differences in the PBSC mobilization rate between patients treated with vs. without GO. The mobilization success in a first attempt directly following cycle 2 was 65% vs. 70% ( p = 0.92); and the mobilization success in a subsequent second attempt after hematologic recovery and repeated stimulation procedure was 24% vs. 19% ( p = 0.56). No significant impact on treatment outcome in terms of EFS ( p = 0.31) or OS ( p = 0.99) was observed. Thus, our results suggest that the addition of GO to induction regimens does not negatively impact PBSC mobilization in favorable-risk AML patients. To our best knowledge, this is the first study comparing the stem cell mobilization potential in favorable-risk AML patients treated with vs. without GO.

Blood tests are widely used in modern medicine to diagnose certain illnesses and evaluate the overall health of a patient. To enable testing in resource-limited areas, there has been increasing interest in point-of-care (PoC) testing devices. To process blood samples, liquid mixing with active pumps is usually required, making PoC blood testing expensive and bulky. We explored the possibility of processing approximately 2 μL of whole blood for image flow cytometry using capillary structures that allowed test times of a few minutes without active pumps. Capillary pump structures with five different pillar shapes were simulated using Ansys Fluent to determine which resulted in the fastest whole blood uptake. The simulation results showed a strong influence of the capillary pump pillar shape on the chip filling time. Long and thin structures with a high aspect ratio exhibited faster filling times. Microfluidic chips using the simulated pump design with the most efficient blood uptake were fabricated with polydimethylsiloxane (PDMS) and polyethylene oxide (PEO). The chip filling times were tested with 2 μL of both water and whole blood, resulting in uptake times of 24 s for water and 111 s for blood. The simulated blood plasma results deviated from the experimental filling times by about 35% without accounting for any cell-induced effects. By comparing the flow speed induced by different pump pillar geometries, this study offers insights for the design and optimization of passive microfluidic devices for inhomogenous liquids such as whole blood in sensing applications.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN), a rare malignancy derived from plasmacytoid dendritic cells, can mimic both acute leukemia and aggressive T-cell lymphoma. Therapy of this highly aggressive hematological disease should be initiated as soon as possible, especially in light of novel targeted therapies that have become available. However, differential diagnosis of BPDCN remains challenging. This retrospective study aimed to highlight the challenges to timely diagnoses of BPDCN. We documented the diagnostic and clinical features of 43 BPDCN patients diagnosed at five academic hospitals from 2001-2022. The frequency of BPDCN diagnosis compared to AML was 1:197 cases. The median interval from the first documented clinical manifestation to diagnosis of BPDCN was 3 months. Skin (65%) followed by bone marrow (51%) and blood (45%) involvement represented the most common sites. Immunophenotyping revealed CD4 + , CD45 + , CD56 + , CD123 + , HLA-DR + , and TCL-1 + as the most common surface markers. Overall, 86% (e.g. CD33) and 83% (e.g., CD7) showed co-expression of myeloid and T-cell markers, respectively. In the median, we detected five genomic alterations per case including mutational subtypes typically involved in AML: DNA methylation (70%), signal transduction (46%), splicing factors (38%), chromatin modification (32%), transcription factors (32%), and RAS pathway (30%), respectively. The contribution of patients (30%) proceeding to any form of upfront stem cell transplantation (SCT; autologous or allogeneic) was almost equal resulting in beneficial overall survival rates in those undergoing allogeneic SCT (p = 0.0001). BPDCN is a rare and challenging entity sharing various typical characteristics of other hematological diseases. Comprehensive diagnostics should be initiated timely to ensure appropriate treatment strategies., (© 2024. The Author(s).)

(1) Background: High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT) is the standard consolidation strategy for patients with newly diagnosed multiple myeloma (MM) and for a subset of patients with relapsed/refractory disease. For stem cell mobilization, G-CSF alone or in combination with chemotherapy mobilizing agents and/or plerixafor are commonly used. Ixazomib is an oral proteasome inhibitor with less neurotoxic potential, which previously showed the ability to mobilize stem cells in preclinical studies. (2) Methods: Prospective single-center phase 1 study assessing the efficacy and safety of stem cell mobilization with ixazomib and G-CSF in patients with newly diagnosed or relapsed/refractory MM undergoing HDCT and ASCT. Primary endpoint was percentage of patients achieving a yield of at least 6.0 × 10 6 /kg CD34+ cells within the first apheresis. G-CSF (filgrastim) 10 μg/kg/day was administered subcutaneously (s.c.) from day 1 to day 5 (planned apheresis) and ixazomib 4 mg orally at day 4. Plerixafor 24 mg s.c. was administered if the stem cell mobilization with ixazomib and G-CSF was not sufficient. (3) Results: 19 patients were treated within the study between 06/2020 and 02/2021. The primary endpoint was reached in 17 (89%) patients, with a median of 7.1 × 10 6 /kg CD34+ cells collected within the first apheresis, comparable to previously published results, and only 2 (11%) patients required a second apheresis. Median number of circulating CD34+ cells was 14.0 × 10 6 /L (2.0-95.2) before the administration of ixazomib, and 33.0 × 10 6 /L (4.2-177.0) pre-apheresis. However, 9 (47%) patients required the addition of plerixafor to ensure optimal stem cell collection. (4) Conclusions: The combination of ixazomib and G-CSF showed promising stem cell mobilizing activity in patients with MM prior to HDCT and ASCT. Future larger studies might further investigate the role of ixazomib in stem cell mobilization regimens for MM.

Background: Next-generation sequencing (NGS) detects somatic mutations in a high proportion of plasma cell dyscrasias (PCD), but is currently not integrated into diagnostic routine. We correlated NGS data with degree of bone marrow (BM) involvement by cytomorphology (BMC), histopathology (BMH), and multiparameter flow cytometry (MFC) in 90 PCD patients., Methods: Of the 90 patients the diagnoses comprised multiple myeloma (n = 77), MGUS (n = 7), AL-amyloidosis (n = 4) or solitary plasmocytoma (n = 2). The NGS panel included eight genes CCND1, DIS3, EGR1, FAM46C (TENT5C), FGFR3, PRDM1, TP53, TRAF3, and seven hotspots in BRAF, IDH1, IDH2, IRF4, KRAS, NRAS., Results: Mutations were detected in 64/90 (71%) of cases. KRAS (29%), NRAS (16%) and DIS3 (16%) were most frequently mutated. At least one mutation/sample corresponded to a higher degree of BM involvement with a mean of 11% pathologic PC by MFC (range, 0.002-62%), and ~ 50% (3-100%) as defined by both BMC and BMH., Conclusions: The probability of detecting a mutation by NGS in the BM was highest in samples with > 10% clonal PC by MFC, or > 20% PC by BMC/ BMH. We propose further evaluation of these thresholds as a practical cut-off for processing of samples by NGS at initial PCD diagnosis., (© 2022. The Author(s).)

Mantle cell lymphoma (MCL) is a rare type of B-cell Non-Hodgkin lymphoma (NHL) affecting predominantly male patients. While complete remissions following first-line treatment are frequent, most patients ultimately relapse, with a usually aggressive further disease course. The use of cytarabine-comprising induction chemotherapy and autologous stem cell transplantation, Rituximab maintenance, Bruton's tyrosine kinase (BTK) inhibitors and CAR T therapy has substantially improved survival. Still, options for patients relapsing after CAR T therapy are limited and recommendations for the treatment of these patients are lacking. We report two cases of patients with mantle cell lymphoma who relapsed after CAR T therapy and were treated with the bispecific CD20/CD3 T cell engaging antibody glofitamab. Both patients showed marked increases of circulating CAR T cells and objective responses after glofitamab administration. Therapy was tolerated without relevant side effects in both patients. One patient completed all 12 planned cycles of glofitamab therapy and was alive and without clinical progression at the last follow-up. The second patient declined further treatment after the first cycle and succumbed to disease progression. We review the literature and investigate possible mechanisms involved in the observed responses after administration of glofitamab, such as proliferation of CAR T cells, anti-tumor effects of the bispecific antibody and the role of other possibly contributing factors. Therapy with bispecific antibodies might offer an effective and well-tolerated option for patients with mantle cell lymphoma relapsing after CAR T therapy.

Definite cure remains exceptional in myeloma patients even after high-dose chemotherapy (HDCT) with melphalan (Mel) and autologous stem cell transplantation (ASCT). Thus, improving efficacy of HDCT in MM remains an unresolved issue. This randomized phase II trial compared standard 200 mg/m 2 Mel HDCT to experimental HDCT with 200 mg/m 2 bendamustine, given both at days -4 and -3, combined with 100 mg/m 2 melphalan at days -2 and -1 (BenMel) before ASCT as first-line consolidation in myeloma patients. The primary endpoint aimed to identify at least a 15% improvement in the complete remission rate (stringent CR + CR) after HDCT with BenMel compared with Mel alone. A total of 120 MM patients were 1:1 randomized. The rate of sCR/CR after ASCT was higher in BenMel than in Mel treated patients (70.0% vs. 51.7%; p = 0.039). Three patients in the BenMel group (5.0%) had reversible acute renal insufficiency compared with none in Mel patients. Minimal residual disease negativity (<10-5) by flow cytometry was observed in 26 (45.6%) BenMel patients and 22 (37.9%) in the Mel group (p = 0.375). Our data suggest that BenMel HDCT is safe and improves the sCR/CR rate compared with standard Mel alone., (© 2022. The Author(s).)

Autologous hematopoietic cell transplantation (HCT) is suitable for consolidation of favorable-/intermediate-risk AML patients in CR1. However, ~50% of AML patients relapse after autologous HCT, and efficacy of subsequent salvage strategies including allogeneic HCT remains unclear. We studied 123 consecutive patients with newly diagnosed AML undergoing high-dose chemotherapy (HDCT)/autologous HCT in CR1. In relapsing patients afterwards, we analyzed salvage treatments and outcomes focusing particularly on salvage allogeneic HCT. Of 123 patients, 64 (52%) relapsed after autologous HCT. Subsequently, 13 (21%) received palliative therapy, whereas 51 (79%) proceeded to salvage therapy with a curative intent. Of the 47 patients with a curative intent and who did not proceed directly to allogeneic HCT, 23 (49%) achieved CR2 or had ongoing hematologic CR1 despite molecular relapse. Finally, 30 patients (47%) received allogeneic HCT with estimated 3-year leukemia-free and overall survival rates of 33% and 43%. Hematologic remission at allogeneic HCT and lack of acute GvHD had a positive impact on OS and LFS (p < 0.05). Our study suggests that almost 80% of AML patients can undergo salvage therapy following relapse after front-line HDCT/autologous HCT. Allogeneic HCT can provide cure in one third of patients relapsing after front-line HDCT/autologous HCT., (© 2021. The Author(s).)

Background: Intraocular lymphoma (IOL) presents a real challenge in daily diagnostics. Cyto- and/or histopathology of vitreous body represent the diagnostic cornerstones. Yet, false negative results remain common. Therefore, we analyzed the diagnostic significance of flow cytometry (FC) within the workup algorithm of IOL and compared its sensitivity with the results obtained from routine cytopathology and molecular genetics; Methods: Seven patients undergoing vitrectomy due to suspected IOL were investigated by FC and parallel cytopathology and, if available, digital droplet PCR (ddPCR) for MYD88 L265P; Results: Four out of seven patients were finally diagnosed with IOL. Among the IOL patients, cytopathology confirmed the presence of lymphoma cells in only two cases. In contrast, FC was positive for IOL in all four cases, and FC additionally confirmed the lack of IOL in the remaining patients. In IOL patients diagnosed by FC and with available ddPCR, the diagnosis of IOL was confirmed by the presence of the MYD88 L265P mutation in all three patients; Conclusions: The combination with FC was superior to cytopathology alone in the diagnostic work-up of IOL, and it showed an excellent correlation with ddPCR results. A comprehensive diagnostic panel consisting of cytopathology, FC and molecular genetics should be considered for the work-up of suspected IOL.

(1) Background: CAR-T-cell therapy is a novel therapeutic option for patients with relapsed/refractory diffuse large B-cell lymphoma ( r / r DLBCL). The parameters that predict a favorable outcome after CAR-T-cell treatment are a matter of ongoing exploration. (2) Methods: We analyzed 36 consecutive patients with r / r DLBCL receiving tisagenlecleucel or axicabtagene ciloleucel at a single academic institution. We hypothesized that lymphoma subtypes (transformed versus de novo DLBCL) are of prognostic importance. We also assessed age, previous treatment, bridging therapy, remission status at the time of CAR-T treatment and at six months, LDH, the occurrence of CRS or ICANS, and CAR-T-DNA ddPCR kinetics for their prognostic impact. (3) Results: CRS was observed in 24 (67%) patients, and ICANS was observed in 14 (39%) patients. CR was achieved in 20 (56%) patients. Achievement of CR within six months after CAR-T was associated with better PFS ( p < 0.0001) and OS ( p < 0.0001). Remarkably, transformed (=secondary) lymphoma was associated with a better outcome than de novo disease for PFS ( p = 0.0093) and OS ( p = 0.0209), and the CR rate was 78% versus 33% ( p = 0.0176). Mortality in patients with transformed DLBCL was 23% compared with 56% in de novo patients ( p = 0.0209). (4) Conclusion: The presence of transformed DLBCL seems to be associated with a more favorable course after CAR-T treatment than that observed in the de novo DLBCL patients.

Engraftment syndrome (ES) following autologous stem cell transplantation (ASCT) at the time of neutrophil recovery may comprise fever, rash, pulmonary edema, or diarrhea. Usually, ES is easily manageable using corticosteroids but may prolong hospitalization. In two consecutive cohorts of subsequent patients with myeloma, lymphomas, and testicular/germ cell cancer, we assessed the benefit of corticosteroid use to prevent incidence and severity of ES following ASCT. Whereas Cohort A (82 patients) received no prophylactic corticosteroids, corticosteroids (4 mg dexamethasone oral daily) were started in Cohort B (60 patients) at day +9 until day +13 following ASCT. Steroid prophylaxis significantly reduced the incidence of ES (6/60; 10% vs. 33/82; 40%; p < 0.001). Hospitalization duration was longer in patients with ES than in patients without ES within both cohorts (in Cohort A: p = 0.007; and B: p = 0.011), but did not differ significantly between cohorts A and B. Finally, in Cohort A, there was a trend to an inferior 2-year overall survival rate in patients without ES compared to patients with ES (p = 0.067), but definite conclusions are not yet allowed. Our results suggest that corticosteroid prophylaxis from days +9 to +13 following ASCT significantly reduces the risk of ES and shortens hospitalization duration., (© 2020 John Wiley & Sons Ltd.)

Relapse of acute myeloid leukemia (AML) remains a major determinant of outcome. A number of molecularly directed treatment options have recently emerged making comprehensive diagnostics an important pillar of clinical decision making at relapse. Acknowledging the high degree of individual genetic variability at AML relapse, next-generation sequencing (NGS) has opened the opportunity for assessing the unique clonal hierarchy of individual AML patients. Knowledge on the genetic makeup of AML is reflected in patient customized treatment strategies thereby providing improved outcomes. For example, the emergence of druggable mutations at relapse enable the use of novel targeted therapies, including FLT3 inhibitors or the recently approved IDH1/2 inhibitors ivosidenib and enasidenib, respectively. Consequently, some patients may undergo novel bridging approaches for reinduction before allogeneic stem cell transplantation, or the identification of an adverse prognostic marker may initiate early donor search. In this review, we summarize the current knowledge of NGS in identifying clonal stability, clonal evolution, and clonal devolution in the context of AML relapse. In light of recent improvements in AML treatment options, NGS-based molecular diagnostics emerges as the basis for molecularly directed treatment decisions in patients at relapse., (© 2020 John Wiley & Sons Ltd.)

Competing Interests: Competing interests: The authors declare no conflict of Interest.

Introduction: Chimeric antigen receptor T-cell (CAR-T) therapies are increasingly used to treat relapsed B-cell lymphomas and acute lymphoblastic leukemia. Considering the frequency of cytokine release syndrome and CAR-T-related encephalopathy syndrome (CRS/CRES) after CAR-T administration, strategies enabling timely prediction of impending CRS/CRES are a clinical need., Methods: We evaluated the dynamics of serum interleukin (IL)-6 levels and CAR-T transgene copy numbers by digital droplet polymerase chain reaction in the peripheral blood of 11 consecutive patients with aggressive B-cell malignancies., Results: Four of 11 patients developed CRS, and 3 patients had CRES (33%), 2 of them had previous CRS. IL-6 levels increased on the day of clinical manifestation of CRS. All CRS patients had increased IL-6 peak levels (median IL-6 peak 606 pg/mL in CRS patients vs. 22 pg/mL in non-CRS patients, p = 0.0061). Different patterns emerged from the dynamics of CAR-T/µg genomic DNA: "rapid increase and rapid decrease with complete disappearance," "rapid increase and slow decrease with higher persistence," "rapid increase and rapid decrease with lower persistence," and "slow increase and rapid decrease with almost disappearance." Patients with the pattern "rapid increase and slow decrease with higher persistence" of CAR-T/µg genomic DNA concentration seemed to be at higher risk of developing CRS/CRES., Conclusion: Thus, the dynamics of CAR-T transgene copy numbers merits further evaluation for a possible association with manifestation of CRS. Increased IL-6 serum levels at CRS manifestation may contribute to the interpretation of symptoms., Competing Interests: Conflict of interest disclosure The authors declare no conflicts of interest., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Reciprocal RUNX1 fusions are traditionally found in up to 10% of acute myeloid leukemia (AML) patients, usually associated with a translocation (8;21)(q22;q22) corresponding to the RUNX1-RUNX1T1 fusion gene. So far, alternative RUNX1 rearrangements have been reported only rarely in AML, and the few reports so far have focused on results based on cytogenetics, fluorescence in situ hybridization, and polymerase chain reaction. Acknowledging the inherent limitations of these diagnostic techniques, the true incidence of rare RUNX1 rearrangements may be underestimated. In this report, we present two cases of adult AML, in which we detected rare RUNX1 rearrangements not by conventional cytogenetics but rather by next-generation panel sequencing. These include t(16;21)(q24;q22)/RUNX1-CBFA2T3 and t(7;21)(p22;q22)/RUNX1-USP42, respectively. In both patients the AML was therapy-related and associated with additional structural and numerical alterations thereby conferring bad prognosis. This is in line with previous reports on rare RUNX1 fusions in AML and emphasizes the clinical importance of their detection. In summary, our report not only confirms the clinical utility of NGS for diagnostics of rare reciprocal rearrangements in AML in a real-life scenario but also sheds light on the variety and complexity within AML. It further emphasizes the need for collection of additional cases for deepening insights on their clinical meaning as well as their frequency., (© 2019 Wiley Periodicals, Inc.)