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.

Analysis of stem cell collections in adult patients with Ewing sarcoma.

BACKGROUND: Ewing sarcoma is one of the most frequent soft-tissue tumors in pediatric patients. The current treatment protocols recommend stem cell apheresis (SCA) after completion of the second course of induction therapy with vincristine, ifosfamide, doxorubicine, and etoposide (VIDE). The feasibility of SCA and graft compositions in adult patients with Ewing sarcoma have not been previously analyzed. METHODS AND MATERIALS: The authors analyzed 29 stem cell collections of 19 adult patients (9 male, 10 female) at a median age of 27 (range 19-53) years mobilized after VIDE (n = 17), cyclophosphamide/topotecan (n = 1) or vincristine, dactinomycin and ifosfamide (n = 1) chemotherapy. All patients were mobilized with filgrastim 5 µg/kg twice daily from day +7 of chemotherapy. The collections were performed if CD34+ cell count in peripheral blood was >10/µL. The target yields were ≥4×106 CD34+ cells/kg body weight. RESULTS: Median CD34+ cells/µL in peripheral blood before SCA were 45.8 (range 6.7-614.4)/µL. The median cumulative yields were 10.6 (range 1.5-38.8) CD34+ cells/kg body weight and ≥2×106 in all but two patients (89%). CD34, CD3, and CD56 yields in collections after the third VIDE and after later courses did not differ. Four patients underwent high-dose therapy with autologous transplantation, and all were engrafted. DISCUSSION: Stem cell mobilization is feasible in most Ewing sarcoma patients. Additionally, the present study's data suggest that it is safe to postpone stem cell collection to a later VIDE chemotherapy cycle if medically indicated.

Stem-cell mobilization of healthy sibling donors with pegfilgrastim-A prospective open-label phase II trial (EudraCT no: 2005-004971-39).

BACKGROUND: Pegfilgrastim is a covalently bound conjugate of filgrastim and mono-methoxypolyethylene glycol with a longer half-life. STUDY DESIGN AND METHODS: We report on phase II prospective monocentric trial examining the feasibility of stem cell mobilization with 12 mg single dose pegfilgrastim in related donors. The objectives were to determine the optimal collection day, defined as CD34+ concentration in peripheral blood (PB) >50 cells/µl, the number of donors collected with single leukapheresis, and the peak level of pegfilgrastim in donor-serum. Furthermore, the cell composition of grafts was assessed and compared to published data. RESULTS: The results included about 28 matched related donors. The median pegfilgrastim serum level remained >200 ng/mL for 48 hours before declining, with the maximal measured concentration of 259.49 ng/ml 24 h after application. The median white blood cell count and CD34 count in PB peaked on day four with 52.6 (range 22.8-85.0) Gpt/l and 66.25 (range 22.9-136.6) cells/µl, respectively. A CD34+ count >50 cells/µl on day four was detected in 75% of donors. 79% of the donors underwent a single collection. Conventional filgrastim was administered additionally in two donors, due to insufficient CD 34+ concentration in PB. 89% of donors showed CD34+ yields ≥4 (median 6.5, range 4.6-14.5) × 10/kg body weight of the recipient. All grafts were administered without rejections. DISCUSSION: The results of this trial showed that stem cell mobilization with pegfilgrastim is a feasible, and attractive option. This is the first trial presenting the kinetics of pegfilgrastim serum levels in healthy donors.

Use of Minimal Residual Disease in Acute Myeloid Leukemia Therapy.

OPINION STATEMENT: The expanding availability of minimal or more precisely measurable residual disease (MRD) assessment in acute myeloid leukemia (AML) with its possible implications for therapeutic decisions is of high interest to clinicians treating AML patients. A variety of mostly retrospective studies have shown that AML patients with a positive MRD test, assessed by different techniques at defined cutoffs and time-points, are at significantly higher risk of relapse and experience shorter overall survival compared to MRD-negative patients. How this valuable information may be adapted in the daily routine of patients' treatment to distinguish individuals who need more aggressive therapy from the ones who can be spared additional therapy to avoid treatment-related toxicities is still being investigated. With the exception of MRD analyses in acute promyelocitic leukemia (APL), the clinical implications of MRD tests for the individual AML patient are still mostly unknown. We currently lack hard evidence that MRD-based therapy modulation during treatment or pre-emptive intervention in MRD-positive patients after therapy would improve outcomes in non-APL AML patients. These questions will be evaluated in prospective randomized clinical trials. Today, however, some conclusions with regard to MRD assessment in AML can be drawn from the published data and are reviewed in this article.

Impact of Previous Allogeneic Hematopoietic Stem Cell Transplantation on Chimeric Antigen Receptor (CAR) T Cell Treatment for Relapsed/Refractory Multiple Myeloma.

BACKGROUND: Anti-BCMA-directed chimeric antigen receptor (CAR) T cells are effective treatment for patients with refractory/relapsed multiple myeloma (RRMM). However, little is known about the impact of previous allogeneic hematopoietic stem cell transplantation (allo-HSCT) on lymphocyte collection for production of CAR T cells and subsequent treatment with CAR T cells. PATIENTS AND METHODS: We performed a retrospective analysis of cellular composition of lymphocyte collections, CAR T cell expansion and treatment outcomes of RRMM patients undergoing therapy with idecabtagene vicleucel (ide-cel) with and without history of allo-HSCT. 27 patients (11/27 female) with median age 63 (range 39-75) years were analyzed. Five patients (19%) had the history of allo-HSCT median of 5.5 years before ide-cel. RESULTS: Prior to apheresis, the white blood cell, absolute lymphocyte counts, CD3+ cells and monocytes did not differ in patients with and without prior allo-HSCT. We also noticed no differences in the collected CD3+ yields or cellular compositions of lymphocyte collections. One year after ide-cel infusion, the progression-free survival and overall survival of patients with and without previous allo-HSCT did not differ with 60% and 45% respectively (P = .58) and 66.7% and 74% respectively (P = .84). The highest expansion of CAR T was detected between day 7 after infusion and showed no difference regarding previous allo-HSCT (P = .71). No graft-versus-host disease during the follow-up was detected. CONCLUSION: Our data confirm that the treatment with ide-cel is feasible for patients with prior allo-HSCT. Furthermore, allo-HSCT did not influence cellular composition of lymphocyte collections, clinical outcome or in vivo expansion of ide-cel.

Cellular dynamics following CAR T cell therapy are associated with response and toxicity in relapsed/refractory myeloma.

B-cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T cells revolutionized the treatment of relapsed/refractory multiple myeloma (RRMM). However, data on cellular (CAR) T cell dynamics and the association with response, resistance or the occurrence of cytokine release syndrome (CRS) are limited. Therefore, we performed a comprehensive flow cytometry analysis of 27 RRMM patients treated with Idecabtagene vicleucel (Ide-cel) to assess the expansion capacity, persistence and effects on bystander cells of BCMA-targeting CAR T cells. Additionally, we addressed side effects, like cytokine release syndrome (CRS) and cytopenia. Our results show that in vivo expansion of CD8+ CAR T cells is correlated to response, however persistence is not essential for durable remission in RRMM patients. In addition, our data provide evidence, that an increased fraction of CD8+ T cells at day of leukapheresis in combination with successful lymphodepletion positively influence the outcome. We show that patients at risk for higher-grade CRS can be identified already prior to lymphodepletion. Our extensive characterization contributes to a better understanding of the dynamics and effects of BCMA-targeting CAR T cells, in order to predict the response of individual patients as well as side effects, which can be counteracted at an early stage or even prevented.

Single-cell multiomic dissection of response and resistance to chimeric antigen receptor T cells against BCMA in relapsed multiple myeloma.

Markers that predict response and resistance to chimeric antigen receptor (CAR) T cells in relapsed/refractory multiple myeloma are currently missing. We subjected mononuclear cells isolated from peripheral blood and bone marrow before and after the application of approved B cell maturation antigen-directed CAR T cells to single-cell multiomic analyses to identify markers associated with resistance and early relapse. Differences between responders and nonresponders were identified at the time of leukapheresis. Nonresponders showed an immunosuppressive microenvironment characterized by increased numbers of monocytes expressing the immune checkpoint molecule CD39 and suppressed CD8+ T cell and natural killer cell function. Analysis of CAR T cells showed cytotoxic and exhausted phenotypes in hyperexpanded clones compared to low/intermediate expanded clones. We identified potential immunotherapy targets on CAR T cells, like PD1, to improve their functionality and durability. Our work provides evidence that an immunosuppressive microenvironment causes resistance to CAR T cell therapies in multiple myeloma.

Two rare cases of bronchus-associated lymphoid tissue lymphoma successfully treated with rituximab-bendamustine.

BALT lymphoma is a rare B-NHL with a favorable prognosis. We here report on two patients with nonspecific symptoms: one showed as major symptom severe thrombocytopenia and the other dyspnea and dry cough, thereby suggesting an inflammatory focus in the lungs. There is no standard of care established yet. Both patients were successfully treated with rituximab and bendamustine. Thus, combined immunochemotherapy should be considered as first-line therapy as in other MALT lymphomas, if the treatment/eradication of an underlying chronic inflammatory disorder/trigger factor can be excluded.

Salvage Therapy With Polatuzumab Vedotin, Bendamustine, and Rituximab Prior to Allogeneic Hematopoietic Transplantation in Patients With Aggressive Lymphomas Relapsing After Therapy With Chimeric Antigen Receptor T-Cells-Report on Two Cases.

Up to 60% of patients with aggressive B-cell lymphoma who receive chimeric antigen receptor (CAR) T-cell therapy experience treatment failure and subsequently have a poor prognosis. Allogeneic hematopoietic stem cell transplantation (alloHSCT) remains a potentially curative approach for patients in this situation. Induction of a deep response prior to alloHSCT is crucial for long-term outcomes, but the optimal bridging strategy following relapse after CAR T-cell therapy has not yet been established. Polatuzumab vedotin, an antibody drug conjugate targeting CD79b, is a novel treatment option for use in combination with rituximab and bendamustine (Pola-BR) in relapsed or refractory disease. Patients: We report two heavily pretreated patients with primary refractory diffuse large B-cell lymphoma (DLBCL) and primary mediastinal B-cell lymphoma (PMBCL) respectively who relapsed after therapy with CAR T-cells with both nodal and extranodal manifestations of the disease. After application of three courses of Pola-BR both patients achieved a complete metabolic remission. Both patients underwent alloHSCT from a human leukocyte antigen (HLA)-mismatched donor following conditioning with busulfan and fludarabine and are disease free 362 days and 195 days after alloHSCT respectively. We conclude that Pola-BR can be an effective bridging therapy before alloHSCT of patients relapsing after CAR T-cell therapy. Further studies will be necessary to define the depth and durability of remission of this salvage regimen before alloHSCT.

Case Report: Large Granular Lymphocyte Leukemia (LGLL)-A Case Series of Challenging Presentations.

Large granular lymphocyte leukemia (LGLL) represents a rare group of diseases with considerable difficulties in their correct diagnostic workup and therapy. The major challenges lie in their distinction from reactive (including autoimmune) lymphoproliferations. Moreover, monoclonal LGL proliferative diseases are in fact a heterogeneous group of disorders, as recognized by the three subtypes in the current WHO classification. It distinguishes two chronic forms (the focus of this case series), namely T-LGLL and chronic lymphoproliferative disorders of Natural Killer cells (CLPD-NK) as well as aggressive NK-cell leukemia. In the clinical routine, the variable presentations and phenotypes of T-LGLL and CLPD-NK are underappreciated. The relevant differential diagnoses range from benign reactive T-cell expansions to other mature T-cell leukemias to highly aggressive γδ-lymphomas. T-LGLL or CLPD-NK patients suffer from a wide variety of symptoms often including, but not limited to, cytopenias or classical autoimmune phenomena. They receive treatments ranging from mere supportive measures (e.g. antibiotics, growth factors, transfusions) over strategies of immunosuppression up to anti-leukemic therapies. The diagnostic pitfalls range from recognition of the subtle T-cell proliferation, repeated establishment of monoclonality, assignment to a descript immunophenotypic pattern, and interpretations of molecular aberrancies. Here, we report a series of selected cases to represent the spectrum of LGLL. The purpose is to raise awareness among the scientifically or practically interested readers of the wide variety of clinical, immunological, and phenotypic features of the various forms of LGLL, e.g. of T-cell type, including its γδ forms or those of NK-lineage. We highlight the characteristics and courses of four unique cases from two academic centers, including those from a prospective nationwide LGLL registry. Each case of this instructive catalogue serves to transport a key message from the areas of (chronic inflammatory) contexts in which LGLL can arise as well as from the fields of differential diagnostics and of various treatment options. Implications for optimization in these areas are discussed.

Case Report: Allogeneic Stem Cell Transplantation Following Induction With CPX-351 in Patients With Acute Myeloid Leukemia Is Feasible.

Acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) and treatment-related acute myeloid leukemia (tAML) after chemotherapy or radiation therapy for other neoplasms are associated with poor outcomes. CPX-351, a dual-drug liposomal encapsulation of daunorubicin and cytarabine, has been shown to improve outcomes in AML-MRC and tAML compared with standard 7+3 regimens. Here we report the cases of four consecutive patients with AML-MRC or tAML who received CPX-351 as outpatient induction therapy immediately followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT). Two patients received allo-HSCT in remission (one in complete remission and one in partial remission) and two patients received allo-HSCT in aplasia (one at 11 days and one at 52 days after the start of induction therapy with CPX-351). With a median follow-up of 188 days after allo-HSCT, all but one patient are alive and two are in remission. Further studies will help define and expand the role of CPX-351 in the treatment of AML-MRC and tAML, especially in patients expected to undergo allo-HSCT.

Clinical Challenges and Consequences of Measurable Residual Disease in Non-APL Acute Myeloid Leukemia.

The ability to detect residual levels of leukemic blasts (measurable residual disease, MRD) has already been integrated in the daily routine for treatment of patients with chronic myeloid and acute lymphoblastic leukemia. In acute myeloid leukemia (AML), a variety of mostly retrospective studies have shown that individuals in AML remission who tested positive for MRD at specific time-points or had increasing MRD levels are at significantly higher risk of relapse and death compared to MRD-negative patients. However, these studies differ with respect to the "MRD-target", time-point of MRD determination, material analyzed, and method applied. How this probably very valuable MRD information in individual patients may be adapted in the daily clinical routine, e.g., to separate patients who need more aggressive therapies from those who may be spared additional-potentially toxic-therapies is still a work-in-progress. With the exception of MRD assessment in acute promyelocytic leukemia (APL), the lack of randomized, prospective trials renders MRD-based decisions and clinical implications in AML a difficult task. As of today, we still do not have proof that early intervention in MRD-positive AML patients would improve outcomes, although this is very likely. In this article, we review the current knowledge on non-APL AML MRD assessment and possible clinical consequences.

Elucidating functional heterogeneity in hematopoietic progenitor cells: a combined experimental and modeling approach.

A detailed understanding of the mechanisms maintaining the hierarchical balance of cell types in hematopoiesis will be important for the therapeutic manipulation of normal and leukemic cells. Mathematical modeling is expected to make an important contribution to this area, but the iterative development of increasingly accurate models will rely on repeated validation using experimental data of sufficient resolution to distinguish between alternative model scenarios. The multipotent hematopoietic progenitor FDCP-Mix cells maintain a hierarchy from self-renewal to post-mitotic differentiation in vitro and are accessible to detailed analysis. Here, we report the development of a combined mathematical modeling and experimental approach to study the principles underlying heterogeneity in FDCP-Mix cultures. We adapt a single-cell based model of hematopoiesis to the conditions of cell culture and describe an association between proliferative history and phenotype of FDCP-Mix cells. While data derived from population studies are incapable of distinguishing between three mechanistically different model scenarios, statistical analysis of single cell tracking data provides a resolution sufficient to select one of them. This scenario favors differences between granulocytic and monocytic lineage with respect to their proliferative behavior and death rates as a mechanistic explanation for the observed heterogeneity. Our results demonstrate the power of a combined experimental/modeling approach in which single cell fate analysis is the key to revealing regulatory principles at the cellular level.

Pretreatment long interspersed element (LINE)-1 methylation levels, not early hypomethylation under treatment, predict hematological response to azacitidine in elderly patients with acute myeloid leukemia.

BACKGROUND: Epigenetic modulations, including changes in DNA cytosine methylation, are implicated in the pathogenesis and progression of acute myeloid leukemia (AML). Azacitidine is a hypomethylating agent that is incorporated into RNA as well as DNA. Thus, there is a rationale to its use in patients with AML. We determined whether baseline and/or early changes in the methylation of long interspersed element (LINE)-1 or CDH13 correlate with bone marrow blast clearance, hematological response, or survival in patients with AML treated with azacitidine. METHODS: An open label, phase I/II trial was performed in 40 AML patients (median bone marrow blast count was 42%) unfit for intensive chemotherapy treated with azacitidine 75 mg/m(2)/day subcutaneously for 5 days every 4 weeks. Bone marrow mononuclear cell samples were taken on day 0 (pretreatment) and day 15 during the first treatment cycle; LINE-1 and CDH13 methylation levels were quantified by methylation-specific, semiquantitative, real-time polymerase chain reaction. RESULTS: Treatment with azacitidine significantly reduced LINE-1 but not CDH13 methylation levels over the first cycle (P < 0.0001). Absolute LINE-1 methylation levels tended to be lower on day 0 (P = 0.06) and day 15 of cycle 1 (P = 0.03) in patients who went on to achieve subsequent complete remission, partial remission or hematological improvement versus patients with stable disease. However, the decrease in LINE-1 methylation over the first treatment cycle did not correlate with subsequent response (P = 0.31). Baseline methylation levels of LINE-1 or CDH13 did not correlate with disease-related prognostic factors, including cytogenetic risk, relapsed/refractory AML, or presence of NPM1 or FLT3 mutations. No correlation was observed between LINE-1 or CDH13 methylation levels and overall survival. CONCLUSION: Analysis of baseline LINE-1 methylation levels may help identify elderly AML patients who are most likely to respond to azacitidine therapy.

Delayed processing of bone marrow samples reveals a prognostic pattern of NME mRNA expression in cytogenetically normal acute myeloid leukemia.

Improvements in the therapy of cytogenetically normal acute myeloid leukemia (CN-AML) will depend largely on the characterization of functional subtypes identified by prognostic markers. Exposing leukemic cells to stress ex vivo may reveal relevant phenotypic markers not apparent in freshly explanted cells. Here, we assess the prognostic relevance of expression of the nucleoside diphosphate kinase genes NME1 and NME2 in a cohort of 78 patients with CN-AML aged < 60 years using archived mononuclear cell samples originally prepared from bone marrow either directly (n = 25) or following 2-3 days of transport (n = 53). The stress conditions arising during transport resulted in the development of a prognostic pattern of NME mRNA with maintenance of high NME2 mRNA being a strong indicator of increased event-free survival independent of FLT3-internal tandem duplication. Prospective analysis of CN-AML bone marrow (n = 7) confirmed that NME1 mRNA is always decreased during storage, while NME2 mRNA is either decreased or maintained. We conclude that ex vivo stress can reveal novel prognostic markers.

Bcr-Abl dependent post-transcriptional activation of NME2 expression is a specific and common feature of chronic myeloid leukemia.

We have previously identified NME2 (Nm23-H2) as a tumor antigen in a patient with chronic myeloid leukemia (CML). Here we investigated the association between NME2 and Bcr-Abl. NME2 protein was highly overexpressed in the cytoplasm of peripheral blood mononuclear cells from 29/30 patients with CML at diagnosis and 10/10 patients resistant to imatinib. Protein was overexpressed in the absence of increased levels of mRNA and was limited to Bcr-Abl + populations, being absent from Bcr-Abl - patient cells, normal donors and 14/15 acute myeloid leukemia (AML) samples. Furthermore, the Bcr-Abl dependent overexpression of NME2 protein was reversed specifically by tyrosine kinase inhibitor (TKI) treatment of Ba/F3 expressing wild-type and TKI-sensitive, but not TKI-resistant, mutants of Bcr-Abl. The post-transcriptional up-regulation of the tumor antigen NME2 is therefore a common and specific property of CML closely associated with Bcr-Abl activity.