Analysis of nonleukemic cellular subcompartments reconstructs clonal evolution of acute myeloid leukemia and identifies therapy-resistant preleukemic clones.

To acquire a better understanding of clonal evolution of acute myeloid leukemia (AML) and to identify the clone(s) responsible for disease recurrence, we have comparatively studied leukemia-specific mutations by whole-exome-sequencing (WES) of both the leukemia and the nonleukemia compartments derived from the bone marrow of AML patients. The T-lymphocytes, B-lymphocytes and the functionally normal hematopoietic stem cells (HSC), that is, CD34+ /CD38- /ALDH+ cells for AML with rare-ALDH+ blasts (<1.9% ALDH+ cells) were defined as the nonleukemia compartments. WES identified 62 point-mutations in the leukemia compartment derived from 12 AML-patients at the time of diagnosis and 73 mutations in 3 matched relapse cases. Most patients (8/12) showed 4 to 6 point-mutations per sample at diagnosis. Other than the mutations in the recurrently mutated genes such as DNMT3A, NRAS and KIT, we were able to identify novel point-mutations that have not yet been described in AML. Some leukemia-specific mutations and cytogenetic abnormalities including DNMT3A(R882H), EZH2(I146T) and inversion(16) were also detectable in the respective T-lymphocytes, B-lymphocytes and HSC in 5/12 patients, suggesting that preleukemia HSC might represent the source of leukemogenesis for these cases. The leukemic evolution was reconstructed for five cases with detectable preleukemia clones, which were tracked in follow-up and relapse samples. Four of the five patients with detectable preleukemic mutations developed relapse. The presence of leukemia-specific mutations in these nonleukemia compartments, especially after chemotherapy or after allogeneic stem cell transplantation, is highly relevant, as these could be responsible for relapse. This discovery may facilitate the identification of novel targets for long-term cure.

Patients With Malignant Lymphoma and HIV Infection Experiencing Remission After First-Line Treatment Have an Excellent Prognosis.

BACKGROUND: Malignant lymphoma is still the leading cause of death among AIDS-related diseases. PATIENTS AND METHODS: We performed a retrospective analysis of 50 HIV-positive lymphoma patients. The median interval between HIV and malignant lymphoma diagnosis was 4 years. Eight patients (16%) had Hodgkin lymphoma and 42 (84%) non-Hodgkin lymphoma. Among non-Hodgkin lymphoma patients, diffuse large B-cell lymphoma (n = 18, 42%), Burkitt lymphoma (n = 11, 26%), and plasmoblastic lymphoma (n = 5, 12%) were the most frequent entities. RESULTS: Lymphoma was treated according to standard protocols. Forty-four patients (88%) received combination antiretroviral therapy, 2 (4%) were not treated, and in 4 (8%) the HIV treatment status was not clarified. Response to first-line therapy was complete response (CR) in 24 (56%), partial response (PR) in 15 (35%), and stable disease in 1 (2%). Three patients (7%) developed progressive disease, and 9 (18%) experienced relapse after CR or PR. At a median observation period of 31 (range, 0.4-192) months, the 1-, 2-, and 5-year overall survival was 87%, 79%, and 76%, respectively. At univariate analysis, remission status after first-line treatment was predictive of outcome, as the 2-year overall survival was 95%, 66%, and 0 for patients with CR, with PR, and with progressive disease (P < .001). Results of the multivariate analysis revealed lactate dehydrogenase concentration at lymphoma diagnosis (P = .046) and relapse (P = .050) to be independent factors for overall survival. CONCLUSION: First-line treatment of lymphoma in HIV positive patients is crucial. Patients who experienced and maintained a first CR had a favorable prognosis.

Human haematopoietic stem cell lineage commitment is a continuous process.

Blood formation is believed to occur through stepwise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a 'continuum of low-primed undifferentiated haematopoietic stem and progenitor cells' (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady-state haematopoiesis downstream of HSCs and provide a basis for the understanding of haematopoietic malignancies.

Plerixafor is effective given either preemptively or as a rescue strategy in poor stem cell mobilizing patients with multiple myeloma.

BACKGROUND: Harvest of more than one CD34+ stem cell transplant has become the standard, to ensure the option for a second autologous transplantation in patients with relapsed or progressive multiple myeloma (MM). Additional administration of the CXCR-4 inhibitor plerixafor has been shown to increase the efficiency of CD34+ stem cell harvest. However, the algorithm when to apply plerixafor is still under debate. STUDY DESIGN AND METHODS: In this retrospective study, 46 MM patients were categorized into four groups according to their CD34+ stem cell count in peripheral blood (PB) and mobilization with or without plerixafor: Group A comprised poor mobilizers with CD34+ cell counts of fewer than 20 × 10(6) /L in PB. Group B included inadequate mobilizers with CD34+ cell counts of 20 × 10(6) /L or more in PB and a low CD34+ stem cell yield in the first leukapheresis session. Patients receiving plerixafor preemptively (Group A1) and as a rescue strategy (Group B1) were compared to patients continuing stem cell collection with granulocyte-colony-stimulating factor alone (Groups A2 and B2). RESULTS: In both, the preemptive and the rescue settings, plerixafor enhanced the CD34+ stem cell yield significantly. Poor mobilization and administration of plerixafor was not associated with delayed engraftment. CONCLUSION: Our data demonstrate that administration of plerixafor is safe and effective and facilitates a significantly higher CD34+ stem cell harvest. Based on the presented data, we propose an algorithm for the use of plerixafor for CD34+ stem cell mobilization and harvesting in poor mobilizing myeloma patients.

The rarity of ALDH(+) cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients.

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH(+) cells; <1.9%; ALDH-rare AML), whereas 24 patients had relatively numerous ALDH(+) cells (≥1.9%; ALDH-numerous AML). In patients with ALDH-rare AML, normal HSC could be separated by their CD34(+) ALDH(+) phenotype, whereas LSC were exclusively detected among CD34(+) ALDH(-) cells. For patients with ALDH-numerous AML, the CD34(+) ALDH(+) subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH(+) cells from ALDH-numerous AML were quiescent, refractory to ARA-C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long-term outcome were also characteristic for patients with ALDH-numerous AML providing an additional risk-stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible.

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

BACKGROUND AIMS: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. METHODS: This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. RESULTS: The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. CONCLUSIONS: This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Targeting CDK9 by wogonin and related natural flavones potentiates the anti-cancer efficacy of the Bcl-2 family inhibitor ABT-263.

Tumor initiation, progression and resistance to therapies are tightly associated with over-expression of anti-apoptotic proteins Bcl-2, Bcl-x(L), Bcl-w and Mcl-1. ABT-263 (Navitoclax), an orally bio-available small-molecule mimetic of the Bcl-2 homology domain 3, inhibits Bcl-2, Bcl-x(L), and Bcl-w and has shown anti-cancer effects mainly on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of ABT-263 in patients is dose-limited due to causing thrombocytopenia via inhibition of Bcl-x(L) in platelets. ABT-199 specifically inhibits Bcl-2; however, its use is limited to tumors over-expressing only Bcl-2. Besides, many tumors resist treatment due to high levels of Mcl-1 expression or develop resistance via up-regulation of Mcl-1 during long-term exposure. These obstacles highlight the demand to improve the ABT-263-based therapy. In this study, we show that anti-cancer flavones, e.g., wogonin, baicalein, apigenin, chrysin and luteolin enhance ABT-263-induced apoptosis in different cancer cell lines and in primary AML and ALL cells by down-regulation of Mcl-1 expression. Importantly, wogonin does not enhance the toxicity of ABT-263 to proliferating normal T cells and thrombocytes. Wogonin also potentiates the lethality of ABT-263 in cancer cells which have acquired resistance to ABT-263. Furthermore, we show that combination of wogonin with ABT-263 promotes in vivo tumor regression in a human T-cell leukemia xenograft mouse model. Our study demonstrates that wogonin (and related flavones) reduce the effective dose of ABT-263 thereby possibly decreasing the risk of adverse side effects.

Identification and separation of normal hematopoietic stem cells and leukemia stem cells from patients with acute myeloid leukemia.

Mounting evidences indicate that leukemic cells in patients with acute myeloid leukemia (AML) are derived from leukemia stem cells (LSC). In analogy to normal hematopoietic stem cells (HSC), LSC remain mostly dormant and are hence resistant to conventional chemotherapy. Residual, physiological HSC exist alongside with LSC, with heterogeneous dominance of LSC over HSC in individual patients. We have devised a flow cytometric method for the identification and separation of these two stem cell populations based on surface antigen markers such as CD34, CD38, lineage aberrant markers, and aldehyde dehydrogenase (ALDH) enzyme activity.

Leukemia stem cells.

Leukemia stem cells (LSCs) might originate from malignant transformation of normal hematopoietic stem cells (HSCs), or alternatively, of progenitors in which the acquired mutations have re-installed a dysregulated self-renewal program. LSCs are on top of a hierarchy and generate leukemia cells with more differentiated characteristics. While most leukemia cells are initially sensitive to chemo- and radiotherapy, LSCs are resistant and are considered to be the basis for disease relapse after initial response. Albeit important knowledge on LSC biology has been gained from xenogeneic transplantation models introducing human leukemia cells into immune deficient mouse models, the prospective identification and isolation of human LSC candidates has remained elusive and their prognostic and therapeutic significance controversial. This review focuses on the identification, enrichment and characterization of human LSC derived from patients with acute myeloid leukemia (AML). Experimental data demonstrating the clinical significance of estimating LSC burden and strategies to eliminate LSC will be summarized. For long-term cure of AML, it is of importance to define LSC candidates and to understand their tumor biology compared to normal HSCs. Such comparative studies might provide novel markers for the identification of LSC and for the development of treatment strategies that might be able to eradicate them.

Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells.

Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine stromal cell derived factor-1 (SDF-1) to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4(+) human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, whereas ß2-adrenergic antagonist inhibited both steady state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady-state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.

Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling.

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1(+)/c-Kit(+)/Lin(-) cells and interfered with their enhanced chemotactic migration to stromal cell-derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met-mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.

A crosstalk between intracellular CXCR7 and CXCR4 involved in rapid CXCL12-triggered integrin activation but not in chemokine-triggered motility of human T lymphocytes and CD34+ cells.

The chemokine CXCL12 promotes migration of human leukocytes, hematopoietic progenitors, and tumor cells. The binding of CXCL12 to its receptor CXCR4 triggers Gi protein signals for motility and integrin activation in many cell types. CXCR7 is a second, recently identified receptor for CXCL12, but its role as an intrinsic G-protein-coupled receptor (GPCR) has been debated. We report that CXCR7 fails to support on its own any CXCL12-triggered integrin activation or motility in human T lymphocytes or CD34(+) progenitors. CXCR7 is also scarcely expressed on the surface of both cell types and concentrates right underneath the plasma membrane with partial colocalization in early endosomes. Nevertheless, various specific CXCR7 blockers get access to this pool and attenuate the ability of CXCR4 to properly rearrange by surface-bound CXCL12, a critical step in the ability of the GPCR to trigger optimal CXCL12-mediated stimulation of integrin activation in T lymphocytes as well as in CD34(+) cells. In contrast, CXCL12-triggered CXCR4 signaling to early targets, such as Akt as well as CXCR4-mediated chemotaxis, is insensitive to identical CXCR7 blocking. Our findings suggest that although CXCR7 is not an intrinsic signaling receptor for CXCL12 on lymphocytes or CD34(+) cells, its blocking can be useful for therapeutic interference with CXCR4-mediated activation of integrins.

Imatinib combined with mitoxantrone/etoposide and cytarabine is an effective induction therapy for patients with chronic myeloid leukemia in myeloid blast crisis.

BACKGROUND: Despite advances in drug therapy and allogeneic stem cell transplantation (allo-SCT), the prognosis of patients with chronic myeloid leukemia (CML) in blast crisis remains poor. Imatinib has demonstrated synergistic effects in vitro with mitoxantrone, etoposide, and cytarabine. METHODS: A Phase I/II trial was performed in patients with CML myeloid blast crisis. Patients were treated with imatinib + mitoxantrone/etoposide in four cohorts: mitoxantrone 10 mg/m(2)/day and etoposide 100 mg/m(2)/day for 2 or 3 consecutive days and imatinib 600 mg/day from Day 15 (cohorts 1 and 2) or from Day 1 (cohorts 3 and 4). After hematologic reconstitution after the cytopenic phase, cytarabine was given at a dose of 10 mg/m(2)/day in addition to imatinib as maintenance treatment. RESULTS: A total of 16 patients were available for analysis, median age 59 years (range, 37-74). All patients who received more intensive induction treatment (cohorts 3 and 4, n = 7) achieved a hematologic response (HR). In contrast, HR was achieved in only 6 of 9 patients treated in cohorts 1 and 2. The induction treatment was well tolerated. Six patients who achieved HR received an allo-SCT with myeloablative conditioning. The median survival in the transplant group was 16.2 months vs 4.7 months in the group with conventional treatment only (P = .067). CONCLUSIONS: The combination of mitoxantrone/etoposide and imatinib is well tolerated, with mild nonhematologic toxicity even in older patients. Eligible patients benefit from allo-SCT after response to the induction treatment.

Myeloablative conditioning in myelofibrosis using i.v. treosulfan and autologous peripheral blood progenitor cell transplantation with high doses of CD34+ cells results in hematologic responses - follow-up of three patients.

Autologous transplantation after myeloablation for myelofibrosis with myeloid metaplasia provides a palliative therapy with a long term relief of symptoms. We have transplanted three patients with more than 5 x 10(6) CD34+ cells/kg body weight after myeloablation with treosulfan (total dose 42 g/m(2)) with a 18 months follow-up. Two of the patients had symptomatic splenomegaly and severe anemia. One patient had symptomatic splenomegaly and thrombocytopenia (< 100x10(9)/L). Granulocyte colony-stimulating factor-supported peripheral blood progenitor cell mobilization and collection was not associated with increased toxicity. Following transplantation we observed a prolonged reconstitution period of 28-38 days without fever or severe mucositis. All patients became free of erythrocyte transfusions or recovered to normal thrombocyte counts. There was a significant reduction of max. spleen size in one patient. We conclude that myeloablation with treosulfan and autologous PBPCT in these three patients with myelofibrosis was safe and useful.

Clonal analysis of individual marrow-repopulating cells after experimental peripheral blood progenitor cell transplantation.

Methods to analyze the clonality of an adverse event in preclinical or clinical retroviral stem cell gene therapy protocols are needed. We analyzed the progeny of retrovirally transduced human peripheral blood progenitor cells (PBPCs) after transplantation and engraftment in immune-deficient mice. The integration site of the provirus serves as a unique tag of the individual transduced PBPC. A plasmid library of junctions between proviral and human genomic DNA was generated. We were able to detect individual transduced cell clones that amounted to 0.14%-0.0001% of chimeric bone marrow cells. This is the first report in which the contribution of individual marrow-repopulating cells to human hematopoiesis is directly quantified.

Secondary follicular lymphoma of the bone, transformed into large cell lymphoma, in a patient with chronic lymphocytic leukaemia: an uncommon manifestation of Richter's syndrome.

Patients with chronic lymphocytic leukemia (CLL) are at a significantly increased risk of developing a second malignant neoplasm in the course of their disease. The occurrence of large cell lymphoma [Richter's syndrome (RS)] has been described in approximately 3-5% of CLL patients. Other types of secondary lymphoid malignancies are extremely rare. Here we describe a patient, heavily pretreated, with long history of CLL who developed a secondary follicular lymphoma, transformed into a diffuse large cell lymphoma (LCL), with isolated manifestation in the bone, a very rare manifestation of RS. CLL and LCL cells were of distinct clonal origin, as documented by DNA sequencing of the CDR3 regions. Twelve months after the completion of chemo- and local radiotherapy, the patient is still in remission.