Endogenous retrovirus induces leukemia in a xenograft mouse model for primary myelofibrosis.

The compound immunodeficiencies in nonobese diabetic (NOD) inbred mice homozygous for the Prkdc(scid) and Il2rg(null) alleles (NSG mice) permit engraftment of a wide-range of primary human cells, enabling sophisticated modeling of human disease. In studies designed to define neoplastic stem cells of primary myelofibrosis (PMF), a myeloproliferative neoplasm characterized by profound disruption of the hematopoietic microenvironment, we observed a high frequency of acute myeloid leukemia (AML) in NSG mice. AML was of mouse origin, confined to PMF-xenografted mice, and contained multiple clonal integrations of ecotropic murine leukemia virus (E-MuLV). Significantly, MuLV replication was not only observed in diseased mice, but also in nontreated NSG controls. Furthermore, in addition to the single ecotropic endogenous retrovirus (eERV) located on chromosome 11 (Emv30) in the NOD genome, multiple de novo germ-line eERV integrations were observed in mice from each of four independent NSG mouse colonies. Analysis confirmed that E-MuLV originated from the Emv30 provirus and that recombination events were not necessary for virus replication or AML induction. Pathogenicity is thus likely attributable to PMF-mediated paracrine stimulation of mouse myeloid cells, which serve as targets for retroviral infection and transformation, as evidenced by integration into the Evi1 locus, a hotspot for retroviral-induced myeloid leukemia. This study thus corroborates a role of paracrine stimulation in PMF disease progression, underlines the importance of target cell type and numbers in MuLV-induced disease, and mandates awareness of replicating MuLV in NOD immunodeficient mice, which can significantly influence experimental results and their interpretation.

JAK1/2 inhibition impairs T cell function in vitro and in patients with myeloproliferative neoplasms.

Ruxolitinib (INCB018424) is the first JAK1/JAK2 inhibitor approved for treatment of myelofibrosis. JAK/STAT-signalling is known to be involved in the regulation of CD4(+) T cells, which critically orchestrate inflammatory responses. To better understand how ruxolitinib modulates CD4(+) T cell responses, we undertook an in-depth analysis of CD4(+) T cell function upon ruxolitinib exposure. We observed a decrease in total CD3(+) cells after 3 weeks of ruxolitinib treatment in patients with myeloproliferative neoplasms. Moreover, we found that the number of regulatory T cells (Tregs), pro-inflammatory T-helper cell types 1 (Th1) and Th17 were reduced, which were validated by in vitro studies. In line with our in vitro data, we found that inflammatory cytokines [tumour necrosis factor-α (TNF), interleukin (IL)5, IL6, IL1B] were also downregulated in T cells from patients (all P < 0·05). Finally, we showed that ruxolitinib does not interfere with the T cell receptor signalling pathway, but impacts IL2-dependent STAT5 activation. These data provide a rationale for testing JAK inhibitors in diseases triggered by hyperactive CD4(+) T cells, such as autoimmune diseases. In addition, they also provide a potential explanation for the increased infection rates (i.e. viral reactivation and urinary tract infection) seen in ruxolitinib-treated patients.

CD133 marks a stem cell population that drives human primary myelofibrosis.

Primary myelofibrosis is a myeloproliferative neoplasm characterized by bone marrow fibrosis, megakaryocyte atypia, extramedullary hematopoiesis, and transformation to acute myeloid leukemia. To date the stem cell that undergoes the spatial and temporal chain of events during the development of this disease has not been identified. Here we describe a CD133(+) stem cell population that drives the pathogenesis of primary myelofibrosis. Patient-derived circulating CD133(+) but not CD34(+)CD133(-) cells, with a variable burden for JAK2 (V617F) mutation, had multipotent cloning capacity in vitro. CD133(+) cells engrafted for up to 10 months in immunocompromised mice and differentiated into JAK2-V617F(+) myeloid but not lymphoid progenitors. We observed the persistence of human, atypical JAK2-V617F(+) megakaryocytes, the initiation of a prefibrotic state, bone marrow/splenic fibrosis and transition to acute myeloid leukemia. Leukemic cells arose from a subset of CD133(+) cells harboring EZH2 (D265H) but lacking a secondary JAK2 (V617F) mutation, consistent with the hypothesis that deregulation of EZH2 activity drives clonal growth and increases the risk of acute myeloid leukemia. This is the first characterization of a patient-derived stem cell population that drives disease resembling both chronic and acute phases of primary myelofibrosis in mice. These results reveal the importance of the CD133 antigen in deciphering the neoplastic clone in primary myelofibrosis and indicate a new therapeutic target for myeloproliferative neoplasms.

Atovaquone for Prophylaxis of Toxoplasmosis after Allogeneic Hematopoietic Stem Cell Transplantation.

Toxoplasmosis and infections by other opportunistic agents such as Pneumocystis jirovecii constitute life-threatening risks for patients after allogeneic hematopoietic stem cell transplantation. Trimethoprim/sulfamethoxazole (TMP-SMX) has been well established for post-transplant toxoplasmosis and pneumocystis prophylaxis, but treatment may be limited due to toxicity. We explored atovaquone as an alternative and compared it with TMP-SMX regarding toxicity and efficacy during the first 100 days after transplantation in 155 consecutive adult stem cell recipients. Eight patients with a prior history of TMP-SMX intolerance received atovaquone as first-line prophylaxis. TMP-SMX was used for 141 patients as first-line strategy, but 13 patients (9.2%) were later switched to atovaquone due to TMP-SMX toxicity or gastrointestinal symptoms. No active toxoplasmosis or active P. jirovecii infection developed under continued prophylaxis with either TMP-SMX or atovaquone. However, for reasons of TMP-SMX and/or atovaquone toxicity, 7 patients were unable to tolerate any efficacious toxoplasmosis prophylaxis and therefore obtained inhalative pentamidine as P. jirovecii prophylaxis but no toxoplasmosis prophylaxis. Importantly, 2 of these patients developed severe toxoplasmosis. In summary, atovaquone appears as a valid alternative for at least some post-transplant patients who cannot tolerate TMP-SMX. This should be further confirmed by multicenter trials.

Immune-modulating drugs and hypomethylating agents to prevent or treat relapse after allogeneic stem cell transplantation.

Allogeneic stem cell transplantation is a curative treatment option for many hematological diseases, and the numbers of transplantations are steadily increasing worldwide. Major progress has been made in lowering treatment-related mortality by reducing intensity of the conditioning regimen and by improving supportive care (eg, for infectious complications). Accordingly, relapse after allogeneic stem cell transplantation has become the major cause for treatment failure. Major efforts to prevent or treat relapse are focused on cellular- (T cell, natural killer cell), cytokine-, or antibody-based strategies to enhance the graft-versus-tumor effect or circumvent immunoescape. In the more recent years, new classes of agents have shown activity in several hematological malignancies, and besides their immediate antitumor activity, most of them also possess immune-modulatory qualities that may be useful alone or in combination with adoptive immunotherapy after allogeneic stem cell transplantation to enhance graft-versus-tumor effects. Here, we summarize the current knowledge and potential use of 2 of these compounds in preventing or treating relapse after allogeneic stem cell transplantation, namely immune-modulating drugs and hypomethylating agents.

Dynamic of bone marrow fibrosis regression predicts survival after allogeneic stem cell transplantation for myelofibrosis.

We correlate regression of bone marrow fibrosis (BMF) on day 30 and 100 after dose- reduced allogeneic stem cell transplantation (allo-SCT) in 57 patients with primary or post-essential thrombocythemia/polycythemia vera myelofibrosis with graft function and survival. The distribution of International Prognostic Scoring System (IPSS) risk score categories was 1 patient with low risk, 5 patients with intermediate-1 risk, 18 patients with intermediate-2 risk, and 33 patients with high risk. Before allo-SCT, 41 patients (72%) were classified as XXX [myclofibrosis (MF)]-3 and 16 (28%) were classified as MF-2 according to the World Health Organization criteria. At postengraftment day +30 (±10 days), 21% of the patients had near-complete or complete regression of BMF (MF-0/-1), and on day +100 (±20 days), 54% were MF-0/-1. The 5-year overall survival rate at day +100 was 96% in patients with MF-0/-1 and 57% for those with MF-2/-3 (P = .04). There was no difference in BMF regression at day +100 between IPSS high-risk and low/intermediate-risk patients. Complete donor cell chimerism at day +100 was seen in 81% of patients with MF-0/-1 and in 31% of those with MF-2/-3. Patients with MF-2/-3 at day +100 were more likely to be transfusion-dependent for either RBCs (P = .014) or platelets (P = .018). Rapid BMF regression after reduced-intensity conditioning allo-SCT resulted in a favorable survival independent of IPSS risk score at transplantation.

Allogeneic stem cell transplantation for myelofibrosis with leukemic transformation: a study from the Myeloproliferative Neoplasm Subcommittee of the CMWP of the European Group for Blood and Marrow Transplantation.

Transformed acute myeloid leukemia in myelofibrosis results in a median survival of less than 5 months. We identified 46 of 1048 myelofibrosis patients in the European Group for Blood and Marrow Transplantation registry who received allogeneic stem cell transplantation for acute leukemia evolving from myelofibrosis. The cumulative incidence of treatment-related mortality at 1 year was 28% (95% confidence interval, 14 to 42) and of relapse at 3 years was 47% (95% confidence interval, 31 to 63). The 3-year progression-free (PFS) and overall survival (OS) rates were 26% and 33%, respectively. The only significant factor for survival was complete remission versus no complete remission before transplantation (69% versus 22%, P = .008); however, complete remission was achieved only in 8 patients. Allogeneic stem cell transplantation can cure myelofibrosis patients transformed to leukemia.

CD34(+)-selected stem cell boost without further conditioning for poor graft function after allogeneic stem cell transplantation in patients with hematological malignancies.

We retrospectively analyzed outcomes of a CD34(+)-selected stem cell boost (SCB) without prior conditioning in 32 patients (male/22; median age of 54 years; range, 20 to 69) with poor graft function, defined as neutrophils ≤1.5 x 10(9)/L, and/or platelets ≤30 x 10(9)/L, and/or hemoglobin ≤8.5 g/dL). The median interval between stem cell transplantation and SCB was 5 months (range, 2 to 228). The median number of CD34(+) and CD3(+) cells were 3.4 x 10(6)/kg (.96 to 8.30) and 9 x 10(3)/kg body weight (range, 2 to 70), respectively. Hematological improvement was observed in 81% of patients and noted after a median of 30 days (range, 14 to 120) after SCB. The recipients of related grafts responded faster than recipients of unrelated grafts (20 versus 30 days, P = .04). The cumulative incidence of acute (grade II to IV) and chronic graft-versus-host disease (GVHD) after SCB was 17% and 26%, respectively. Patients with acute GVHD received a higher median CD3(+) cell dose. The 2-year probability of overall survival was 45%. We suggest that SCB represents an effective approach to improve poor graft function post transplantation, but optimal timing of SCB administration, anti-infective, and GVHD prophylaxis needs further evaluation.

5-azacytidine promotes an inhibitory T-cell phenotype and impairs immune mediated antileukemic activity.

Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ + T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza.

Impact of high-risk cytogenetics and achievement of molecular remission on long-term freedom from disease after autologous-allogeneic tandem transplantation in patients with multiple myeloma.

Within a prospective protocol, the incidence and impact of achievement of molecular remission (mCR) and high-risk cytogenetics was investigated in 73 patients with multiple myeloma (MM) after autologous (auto)-allogeneic (allo) tandem stem cell transplantation (SCT). After induction chemotherapy, patients received melphalan 200 mg/m(2) before undergoing auto-SCT, followed 3 months later by melphalan 140 mg/m(2) and fludarabine 180 mg/m(2) before allo-SCT. Sixteen patients had high-risk cytogenetic features, defined by positive FISH for del(17p13) and/or t(4;14). Overall, 66% of the patients achieved CR or near-CR, and 41% achieved mCR, which was sustained negative (at least 4 consecutive samples negative) in 15 patients (21%), with no significant difference in incidence between the patients with high-risk cytogenetics and others (P = .70). After a median follow-up of 6 years, overall 5-year progression-free survival was 29%, with no significant difference between del 17p13/t(4;14)-harboring patients and others (24% versus 30%; P = .70). The 5-year progression-free survival differed substantially according to the achieved remission: 17% for partial remission, 41% for CR, 57% for mCR, and 85% for sustained mCR. These results suggest that auto-allo tandem SCT may overcome the negative prognostic effect of del(17p13) and/or t(4;14) and that achievement of molecular remission resulted in long-term freedom from disease.

Outcome of allogeneic SCT in patients with chronic myeloid leukemia in the era of tyrosine kinase inhibitor therapy.

The introduction of tyrosine kinase inhibitors (TKIs) for chronic myeloid leukemia (CML) led to a dramatic change in the role of allogeneic stem cell transplantation (SCT) with a rapid decline in the number of patients receiving SCT in first chronic phase (CP1). We evaluated 68 consecutive patients in all phases of CML (male/female = 39:29, 27 in CP1), who received SCT from related/unrelated donors (related/unrelated = 23:45) under myeloablative or reduced intensity conditioning (MAC/RIC = 45:23). Forty-eight patients (71 %) received TKIs pre-SCT, 20 patients post-SCT (29 %). Overall survival (OS) of CP1 patients achieved a plateau of 85 % at 10 months. Relapse-free survival (RFS) of CP1 patients was 85 % at 1 and 2 years, and 81 % at 5 years. Multivariate analysis showed adverse OS and RFS for patients transplanted >CP1 (hazard ratio (HR) = 6.61 and 4.62) and those who had grade III-IV aGvHD (HR = 2.45 and 1.82). Patients with advanced CML had estimated OS of 65 and 47 %; and RFS of 41 and 32 % at 1 and 2 years respectively. Therefore, for patients with advanced CML phases, allogeneic SCT provides an acceptable chance of cure. Transplant research should focus on improving conditioning regimens and post-SCT management for this subgroup of CML patients.

Comparative diagnostic performance of ¹8F-FDG PET/CT versus whole-body MRI for determination of remission status in multiple myeloma after stem cell transplantation.

OBJECTIVES: To compare the diagnostic performance of whole-body magnetic resonance imaging (WBMRI) versus (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography/computed tomography (PET/CT) for determination of remission status in patients with multiple myeloma (MM) after stem cell transplantation (SCT). METHODS: Thirty-one patients were examined by both WBMRI and PET/CT after SCT. Imaging results and clinical remission status as determined by the clinical gold standard (Uniform Response Criteria) were compared. RESULTS: One hundred four lesions were detected in 21 patients. PET/CT had a sensitivity of 50.0 %, a specificity of 85.7 %, a positive predictive value of 62.5 %, a negative predictive value of 78.3 %, and an overall accuracy of 74.2 % for determination of remission status. MRI had a sensitivity of 80.0 %, a specificity of 38.1 %, a positive predictive value of 38.1 %, a negative predictive value of 80 %, and an overall accuracy of 51.6 %. Concordant results were observed in only 12 (11.5 %) of the 104 lesions. CONCLUSIONS: In the post-treatment setting, both FDG PET/CT and WBMRI provide information about the extent of disease, allowing for a more comprehensive evaluation of persisting or recurrent myeloma. MRI may often be false positive because of persistent non-viable lesions. Therefore, PET/CT might be more suitable than MRI for determination of remission status.

Diagnostic performance of whole-body MRI for the detection of persistent or relapsing disease in multiple myeloma after stem cell transplantation.

OBJECTIVES: To compare the diagnostic performance of whole-body MRI (WBMRI) with haematological parameters for detecting persistent or relapsing disease in patients with multiple myeloma after stem cell transplantation. METHODS: Sixty-six WBMRI acquisitions were performed in 33 patients with multiple myeloma at two time points after stem cell transplantation. Extent of disease and inter-test dynamics of intra- and extramedullary myeloma manifestations were compared (kappa statistics) with Uniform Response Criteria, comprising haematological parameters. RESULTS: Using data from 66 sequential WBMRI acquisitions in 33 patients, 10 patients (30.3 %) were classified as having progressive disease and 23 (69.7 %) as being in remission. Eight (80 %) of the ten patients with progressive disease revealed intramedullary lesions, and two patients (20 %) had intra- and extramedullary lesions. WBMRI and laboratory tests were concordant in 26/33 (78.8 %) patients. We found an agreement of 51.2 %, 95 % confidence interval 19.8 %-82.6 %, between results from WBMRI and haematological parameters. WBMRI had a sensitivity of 63.6 %, specificity of 86.4 %, PPV of 70.0 %, NPV of 82.6 % and accuracy of 78.8 % for detection of remission. CONCLUSIONS: WBMRI allows the detection and exact localisation of intra- and extramedullary myeloma manifestations after stem cell transplantation, but shows only moderate agreement with routinely performed laboratory tests for determination of remission.

Impact of the NK cell receptor LIR-1 (ILT-2/CD85j/LILRB1) on cytotoxicity against multiple myeloma.

The role of different receptors in natural-killer- (NK-) cell-mediated cytotoxicity against multiple myeloma (MM) cells is unknown. We investigated if an enhancement of NK-cell-mediated cytotoxicity against MM could be reached by blocking of the inhibitory leukocyte immunoglobulin-like receptor 1 (LIR-1). Our investigations revealed high levels of LIR-1 expression not only on the NK cell line NK-92, but also on myeloma cells (MOLP-8, RPMI8226) as well as on a lymphoblastoid cell line (LBCL; IM-9). Subsequent cytotoxicity assays were designed to show the isolated effects of LIR-1 blocking on either the effector or the tumor side to rule out receptor-receptor interactions. Although NK-92 was shown to be capable of myeloma cell lysis, inhibition of LIR-1 on NK-92 did not enhance cytotoxicity. Targeting the receptor on MM and LBCL did not also alter NK-92-mediated lysis. We come to the conclusion that LIR-1 alone does not directly influence NK-cell-mediated cytotoxicity against myeloma. To our knowledge, this work provides the first investigation of the inhibitory capability of LIR-1 in NK-92-mediated cytotoxicity against MM and the first functional evaluation of LIR-1 on MM and LBCL.

Purification of CD4+ T cells for adoptive immunotherapy after allogeneic hematopoietic stem cell transplantation.

Donor lymphocyte infusions (DLIs) are used for adoptive immunotherapy to prevent or treat relapse and infectious complications after allogeneic hematopoietic stem cell transplantation (HSCT). Unmanipulated DLIs are associated with a risk of graft-versus-host disease (GVHD), probably related to CD8(+) T cell activity. We investigated an automated clinical-scale human-CD4(+)-cell purification method to deplete CD8(+) cells. Twenty-four stem cell recipients received a total of 24 leukapheresis products being enriched for CD4(+) cells using magnetic associated cell sorting (MACS) with an automated device (CliniMACS(®)) before DLIs. MACS resulted in a mean CD4(+) cell count of 16 × 10(6)/kg bw corresponding to 3.4-fold CD4(+) cell enrichment. Mean yield and purity of CD45(+)CD3(+)CD4(+)CD14(-)7AAD(-) were 74% ± 23% and 82% ± 11%, respectively. Median initial dose of DLIs was 1.1 × 10(6) CD4(+)/kg. During a median follow-up of 25 months, 7 (30%) patients experienced GVHD (acute II-IV: n = 4, 17%; acute III-IV: n = 2, 8%; chronic limited: n = 2, 8%; chronic extensive: n = 1, 4%). Thirteen of 21 further evaluable patients (62%) showed measurable clinical response, 2 patients with therapy refractory infectious complications (HSV) showed remarkable immunologic improvement. Automated enrichment of CD4(+) by magnetic cell sorting provides an efficient and rapid method for processing donor lymphocytes. Additional studies should further investigate this approach in terms of efficacy and the risk of GVHD.

Postallograft lenalidomide induces strong NK cell-mediated antimyeloma activity and risk for T cell-mediated GvHD: Results from a phase I/II dose-finding study.

Lenalidomide may prevent relapses after allogeneic stem cell transplantation by promoting the immune-mediated graft-versus-tumor effect. We performed a prospective phase I/II study to define the dose-limiting toxicity and the immunologic effects of lenalidomide given early (day 100-180) after allograft for four cycles in patients with multiple myeloma. According to the Fibonacci design, 24 patients with a median age of 53 years were included. Dose-limiting toxicity was organ toxicity owing to graft-versus-host disease, and the maximum tolerable dose was 5 mg. The incidence of graft-versus-host disease after lenalidomide was 38%, occurring after a median of 22 days, and was beside organ toxicity, a leading cause to discontinue the study in 29% of the patients. Immune monitoring revealed a significant increase in peripheral γ-interferon-secreting CD4(+) and CD8(+) T cells within the first week of lenalidomide treatment followed by a delayed increase in T regulatory cells. Furthermore, natural killer (NK) cells isolated from the peripheral blood of patients evidenced a significantly improved antimyeloma activity after lenalidomide treatment. The immune effect might have contributed to the increased CR rate from 24-42% after lenalidomide treatment because nonresponding patients showed significantly less natural killer and T cell activation. (Study registered under: NCT 00778752.).

Osteoprotegerin expression in dendritic cells increases with maturation and is NF-kappaB-dependent.

Dendritic cells (DC) comprise a unique leukocyte population which controls primary immune responses. Recent studies indicate that DC express osteoprotegerin (OPG), a secreted tumor necrosis factor receptor homolog, which regulates DC survival, monocyte chemotaxis, and B cell development and function by ligating TNF family member receptor activator of NF-kappaB ligand (RANKL). The precise regulators of OPG expression in DC have not been investigated. In this study, we assessed OPG mRNA steady state levels by Northern blot analysis and OPG protein secretion by an immunoassay in monocyte-derived DC of different maturation, and the effect of different cytokines and hormones on OPG expression. OPG was upregulated with maturation of DC, whereas pretreatment of DC with 1alpha,25(OH)(2) vitamin D(3), tamoxifen, or dexamethasone, agents that inhibit differentiation of DC, decreased OPG expression. In vivo, OPG was found to be colocalized with mature CD83(+) DC in human tonsils by immunofluorescence confocal microscopy analysis. Furthermore, OPG was upregulated by TNF superfamily members TNF-alpha, anti-CD40, and RANKL, and by ligands of the Toll-like/IL-1 receptor family including IL-1beta, double-stranded RNA (poly I:C), or lipopolysaccharide (LPS), all of which induce maturation of DC. Gene silencing by small interfering RNA (siRNA) directed against transcription factor NF-kappaB abrogated the expression of OPG as demonstrated by real-time PCR. In summary, we describe that the expression of OPG by DC increases with maturation and is NF-kappaB-dependent, possibly regulating immune responses in lymphoid tissues.

C-Raf controlled pathways in the protection of tumor cells from apoptosis.

The Raf serine-threonine kinase is upregulated in many human tumors and plays a pivotal role in tumor cell proliferation and survival. Abrogation of c-Raf expression by specific antisense oligonucleotides (Raf-AS-ODN) efficiently blocks tumor cell growth and induces apoptosis in human cancer cells. The signaling pathways and molecular mechanisms c-Raf utilizes to mediate the survival of tumor cells are, however, not well understood. Here we show that apoptosis triggered by Raf depletion cannot be overcome by ectopic Bcl-2 expression and occurs in the absence of cytochrome c release, arguing against a direct impact of c-Raf on mitochondrial pathways of apoptosis regulation. We also show that c-Raf depletion leads to a clearly decreased expression of different epidermal growth factor (EGF) receptor ligands, suggesting that the autocrine stimulation of an EGF receptor-mediated survival pathway might be involved in the blockade of tumor cell apoptotis by c-Raf.