Coexisting driver mutations in MPN: clinical and molecular characteristics of a series of 11 patients.

OBJECTIVES: CML, PV, ET and PMF are so called classical MPN with distinct clinical phenotypes. The discovery of the BCR-ABL1 translocation and mutations in driver genes JAK2, MPL and CALR has provided novel insights in their pathogenesis. While these mutations are thought to be mutually exclusive, rare cases of MPN with coexisting driver mutations have been reported. However, little is known about the clinical, biological and molecular characteristics of these patients and the interaction of the neoplastic clones. METHODS: We retrospectively studied 11 MPN patients with coexisting driver mutations (JAK2 V617F + BCR-ABL1: n = 8; CALR type 2 + BCR-ABL1: n = 1; JAK2 V617F + MPL W515: n = 1; JAK2 V617F + CALR type 1: n = 1). To assess possible associated molecular aberrations, we analysed DNA of six patients using NGS. RESULTS: In four CML patients, decreasing BCR-ABL1 transcript levels with increasing JAK2 V617F allele burden under TKI were observed. This strongly suggests that the coexistence of driver mutations originates from two different clones growing independently. Additional somatic mutations were detected in 5 out of 6 (83%) patients affecting 4 different genes, confirming the heterogeneity of this study cohort. Suboptimal response to TKI was observed with a higher frequency (4/8 patients) than reported in conventional series of CML and the overall tolerance of treatment with hydroxyurea and/or imatinib in our series was poor. CONCLUSION: Given the emergence of NGS in clinical practice, more similar cases will be identified in the coming years. The optimal treatment strategy for this rare group of patients is uncertain and toxicity of combination treatment may have to be considered.

A clinical-molecular prognostic model to predict survival in patients with post polycythemia vera and post essential thrombocythemia myelofibrosis.

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms with variable risk of evolution into post-PV and post-ET myelofibrosis, from now on referred to as secondary myelofibrosis (SMF). No specific tools have been defined for risk stratification in SMF. To develop a prognostic model for predicting survival, we studied 685 JAK2, CALR, and MPL annotated patients with SMF. Median survival of the whole cohort was 9.3 years (95% CI: 8-not reached-NR-). Through penalized Cox regressions we identified negative predictors of survival and according to beta risk coefficients we assigned 2 points to hemoglobin level <11 g/dl, to circulating blasts ⩾3%, and to CALR-unmutated genotype, 1 point to platelet count <150 × 109/l and to constitutional symptoms, and 0.15 points to any year of age. Myelofibrosis Secondary to PV and ET-Prognostic Model (MYSEC-PM) allocated SMF patients into four risk categories with different survival (P<0.0001): low (median survival NR; 133 patients), intermediate-1 (9.3 years, 95% CI: 8.1-NR; 245 patients), intermediate-2 (4.4 years, 95% CI: 3.2-7.9; 126 patients), and high risk (2 years, 95% CI: 1.7-3.9; 75 patients). Finally, we found that the MYSEC-PM represents the most appropriate tool for SMF decision-making to be used in clinical and trial settings.

Diagnosing nocturnal paroxysmal hemoglobinuria: a single-center 4-year experience.

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease and can present as a wide range of signs and symptoms. As such, the indication for diagnostic testing for PNH is not always straightforward. Therefore, we analyzed all first-time samples tested over a 56-month period to determine the clinical settings with a high probability of detecting a PNH clone. METHODS: We retrospectively analyzed 323 first-time PNH flow cytometry tests, including LDH, cytopenias, direct antiglobulin test (DAT), and clinical indication for testing as available at the time of testing. RESULTS: The probability of finding a PNH clone was 47% in patients tested because of aplastic/hypoplastic bone marrow disorders, 10% in DAT-negative hemolytic anemia (HA), 5% in myelodysplastic syndromes (MDS), 3% in cytopenias other than HA, and 2% in thrombosis. When testing for another reason than the indications described before, there were no positive samples. CONCLUSION: Our findings reinforce guidelines from the International PNH Interest Group which suggest testing for PNH in the setting of unusual thrombosis, HA, aplastic/hypoplastic bone marrow disorders, or MDS, as these have a higher pretest probability. This probability drops to zero in our study in nonrecommended indications. This reflects the need for better education of clinicians about the disease PNH and the indications for diagnostic testing.

A randomized study of pomalidomide vs placebo in persons with myeloproliferative neoplasm-associated myelofibrosis and RBC-transfusion dependence.

RBC-transfusion dependence is common in persons with myeloproliferative neoplasm (MPN)-associated myelofibrosis. The objective of this study was to determine the rates of RBC-transfusion independence after therapy with pomalidomide vs placebo in persons with MPN-associated myelofibrosis and RBC-transfusion dependence. Two hundred and fifty-two subjects (intent-to-treat (ITT) population) including 229 subjects confirmed by central review (modified ITT population) were randomly assigned (2:1) to pomalidomide or placebo. Trialists and subjects were blinded to treatment allocation. Primary end point was proportion of subjects achieving RBC-transfusion independence within 6 months. One hundred and fifty-two subjects received pomalidomide and 77 placebo. Response rates were 16% (95% confidence interval (CI), 11, 23%) vs 16% (8, 26%; P=0.87). Response in the pomalidomide cohort was associated with ⩽4 U RBC/28 days (odds ratio (OR)=3.1; 0.9, 11.1), age ⩽65 (OR=2.3; 0.9, 5.5) and type of MPN-associated myelofibrosis (OR=2.6; 0.7, 9.5). Responses in the placebo cohort were associated with ⩽4 U RBC/28 days (OR=8.6; 0.9, 82.3), white blood cell at randomization >25 × 109/l (OR=4.9; 0.8, 28.9) and interval from diagnosis to randomization >2 years (OR=4.9; 1.1, 21.9). Pomalidomide was associated with increased rates of oedema and neutropenia but these adverse effects were manageable. Pomalidomide and placebo had similar RBC-transfusion-independence response rates in persons with MPN-associated RBC-transfusion dependence.

A novel approach for BCR-ABL1 standardization to improve International Scale estimation.

INTRODUCTION: Standardization of BCR-ABL1 messenger RNA quantification by real-time PCR on the International Scale (IS) is critical for monitoring therapy response in chronic myelogenous leukaemia. Since 2006, BCR-ABL1 IS standardization is propagated along reference laboratories by calculating a laboratory-specific conversion factor (CF), co-ordinated in Europe through the European Treatment and Outcome Study project. Although this process has proven successful to some extent, it has not been achievable for all laboratories due to the complexity of the process and the stringent requirements in terms of numbers of samples to be exchanged. In addition, several BCR-ABL1 IS quantification methods and secondary reference materials became commercially available. However, it was observed that different IS methods generate consistently different results. METHODS: To overcome these difficulties, we have developed an alternative and simple approach of CF calculation, based on the retrospective analysis of existing external quality assessment (EQA) data. Our approach does not depend on the exchange of samples and is solely based on the mathematical CF calculation using EQA results. RESULTS AND CONCLUSION: We have demonstrated by thorough statistical validation that this approach performs well in converting BCR-ABL1 measurements to improve IS estimation. In expectation of a true golden standard method for BCR-ABL1 IS quantification, the proposed method is a valuable alternative.

Budd-Chiari syndrome: reassessment of a step-wise treatment strategy.

BACKGROUND AND STUDY AIMS: The Budd-Chiari syndrome is a rare disorder characterized by hepatic venous outflow obstruction. A step-wise management was recently proposed. The aim of this study is to reassess our treatment approach and long-term outcome. PATIENTS AND METHODS: The data of 37 Budd-Chiari patients, seen in our unit, were critically analyzed and compared with the ENVIE (European Network For Vascular Disorders of the Liver) data. RESULTS: Most patients had multiple prothrombotic conditions (41%), of which an underlying myeloproliferative neoplasm was the most frequent (59%). The JAK2V617F mutation was associated with more complete occlusion of all hepatic veins (JAK2 mutation +: 70% vs JAK2 mutation -: 23% and a higher severity score. The step-wise treatment algorithm used in our unit, in function of the severity of the liver impairment and the number and the extension of hepatic veins occluded, resulted in the following treatments: only anticoagulation (n = 7.21%), recanalization procedure (n = 4.21%), portosystemic shunts (n = 9.26%) and liver transplantation (n = 14.44%). This resulted in a 10 year survival rate of 90%. Treatment of the underlying hemostatic disorder offered a low recurrence rate. None of the 21 patients with a myeloproliferative neoplasm died in relation to the hematologic disorder. CONCLUSIONS: An individualized treatment regimen consisting of anticoagulation and interventional radiology and/or transplantation when necessary and strict follow-up of the underlying hematologic disorder, provided an excellent long-term survival, which confirm the data of the ENVIE study.

Myelofibrosis patients in Belgium: disease characteristics.

OBJECTIVE: To date, only a small number of epidemiological studies on myelofibrosis have been performed. The current study aimed to characterize the myelofibrosis patient population in Belgium according to pre-defined disease parameters (diagnosis, risk categories, hemoglobin <10 g/dl, spleen size, constitutional symptoms, platelet count, myeloblast count), with a view to obtaining a deeper understanding of the proportion of patients that may benefit from the novel myelofibrosis therapeutic strategies. METHODS: A survey was used to collect data on prevalence and disease parameters on all myelofibrosis patients seen at each of 18 participating hematologic centers in 2011. Aggregated data from all centers were used for analysis. Analyses were descriptive and quantitative. RESULTS: A total of 250 patients with myelofibrosis were captured; of these, 136 (54%) were male and 153 (61%) were over 65 years old. One hundred sixty-five (66%) of myelofibrosis patients had primary myelofibrosis and 85 (34%) had secondary myelofibrosis. One hundred ninety-three myelofibrosis patients (77%) had a palpable spleen. About a third of patients (34%) suffered from constitutional symptoms. Two hundred twenty-two (89%) myelofibrosis patients had platelet count ≧50 000/µl and 201 (80%) had platelet count ≧100 000/µl. Of 250 patients, 85 (34%) had a myeloblast count ≧1%. Six (2%) patients had undergone a splenectomy. Thirteen (5·2%) patients had undergone radiotherapy for splenomegaly. CONCLUSIONS: The results of this survey provide insight into the characteristics of the Belgian myelofibrosis population. They also suggest that a large proportion of these patients could stand to benefit from the therapies currently under development.

Treatment with lenalidomide (Revlimid®), cyclophosphamide (Endoxan®) and prednisone (REP) in relapsed/refractory multiple myeloma patients: results of a single centre retrospective study.

Lenalidomide (Revlimid®) combined with intermittent dexamethasone (the RD regimen) is one of the current standards for treatment of patients with relapsed/refractory multiple myeloma (MM). However, since the disease in the majority of patients will become resistant to RD, or treatment with RD needs to be discontinued due to side effects, we evaluated the combination lenalidomide, low-dose oral cyclophosphamide, with prednisone (REP) in patients with relapsed/refractory MM previously exposed to RD. For this purpose, we performed a single centre retrospective study of the efficacy of REP in 19 patients with relapsed/refractory MM. Overall response rate (partial response or better) with REP was 68% compared with 83% with RD, but with a shorter time to response with the triplet REP. Time to progression after REP was 6 months. Overall the REP regimen was better tolerated compared to RD. We conclude that the REP regimen is an effective treatment regimen for patients with relapsed/refractory MM with good tolerance, warranting further exploration in prospective randomized trials.

A 75-year-old missionary with fever, splenomegaly and pancytopenia.

We present a case of hyperreactive malarial splenomegaly, a disease which is exceptional in Caucasian people, but which is expected to become more important since the increasing number of travelling to tropical areas. It is the chronic stage of an abnormal long-term stimulation of the immune system secondary to plasmodial infection. Diagnostic criteria include long-term stay in an endemic zone, large splenomegaly and overproduction of both IgM and IgG antibodies. The disease can be treated by a short-term antimalarial therapy as long as the patient resides out of a malarial endemic country.

Subset characterization of myeloid-derived suppressor cells arising during induction of BM chimerism in mice.

To date, myeloid-derived suppressor cells (MDSC) have been best studied in cancer, where they represent an escape mechanism for immune surveillance. MDSC are now also gaining interest in the context of transplantation. Suppressive CD11b(+) myeloid progenitor cells have been reported to expand endogenously during BM chimerism induction in mice; in particular, in irradiated MHC-matched BM chimeras and in parent-in-F1 BM chimeras. Myeloid cell expansion coincided with a time frame where donor lymphocyte infusion (DLI) therapy-mediated GVL effects without GVHD. Hypothesizing that regulatory myeloid cells may have a role in regulating post-transplant T-cell alloreactivity, we performed a detailed phenotypic and functional characterization of these cells in the parent-in-F1 C57BL/6 → [C57BL/6xDBA2] model. We found that transiently expanding CD11b(+) myeloid progenitor cells comprise the two phenotypically and functionally distinct mononuclear and polymorphonuclear MDSC subsets that were recently described in tumor-bearing mice. Both MDSC subsets suppressed in vitro and in vivo alloreactive T-cell proliferation. Also, both the subsets mediated enhanced in vitro suppression when harvested from chimeras, given a prior in vivo challenge with non-tolerant donor T cells, indicating that allo-activated T cells can activate MDSC in vivo. This study provides the basis to investigate the-potentially beneficial-role of expanding MDSC in influencing the risk of GVHD during chimerism induction.

Thrombotic microangiopathy following intestinal transplantation: a single center experience.

BACKGROUND: Transplant-related thrombotic microangiopathy (TMA) is a well-recognized complication of all types of transplantations. Despite its known relationship with immunosuppressive therapy, only a few cases have been reported following intestinal transplantation. METHODS: We retrospectively reviewed the medical files of nine consecutive intestinal transplant patients between 2000 and 2008. RESULTS: The diagnosis of TMA was established in 3 patients (33%). At diagnosis the immunosuppressive therapy consisted of tacrolimus (n = 3), combined with azathioprine (n = 1) or sirolimus (n = 2) and steroids (n = 2). The median time between transplantation and TMA was 104 days (range, 55-167 days). Levels of ADAMTS13, a von Willebrand protease, were within normal ranges in all 3 patients. Treatment consisted of stopping/tapering of tacrolimus, together with initiation of plasma therapy, leading to complete remission in all 3 patients. During further follow-up, all 3 patients showed severe graft rejection necessitating more profound immunosuppressive therapy, leading to graft loss in 1 patient and infection-related death in the 2 others. At a median follow-up of 52 months (range, 9-100 months) all remaining TMA-free patients (n = 6) were alive with functioning grafts under minimal immunosuppression. CONCLUSION: Herein we have described 3 intestinal transplant patients who were diagnosed with transplantation-related TMA. Despite excellent disease control the final outcomes were dismal, which clearly contrasts with the outcome among TMA-free patients, who were all well with functioning grafts at last follow-up.

KIR-ligand incompatibility in the graft-versus-host direction improves outcomes after umbilical cord blood transplantation for acute leukemia.

Donor killer cell immunoglobulin-like receptor (KIR)-ligand incompatibility is associated with decreased relapse incidence (RI) and improved leukemia-free survival (LFS) after haploidentical and HLA-mismatched unrelated hematopoietic stem cell transplantation. We assessed outcomes of 218 patients with acute myeloid leukemia (AML n=94) or acute lymphoblastic leukemia (n=124) in complete remission (CR) who had received a single-unit unrelated cord blood transplant (UCBT) from a KIR-ligand-compatible or -incompatible donor. Grafts were HLA-A, -B or -DRB1 matched (n=21) or mismatched (n=197). Patients and donors were categorized according to their degree of KIR-ligand compatibility in the graft-versus-host direction by determining whether or not they expressed HLA-C group 1 or 2, HLA-Bw4 or HLA-A3/-A11. Both HLA-C/-B KIR-ligand- and HLA-A-A3/-A11 KIR-ligand-incompatible UCBT showed a trend to improved LFS (P=0.09 and P=0.13, respectively). Sixty-nine donor-patient pairs were HLA-A, -B or -C KIR-ligand incompatible and 149 compatible. KIR-ligand-incompatible UCBT showed improved LFS (hazards ratio=2.05, P=0.0016) and overall survival (OS) (hazards ratio=2.0, P=0.004) and decreased RI (hazards ratio=0.53, P=0.05). These results were more evident for AML transplant recipients (2-year LFS and RI with or without KIR-ligand incompatibility 73 versus 38% (P=0.012), and 5 versus 36% (P=0.005), respectively). UCBT for acute leukemia in CR from KIR-ligand-incompatible donors is associated with decreased RI and improved LFS and OS.

Role of CD4+ and CD8+ T cells in the rejection of heart or islet xenografts in recipients with xenotolerance in the innate immune compartment.

UNLABELLED: To further study the interactions between innate and adaptive immunity in xenotransplantation, we explored the relative contribution of T-cell subsets in vascularized (heart) and cellular (islets) xenografts in a model with established xeno-non-reactivity of the innate system. MATERIALS: Specific innate xenotolerance was induced in xenoheart (hamster) recipients (nude rats) by a tolerizing regimen (TR), consisting of donor antigen infusion, temporary natural killer (NK)-cell depletion and a 4-week administration of leflunomide. Hamster pancreatic islets were transplanted either 1 week after heart transplantation or alone and syngeneic T-cell adoptive transfer was performed 10 days later. Purified CD3(+), CD4(+), and CD8(+) T cells were given 2 weeks after withdrawal of all drugs. At the day of rejection, xenografts were removed for histology. Serum was taken and IgM and IgG xenoantibody titers were measured by flow cytometry. RESULTS: Both heart and islet grafts were rejected after CD4(+) reconstitution. After CD8(+) T-cell adoptive transfer, cellular grafts were not rejected but vascularized grafts were rejected, although only after several months. Rejection in CD4(+) reconstituted nude rats was accompanied by the generation of predominantly IgG xenoantibodies. CONCLUSION: CD4(+) T lymphocytes are able to rapidly initiate the rejection of islet xenografts in the presence of a xenotolerant innate immune system either by breaking the "innate tolerance" (e.g., by activating macrophages and NK-cells) or through a mechanism without any involvement of the innate tolerance (e.g., T-dependent IgG antibody production). In contrast, CD8(+) T cells provoke a late rejection of only xenoheart grafts.

Intergroup emotions: explaining offensive action tendencies in an intergroup context.

Three studies tested the idea that when social identity is salient, group-based appraisals elicit specific emotions and action tendencies toward out-groups. Participants' group memberships were made salient and the collective support apparently enjoyed by the in-group was measured or manipulated. The authors then measured anger and fear (Studies 1 and 2) and anger and contempt (Study 3), as well as the desire to move against or away from the out-group. Intergroup anger was distinct from intergroup fear, and the inclination to act against the out-group was distinct from the tendency to move away from it. Participants who perceived the in-group as strong were more likely to experience anger toward the out-group and to desire to take action against it. The effects of perceived in-group strength on offensive action tendencies were mediated by anger.

Cloning and characterization of Leishmania donovani telomeres.

We describe here the cloning and sequence characterization of the absolute termini of several telomeres from the human parasite Leishmania donovani using a vector-adapter protocol. The 3' protruding strand of L. donovani telomeres terminates with the sequence 5'-GGTTAGGGT-OH 3'. This single-stranded sequence is adjacent to tandemly repeated blocks of double-stranded sequence consisting of variable numbers of the hexameric repeat 5'-TAGGGT-3', variable numbers of an octameric repeat 5'-TGGTCATG-3', and a single 62-bp sequence, in that order. A number of additional, more chromosome-internal, nonrepeated sequences were found adjacent to the telomere sequences. Hybridization analyses indicated that some of these telomere adjacent sequences are found on all L. donovani chromosomes, some are more abundant on certain subsets of chromosomes, and some are unique to individual chromosomes.

LdARL-3A, a Leishmania promastigote-specific ADP-ribosylation factor-like protein, is essential for flagellum integrity.

The small G protein-encoding LdARL-3A gene, a homologue of the human ARL-3 gene, was isolated from Leishmania donovani, and its protein product characterised. It is unique in the Leishmania genome and expressed only in the extracellular promastigote insect form, but not in the intracellular amastigote mammalian form, as shown by northern blots and western blots developed with a specific anti-C terminus immune serum. Indirect immunofluorescence microscopy revealed distinct labelled spots regularly distributed on the plasma membrane, including the part lining the flagellum and the flagellar pocket. By transfection experiments, it was found that wild-type LdARL-3A-overexpressing promastigotes reached higher densities in culture, but released significantly less secreted acid phosphatase in the extracellular medium than the parental strain. When LdARL-3A blocked under the GDP-bound 'inactive' form or with an inactivated potential myristoylation site was overexpressed, the cells displayed an apparent wild-type phenotype, but died earlier in the stationary phase; in contrast to parental cells, they showed a diffuse pattern of fluorescence labelling in the cytoplasm and on the cell membrane. Strikingly, when a constitutively 'active' form of LdARL-3A (blocked under the GTP-bound form) was overexpressed, the promastigotes were immobile with a very short flagellum, a slow growth rate and a low level of acid phosphatase secretion; the length of the flagellum was inversely proportional to mutant protein expression. We concluded that LdARL-3A could be an essential gene involved in flagellum biogenesis; it may provide new approaches for control of the parasite at the insect stage.