Jean-Loup Demory

Elevated levels of basic fibroblast growth factor in megakaryocytes and platelets from patients with idiopathic myelofibrosis

Idiopathic myelofibrosis, or agnogenic myeloid metaplasia, is a chronic myeloproliferative disorder characterized by clonal expansion and marrow fibrosis. Although marrow fibrosis appears to be a reactive process, it substantially contributes to impaired haemopoiesis. During the last few years the implication of megakaryocyte‐derived growth factors in its pathogenesis has been documented.

Dysregulation and overexpression of HMGA2 in myelofibrosis with myeloid metaplasia.

Among cytogenetic studies of patients affected with myelofibrosis with myeloid metaplasia (MMM), a rare chronic myeloproliferative disorder, we found several reports of structural abnormalities of the long arm of chromosome 12. Two MMM patients had a balanced translocation involving 12q: t(4;12)(q32;q15) and t(5;12)(p14;q15), respectively. FISH (fluorescence in situ hybridization) analysis showed that BAC (bacterial artificial chromosome) RP11‐366L20 overlaps the breakpoint in both cases. A gene, HMGA2, most of which is included in that BAC, thus was identified as a potential candidate. Using reserves transcriptase–polymerase chain reaction (RT‐PCR), we looked for expression of HMGA2 in blood mononuclear cells from these 2 patients and demonstrated a transcript in both. Moreover, we found the gene expressed in the hematopoietic cells of 10 of 10 additional patients bearing no 12q anomalies. HMGA2, not expressed in normal subjects, is implicated in benign solid tumors such as lipomas, leiomyomas, and other rare tumors of mesenchymal origin. We postulate that its dysregulation and overexpression in myeloid progenitors contribute also to the pathogenesis of MMM.

Diagnosis, pathogenesis and treatment of the myeloproliferative disorders essential thrombocythemia, polycythemia vera and essential megakaryocytic granulocytic metaplasia and myelofibrosis

According to strict clinical, hematological and morphological criteria, the Philadelphia (Ph) chromosome negative chronic myeloproliferative disorders essential thrombocythemia (ET), polycythemia vera (PV), and agnogenic myeloid (megakaryocytic/granulocytic) metaplasia (AMM) or idiopathic myelofibrosis (IMF) are three distinct disease entities with regard to clinical manifestations, natural history and outcome in terms of life expectancy. As clonality studies have clearly demonstrated that fibroblast proliferation in AMM, as well as in many other conditions such as advanced stages of Ph(+)-essential thrombocythemia, Ph(+)-granulocytic leukemia, and Ph(-)-polycythemia vera, is polyclonal indicating that myelofibrosis is secondary to the megakaryocytic granulocytic metaplasia in these various conditions, AMM is illogically labeled as IMF. As abnormal megakaryocytic granulocytic metaplasia is the essential feature preceding the early prefibrotic stage of AMM, the term essential megakaryocytic granulocytic metaplasia (EMGM) can readily be used to characterize this condition more appropriately at the biological level. Clinical, hematological and morphological characteristics, in particular megakaryocytopoiesis and bone marrow cellularity, reveal diagnostic features, which enable a clear-cut distinction between ET, PV and EMGM or classical IMF. The characteristic increase and clustering of enlarged megakaryocytes with mature cytoplasm and multilobulated nuclei and their tendency to cluster in a normal or only slightly increased cellular bone marrow represent the hallmark of ET. The characteristic increase and clustering of enlarged mature and pleiomorphic megakaryocytes with multilobulated nuclei and proliferation of erythropoiesis in a moderate to marked hypercellular bone marrow with hyperplasia of dilated sinuses are the specific diagnostic features of untreated PV. EMGM, including the early prefibrotic stages as well as the various myelofibrotic stages of classical IMF appear to be a distinct neoplastic dual proliferation of abnormal megakaryopoiesis and granulopoiesis. The histopathology of the bone marrow in prefibrotic EMGM and in classical IMF is dominated by atypical, enlarged and immature megakaryocytes with cloud-like immature nuclei, which are not seen in ET and PV at diagnosis and during follow-up. Myelofibrosis in ET, PV and EMGM is graded into: no reticulin fibrosis (MF0), early reticulin fibrosis (MF1), advanced reticulin sclerosis with minor or moderate collagen fibrosis (MF2) and advanced collagen fibrosis with osteosclerosis (MF3). Myelofibrosis is not a feature of ET at diagnosis and during long-term follow-up. Myelofibrosis may be present in a minority of PV-patients at diagnosis and usually becomes apparent during long-term follow-up in the majority of PV-patients. Myelofibrosis secondary to the abnormal megakaryocytic and granulocytic myeloproliferation constitutes a prominent feature in the majority of EMGM/IMF at time of diagnosis and usually progresses more or less rapidly during the natural history of the disease. Life expectancy is normal in ET, normal during the 1st ten years and compromised during the 2nd ten years follow-up in PV, but significantly shortened in the prefibrotic stage of EMGM as well as in the various myelosclerotic stages of classical IMF. First line treatment options in prospective randomized clinical trials of newly diagnosed MPD-patients are control of platelet function with low-dose aspirin versus reduction of platelet count with anagrelide, interferon or hydroxyurea in ET; control of platelet and erythrocyte counts by interferon alone versus bloodletting plus hydroxyurea on indication in PV; interferon versus no treatment in the early stages of EMGM; a wait and see strategy in the fibrotic stages of EMGM or classical IMF with favorable prognostic factors, and bone marrow transplantation in classical IMF with poor prognostic factors at presentation or during short-term follow-up.

Efficacy and safety of pegylated-interferon α-2a in myelofibrosis: a study by the FIM and GEM French cooperative groups.

Myeloproliferative neoplasm‐related myelofibrosis is associated with cytopenic or proliferative phases, splenomegaly and constitutional symptoms. Few effective treatments are available and small series suggested that interferon could be an option for myelofibrosis therapy. We performed a retrospective study of pegylated‐interferon α‐2a (Peg‐IFNα‐2a) therapy in myelofibrosis. Sixty‐two patients treated with Peg‐IFNα‐2a at 17 French and Belgian centres were included. Responses were determined based on the criteria established by the International Working Group for Myelofibrosis Research and Treatment. Mean follow‐up was 26 months. Sixteen of 25 anaemic patients (64%) (eight concomitantly receiving recombinant erythropoietin) achieved a complete response and transfusion‐independence was obtained in 5/13 patients (38·5%). Constitutional symptoms resolved in 82% of patients. All five leucopenic patients normalized their leucocyte counts, whereas a normal platelet count was obtained in 5/8 thrombocytopenic patients. Splenomegaly was reduced in 46·5% of patients, and complete resolution of thrombocytosis and leucocytosis were observed in 82·8% and 68·8% of patients, respectively. Side effects (mostly haematological) were mainly of grade 1–2. The only factor independently associated with treatment failure was a spleen enlargement of more than 6 cm below the costal margin. In conclusion, Peg‐IFNα‐2a induced high response rates with acceptable toxicity in a large proportion of patients with primary and secondary myelofibrosis, especially in early phases.

Identification during the follow-up of time-dependent prognostic factors for the competing risks of death and blast phase in primary myelofibrosis: a study of 172 patients

The median survival of patients with primary myelofibrosis ranges from 3.5 to 5.5 years, and most patients die from cause related to the disease, including blast phase (BP, in 5%-30% of cases). Because identification of high-risk patients might use information collected during the clinical course, we assessed the prognostic value of time-dependent covariates for 2 competing risks (death and BP) in a series of 172 patients. Significant (P < .01) adverse time-dependent prognostic factors for the risk of death were the time to onset of anemia (hemoglobin < 100 g/L [10 g/dL]), leukocytosis (leukocyte count > 30 x 10(9)/L), thrombocytopenia (platelet count < 150 x 10(9)/L), presence of circulating blasts, intermediate-high or high International Working Group for Myelofibrosis Research and Treatment score, and time to splenectomy. The first 3 dependent covariates and the time to chemotherapy initiation (P = .05) were prognostic factors for the risk of BP. The prognostic effect of onset of leukocytosis was significantly more pronounced for BP than for death. Thus, occurrence during the follow-up of characteristics associated with an adverse prognostic value at diagnosis also influenced the risks of death and BP. Patients with leukocytosis should be closely monitored. These data might efficiently help to evaluate the severity of the disease before treatment decision during the clinical course.

Expression of HMGA2 in PB leukocytes and purified CD34+ cells from controls and patients with Myelofibrosis and myeloid metaplasia.

INSERM Unité 524, Institut de Recherche sur le Cancer de Lille, Lille, France, Laboratoire de Génétique Médicale, Hôpital Jeanne de Flandre, CHRU de Lille, France, Laboratoire d’Hématologie, CHU de Bordeaux, France, INSERM U602, Hôpital Paul Brousse, Villejuif, France, Service des Maladies du Sang, CHRU de Lille, France, Service d’Hématologie, Hôpital Calmette, CHRU de Lille, France, and Département d’Hématologie, Université Catholique de Lille, France

Tetraspanin CD9 participates in dysmegakaryopoiesis and stromal interactions in primary myelofibrosis

Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the “bad seed in bad soil” hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis.

Benefits and pitfalls of pegylated interferon-α2a therapy in patients with myeloproliferative neoplasm-associated myelofibrosis: a French Intergroup of Myeloproliferative neoplasms (FIM) study

We have previously described the safety and efficacy of pegylated interferon-α2a therapy in a cohort of 62 patients with myeloproliferative neoplasm-associated myelofibrosis followed in centers affiliated to the French Intergroup of Myeloproliferative neoplasms. In this study, we report their long-term outcomes and correlations with mutational patterns of driver and non-driver mutations analyzed by targeted next generation sequencing. The median age at diagnosis was 66 years old, the median follow-up since starting pegylated interferon was 58 months. At the time of analysis, 30 (48.4%) patients were alive including 16 still being treated with pegylated interferon. The median survival of patients with intermediate and high-risk prognostic Lille and dynamic International Prognostic Scoring System scores treated with pegylated interferon was increased in comparison to that of historical cohorts. In addition, overall survival was significantly correlated with the duration of pegylated interferon therapy (70 versus 30 months after 2 years of treatment, P<10−12). JAK2V617F allele burden was decreased by more than 50% in 58.8% of patients and two patients even achieved complete molecular response. Next-generation sequencing analyses performed in 49 patients showed that 28 (57.1%) of them carried non-driver mutations. The presence of at least one additional mutation was associated with a reduction of both overall and leukemia-free survival. These findings in a large series of patients with myelofibrosis suggest that pegylated interferon therapy may provide a survival benefit for patients with intermediate- or high-risk Lille and dynamic International Prognostic Scoring System scores. It also reduced the JAK2V617F allele burden in most patients. These results further support the use of pegylated interferon in selected patients with myelofibrosis.

Microparticle phenotypes are associated with driver mutations and distinct thrombotic risks in essential thrombocythemia

Essential thrombocythemia (ET) is a Philadelphia-negative myeloproliferative neoplasm with high platelet counts and an increased risk of thrombosis. Acquired somatic mutations delineate four ET subtypes: JAK2-V617F , CALR -mutated, MPL -mutated, and “Triple-Negative” (TN).[1][1] CALR mutations