Imatinib mesylate is the sole BCR–ABL tyrosine kinase inhibitor approved as first-line treatment of accelerated-phase (AP) chronic myeloid leukemia (CML). Indication was based on the STI571 0109 study, in which imatinib favorably compared to historical treatments in patients failing prior therapies. The relevance of these results to currently newly diagnosed AP-CML patients remains unknown. We evaluated the benefit of imatinib in 42 newly diagnosed AP-CML patients. In all, 16 patients had hematological acceleration without chromosomal abnormalities in addition to the Philadelphia chromosome (ACAs; HEM-AP), 16 solely had ACAs (ACA-AP) and 10 had hematological acceleration plus ACAs (HEM-AP+ACA). Major cytogenetic responses were achieved in 93.7% of HEM-AP patients, 75% of patients with ACA-AP ( P =NS) and 40% of patients with HEM-AP+ACA ( P =0.0053). The 24-month failure-free survival rate was 87.5% in HEM-AP patients, 43.8% in ACA-AP patients and 15% in HEM-AP+ACA patients ( P =0.022). The 24-month estimate of progression-free survival was 100% in HEM-AP patients, 92.8% in ACA-AP patients and 58.3% in HEM-AP+ACA patients ( P =0.0052). In conclusion, frontline imatinib allows favorable outcomes in HEM-AP and ACA-AP patients but appears insufficient for patients with HEM-AP+ACA. Broader-target and/or more potent BCR–ABL tyrosine kinase inhibitors alone or in combination may be considered in this setting.

Le nilotinib (Tasigna®) est un inhibiteur de tyrosine-kinase de deuxième génération (ITK2) d'introduction récente, indiqué dans le traitement des patients atteints de leucémie myéloïde chronique (LMC) en phase chronique (PC) ou accélérée (PAcc), intolérants ou résistants à l'imatinib. Ce traitement a permis une avancée importante dans la maîtrise de la maladie chez de tels patients, mais peut être source d'effets indésirables gênants, parfois sévères, qui peuvent limiter son utilisation optimale. Nous proposons dans ce travail des recommandations pratiques qui sont susceptibles d'aider le prescripteur dans la gestion de ces effets indésirables.

Nilotinib (Tasigna) is a second-generation BCR-ABL kinase inhibitor, recently introduced and used for the treatment of chronic or accelerated phase CML patients, intolerant or resistant to imatinib. This treatment represents and important step forward for the disease control of such patients but can lead to side effects, sometimes serious, which can limit its optimal use. We propose here some guidelines that might be of help in daily practice, in order to manage properly these side effects.

The emergence of ABL point mutations is the most frequent cause for imatinib resistance in chronic myelogenous leukemia (CML) patients and can occur during any phase of the disease; however, their clinical impact remains controversial. In this study, we retrospectively analyzed the predictive impact of 94 BCR-ABL kinase domain mutations (18 T315I, 26 P-loop, 50 in other sites) found in 89 imatinib-resistant CML patients. At imatinib onset, 64% of patients (57/89) were in chronic phase (CP), 24% (21/89) in accelerated phase (AP) and 12% (11/89) in blastic phase (BP). T315I and P-loop mutations were preferentially discovered in accelerated phase of BP CML, and other types of mutations in CP ( P =0.003). With a median follow-up of 39.2 months (6.3–67.2), since imatinib initiation, overall survival (OS) was significantly worse for P-loop (28.3 months) and for T315I (12.6 months), and not reached for other mutations ( P =0.0004). For CP only, multivariate analysis demonstrated a worse OS for P-loop mutations ( P =0.014), and a worse progression-free survival (PFS) for T315I mutations ( P =0.014). Therefore, P-loop and T315I mutations selectively impair the outcome of imatinib-resistant CML patients, in contrast to other mutations, which may benefit from dose escalation of imatinib, able to improve or stabilize disease response.

Between October 1985 and October 1989, 75 previously untreated patients with stage III and IV non Hodgkin's lymphoma, large cell type, were treated with an alternating weekly chemotherapy regimen including the following drugs: week 1: Doxorubicin, vincristine, cyclophosphamide, bleomycin, and intrathecal (i.th.) methotrexate and cytarabine; week 2: Methotrexate with leucovorin rescue: week 3: Doxorubicin, ifosfamide with mesna, etoposide, and i.th. methotrexate and cytarabine; week 4: Methotrexate with leucovorin rescue. Complete responders after three cycles according to this schedule (12 weeks) were given 18 gys cranial irradiation and randomized between one additional cycle or three monthly CHOP (consolidation treatment). Among 66 evaluable patients, 53 achieved a complete remission (CR 80 per cent) and seven a partial remission (11 per cent). There were six failures, and nine early deaths during the initial phase, mostly due to septic problems. Forty-one of the 53 CR patients (77-3 per cent) have remained free of disease with a median follow-up of 15 months (1-49). Eight of the 12 relapses occurred during the first year, the four others at 13, 14, 16 and 38 months respectively. The 2-year survival was 63 per cent for the whole group, and 77 per cent for the CR group. No difference has been observed up until now between the two groups with different consolidation treatment. Therefore, this protocol seems to be able to produce a high rate of complete and durable remission. The analysis of prognostic factors suggests that some high-risk patients should be considered for intensification therapy with the support of autologous bone marrow transplantation.

Several lines of evidence obtained from idiopathic myelofibrosis (IM) studies are in favour of a crucial role of the NF-κB pathway activation in myelofibrosis induction. It has been demonstrated that megakaryocytes, monocytes but also CD34+ cells from IM patients present a spontaneous NF-κB pathway activation associated with transforming growth factor-β1 (TGF-β1) secretion. This growth factor has been previously shown as the main fibrogenic cytokine involved in the myelofibrosis development. Mice exposed to high systemic levels of thrombopoietin (TPO) mediated by a retroviral vector (TPOhigh mice) develop a myeloproliferative disorder featuring numerous aspects of the human disease including bone marrow fibrosis, extramedullary hematopoiesis and dysmegakaryopoiesis. Moreover, TPOhigh mice display high plasma levels of IL-1α suggesting that the NF-κB pathway may play a role in this model of fibrosis. We then conducted a study to investigate whether NF-κB inhibition in this mice model could have an impact on myelofibrosis development using the proteasome inhibitor bortezomib. One month after engraftment with TPO-overexpressing hematopoietic cells, 2 groups of 20 immunocompetent C57BL/6J mice displaying similar myeloproliferation induced by TPO overexpression were constituted. Mice engrafted were treated twice a week, with either bortezomib (1 mg/kg) or a placebo for 4 weeks. At the end of this protocol, mice from both groups were examined for histological and haematological analysis including TGF-β1 and IL-1α levels determination. Here, we demonstrate that: i) the NF-κB pathway is activated in TPOhigh spleen cells and bortezomib treatment is able to inhibit this activation; ii) bortezomib treatment is able to decrease plasma concentration of TGF-β1 and IL-1α in mice as well as TGF-β1 content in extracellular fluids of marrow and spleen; iii) myelofibrosis development is inhibited after bortezomib treatment. These results emphasize the interest of developing bortezomib treatment in IM patients.

Polycythemia Vera (PV) is a myeloproliferative disorder (MPD), whose diagnosis is currently based on an association of clinical and biological criteria following the WHO or the PVSG classification. It is characterized by a primitive absolute erythrocytosis, and formation of endogenous erythroid colonies (EEC). Recently, we described a point mutation in JAK2 (JAK2 V617F) and showed that this activating mutation was the cause of the disease (James et al, Nature, 2005). This molecular abnormality was therefore likely to be a diagnostic marker of PV, as bcr-abl for chronic myeloid leukemia (CML). Nevertheless, JAK2 V617F is also found in other MPDs, sharing some common features with PV, as essential thrombocythemia, idiopathic myelofibrosis, and other rare MPDs. One major criterion for the diagnosis of PV requires the demonstration of increased red cell mass as measured by isotopic methods. We assessed the value of detection of JAK2 V617F as a first intention diagnostic test in 88 patients with hematocrit values above 51% (=erythrocytosis) and showed that the mutation correlated with the diagnosis of PV according to the WHO (R=0.879) and the PVSG (R=0.717) criteria, with a positive predictive value of 100% in the context of erythrocytosis. Besides, the presence of the mutation strongly correlated with EEC formation in 81/87 patients (R=0.824) and only weakly with the serum erythropoietin level (R=0,416). PCR-based genotyping techniques are less time-consuming, less expensive, than DNA sequencing and are easier to perform in hematology diagnostic laboratories. Therefore, we studied the feasibility of the detection of JAK2 V617F with widespread instruments commonly used in routine. We analyzed 119 samples from patients with a suspicion of myeloproliferative disease and showed that JAK2 V617F was efficiently detected by LightCycler® and TaqMan® genotyping technologies, these latter being a little more sensitive than sequencing. For 50 patients, peripheral blood and bone marrow samples were both available. In all cases (34 mutated, 16 non-mutated) the mutation was identically detected. Based on these results, we propose that the detection of JAK2 V617F in granulocytes has a first place in the diagnostic chart of an erythrocytosis, as bcr-abl in CML, avoiding, if positive, an isotopic red cell mass measurement and bone marrow EEC assays. The presence of JAK2 V617F in a patient with erythrocytosis would then lead to the diagnosis of MPD of PV type. Further prospective studies will be necessary to assess if all the MPDs patients bearing JAK2 V617F can be grouped in a new subset within the MPD entity, especially in term of thrombotic and neoplasic risk.

The diagnosis of polycythemia vera (PV) is currently based on clinical and biological criteria defined either by the World Health Organization (WHO) or the Polycythemia Vera Study Group (PVSG).1, 2 Both of these classifications use clinical and biological markers organized in major and minor criteria, allowing to diagnose a PV when a define combination of major and minor criteria is present. Recently, a somatic point mutation of the tyrosine kinase JAK2 (JAK2 V617F) has been described in about 80% of PV patients as well as 30% of essential thrombocythemia (ET) and 50% of idiopathic myelofibrosis (IMF)3, 4, 5, 6 and several in vitro and in vivo experiments demonstrated that this mutation of JAK2 was the molecular event at the origin of PV.3 It was therefore tempting to use the detection of JAK2V617F as a diagnostic test for erythrocytosis. Such an attitude supposes the development of other techniques than sequencing, as this latter is time consuming and not always feasible in hematology laboratories. We thus compared sequencing with two techniques of real-time PCR-based mutation detection (one using the LightCycler® instrument, the other the Taqman® ABI Prism 7500), for the efficiency to detect the JAK2 V617F mutation in 119 samples from patients with suspicion of myeloproliferative disorder (MPD). The three techniques were equivalent in all samples but one, where sequencing failed to detect the mutation revealed by both LightCycler® and Taqman® technologies. To evaluate the sensitivity of these techniques, we tested serial dilutions of the homozygously mutated HEL cell line DNA in the nonmutated TF-1 cell line DNA, and serial dilutions of the genomic DNA from a patient homozygous for the JAK2 V617F mutation in normal DNA. Sequencing failed to detect the mutated allele under 5% of HEL cell line DNA diluted in TF-1 cell line DNA, and under 10% of the homozygously mutated patient's DNA diluted in normal DNA. The sensitivity of LightCycler® and Taqman® techniques were equivalent, slightly higher than sequencing, reaching 0.5–1% of HEL cell line DNA diluted in TF-1 cell line DNA (Figure 1), and 2–4% of a homozygously mutated patient's DNA diluted in normal DNA.

BACKGROUND AND OBJECTIVES:Myelofibrotic bone marrow displays abnormal angiogenesis but the pathogenic mechanisms of this are poorly understood. Since pericyte abnormalities are described on solid tumor vessels we studied whether vessel morphology and pericyte coverage in bone marrow samples from patients with myelofibrosis differed from that in samples from controls.DESIGN AND METHODS:We assessed the microvascular density (MVD), vessel morphology and pericyte coverage in bone marrows from 19 myelofibrosis patients and nine controls. We also studied the same parameters in two mouse models of myelofibrosis, with genetic alterations affecting megakaryocyte differentiation (i.e. one model with low GATA-1 expression and the other with over-expression of thrombopoietin).RESULTS:In myelofibrotic marrows, MVD was 3.8-fold greater than in controls (p<0.001) and vessels displayed 5.9-fold larger mean perimeters (p<0.001). MVD was 1.8-fold greater in JAK2 V617F-positive than in negative patients (p=0.026). Moreover, 92+/-11 % of vessels in patients with myelofibrosis were pericyte-coated but only 51+/-20 % of vessels in controls (p<0.001). In the two mouse models of myelofibrosis caused by targeting megakaryocytopoesis, wide, pericyte-coated and morphologically aberrant vessels were detected. MVD was significantly greater in bone marrow and spleen samples from animals with myelofibrosis than in wild-type mice.INTERPRETATION AND CONCLUSIONS:We conclude that angiogenesis is similarly abnormal in human and murine myelofibrosis with intense pericyte coating, presumably related to abnormal megakaryocytopoiesis.

In CML, the most frequent mechanism responsible for IM resistance is the onset of BCR-ABL mutations. Mutations interfering with IM binding to the ABL-kinase domain that restore the tyrosine kinase activity of BCR-ABL (T315I and P-loop mutations) are of poor prognosis. In a retrospective analysis, we analysed the features and clinical outcomes of 50 CML patients and presenting IM resistance, and harbouring 33 P-loop (1 patient had mutations) [Group 1 (G1)] and 18 T315I mutations [Group 2 (G2)] detected by direct sequencing. Twenty-eight patients were in chronic phase (CP), 12 in accelerated phase and 9 in blastic phase when IM was started, with no difference between P-loop and T315I groups. There were 23 M and 9 F, with a median age of 53 (13–74) for G1; and 14 M and 4 F, with a median age of 52 (28–70) for G2. The median duration of IM was 25 Mo (2.4–50) for G1 and 20 (0.5–145) for G2 (p=ns). The duration of IFN prior to IM was equivalent for both groups (12 Mo (0–86) for G1 vs 10 Mo (4–131) for G2, p=ns). The median interval between diagnosis and day 1 of IM was 50 Mo(0.4–128) for G1 and 20 Mo (0.5–145) (p=ns) for G2. Multivariate analysis for gender, age, prior IFN, interval diagnosis-IM start, and major cytogenetic remission achievement with IM, did not show any significant impact of these variables. Overall and Progression free survival (PFS) Kaplan Meier curves for all phases did not show any difference, however, PFS curves for CP only showed a somewhat worse survival for Y253H+E255K P-loop mutations than for other P-loop mutations or T315I (p=0.05).

Background. Post-transplantation lymphoproliferative disorders develop in 1 to 10p. 100 of organ transplant recipients and are frequently associated with Epstein-Barr virus (EBV). Among post-transplantation lymphoproliferative disorders, plasmacytoma with cutaneous involvement is exceptional. Association with EBV has been rarely reported in post-transplantation plasmacytomas and the latency type of EBV has never been characterized. We report. 2 new cases cutaneous monotype EBV-related plasmacytomas.Case-reports. Clinical presentation was a sub-cutaneous tumor on the thigh in the first case and an ulcerated nodule of the leg in the second case, occurring respectively 7 and 8 years after organ transplantation (liver transplantation and heart transplantation). In both lesions, tumor cells exhibited lambda light chain restriction and the association with EBV was confirmed using immunohistochemistry and it situ hybridization. The expression of EBV genes in tumor cells demonstrated type III latency. Discussion. The classification of post-transplantation lymphoproliferative disorders is not well defined and some authors retain 3 categories. Among the latter, plasmacytomas have been rarely described. Cutaneous involvement is reported it 4 cases and an association with EBV in only 2 cases without description of viral latency. Clinical and histological features of post-transplantation plasmacytomas appear polymorphic. We report EBV-association it both cases, with a type III latency clearly demonstrated in one case, as has been reported in other lymphoproliferative diseases in patients with congenital or acquired immunodeficiency. We also discuss various possible theraputic strategies for post-transplantation lymphoproliferative disorders.

Human herpesvirus 8 (HHV-8) is associated with the development of Kaposi's sarcoma (KS) and rare lymphoproliferative disorders in immunosuppressed patients. The risk of HHV-8 transmission by liver transplantation and the clinical manifestations of primary infection in this setting have yet to be determined. In order to evaluate this risk, we measured the seroprevalence of HHV-8 among 122 liver donors and their respective recipients before and after transplantation. Molecular methods and immunohistochemical analyses were performed to study the features of HHV-8 infection. Antibodies to HHV-8 were detected in sera of 4 donors before transplantation (3.3%) and of 3 recipients (2.4%). None of the 3 recipients, who were HHV-8 seropositive before transplantation, developed a KS during the follow-up. Four primary HHV-8 infections were detected among the 4 HHV-8 seronegative recipients who received a liver from an HHV-8 positive donor. Among these 4 recipients, 2 particularly immunosuppressed patients developed symptomatic diseases and died a few months after transplantation, harboring disseminated KS and HHV-8 positive lymphoproliferation. In these 2 patients, HHV-8 DNA genome sequences were detectable in peripheral blood mononuclear cells and other tissues with high viremia levels before and at the beginning of HHV-8-related diseases. In conclusion, in liver transplantation recipients, HHV-8 primary infection can be associated with fatal outcome. This study raises the question of screening liver donors for HHV-8--even in low HHV-8 infection prevalence countries--not systematically to exclude the graft but to monitor, clinically and biologically, patients who received a graft from an HHV-8-infected donor.

Myelofibrosis is a clinical feature of several hematopoietic disorders and is most prominent in idiopathic myelofibrosis. It is characterized by excessive deposits of extracellular matrix proteins which occur as a marrow microenvironment reactive response to cytokines released from the clonal malignant myeloproliferation. The observation that mice exposed to high systemic levels of thrombopoietin (TPO) invariably develop a myelofibrosis within a period of 4 to 8 weeks has allowed the demonstration of the crucial role of TGF-b1 released by hematopoietic cells in the promotion of myelofibrosis (Chagraoui et al Blood, 2002 ; 100 :3495). The aim of this study was to investigate whether TGF-b1 inhibition could directly inhibit the fibrosis development in a curative approach of this murine model.