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Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/beta-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT 10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated beta-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)gamma c(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.

Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/beta-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT 10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated beta-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)gamma c(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration. Neoplasia (2012) 14, 1236-1248, AuthorCount:17

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Alessandra Tedeschi, Eleonora Vismara, Francesca Ricci, Enrica Morra, Marco MontilloDepartment of Hematology, Niguarda Ca’ Granda Hospital, Milano, ItalyAbstract: The monoclonal chimeric anti-CD20 antibody, rituximab, has considerably improved therapeutic outcome in B-cell chronic lymphocytic leukemia. Rituximab has limited clinical activity when used as a single agent. The combination of the monoclonal antibody with fludarabine-based regimens clearly demonstrated, in Phase II and randomized trials, an increase in clinical efficacy in previously untreated and pretreated patients. Furthermore the addition of rituximab enabled the eradication of minimal residual disease, which is correlated with the prognosis in a high proportion of patients. Although the combination of rituximab with fludarabine-based regimens increased myelosuppression and immunosuppression, incidence of infections did not increase. The benefit of adding rituximab to other purine analogs or other chemotherapeutic combination regimens has also been explored. Moreover there could be a role for achieving better quality of responses with the combination of different monoclonal antibodies, considering that they target different antigens and exert different mechanism of action. Although the role of rituximab as maintenance therapy in low grade non-Hodgkin’s lymphomas has been determined, the benefit and optimal schedule in chronic lymphocytic leukemia are still under investigation. This review brings together knowledge of the pharmacokinetics, mechanism of action and clinical use of rituximab in chronic lymphocytic leukemia.Keywords: rituximab, B-cell chronic lymphocytic leukemia, first-line treatment, refractory/relapsed

As many options are now available to treat patients with de novo acute myeloid leukemia; the Italian Society of Hematology and two affiliated societies (SIES and GITMO) commissioned a project to an Expert Panel aimed at developing clinical practice guidelines for acute myeloid leukemia treatment. After a systematic comprehensive literature review, the Expert Panel formulated recommendations for the management of primary acute myeloid leukemia (with the exception of acute promyelocytic leukemia) and graded them according to the supporting evidence. When evidence was lacking, consensus-based statements have been added. First-line therapy for all newly diagnosed patients eligible for intensive treatment should include one cycle of induction with standard dose cytarabine and an anthracycline. After achieving complete remission, patients aged less than 60 years should receive consolidation therapy including high-dose cytarabine. Myeloablative allogeneic stem cell transplantation from an HLA-compatible sibling should be performed in first complete remission: 1) in children with intermediate-high risk cytogenetics or who achieved first complete remission after the second course of therapy, 2) in adults less than 40 years with an intermediate-risk; in those aged less than 55 years with either high-risk cytogenetics or who achieved first complete remission after the second course of therapy. Stem cell transplantation from an unrelated donor is recommended to be performed in first complete remission in adults 30 years old or younger, and in children with very high-risk disease lacking a sibling donor. Alternative donor stem cell transplantation is an option in high-risk patients without a matched donor who urgently need transplantation. Patients aged less than 60 years, who either are not candidate for allogeneic stem cell transplantation or lack a donor, are candidates for autologous stem cell transplantation. We describe the results of a systematic literature review and an

Francesca Ricci, Alessandra Tedeschi, Enrica Morra, Marco MontilloDepartment of Oncology/Haematology, Niguarda Ca’Granda Hospital, Milan, ItalyAbstract: Fludarabine (FAMP) is the most effective and most extensively studied purine analog in indolent B-cell malignancies. Its use is indicated for first- and second-line treatment of B-cell chronic lymphocytic leukemia (B-CLL). FAMP as a single agent has produced superior response rates and progression-free survival than standard therapy with chlorambucil and alkylator-based regimen. Efficacy of FAMP may be increased by combining this purine analog with other chemotherapeutic and non-chemotherapeutic agents. FAMP and cyclophosphamide combination (FC) has shown promising results with higher overall response and complete response rates than FAMP in monotherapy, although no difference has been detected in survival. Quality of response and eradication of minimal residual disease (MRD) have been reported to be associated with prolonged survival. Eradication of MRD has been achieved by combining FC with mitoxantrone or monoclonal antibody including alemtuzumab or rituximab or both. FAMP has been widely used in non-myeloablative conditioning regimens, often combined with a variety of other cytotoxic agents, with the aim of inducing enough immunosuppression to allow successful engraftment and to exert some pretransplant anti-tumor activity. The current paper provides an overview of use of FAMP as a single agent or as a cornerstone of different therapeutic strategies for treatment of B-CLL patients.Keywords: fludarabine, chronic lymphocytic leukemia, cyclophosphamide

Marco Montillo, Francesca Ricci, Sara Miqueleiz, Alessandra Tedeschi, Enrica MorraDepartment of Oncology/Hematology, Division of Hematology and Bone Marrow Transplant Unit, Niguarda Ca’ Granda Hospital, Milan, ItalyAbstract: The introduction of immunotherapeutic agents has provided renewed hope for Chronic lymphocytic leukemia fludarabine-refractory patients. Several clinical trials have shown that alemtuzumab is a more effective option compared to combination chemotherapy for treatment of patients who have relapsed or who are refractory to fludarabine, including those with poor prognostic factors. Although there are significant potential toxicities associated with alemtuzumab, such as infusional reactions and the risk of cytomegalovirus (CMV) reactivation, most are manageable. Pre-treatment anti-pyretics and anti-histamines are recommended to prevent or mitigate the acute infusional reactions associated with intravenous infusion. Recent use of alemtuzumab via the subcutaneous route has been shown to be well tolerated and has yielded similar response rates to the infusional method of administration. Prophylaxis with thrimethoprim/sulphamethoxazole (TMP/SMZ) as well as valacyclovir or a similar anti-viral can prevent many of the opportunistic infections seen in early trials. Reactivation of CMV infection can be effectively managed with monitoring and early treatment. Chemo-immunotherapy combination with alemtuzumab has been tested and demonstrated unprecedented clinical results in relapsed and refractory patients. The use of this agent earlier in the algorithm of patients with these characteristics should be considered. Future areas of research will include the use of alemtuzumab in combination with other monoclonal antibodies and other targeted therapies.Keywords: chronic lymphocytic leukemia, fludarabine, alemtuzumab

Background. Several studies showed the effectiveness of fibrin glue (FG) in reducing bleeding from cut surface in liver resection. This study compares the efficacy in reducing bleeding of a homemade FG, in comparison with a commercial product. Methods. Thirthy-four patients submitted to liver resection for carcinoma were enrolled. Fourteen of them received homemade FG, while the others the commercial product (Tissucol, Baxter). Homemade FG was obtained from allogeneic fresh frozen plasma by using the CryoSeal CS-1 system (Thermogenesis Corp.) device. Fibrinogen concentration in homemade FG was assessed according to the Clauss method. In all the patients the same amount of FG (5 mL) was sprayed on liver cut surface, at the end of the liver resection. Results. Mean fibrinogen concentration in homemade FG was 23.16±5.93mg/mL. In homemade group the baseline haematocrit (Hct) was 40.3±4.1%, in the Tissucol group 40.1±5.4% (p NS). The Hct in 5thpostoperative day was 35±4.1 in group treated with homemade FG, and 32.8±3.8 in pts receiving Tissucol (p<0.05). Blood loss in patients treated by Tissucol was superior to that observed in patients receiving homemade FG (527±251.6 vs 305.6±253.7mL, p<0.05). It has been observed that out of 14 patients treated with homemade FG, 2 (14.3%) received blood transfusion with a mean RBC volume of 500±141.4 mL, in Tissucol group 35% of the patients (p NS) have been transfused and received a mean RBC volume of 542±222.5mL (p NS). Conclusions. Despite the fibrinogen concentration in homemade FG was significantly lower than that reported to be present in Tissucol (75-115mg/mL), the use of homemade product was associated with a statistically significant reduction in blood loss

Tacrolimus (FK506)-induced hematological toxicity, which has rarely been reported in transplant recipients, may result in anemia episodes, reported mainly in kidney and heart transplant recipients, sporadic cases of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome, red cell aplasia (4 reported cases), and generalized bone marrow suppression (only 1 reported case). We describe a case of a liver transplant recipient with pancytopenia that appeared during immunosuppressive therapy with tacrolimus. This patient suffered from progressive anemia, leukopenia with severe neutropenia, and mild thrombocytopenia; bone marrow biopsy showed hypoplasia (20% of cellularity) without dysplasia. Bone marrow recovery was made possible by suspending tacrolimus and changing to immunosuppression with cyclosporine A, despite the two drugs being very similar in their mechanism of immunosuppression. Contrary to previously reported cases (pure red cell aplasia and bone marrow hypoplasia), the recovery of hemoglobin and neutrophil values was slow after tacrolimus suspension, even though in the first month transfusions were no longer necessary. ©2004 The Japanese Society of Hematology, Indications:Graft rejection prophylaxis in 1 patient who underwent orthotopic liver transplantation. Concomitant disease: bone marrow hypoplasia caused by tacrolimus.Patients:One 17-year-old girl.TypeofStudy:Bone marrow hypoplasia in liver transplant recipient during tacrolimus therapy, characterized by a slow but complete recovery with replacement of tacrolimus with Sandimmun was described. Case report.DosageDuration:Dosage not stated. Duration for 15 months.ComparativeDrug:Tacrolimus 0.15 mg/kg, bid. Duration not stated.Results:In the following month after replacement of tacrolimus with Sandimmun, hemoglobin levels increased slowly but constantly, and the patient was no longer blood-transfusion dependent within a month after suspension. At the 12th month after tacrolimus suspension, her hemoglobin level reached 12.5 g/dL; neutrophils were 2.9 x 109/L, and platelets 245 x 109/L. A bone marrow biopsy performed 15 months after discontinuation of tacrolimus showed normal cellularity (>70%).AdverseEffects:No adverse events were mentioned.AuthorsConclusions:Finally, it is important to emphasize that the change to cyclosporin A permitted recovery from pancytopenia, a result showing that, despite the two drugs being very similar as to mechanism of immunosuppression, the side effects impacting on bone marrow probably do not have the same etiologic mechanism, as in patients affected by tacrolimus-induced TTP [thrombocytopenic purpura].FreeText:The patient underwent urgent orthotopic liver transplantation. Postoperative course was regular; the prophylactic treatment, consisted of gancyclovir, 15 mg/kg daily for 2 weeks, and oral pyrimethamine-sulfadiazine 500 mg daily with coadministration of folate for 1 month. Immunosuppression therapy with steroids, tacrolimus (0.15 mg/kg, bid), and azathioprine (50 mg daily). Thirteen days after transplantation, normochromic normocytic anemia (hemoglobin 7.2 g/dL) with low reticulocytes (10 x 109/L ) appeared. Azathioprine was suspended

Udgivelsesdato: 2002-Dec, BACKGROUND AND OBJECTIVES: Novel therapeutic agents and strategies have been introduced into the management of myelodysplastic syndromes (MDS) in the last years. This has led to more treatment options and a better chance of long-term survival for MDS patients, but also to uncertainty regarding the optimal use and possible side effects of these treatments. The Italian Society of Hematology commissioned a project to develop guidelines for the therapy of MDS using evidence-based knowledge and consensus-formation techniques. DESIGN AND METHODS: An Advisory Council (AC) shaped the project around a series of key clinical questions, performed a systematic search for evidence and graded the available evidence according to the Scottish Intercollegiate Guidelines Network (SIGN). A list of clinical questions was mailed to each of 10 senior hematologists composing the Expert Panel (EP): the panelists were asked to rank the most relevant questions, and to formulate answers to the questions according to the tables of evidence. A scenario phase followed, so as to reach a consensus on the three top ranked questions. The EP was asked to score patient profiles as appropriate or not appropriate for the therapeutic strategy under scrutiny, according to the RAND technique. Finally, from September 2001 to January 2002, four Consensus Conferences conducted according to the Nominal Group Technique were held in Milan, Italy. The overall goal of the conferences was to take a final decision upon the appropriateness of the uncertain scenarios and of the uncertain responses to the clinical questions. RESULTS: Evidence was judged sufficient for providing recommendations on the use of allogeneic stem cell transplantation, leukemia-like chemotherapy, autologous stem cell transplantation, low-dose chemotherapy, danazol, immunosuppressive therapy, hypomethylating agents and hematopoietic growth factors. Specific recommendations for supportive therapy, including iron chelation, were issued. Allogenei

Udgivelsesdato: 2002-Dec, BACKGROUND AND OBJECTIVES: Novel therapeutic agents and strategies have been introduced into the management of myelodysplastic syndromes (MDS) in the last years. This has led to more treatment options and a better chance of long-term survival for MDS patients, but also to uncertainty regarding the optimal use and possible side effects of these treatments. The Italian Society of Hematology commissioned a project to develop guidelines for the therapy of MDS using evidence-based knowledge and consensus-formation techniques. DESIGN AND METHODS: An Advisory Council (AC) shaped the project around a series of key clinical questions, performed a systematic search for evidence and graded the available evidence according to the Scottish Intercollegiate Guidelines Network (SIGN). A list of clinical questions was mailed to each of 10 senior hematologists composing the Expert Panel (EP): the panelists were asked to rank the most relevant questions, and to formulate answers to the questions according to the tables of evidence. A scenario phase followed, so as to reach a consensus on the three top ranked questions. The EP was asked to score patient profiles as appropriate or not appropriate for the therapeutic strategy under scrutiny, according to the RAND technique. Finally, from September 2001 to January 2002, four Consensus Conferences conducted according to the Nominal Group Technique were held in Milan, Italy. The overall goal of the conferences was to take a final decision upon the appropriateness of the uncertain scenarios and of the uncertain responses to the clinical questions. RESULTS: Evidence was judged sufficient for providing recommendations on the use of allogeneic stem cell transplantation, leukemia-like chemotherapy, autologous stem cell transplantation, low-dose chemotherapy, danazol, immunosuppressive therapy, hypomethylating agents and hematopoietic growth factors. Specific recommendations for supportive therapy, including iron chelation, were issued. Allogenei

Background and objectives: Novel therapeutic agents and strategies have been introduced into the management of myelodysplastic syndromes (MDS) in the last years. This has led to more treatment options and a better chance of long-term survival for MDS patients, but also to uncertainty regarding the optimal use and possible side effects of these treatments. The Italian Society of Hematology commissioned a project to develop guidelines for the therapy of MDS using evidence-based knowledge and consensus-formation techniques. Design and methods: An Advisory Council (AC) shaped the project around a series of key clinical questions, performed a systematic search for evidence and graded the available evidence according to the Scottish Intercollegiate Guidelines Network (SIGN). A list of clinical questions was mailed to each of 10 senior hematologists composing the Expert Panel (EP): the panelists were asked to rank the most relevant questions, and to formulate answers to the questions according to the tables of evidence. A scenario phase followed, so as to reach a consensus on the three top ranked questions. The EP was asked to score patient profiles as appropriate or not appropriate for the therapeutic strategy under scrutiny, according to the RAND technique. Finally, from September 2001 to January 2002, four Consensus Conferences conducted according to the Nominal Group Technique were held in Milan, Italy. The overall goal of the conferences was to take a final decision upon the appropriateness of the uncertain scenarios and of the uncertain responses to the clinical questions. Results: Evidence was judged sufficient for providing recommendations on the use of allogeneic stem cell transplantation, leukemia-like chemotherapy, autologous stem cell transplantation, low-dose chemotherapy, danazol, immunosuppressive therapy, hypomethylating agents and hematopoietic growth factors. Specific recommendations for supportive therapy, including iron chelation, were issued. Allogenei