High serum levels of extracellular vesicles expressing malignancy-related markers are released in patients with various types of hematological neoplastic disorders.

Many cell types release extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic bodies, which play a role in physiology and diseases. Presence and phenotype of circulating EVs in hematological malignancies (HMs) remain largely unexplored.The aim of this study was to characterize EVs in peripheral blood of HM patients compared to healthy subjects (controls). We isolated serum EVs from patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), Waldenstrom's macroglobulinemia (WM), Hodgkin's lymphoma (HL), multiple myeloma (MM), acute myeloid leukemia (AML), myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS), and controls. EVs were isolated from serum of peripheral blood by ultracentrifuge steps and analyzed by flow cytometry to define count, size, and immunophenotype. MV levels were significantly elevated in WM, HL, MM, AML, and some MPNs and, though at a lesser degree, in CLL and NHL as compared to healthy controls. HL, MM, and MPNs generated a population of MVs characterized by lower size (below 0.3 µm) when compared to controls. MVs from patients specifically expressed tumor-related antigens, such as CD19 in B cell neoplasms, CD38 in MM, CD13 in myeloid tumors, and CD30 in HL. Both total and antigen-specific count of MVs significantly correlated with different HM clinical features such as Rai stage in CLL, International Prognostic Scoring System in WM, International Staging System in MM, and clinical stage in HL. MVs may represent a novel biomarker in HMs.

HDAC inhibitors repress BARD1 isoform expression in acute myeloid leukemia cells via activation of miR-19a and/or b.

Over the past years BARD1 (BRCA1-associated RING domain 1) has been considered as both a BRCA1 (BReast Cancer susceptibility gene 1, early onset) interactor and tumor suppressor gene mutated in breast and ovarian cancers. Despite its role as a stable heterodimer with BRCA1, increasing evidence indicates that BARD1 also has BRCA1-independent oncogenic functions. Here, we investigate BARD1 expression and function in human acute myeloid leukemias and its modulation by epigenetic mechanism(s) and microRNAs. We show that the HDACi (histone deacetylase inhibitor) Vorinostat reduces BARD1 mRNA levels by increasing miR-19a and miR-19b expression levels. Moreover, we identify a specific BARD1 isoform, which might act as tumor diagnostic and prognostic markers.

Comparison of fixed dose pegfilgrastim and daily filgrastim after autologous stem cell transplantation in patients with multiple myeloma autografted on a outpatient basis.

Different authors have explored the feasibility of autografting patients with multiple myeloma (MM) on an outpatient basis. Peg-filgrastim (PEG), a long-acting recombinant G-CSF, has similar efficacy when compared to conventional G-CSF for chemotherapy-induced neutropenia, but little is known about its use in the autologous stem-cell transplantation (ASCT) setting, namely in patients programmed to be autografted on outpatient basis. In this study, we compared therapeutic results in terms of hematopoietic recovery, non-hematologic toxicity, duration of hospitalization and percentage of hospital readmission between patients receiving either conventional G-CSF or PEG. Thirty-eight MM patients (48 autografts) received PEG, given at a single dose of 6 mg at day +5 from stem cell infusion, while 81 (113 autografts) received G-CSF from day + 2 up to stable neutrophil recovery. The conditioning regimen was high dose melphalan in all patients. The median age and the median number of CD34 + cell infused were comparable between the two groups. Overall, a second hospital admission was required in 36 procedures out of 161 (32%). Febrile neutropenia (FN) and severe mucositis were the most frequent causes of hospitalization. There was no statistically significant difference as percentage of hospital readmission is concerned: in the PEG group readmission was needed in 6 out of 48 autografts (12%) as opposed to 30 out of 113 (26%) in the G-CSF subgroup, p: 0.06. The median time of hospital stay for readmitted patients was identical for the two subgroups (9 days vs. 9 days, p: 0.94). Finally, one case of transplant related mortality occurred in the whole patient series (0.6%). In conclusion, ASCT on an outpatient basis is feasible and safe in patients with MM, the majority of whom are manageable at home. The administration of single dose PEG results in no different outcome in terms of safety and efficacy as compared to 8 days of G-CSF.

Continuous sequential infusion of fludarabine and cytarabine for elderly patients with acute myeloid leukaemia secondary to a previously diagnosed myelodysplastic syndrome.

Acute myeloid leukaemia (AML) secondary to myelodysplastic syndrome (MDS) is characterized by poor prognosis, namely in older patients. The combination of fludarabine (F) with cytarabine (ARA-C) ± G-CSF was proven as effective in patients with poor risk AML. The efficacy and toxicity of a regimen including F + ARA-C as sequential continuous infusion (CI-FLA) in 64 untreated patients aged >60 years, in which AML arose after a previous MDS, was investigated. Median age was 67 years (61-81). In patients achieving CR, an additional course, followed by G-CSF to mobilize CD34+ cells and subsequent autologous stem cell transplantation (ASCT) were programmed. Overall, 43 patients (67%) achieved complete remission (CR). There were 10 induction deaths (16%), while 11 patients (17%) were refractory to induction treatment. Thirty-four patients (79% of remitters) were eligible for the consolidation and 30 were monitorized for the mobilization of CD34+ cells, collection being successful in 20 of them (67%). Median number of CD34+ cells/kg collected was 6.8 × 10E6. Thirteen patients (20% of the whole population) received ASCT. Median disease free survival (DFS) and overall survival (OS) were 10 and 9 months, respectively. Survival at 5 years is projected to 15%. The only parameter significantly related to either DFS duration or OS duration was unfavourable cytogenetics, which did significantly influence also CR achievement. CI-FLA is effective in elderly patients with AML secondary to previously diagnosed MDS. Best results are achievable in the subgroup of patients with diploid karyotype.

Autologous stem cell transplantation for patients with acute promyelocytic leukemia in second molecular remission.

Relapse still occurs in approximately 20-30% of patients with acute promyelocytic leukemia (APL) and, after achievement of second complete remission (CR), the optimal strategy is still controversial. We describe therapeutic results from a series of 13 patients autografted in second molecular remission (MR) by a molecular negative apheresis product. In all patients, the disease was confirmed at the molecular level and all had received the GIMEMA/AIDA protocol, achieving molecular remission at the end of consolidation. Relapse was hematological in 12 cases and molecular in one. After consolidation with chemotherapy, all patients achieved MR and received a further course plus granulocyte-colony stimulating factor as mobilizing therapy. A median of 7.6×10(6) (range 2.7-10) CD34-positive cells/kg were collected. In all cases, molecular evaluation of the apheresis product was negative for the promyelocytic leukemia/retinoic acid receptor alpha gene. No case of transplant-related mortality was recorded. No maintenance or consolidation therapy after autologous stem cell transplantation (ASCT) was given to any patient. After a median follow-up of 25 months from ASCT, 10 patients are alive in sustained MR, while two relapsed after ASCT and died in the setting of refractory disease; one patient achieved a third CR and is waiting for allogeneic SCT. These results suggest that ASCT performed with a molecularly negative graft in APL patients in second MR offers a valid chance for achieving a cure. Such an approach should also be considered in relapsed patients with an HLA-compatible donor, namely in those with a first CR lasting more than one year or in unfit or elderly individuals.

Day 15 bone marrow driven double induction in young adult patients with acute myeloid leukemia: feasibility, toxicity, and therapeutic results.

The strategy named double induction (DI) in acute myeloid leukemia (AML) consists of two courses of chemotherapy irrespective of the degree of cytoreduction in the bone marrow (BM) after the first course, unless severe complications prohibit its application. We describe treatment results from a series of 33 patients in whom DI was adopted only after demonstration of persistence of more than 10% blast cells at day 15 (D15) examination of BM. All patients received as induction idarubicin, cytarabine, and etoposide. As second induction, we administered the combination of fludarabine, intermediate dose cytarabine, and Granulocyte colony stimulating factor (G-CSF). The median blast count at D15 was 30 (15-90). Overall, 30 of 33 patients were judged as eligible to receive DI, reasons for exclusion being in all cases active infection in the context of severe pancytopenia. Nineteen patients (63%) had unfavorable karyotype and 11 (37%) normal karyotype; seven of these had Fms-like tyrosine kinase gene internal tandem duplication (FLT3/ITD) mutation. Overall, complete remission (CR) was achieved in 20/30 patients (67%), while eight patients (27%) were refractory and two died of infectious complications. All refractory patients had unfavorable cytogenetics. All patients achieving CR were programmed to receive allogeneic stem cell transplantation (allo-SCT), which was actually performed in 11 patients. Our study suggest that D15 driven DI represents a feasible and effective therapeutic strategy in young adult AML patients, improving therapeutic results and not compromising feasibility of allo-SCT. When compared with conventional DI, it offers the potential to avoid unnecessary toxicity in a consistent proportion of patients.

Favorable outcome in patients with acute myelogenous leukemia with the nucleophosmin gene mutation autografted after conditioning with high-dose continuous infusion of idarubicin and busulfan.

Mutations of the nucleophosmin gene (NPM1), in the absence of concurrent FLT3-internal tandem duplication (FLT3-ITD) have impressive prognostic value in patients with acute myelogenous leukemia (AML), carrying normal karyotype (NK). In this study we describe treatment results from a series of 19 patients with NPM+/FLT3- autografted in first complete remission (CR) after conditioning with a regimen, named BuI, based on high-dose continuous infusion of idarubicin and Busulfan. Ninety-nine consecutive patients (median age of 54 years) with NK AML autografted in first CR were analyzed. Nineteen of 99 patients (19%) had NPM1 mutation in the absence of FLT3 mutations. The control group, accounting for 80 patients, included 16 cases (15%) with both mutations, 10 (12%) with FLT3/ITD mutation and no NPM mutation, and 54 (68%) in whom neither NPM1 nor FLT3 mutations were detectable. The median overall survival (OS) for the whole patient population was 34 months, the median disease-free survival (DFS) was 22 months. Median OS and DFS were significantly longer for patients with isolated NPM1 mutation as opposed to controls (OS: not reached versus 25 months, P = .02; DFS: not reached versus 16 months, P = .007, respectively). Of interest, patients with isolated NPM1 mutation had a better outcome in terms of either OS or DFS compared to the group of 16 NMP1+/FLT3+ patients. In conclusion, our study suggest that BuI regimen results in favorable clinical outcome in patients with isolated NPM1 mutation, and could be investigated in a randomized study versus other regimes or repeated courses of high dose cytosine-arabinoside.

Continuous infusion idarubicin and intravenous busulphan as conditioning regimen to autologous stem cell transplantation for patients with acute myeloid leukaemia.

The current study aimed to evaluate the efficacy and toxicity of a combination of intravenous (iv) busulfan (Bu) and continuous infusion Idarubicin (IDA) as a conditioning regimen to autologous haematopoietic stem cell transplantation (ASCT) in patients with acute myeloid leukaemia (AML). The protocol included IDA at 20 mg/sqm daily as 3 days continuous infusion (from day -13 to -11) and intravenous BU at 3.2 mg/kg daily from day -5 to -2. Patients aged over 60 years received a reduced schedule (2 days IDA and 3 days BU at the same dose). Twenty-five patients with a median age of 51 years (28-72) were enrolled. All patients received peripheral blood stem cells (PBSC). The median interval between diagnosis and ASCT was 4 months. The median number of CD34+ cells infused was 5.9 x 10E6/kg. The median number of days to PMN >500/cmm and platelets >20000/cmm was 10 and 13, respectively. In order to perform a comparison in terms of haematological and non haematological toxicity, a group of 30 patients, who were previously autografted after conditioning with IDA and oral Bu was considered. Selection of factors for a matched pair analysis included median age, percentage of subjects aged over 60 years, median CD34+ cell received, cytogenetic and molecular findings and per cent of secondary AML. As compared to previous series, the occurrence of severe mucositis was dramatically reduced (80% vs. 12%, p < 0.0001). In addition, need and duration of total parenteral nutrition (TPN), iv antibiotic therapy and hospitalization were also significantly reduced. We conclude that replacement of oral with intravenous BU results in a more favourable toxicity profile. A longer follow-up is required to assess a potential advantage in terms of disease free survival (DFS).

Autologous stem cell transplantation for elderly patients with acute myeloid leukaemia conditioned with continuous infusion idarubicin and busulphan.

Different studies have suggested the potential utility of autologous stem cell transplantation (ASCT) in acute myeloid leukaemia (AML) of the elderly with encouraging results in selected patients. However, while the introduction of peripheral blood stem cells (PBSC) has consistently reduced morbidity and mortality of the procedure, relapse still represents the major cause of ASCT failure. One possibility to ameliorate therapeutic results could rely on the adoption of conditioning regimens specifically designed for AML. We report therapeutic results from a series of 40 AML patients older than 60 years (median age 67 years) autografted in first complete remission (CR), after conditioning with continuous infusion (c.i.) high dose idarubicin and busulphan. Fourty patients (median age: 67 years) received 2 days c.i. idarubicin at 20 mg/m(2)/day, followed by 3 days oral or intravenous busulphan (4 mg/kg/day) as conditioning. No case of transplant-related mortality occurred. Cardiac toxicity was absent, while 31 patients (77%) had grade 3-4 mucositis. After a median follow-up of 25 months, median disease free and overall survival (OS) for the whole patient population were 13 and 22 months, respectively. Three patients died while in CR from causes unrelated to AML. Better results were achieved in patients with intermediate karyotype as opposed to those with adverse cytogenetics. Our data confirm the feasibility of a conditioning regimen based on high-dose idarubicin plus busulphan in older selected AML patients and suggest clinical improvement in patients with normal cytogenetics.

Thromboembolic events and haematological diseases: a case of stroke as clinical onset of a paroxysmal nocturnal haemoglobinuria.

Some haematological diseases are associated to an increased risk of thromboembolic events. We report a case of paroxysmal nocturnal haemoglobinuria (PNH) in which a cerebrovascular event represented the first clinical manifestation of disease. PNH is associated to thromboembolic events, generally of venous districts often involving unusual locations such as mesenteric vessels, sagittal veins, inferior vena cava and renal veins.To our knowledge arterial thrombotic episodes are rare and the involvement of arterial cerebral vessels is exceptional. Then, our case points out the importance of investigating about haematological disorders in all patients presenting with a stroke, in which the common predisposing conditions are excluded.