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In the present study, we aimed to evaluate the absolute risk of infection in the real-life setting of AML patients treated with CPX-351. The study included all patients with AML from 30 Italian hematology centers of the SEIFEM group who received CPX-351 from July 2018 to June 2021. There were 200 patients included. Overall, 336 CPX-351 courses were counted: all 200 patients received the first induction cycle, 18 patients (5%) received a second CPX-351 induction, while 86 patients (26%) proceeded with the first CPX-351 consolidation cycle, and 32 patients (10%) received a second CPX-351 consolidation. A total of 249 febrile events were recorded: 193 during the first or second induction, and 56 after the first or second consolidation. After the diagnostic work-up, 92 events (37%) were classified as febrile neutropenia of unknown origin (FUO), 118 (47%) were classifiable as microbiologically documented infections, and 39 (17%) were classifiable as clinically documented infections. The overall 30-day mortality rate was 14% (28/200). The attributable mortality–infection rate was 6% (15/249). A lack of response to the CPX-351 treatment was the only factor significantly associated with mortality in the multivariate analysis [p-value: 0.004, OR 0.05, 95% CI 0.01–0.39]. Our study confirms the good safety profile of CPX-351 in a real-life setting, with an incidence of infectious complications comparable to that of the pivotal studies; despite prolonged neutropenia, the incidence of fungal infections was low, as was infection-related mortality.

BackgroundMaintenance treatment after autologous bone marrow transplantation in multiple myeloma improves the outcome of patients. We designed a phase II clinical trial to evaluate the treatment with IL2 and zoledronate after autologous bone marrow transplantation in myeloma patients.MethodsPatients with a histologically proven diagnosis of multiple myeloma become eligible if achieved a very good partial remission in bone marrow samples after 3 months from autologous bone marrow transplantation. IL2 was administered from day 1 to 7. In the first cycle, the daily dose was 2 × 106 IU, whereas, in subsequent ones the IL2 dose was progressively escalated, with +25% increases at each cycle, until evidence of toxicity or up to 8 × 106 IU. Four mg of zoledronic acid were infused on day 2. Flow cytometry analysis of γδ-lymphocytes was performed at days 1 and 8 of treatment cycles.ResultsForty-four patients have been enrolled between 2013 and 2016. The median time to progression was 22.5 months (95% CI 9.7–35.2). A complete remission with a negative immunofixation was obtained in 18% of patients and correlated with a significantly longer time to progression (p = 0.015). Treatment was well tolerated without G3 or 4 toxicities. After a week of treatment with IL2 and zoledronate, γδ lymphocytes, Vγ9δ2, CD57+, effector, late effector, and memory γδ increased but in subsequent cycles, there was a progressive reduction of this expansion.ConclusionsThe maintenance treatment with IL2 and Zoledronate has a modest activity in myeloma patients after autologous bone marrow transplantation.EudraCT Number2013-001188-22.

Graphical abstract, COVID-19 is a medical emergency, with 20 % of patients presenting with severe clinical manifestations. From the pathogenetic point of view, COVID-19 mimics two other well-known diseases characterized by cytokine storm and hyper-activation of the immune response, with consequent organ damage: acute graft-versus-host disease (aGVHD) and macrophage activation syndrome (MAS). Hematologists are confident with these situations requiring a prompt therapeutic approach for switching off the uncontrolled cytokine release; here, we discuss pros and cons of drugs that are already employed in hematology in the light of their possible application in COVID-19. The most promising drugs might be: Ruxolitinib, a JAK1/2 inhibitor, with a rapid and powerful anti-cytokine effect, tyrosine kinase inhibitors (TKIs), with their good anti-inflammatory properties, and perhaps the anti-Cd26 antibody Begelomab. We also present immunological data from gene expression experiments where TKIs resulted effective anti-inflammatory and pro-immune drugs. A possible combined treatment algorithm for COVID-19 is here proposed.

Objective Pre-clinical and uncontrolled studies in patients with systemic lupus erythematosus (SLE) showed that mesenchymal stromal cells (MSCs) have a potential therapeutic role in refractory cases. The optimal therapeutic strategy in these patients remain to be elucidated. Our aim was to test the hypothesis that repeated administrations of 1×106/kg body weight of allogenic MSCs, that is a significantly lower dosage with respect to the fixed 1×106 MSC used in animal models, can be effective in improving the clinical course of a murine SLE model. Methods Bone marrow derived MSCs were obtained from 12-week-old C57BL/6J mice. Seventy-five 8 weeks old female NZ mice were randomly assigned to receive via caudal vein the following alternative treatments: 1) single infusion of 106 MSCs/kg body weight at 18 weeks of age (NZs18) or at at 22 weeks of age (NZs22); 2) multiple monthly infusions of 106 MSCs/kg body weight starting at 18 weeks of age (NZM18) or at 22 weeks of age (NZM22); 3) saline infusions (NZc) Fifteen 8 weeks old C57BL/6J mice (Envigo, Huntingdon, UK) were used as untreated controls (C). Weekly, body weight was recorded and twenty-four hour urines were collected by metabolic cages for each animal; proteinuria was detected by dipstick analysis. At sacrifice, peripheral blood samples were collected from mice and anti-dsDNA antibodies were detected by enzyme immunoassorbent assay (ELISA) method using commercial kits. At sacrifice, kidneys were analyzed for histopathology and immunohistochemical analysis for B220, CD4, MPO, CD4+Foxp3, F40/80 infiltration was performed. Results Proteinuria occurrence was delayed NZS and NZM mice, no differences were observed in anti-dsDNA autoantibody titer among the groups at the different time-points; at 36 weeks, no significant differences were observed in term of nephritis scores. Inflammatory cells deposition (MPO and F4/80 positive cells) in NZM was significantly higher than in NZ and NZS. An overexpression of B lymphocytes (B220) was found in NZM while T regulatory cells (CD4+ Foxp3+ cells) were reduced in both NZS and NZM with respect to NZc. Conclusions Overall, our study failed to show a positive effect of a treatment with murine MSCs in this model and, for some aspects, even deleterious results seem to be observed.

To retrospectively assess the efficacy of bendamustine alone and with rituximab (R-B), 109 patients with relapsed chronic lymphocytic leukaemia (CLL) were enrolled in 24 Italian centres. The median age was 66 years (range 39-85). Forty-three percent of patients had relapsed and 57% were resistant (median previous therapies = 3; range 1-8). Twenty-two patients received bendamustine alone and 87 patients received R-B (median B dosage: 100 mg/m(2) per day, range 90-130 mg/m(2) per day). The overall response rate was 69·6% (complete response 28·6%; partial response 41%), and was significantly higher in patients treated with R-B (P = 0·014) and in those responsive to the previous treatment (P=0·04). After a median follow-up of 7·9 months (range 1-148), the median progression-free survival was 16 months and the median duration of response was 13 months. Median overall survival (OS) was 16·8 months for the whole cohort; patients not responding to the treatment had a significantly worse outcome than those who attained a response (P = 0·0001). In multivariate analysis, only resistant disease status at start of bendamustine treatment (HR 3·2, 95% CI 1·4-7·3, P = 0·006) had an independent prognostic value for OS. Toxicity was manageable and mostly haematological. In conclusion, in our experience R-B was an effective and well-tolerated treatment for relapsed/refractory CLL patients, producing a remarkable high CR rate and mild toxicity.

We have recently identified mesodermal progenitor cells (MPCs) isolated from adult human bone marrow. These cells show unusual phenotypes, having putative embryonic markers and aldehyde dehydrogenase (ALDH) activity. Interestingly, these resting cells, which have been selected by culturing them in the presence of adult human serum, can easily be induced to differentiate into mature mesenchymal stromal cells (MSCs) after substituting the adult human serum for fetal bovine serum (FBS) or human cord serum. MPC-derived MSCs are, in turn, able to differentiate toward osteoblasts, chondrocytes, and adipocytes. Furthermore, MPCs are able to differentiate into endothelial cells. MPCs have been proven to be strongly adherent to plastic culture bottles and to be trypsin-resistant. In the present article, we show a simple and inexpensive method to isolate highly selected mesodermal progenitors from bone marrow or cord blood. The optimization of standard culture conditions (using commercial human AB sera and appropriate concentrations for cell seeding in plastics) allows a pure population of MPCs to be obtained even after a short culture period. We believe that this simple, repeatable, and standardized method will facilitate studies on MPCs.

BACKGROUND: Recently, there has been an increased interest in using mesenchymal stromal cells (MSCs) in bone tissue engineering coupled with a suitable scaffold of both biological and synthetic origin. The cells and these constructs can be combined in vitro or directly in vivo to enhance tissue repair. MSCs are spindle-shaped cells capable of self-renewal and can be induced to differentiate mainly into osteo-, chondro-, and adipogenic-progeny types. Several biomaterials are currently available and, among them, fibrin-based constructs seem to be suitable for guiding the cells during tissue repair or regeneration due to their biocompatibility and biodegradability. STUDY DESIGN AND METHODS: Here, this study describes a simple in vitro system using human mesenchymal stromal cells (hMSCs) and fibrin scaffold prepared at different concentrations in fibrinogen (1.5%-3% and 6%) to evaluate cell proliferation and viability inside these constructs. RESULTS: The data demonstrate that the constructs with 3 percent in fibrinogen resulted in the best scaffolds, because within them the cells were able to proliferate and were uniformly distributed. Finally, analyzing the capability of the clots to support osteogenic differentiation of MSCs, we observed that they differentiated into osteoblasts. CONCLUSION: These results suggest that fibrin gel could be useful as a delivery system for hMSCs. B one regeneration is required for fracture healing, and different surgical and biological procedures have been used to promote osteogenesis. This process involves migration, proliferation, and differentiation of osteocompetent cells. However, successful repair of large bone defects remains a challenge for surgeons, especially in elderly patients or in patients with severe trauma. Moreover, in some clinical cases, the local recruitment of these cells is defective because the wound bed cannot provide them. Various strategies have been used to promote osteogenesis; among them tissue engineering seems to be increasingly promising for orthopedic therapeutic applications. It involves the use of cells coupled with biological or artificial matrices, which guide the cells during tissue repair or regeneration. These materials, loaded with osteocompetent cells, should be able to generate new

PS-341 (Bortezomib) is a dipeptide boronic acid proteasome inhibitor with antitumor activity that induces apoptosis in different human cancer cell lines. We investigated effects of PS-341 (Bortezomib) on cell proliferation, cell cycle progression, induction of apoptosis and differentiation in a megakaryoblastic (MO7-e) cell line. PS-341 was able to retain NF-κB in the cytoplasm and inhibit cell growth (IC 50 = 22.5 nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines (KG1a, K562/R7, HL60/DNR) were unaffected by the PS-341 treatment. Moreover, PS-341 in MO7-e induced a significant pro-apoptotic effect from 10 nM concentration (40% versus 12% in the control, p On the other hand, at lower concentration (5 nM), Bortezomib blocked cell cycle in the G2 phase. Finally, this compound was able to down-regulate WT1 expression. No significant effects on cell differentiation were found. Because a spontaneous NF-κB activation has been reported in megakaryocytes from patients affected by myeloproliferative disorders, Bortezomib would so be an attractive therapeutic tool for these malignancies, including essential thrombocythemia or idiopathic myelofibrosis. Preliminary data show an inhibiting activity of Bortezomib in the megakaryocytic colonies formation. Finally, also down-regulation of the WT1 gene Bortezomib-driven could be relevant, because of the role that this gene would play in the pathogenesis of acute and chronic myeloproliferative disorders.

Mesenchymal stromal cells (MSCs) have been the object of extensive research for decades, due to their intrinsic clinical value. Nonetheless, the unambiguous identification of a unique in vivo MSC progenitor is still lacking, and the hypothesis that these multipotent cells could possibly arise from different in vivo precursors has been gaining consensus in the last years. We identified a novel multipotent cell population in human adult bone marrow that we first named Mesodermal Progenitor Cells (MPCs) for the ability to differentiate toward the mesenchymal lineage, while still retaining angiogenic potential. Despite extensive characterization, MPCs positioning within the differentiation pathway and whether they can be ascribed as possible distinctive progenitor of the MSC lineage is still unclear. In this study, we describe the ex vivo isolation of one novel bone marrow subpopulation (Pop#8) with the ability to generate MPCs. Multicolor flow cytometry in combination with either fluorescence-activated cell sorting or magnetic-activated cell sorting were applied to characterize Pop#8 as CD64(bright)CD31(bright)CD14(neg). We defined Pop#8 properties in culture, including the potential of Pop#8-derived MPCs to differentiate into MSCs. Gene expression data were suggestive of Pop#8 in vivo involvement in hematopoietic stem cell niche constitution/maintenance. Pop#8 resulted over three logs more frequent than other putative MSC progenitors, corroborating the idea that most of the controversies regarding culture-expanded MSCs could be the consequence of different culture conditions that select or promote particular subpopulations of precursors.

It has been proven that mesenchymal stromal cells (MSCs) can differentiate into tenocytes. Attempts to repair tendon lesions have been performed, mainly using scaffold carriers in experimental settings. In this article, we describe the clinical use of undifferentiated MSCs in racehorses. Significant clinical recovery was achieved in 9 of 11 horses evaluated using ultrasound analysis and their ability to return to racing. Our results show that the suspension of a small number of undifferentiated MSCs may be sufficient to repair damaged tendons without the use of scaffold support. Ultrasound scanning showed that fibers were correctly oriented. By using undifferentiated cells, no ectopic bone deposition occurred. A sufficient number of cells was recovered for therapeutic purposes in all but 1 case. We suggest that the use of autologous MSCs is a safe therapeutic method for treating incompletely (i.e., not full-thickness) damaged tendons.

Recent advances in the isolation, expansion, and characterization of human mesenchymal stem cells (hMSCs) have raised the possibility of using them in cell therapies and tissue engineering for bone reconstruction. hMSCs, isolated from the bone marrow of eight normal adult patients, were minimally expanded ex vivo and pulsed twice toward osteogenic lineage. The cells were then included into autologous plasma-derived clots. Cytofluorimetric analysis, immunocytochemistry (osteopontin), histochemistry (alkaline phosphatase, Alcian blue, Von Kossa, and alizarin red staining), and viable/proliferation tests were performed to study both stem and differentiating cells. Although two short inductions increased osteogenic markers in hMSCs, inside the clot the cells were able to terminally differentiate into osteoblasts. Moreover, we show that the clot is able to sustain cell proliferation under appropriate cell culture conditions. Our results suggested that clot could be useful for hMSC delivery into the site of the lesion to promote bone formation. Moreover, the plasticity of this material allowed good in vitro hMSC spreading and proliferation. The advantages of using this autologous biological material are its biocompatibility and reabsorption; furthermore, using a gel as scaffold, it is possible to mold it to the shape of a bone cavity.

Mantle cell lymphoma (MCL) accounts for 3-10% of all non-Hodgkin's lymphomas, with median overall survival not exceeding 3-4 years. Rituximab in combination with the Hyper-CVAD regimen appears the most promising regimen; thus, we adopted it as a first-line treatment strategy in a series of 24 patients. In addition to evaluation of clinical success of the regimen, we investigated a possible role of polymorphism in IgG Fc receptors, FCgammaRIIIa and FCgammaRIIa. The frequencies of FCgammaRIIIa-158 were as follows: V/V=4/24 (17%); V/F=16/24 (66%); F/F=4/24 (17%). Those of the FCgammaRIIa-131 polymorphism were H/H=11/24 (46%), H/R=9/24 (37%), R/R=4/24 (17%). The overall response rate was 62.5%, with 33% of complete responses (CRs) after four cycles of R-Hyper-CVAD. Two-year progression-free survival (PFS) was 78% for 158V/V patients vs 75% for cases carrying phenylalanine (p=0.88). When the FCgammaRIIa polymorphism was assessed, the 2-year PFS was 82% for 131H/H patients vs 75% for those carrying arginine (p=0.26). Eighty-three percent of cases achieved Polymerase Chain Reaction (PCR)-negativity: the progression rate was significantly influenced by the minimal residual disease clearance, with 12% progression in the subgroup of PCR-negative cases versus 67% progression in PCR-positive cases (p=0.008). The achievement of PCRnegativity was not significantly influenced by FCgammaR polymorphisms. Results confirm that rituximab plus Hyper-CVAD is an effective regimen for the induction of prolonged remission in patients with aggressive MCL and suggest that rituximab efficacy is independent of the FCgammaR polymorphisms.

Chronic myeloid leukemia (CML) and hairy cell leukemia (HCL) are two distinct haematological disorders. Only one single case of coexistence of the two pathologies at diagnoses has been previously reported. We present a second case of coexistence at diagnosis, indicating the diagnostic procedures involving morphological, immunophenotyping, and molecular testing. We decided to use Interferon as common first-line therapy and Imatinib and Rituximab (anti-CD20 monoclonal antibody), to improve the first-line therapy result, obtaining a complete molecular remission for CML and clinical remission with molecular minimal residual disease for HCL. After a critical analysis of the results, we speculate on the different clonal origin of the two pathologies.

This study evaluates the prognostic value of molecular monitoring of minimal residual disease (MRD) in 20 patients with multiple myeloma (MM) following autologous (peripheral blood stem cell transplantation, PBSCT) and non-myeloablative allogeneic (NMT) transplant. All patients completed their program, with a treatment-related mortality (TRM) of 20% and a 2-year progression-free survival (PFS) of 51%. After PBSCT, only 3 patients (15%) achieved PCR-negativity, versus 12 (60%) after NMT. The eradication of MRD had a favorable impact on 2-year OS. In fact, 76% of patients with no detectable MRD was still alive versus 34% of persistently IgH-positive cases (p=0.03). PCR status did not correlate with chimerism percentage: Seventy-five percent of patients achieved full donor chimerism, which was more frequently observed in cases presenting cGHVD (p=0.01). These data sustain the relevant role of molecular monitoring in MM patients undergoing NMT. MRD monitoring would assist physicians in making additional therapeutic decisions to better control this hematological malignancy.

Purine analogues have dramatically improved the outcome of patients affected by hairy cell leukemia (HCL), although complete eradication of disease was achieved in few cases. The purpose of this study was to evaluate the role of Rituximab in eradicating minimal residual disease (MRD) in HCL patients after a pre-treatment with 2-chloro-deoxy-adenosine (2-CdA). Ten patients received four cycles of Rituximab after administration of Cladribrine. Before starting anti-CD20 antibody, two patients were in complete remission, six in partial remission and two showed no significant response to Cladribrine. All cases resulted IgH-positive. Median time from the last 2-CdA infusion was 5.7 months. Eight of 10 patients [four in partial remission (PR), two in complete remission (CR) and two unresponsive after 2-CdA] were evaluable for response. Two months after the end of anti-CD20 therapy, all evaluated patients presented a complete haematological remission. Moreover, Rituximab increased percentage of molecular remission up to 100% 1 yr after the end of treatment. Interestingly, in all cases but one, including those persistently polymerase chain reaction (PCR)-positive, semi-quantitative molecular analyses showed MRD levels lower than those found before Rituximab administration. Toxicity was very mild. The present results not only confirm the therapeutic effect of Rituximab, but also show its relevance in eradicating MRD in HCL.

The increase of megakaryocytes and platelets that characterizes essential thrombocythemia (ET) appears to be secondary to a deregulation of megakaryocytopoiesis. The carboxy-terminal fragment of osteogenic growth peptide (OGP10–14) promotes bone formation and hemopoiesis, while it inhibits megakaryocytopoiesis. In this paper we show that treatment with synthetic OGP10–14 (sOGP10–14) induces a significant reduction of mid and large colony-forming unit-megakaryocytes (CFU-Mk) in ET patients as well as in controls, and is associated with a significant inhibition of thrombopoietin (TPO)-primed MO-7e megakaryoblastic cells proliferation. These actions appear to be related to sOGP10–14 modulation of TGF-β 1 synthesis and/or secretion, although a direct effect on TGF-β receptor expression cannot be excluded.

Expression of two MDR genes, BCRP and MDR1, was evaluated by real-time PCR technique in 51 AML patients. Fifty-six percent expressed the BCRP gene, with the 48.2% showing intermediate levels. Eighty-eight percent expressed the MDR1, with 23.8% of cases at high expression. A significant correlation between BCRP and MDR1 values was found by regression analysis. Either levels of BCRP or MDR1 did not correlate with clinical characteristics of patients at diagnosis.

The carboxy-terminal fragment of osteogenic growth peptide, OGP(10-14), is a pentapeptide with bone anabolic effects and hematopoietic activity. The latter activity appears to be largely enhanced by specific growth factors. To study the direct activity of OGP(10-14) on myeloid cells, we tested the pentapeptide proliferating/differentiating effects in HL60 cell line. In this cell line, OGP(10-14) significantly inhibited cell proliferation, and enhanced myeloperoxidase (MPO) activity and nitroblue tetrazolium reducing ability. Moreover, it induced cytoskeleton remodeling and small GTP-binding protein RhoA activation. RhoA, which is known to be involved in HL60 differentiation, mediated these effects as shown by using its specific inhibitor, C3. Treatment with GM-CSF had a comparable OGP(10-14) activity on proliferation, MPO expression, and RhoA activation. Further studies on cell proliferation and RhoA activation proved enhanced activity by association of the two factors. These results strongly suggest that OGP(10-14) acts directly on HL60 cells by activating RhoA signaling although other possibilities cannot be ruled out.

A high expression of Wilms’ tumor gene (WT1) in acute myeloid leukemia (AML) seems to correlate with a poor outcome and its increased levels can be predictive of an impending relapse. WT1 has been shown in vitro to interact with the promoter of the MDR1, a gene involved in the multidrug resistance phenomenon. The aim of this study was to measure, by real-time polymerase chain reaction, levels of WT1 and MDR1 expression, in order to find a possible association between these genes, in a series of 50 newly diagnosed AML cases. Twenty-five percent of patients carried very high (>75° percentile) MDR1- and 23.3%WT1-mRNA levels. Interestingly, high levels of WT1 were significantly correlated with correspondent high levels of MDR1 gene. Nevertheless, the co-expression of these genes did not significantly influence the complete response rate to the induction therapy. Reported data confirm the existence of a co-expression of WT1 and MDR1 genes even in vivo; this may be relevant because one consequence could be the positive selection by chemotherapeutic regimens of cells with higher MDR1 levels already present before treatment. Thus, the association between these genes could suggest avoiding the use of drugs involved in the multidrug resistance (MDR) phenomenon in patients carrying high levels of WT1 at diagnosis.

Summary. Idiopathic myelofibrosis (IMF) is a clonal stem cell disorder characterized by reactive fibrosis of bone marrow sustained by a complex cytokine network. At present, no efficacious therapy for this disease exists. Synthetic carboxy-terminal pentapeptide of osteogenic growth factor (sOGP10-14) can increase bone marrow cellularity and the number of haematopoietic colonies; this study evaluated the activity of sOGP10-14 in IMF. Fragments of bone marrow biopsies from patients affected by IMF were cultured with or without the addition of sOGP10-14. Cellular density was evaluated by image analysis, and transforming growth factor-β1 (TGF-β1) concentration was immunologically assayed in the supernatant of cultured bone marrow biopsies. The proliferation rate of the megakaryoblastic M07-e cell line, cultured in the presence of either granulocyte–macrophage colony stimulating factor or thrombopoietin (TPO), and with or without sOGP10-14, was evaluated. Megakaryocyte colony forming unit (CFU-Mk) assay was performed on bone marrow samples of IMF patients with or without sOGP10-14. After 14 d, bone marrow cellularity was significantly increased in samples cultured with the pentapeptide. Moreover, sOGP10-14 induced a significant increase of TGF-β in culture supernatants. TPO-primed proliferation of M07-e was reduced by sOGP10-14, and the pentapeptide significantly reduced CFU-Mk on IMF bone-marrow-derived cells. sOGP10-14 increased ex vivo bone marrow cellularity in IMF. This action could be related to the megakaryocyte inhibition induced by the interference of this pentapeptide with growth factor activities. These findings suggest that a deficiency of osteoblast-related factors may play a role in bone marrow failure in IMF.

Osteogenic growth peptide (OGP) is a peptide exerting regulatory effects on the bone and on bone marrow. The carboxy-terminal pentapeptide (OGP10-14) is the biologically active portion of OGP. We evaluated OGP10-14 hematopoietic activity performing colony-forming tests on human stem cells derived by bone marrow, peripheral blood and cord blood. Granulocyte-macrophage colony-forming unit (CFU) were significantly increased in OGP10-14-treated samples, while granulocyte-erythrocyte-monocyte-megakaryocyte CFU and burst-forming unit (BFU) erythroid were increased only in the cord blood test.Moreover, OGP10-14 preserves stem cells self renewal potential in long-term culture (LTC) initiating cells and acts directly on CD34+ enriched cells or by increasing activity of stem cell factor (SCF) and granulocyte-megakaryocyte colony-stimulating factor.

Lymph node or bone marrow biopsy from sixty-one patients affected by aggressive non-Hodgkin lymphomas (NHL) were retrospectively evaluated to assess the histology at relapse. Eighteen cases (29.5%) were proven to have relapsed or persistent low-grade lymphoma after conventional therapy. In 5/18 patients association of low and high-grade lymphoma was detectable at diagnosis by bone marrow biopsy. In the remaining 13/18 no evidence of follicular lymphoma was detected at diagnosis. The outcome of these patients was compared to that of 43 patients relapsed without change in histology and treated by a second line therapy. Of these 43 patients, 13 were not responders (NR), 10 achieved a partial remission (PR) and 18 complete remission (CR). Two were lost during follow-up. The 18 patients with residual/relapsed indolent subtype received oral cyclophosphamide (100 mg/day for 15 days every month for six months): 3 of them had NR, 5 CR, and 10 PR. The overall survival (OS) median time was 39 months in low-grade resistant/relapsed patients and 20 months in patients with aggressive histology. OS at 24 months was 71 and 41%, respectively, (p < 0.02). Most of the patients with high-grade disease were refractory or relapsed after a median of five months, whereas cases with low-grade NHL showed a long lasting stable PR. We suggest that the higher grade patients with residual or relapsed low grade lymphoma were, in fact, transformed low-grade at diagnosis and, after removing the more aggressive component by chemotherapy, it is possible to manage these patients by conventional therapy for indolent lymphomas.

Dear Editor, The incidence rate of non-Hodgkin lymphomas (NHL) doubled over the past two decades in both the US and most westernized countries [1]. Etiology remains largely unknown, but herbicide exposure (especially to phenoxyacids) has been considered a risk factor for indolent NHLs since the 1990s [2–4]. Although the fraction of agricultural workers in westernized countries is small and decreasing over time, herbicide exposure of the general population has been increasing [1]. Despite general population exposures may be lower than those in occupational settings, a relative risk as small as 1.2 could explain 15% of the current NHL risk, assuming that over 90% of the general population is exposed [5]. Based on preliminary unpublished observations on the geographical clustering of mortality from NHLs in the province of Pisa (western Tuscany, Italy), we ran an ecological study on the distribution of NHL cases in areas with high exposure to herbicides. We defined exposure to herbicides using an index calculated as follows: the 2,450,000 ha of the province of Pisa were subdivided into geofunctional areas marking autonomous systems characterized by very low intermigration, closed water system, and homogeneous soil composition [6]. Data on spraying of the 13 most used herbicides in the province of Pisa in 1988–1990 were extrapolated from the recordings of the Italian Health Ministry [7], as reported in Table 1. A modification of Mackay and Peterson’s model of fugacity was used to calculate herbicide percolation into the soil [8–11]. Land areas treated with herbicides were estimated on the basis of the 1980 Third General Census of Italian Agriculture [12], and herbicide use was assumed homogeneous throughout the whole province. The theoretical concentration of each active ingredient expected in percolation water in 1988– 1990 was estimated as micrograms per gram of soil. Assuming that all active ingredients had the same lymphomagenic impact, a cumulative concentration index (Cw) was used (millimeter per cubic meter). At first, we retrospectively investigated the correlation between the herbicide soil percolation index in 1988–1990 and mortality from NHLs in the province of Pisa in the period 1987–1992. Overall, 370 deaths from NHLs occurred in the study period. Mortality rates were calculated for each geofunctional area using data obtained from the Tuscan Mortality Registry. Soil percolation by herbicides and mortality rates of NHLs were then correlated using Spearman’s rank test (ρ), showing a moderate correlation (ρ=0.355; p

Arsenic trioxide has recently been reported to be successful in the treatment of promyelocytic leukemia. Several concerns about the use of this toxic agent are currently reducing its potential clinical use even in severely ill patients. In this report we describe the results achieved by As2O3 with all-trans retinoic acid in a patient suffering from secondary, relapsed, resistant promyelocytic leukemia. Several complications, including sepsis and an extensive area of skin necrosis, did not allow us to treat the patient further with chemotherapy. With As2O3 and ATRA therapy, the patient obtained a complete molecular remission without any significant toxicity and, subsequently, it was possible to perform a bone marrow autograft in a state of complete remission.

Mesenchymal stromal cells (MSCs) are a heterogeneous cell population capable of differentiating toward several cell lines in vitro and, possibly, in vivo. Within cultured MSCs, we identified and purified a precursor cell population [mesodermal progenitor cells (MPCs)] retaining robust proliferation potential and ability to differentiate into endothelial or mesenchymal cells. MPC-derived MSCs retain the ability to further differentiate into osteoblasts, cartilage, or fat cells. Here we further characterized MPCs and MSCs by evaluating expression of integrins and adhesion molecules showing their ability to assemble the molecular machinery involved in endothelium adhesion. MPCs were shown to interact with activated and nonactivated endothelium, whereas MSCs exhibited activation of focal adhesion complexes, higher cell motility, and reduced or absent adhesiveness onto endothelial cells, suggesting a matrix remodeling vocation. We also reported a consistent expression of CXCR4 on the MPC cell surface, suggesting that the different phenotypic behavior could be related to specific functions of the cell in each differentiation stage.

Background: Multiple myeloma is still today an incurable disease. The many therapeutic techniques and new therapies proposed in recent years have extended survival but did not allow for healing. Further study allowed to demonstrate that a maintenance could be useful to control the progression of disease. However, there is no clear indication for which maintenance has to be used after a first line of induction therapy. The technique of allograft, used in patients at highest risk, demonstrates that the immune response to the residual disease plays a key role in the success of this technique. Among the major players in response to myeloma, in allogeneic stem cell transplantation, gamma delta lymphocytes play a significant action: complete response after allogeneic few months later (also the molecular level) happen in parallel with the presence in the bone marrow of a significant proportion of lymphocytes with gamma delta oligoclonal expression of TCR rearrangements. Zoledronic acid induces proliferation of these cells by the production of several cytokines, in particular interleukin-2 (IL-2). Furthermore, T lymphocytes Vdelta2 are proved to be crucial antineoplastic mediators and, after expansion in vitro, capable of controlling tumor growth in animal models. These data confirm the hypothesis that gammadelta lymphocytes have a role in controlling the growth of myeloma plasma cells and can be active on the residual disease after autologous stem cell transplant. We planned to evaluate the role of the association of Zoledronate and IL-2 in vivo as post ASCT maintenance therapy in patients with newly diagnosed multiple myeloma (NDMM). Methods: This is a single arm phase II multicenter ongoing study of the combination of IL-2 with zoledronic acid as maintenance therapy for NDMM patients post ASCT. The primary objective was to establish safety and efficacy of IL-2 as maintenance therapy. The secondary objective was to evaluate the immunological expansion of gamma delta lymphocytes. Eligible patients had undergone ASCT, with melphalan as a preparative regimen. At July 2016, forty two patients in very good partial remission (VGPR) have been enrolled in the study (total planned enrollment: 43 pts) and started maintenance therapy 90-180 days post ASCT. Maintenance schedule included IL2 and zoledronic acid. IL2 was administered at a fixed dose of 2 x 106UI from day 1 to day 7 for the first cycle and with the maximum tolerated dose (up to a max of 8 x 106UI) from day 1 to day 7 for subsequent cycles (dose escalation of 25% in each cycle in the absence of toxicity). Zoledronic acid was infused 4 mg iv on day 2. This dosing regimen is repeated every 28 days until disease progression. Adverse events were graded by NCI-CTCAE v4. Response was assessed by the modified International Uniform Response Criteria. Results and toxicity: 42 patients (pts) have been enrolled with a median age of 59 (range 42-72); 50% were male and 50% female. All the 42 pts have received a median of 11 cycles (range 1-23). Of the 42 pts 21 remain on therapy (data at July 2016), 21 pts are off study: 9 due to progressive disease (PD) and 12 due to consent withdrawal. Among the 9 pts with PD, the median PFS post ASCT was 12 months (2-18 months). Of the 42 pts, 33 (79%) not progressed after a median of 13 months (range 1-33) and the median PFS has not been reached. 7/42 patients (17%) reached complete remission. Peripheral and bone marrow analysis of gamma delta lymphocytes expansion to evaluate the level of immune response is still under examination. Grade 1/2 hematologic adverse events (AEs) included: grade 1 (G1) anemia (3 pts), G1 neutropenia (3). Grade 1/2 drug-related non-hematologic AEs included: G1 fever (25) G2 fever (8); G2 constitutional symptoms (joint pains) (20); G2 constipation (4); G1/2 nausea (10); G1 fatigue (15), G1/2 cutaneous rash (2). Conclusions: Long term administration of combination of IL-2/zoledronate as maintenance therapy post ASCT is feasible. The incidence of non hematologic adverse events (in particular fever) were manageable with no dose escalation of IL-2 over 5 x 106UI. This immunological approach, without any chemotherapeutic drug, seems to be able to control the disease and to obtain the complete remission in a subgroup of myeloma patients. Disclosures No relevant conflicts of interest to declare.

Two patients, observed at our institution, developed, after treatment with fludarabine, an early reappearance of metastatic primary solid cancers which were previously in long-lasting, complete remission. Patients had earlier suffered from a solid cancer considered cured and, subsequently, developed a lymphoid disorder treated with fludarabine. The two patients developed histologically confirmed hepatic metastasis from breast cancer and colic adenocarcinoma respectively 11 and 4 months after the beginning of fludarabine-therapy. Purine analogs have been reported to be effective against chronic lymphocytic leukemia and indolent lymphomas. However, these drugs induce severe immunodeficiency. In addition to the infectious diseases related to the treatment, the use of these drugs could facilitate the development of secondary neoplasms, related to the patient's impaired immunosurveillance. The surprisingly short latency between the therapy and the reappearance of non hematological cancers seen in our patients suggests that treatment with purine analogs may be involved in the reappearance of the tumors. In this regard, we suggest a possible role for purine analog-induced immunodeficiency in allowing the growth of previously undetected cancer cells rather than a direct drug-related mutagenic activity.

We investigated the efficacy of alpha recombinant human erythropoietin (α-rHuEPO) administered as single agent to 133 patients affected by myelodysplastic syndromes referring to our Institution in the last 10 years. WPSS score was "very low" in 67%, "low" in 19%, "intermediate" in 14%. The starting schedule was: 40,000 IU bi-weekly, with reduction or suspension, when necessary, in responsive patients. According to new IWG criteria, response rate (RR) was 75%, 66%, 59% after 8, 16, 24 weeks, respectively. Comparing "very low" and "low/intermediate" risk, RR was 81% vs. 43% (P < 0.001); 70% vs. 45% (P = 0.040); 63% vs. 42% (P = NS) after 8, 16, 24 weeks. RR was significantly influenced by transfusion dependence (P = 0.039) and basal serum EPO level (P < 0.001). Mean Hb value was 94 ± 11 g/l before therapy; 114 ± 19 after 8 weeks (P < 0.001); 116 ± 18 after 16 weeks (P < 0.001); 114 ± 17 after 24 weeks (P < 0.001). Reduction or suspension of therapy significantly affected Hb level after 4 (P < 0.001) and 8 weeks (P < 0.01). Conversely, restart of full dosage significantly enhanced again Hb level after 4 (P < 0.01) and 8 weeks (P < 0.001). 65% patients are alive (mean survival: 74 weeks). Seventy percent are alive in the "very low risk" group and 38% in "low/intermediate risk" group (P < 0.001). Overall mean follow-up was 69 weeks (range, 8-376): it was 80 weeks in responsive patients (max 376) and 38 weeks in patients who progressively became unresponsive (max 168) (P < 0.01). Median response was 36 weeks, with 33% of patients still responding after one year. Treatment was well tolerated.

To retrospectively assess the efficacy of bendamustine alone and with rituximab (R-B), 109 patients with relapsed chronic lymphocytic leukaemia (CLL) were enrolled in 24 Italian centres. The median age was 66 years (range 39-85). Forty-three percent of patients had relapsed and 57% were resistant (median previous therapies = 3; range 1-8). Twenty-two patients received bendamustine alone and 87 patients received R-B (median B dosage: 100 mg/m(2) per day, range 90-130 mg/m(2) per day). The overall response rate was 69·6% (complete response 28·6%; partial response 41%), and was significantly higher in patients treated with R-B (P = 0·014) and in those responsive to the previous treatment (P=0·04). After a median follow-up of 7·9 months (range 1-148), the median progression-free survival was 16 months and the median duration of response was 13 months. Median overall survival (OS) was 16·8 months for the whole cohort; patients not responding to the treatment had a significantly worse outcome than those who attained a response (P = 0·0001). In multivariate analysis, only resistant disease status at start of bendamustine treatment (HR 3·2, 95% CI 1·4-7·3, P = 0·006) had an independent prognostic value for OS. Toxicity was manageable and mostly haematological. In conclusion, in our experience R-B was an effective and well-tolerated treatment for relapsed/refractory CLL patients, producing a remarkable high CR rate and mild toxicity.

Background: Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4 + CD105 + CD90 neg phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4 neg CD105 + CD90 bright and MSCA-1 + ), in the primary cultures, resulted lower than 2%. Methodology/Principal Finding: We demonstrate that MPCs differentiate to MSCs through an SSEA-4 + CD105 + CD90 bright early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p,0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4 neg ). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID50=0.5 mM, p,0.01), while endothelial differentiation was unaffected. Conclusion: The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required.

Large granular lymphocyte proliferative status represents a group of clonal and nonclonal lymphoproliferative disorders of natural killer (NK) or T-cell lineages with common morphological features. Cellular differences may sustain the clinical polymorphism observed in these disorders. Here we report a case of large granular lymphocyte disease unusually expressing CD4+CD8+ clonal T cells and atypical cell morphology in bone marrow.

Background We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. Methodology/Principal Findings MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. Conclusions/Significance MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested from human bone marrow.

The genomic profile of mantle cell lymphoma (MCL) has been reported to be significantly different from that of other indolent lymphoproliferative disorders, Topoisomerase IIalpha, glutathione-s-transferasepi (GSTpi) and ABCG2 (BCRP) chemoresistance genes being over-expressed in MCL. In our study, expression levels of the above mentioned genes plus MDR1 were tested on bone marrow samples from 20 patients treated with Rituximab plus hyper-CVAD regimen, in order to evaluate a possible impact of the chemoresistance phenomenon on this promising treatment regimen. All patients expressed ABCG2 and MDR1 genes; 85% of cases expressed GSTpi and topoisomerase IIalpha. Only ABCG2 were over-expressed in comparison both with marrow from healthy donors and tonsilar CD5+/CD20+ lymphocytes (adopted as normal counterpart of the neoplastic population). The overall response rate of the entire series was 87.5%, with 44% of complete responses. Fifty-seven percent of patients achieved the clearance of minimal residual disease. Levels of tested genes did not condition either quality of clinical response or PFS (76% at 24 months). Nevertheless, an ABCG2 higher expression appeared associated with a worse PFS and levels of this gene paralleled the status of minimal residual disease. A further evaluation of ABCG2 expression in larger series of MCL patients would be suitable.

Vorinostat and bortezomib significantly inhibit WT1 gene expression in MO7-e and P39 cell lines

Recent advances in the isolation, expansion, and characterization of human mesenchymal stem cells (hMSCs) have raised the possibility of using them in cell therapies and tissue engineering for bone reconstruction. hMSCs, isolated from the bone marrow of eight normal adult patients, were minimally expanded ex vivo and pulsed twice toward osteogenic lineage. The cells were then included into autologous plasma-derived clots. Cytofluorimetric analysis, immunocytochemistry (osteopontin), histochemistry (alkaline phosphatase, Alcian blue, Von Kossa, and alizarin red staining), and viable/proliferation tests were performed to study both stem and differentiating cells. Although two short inductions increased osteogenic markers in hMSCs, inside the clot the cells were able to terminally differentiate into osteoblasts. Moreover, we show that the clot is able to sustain cell proliferation under appropriate cell culture conditions. Our results suggested that clot could be useful for hMSC delivery into the site of the lesion to promote bone formation. Moreover, the plasticity of this material allowed good in vitro hMSC spreading and proliferation. The advantages of using this autologous biological material are its biocompatibility and reabsorption; furthermore, using a gel as scaffold, it is possible to mold it to the shape of a bone cavity.