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The transcription factor RUNX1, a pivotal regulator of HSCs and haematopoiesis, is a frequent target of chromosomal translocations, point mutations or altered gene/protein dosage. These modifications lead or contribute to the development of myelodysplasia, leukaemia or platelet disorders. A better understanding of how regulatory elements contribute to fine‐tune the RUNX1 expression in haematopoietic tissues could improve our knowledge of the mechanisms responsible for normal haematopoiesis and malignancy insurgence. The cohesin RAD21 was reported to be a regulator of RUNX1 expression in the human myeloid HL60 cell line and during primitive haematopoiesis in zebrafish. In our study, we demonstrate that another cohesin, NIPBL, exerts positive regulation of RUNX1 in three different contexts in which RUNX1 displays important functions: in megakaryocytes derived from healthy donors, in bone marrow samples obtained from adult patients with acute myeloid leukaemia and during zebrafish haematopoiesis. In this model, we demonstrate that alterations in the zebrafish orthologue nipblb reduce runx1 expression with consequent defects in its erythroid and myeloid targets such as gata1a and spi1b in an opposite way to rad21. Thus, also in the absence of RUNX1 translocation or mutations, additional factors such as defects in the expression of NIPBL might induce haematological diseases.

Introduction Mantle-cell lymphoma (MCL) is a B-cell non-Hodgkin Lymphoma (NHL) characterized by heterogenous behavior, ranging from indolent phenotype to highly aggressive and drug resistant one with dismal prognosis. Drug resistance may be generated by Tumor Microenvironment (TME), owing that Tumor-Associated Macrophages (TAM) are pathologically functional in providing survival signals to MCL cells (Pham, Front Oncol. 2018). Recently, "Don't Eat Me" signal (DEMs) blockade with anti-CD47 monoclonal Antibody (moAb) showed promising activity in pretreated NHL, through increase of phagocytosis by TAM (Advani, NEJM. 2019). CD24 was also demonstrated to be involved in DEMs and, in a preclinical model of solid cancer, blocking the CD24/Siglec-10 interaction provided an improvement of M2-like TAM-mediated phagocytosis in vitro and an increase of survival in vivo (Barkal, Nature. 2019). CD24 can be expressed in some phases of B-cell differentiation and MCL derives from a B-cell precursor with upregulated CD24. To date, there are no functional studies showing an improvement of phagocytosis through CD24/Siglec-10 pathway inhibition in hematologic malignancies and MCL. Here, we present our in vitro results of CD24/Siglec-10 DEMs blockade in MCL subset. Methods A panel of MCL cell lines (Jeko-1, Granta-519, Mino) has been analyzed for CD24 surface expression by flow cytometry (FC) (clone SN3). Consequently, we performed co-culture experiments with MCL cell lines and macrophages from healthy donors. Briefly, Peripheral Blood Mononucleated Cells (PBMC) were collected from healthy volunteers through density gradient centrifugation technique. CD14+ monocytes were isolated through CD14 Microbeads isolation kit and cultured in plates with 50 ng/ml human GM-CSF for 7-9 days. In order to create M2-like Siglec-10+ TAM, 50 ng/ml human IL-10 and 50 ng/ml human TGF-β 1 were added on days 3-4 of differentiation until use on days 7-9. Siglec-10 expression on TAM was checked by FC (clone 5G6). M2-like macrophages were then collected and co-cultured with CFSE-labelled MCL target cells for 1-2 hours in a serum-free medium. Anti-CD24 moAb (clone SN3) or the appropriate IgG 1 isotype control were added at a concentration of 10 μg/ml. Phagocytosis was then stopped on ice and CD11b-PE staining (anti-CD11b moAb, clone REA713) was performed to identify human macrophages by FC. Phagocytosis was measured as the number of CD11b+/CFSE+ macrophages, quantified as a percentage of the total CD11b+ macrophages. Each phagocytosis reaction was performed in technical triplicate and phagocytosis was normalized to the highest technical replicate per donor in order to consider raw phagocytic level among donor-derived macrophages. Results MCL cell lines express surface CD24 by FC, with higher levels in Mino cell line (Figure 1A). Differentiated M2-like macrophages showed an upregulation of Siglec-10 expression after immunosuppressive stimuli, which is fundamental owing that Siglec-10 is the ligand of CD24 (Figure 1B). As pertains to the phagocytic assay, we documented an improvement of phagocytosis when M2-like macrophages and MCL cell lines were co-cultured together with anti-CD24 moAb (Figure 2 and Figure 3A). Furthermore, it is worth mentioning that phagocytosis seemed to be much higher in MCL cell lines with higher surface levels of CD24 (e.g., Mino), presenting increased number of CD11b+/CFSE+ M2-like TAM by FC (Figure 3B). Conclusions MCL was found to be sensitive to CD24/Siglec-10 DEMs blockade when co-cultured with M2-like macrophages in vitro. We can argue that most of the observed increase of phagocytosis after the addition of anti-CD24 moAb may be secondary to loss of CD24 signalling rather than Fc-mediated opsonization, as already documented in previous analysis about solid cancer (Barkal, Nature. 2019). We can therefore hypothesize that the blockade of this DEMs pathway can improve phagocytosis in a non-opsonization manner in NHL as well. Furthermore, CD24 surface density seemed to be positively correlated to the intensity of phagocytic activity, suggesting that MCL subtypes expressing higher CD24 levels are much more dependent on this DEMs pathway than others with low CD24 density. Overall, CD24 turned out to be a potential immunotherapeutic target in MCL, aiming at improving innate immune system through DEMs blockade. In vivo studies are needed to confirm the activity we documented in vitro in this NHL subset. Figure 1 Figure 1. Disclosures Gambacorti-Passerini: Bristol-Myers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

We have treated 69 patients with steroid refractory acute graft versus host disease (SR-aGvHD), with an anti-CD26 monoclonal antibody (Begelomab): 28 patients in two prospective studies (EudraCT No. 2007-005809-21; EudraCT No. 2012-001353-19), and 41 patients on a compassionate use study. The median age of patients was 42 and 44 years; the severity of GvHD was as follows: grade II in 8 patients, grade III in 33, and grade IV in 28 patients. There were no adverse events directly attributable to the antibody. Day 28 response was 75% in the prospective studies and 61% in the compassionate use patients, with complete response rates of 11 and 12%. Response for grade III GvHD was 83 and 73% in the two groups; response in grade IV GvHD was 66 and 56% in the two groups. Non relapse mortality (NRM) at 6 months was 28 and 38%. Overall there were 64, 56, 68% responses for skin, liver, and gut stage 3-4 GvHD. The overall survival at 1 year was 50% for the prospective studies and 33% for the compassionate use patients. In conclusion, Begelomab induces over 60% responses in SR-aGvHD, including patients with severe gut and liver GvHD, having failed one or more lines of treatment.

We performed a nationwide registry-based analysis to describe the clinical outcome of adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) who underwent an allogeneic hematopoietic stem cell transplantation (HSCT) after a tyrosine kinase inhibitor (TKI)-based treatment A total of 441 patients were included in the study. The median age at HSCT was 44 years (range, 18 to 70 years). All 441 patients (100%) received TKI before HSCT (performed between 2005 and 2016). Of these 441 patients, 404 (92%) were in cytologic complete remission (CR), whereas the remaining 37 (8%) had active disease at the time of HSCT. Molecular minimal residual disease (MRD) was negative in 147 patients (36%) at the time of HSCT. The donor was unrelated in 46% of patients. The most prevalent source of stem cells was peripheral blood (70%). The conditioning regimen was myeloablative in 82% of cases (total body irradiation-based in 50%) and included antithymocyte globulin in 51% of patients. With a median follow-up after HSCT of 39.4 months (range, 1 to 145 months), the probability of overall survival (OS) at 1, 2, and 5 years was 69.6%, 61.1% and 50.3%, respectively, with a median OS of 62 months. Progression-free survival (PFS) at 1, 2, and 5 years was 60.2%, 52.1% and 43.7%, respectively. OS and PFS were significantly better in patients who were in CR and MRD-negative at the time of HSCT compared with patients who were in CR but MRD-positive (50% OS not reached versus 36 months; P = .015; 50% PFS not reached versus 26 months, P = .003). The subgroup of MRD-negative patients both at HSCT and at 3 months after HSCT had a better outcome (5-year OS, 70%). Conversely, the 37 patients who underwent a HSCT with active Ph+ ALL had a median OS of 7 months and a median PFS of 5 months. The 5-year cumulative incidence of relapse was significantly lower in MRD-negative patients (19.5% versus 35.4%; P = .001). Nonrelapse mortality (NRM) after 1, 2, and 5 years was 19.1% (95% confidence interval [CI], 15.5% to 22.9%), 20.7% (95% CI, 17% to 24.7%), and 24.1% (95% CI, 20% to 28.5%), respectively. NRM was significantly lower with a modified European Society for Blood and Marrow Transplantation (mEBMT) risk score of 0 to 2 compared with ≥3 (15% versus 25%; P = .016). The median OS for Ph+ ALL patients who underwent a TKI-based treatment followed by an allogeneic HSCT, in recent years at the GITMO centers, was 62 months. Evaluation of the mEBMT risk score can be useful to predict NRM. Our data confirm that HSCT is a potentially curative treatment for Ph+ ALL with an excellent outcome for the subgroup of MRD-negative patients both at HSCT and at 3 months after HSCT (5-year OS, 70%).

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow–derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 106/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (

The coexistence or the development of Philadelphia chromosome-negative myeloproliferative neoplasms after a lymphoproliferative disease in the same patient is an extremely rare event. We report the case of a 72-year-old man who developed JAK2V617F polycythemia vera 3 years after the diagnosis and treatment of primary diffuse large B cell non-Hodgkin's lymphoma of the central nervous system. We also review the literature regarding the pathogenesis underlying the association of myeloproliferative and lymphoproliferative chronic disorders.

Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19(+)CD5(+)CD20(dim) B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23. CAR). CD23.CAR(+) T cells showed specific cytotoxic activity against CD23(+) tumor cell lines (average lysis 42%) and primary CD23(+) CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR(+) T cells released inflammatory cytokines (1445-fold more TNF-beta, 20-fold more TNF-alpha, and 4-fold more IFN-gamma). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR(+) T cells. Redirected T cells were also effective in vivo in a CLL Rag2(-/-)gamma(-/-)(c) xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23. CAR(+) T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23. CAR(+) T cells represent a selective immunotherapy for the elimination of CD23(+) leukemic cells in patients with CLL. (Blood. 2011; 117(18): 4736-4745) Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19(+)CD5(+)CD20(dim) B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23. CAR). CD23.CAR(+) T cells showed specific cytotoxic activity against CD23(+) tumor cell lines (average lysis 42%) and primary CD23(+) CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR(+) T cells released inflammatory cytokines (1445-fold more TNF-beta, 20-fold more TNF-alpha, and 4-fold more IFN-gamma). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR(+) T cells. Redirected T cells were also effective in vivo in a CLL Rag2(-/-)gamma(-/-)(c) xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23. CAR(+) T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23. CAR(+) T cells represent a selective immunotherapy for the elimination of CD23(+) leukemic cells in patients with CLL. (Blood. 2011; 117(18): 4736-4745)

Chronic myeloid leukemia (CML) is caused by the BCR-ABL hybrid gene. The molecular mechanisms leading from chronic phase (CP) to blast crisis (BC) are not understood. However, both the presence and the levels of BCR-ABL seem to be important for CML progression. BCR-ABL is under the transcriptional control of BCR promoter. Here we focused on the gene expression control of BCR and BCR-ABL upon myeloid differentiation in healthy donors (HDs), CP and BC patients. As previously reported, BCR-ABL is downregulated during myeloid maturation in CP patients. A similar pattern was detected for BCR (but not for ABL) in CP-CML and in HD, thus suggesting that the two genes may be under a similar transcriptional control. In BC this mechanism is similarly impaired for both BCR-ABL and BCR. These data indicate the presence of an 'in trans' deregulated transcription of both BCR and BCR-ABL promoters, associated with CML progression.

Background. Immune mechanisms of extracorporeal photochernotherapy (ECP) in refractory/resistant graft-versus-host disease (GvHD) are complex. We have previously analyzed the role of CD4(+)CD25(+)Foxp3(+) regulatory T cells (T-regs). Methods. In the current study, we have enlarged the size of the population (n=27; chronic GvHD=18, acute GvHD=9) for a median follow-up of 24 months. T-regs were monitored for CD4, CD25, glucocorticoid-induced tumor necrosis factor receptor (GITR), CD62L, CCR7, Foxp3, and STAT-5. Immune analysis by interleukin (IL)-17 Elispot was carried out on circulating T-helper CD4(+) cells secreting IL-17, a subset of T cells considered relevant in the pathogenesis of GvHD. Results. We confirm that ECP is accompanied by a significant increase of CD4(+)CD25(+)Foxp3(+)GITR(+)CD62L(+)CCR7(+) T-regs. Sorted T-regs show augmented phosphorylation of STAT-5. Only ECP-responding patients demonstrate a raise of circulating T-regs, being mostly affected by chronic GvHD. Moreover, this phenomenon corresponds to a diminished secretion of IL-17. Discussion. In conclusion, our study shows that T-regs represent important immune mediators of the clinical benefits of ECP in patients affected by GvHD.

Acute Graft-versus-Host Disease (GvHD) remains a major complication of allogeneic haematopoietic stem cell transplantation, with a significant proportion of patients failing to respond to first-line systemic corticosteroids. Reliable biomarkers predicting disease severity and response to treatment are warranted to improve its management. Thus, we sought to determine whether pentraxin 3 (PTX3), an acute-phase protein produced locally at the site of inflammation, could represent a novel acute GvHD biomarker. Using a murine model of the disease, we found increased PTX3 plasma levels after irradiation and at GvHD onset. Similarly, plasma PTX3 was enhanced in 115 pediatric patients on day of transplantation, likely due to conditioning, and at GvHD onset in patients experiencing clinical symptoms of the disease. PTX3 was also found increased in skin and colon biopsies from patients with active disease. Furthermore, PTX3 plasma levels at GvHD onset were predictive of disease outcome since they resulted significantly higher in both severe and therapy-unresponsive patients. Multiple injections of rhPTX3 in the murine model of GvHD did not influence the disease course. Taken together, our results indicate that PTX3 constitutes a biomarker of GvHD severity and therapy response useful to tailor treatment intensity according to early risk-stratification of GvHD patients.

Background. Extracorporeal photochemotherapy (ECP) produces clinical improvements in refractory/resistant graft-versus-host disease (GvHD). Immunological mechanisms of ECP are still under investigation. Methods. We have evaluated the changes in frequency and immunophenotype of circulating regulatory T cells (T-regs) in 10 patients undergoing allogeneic hematopoietic stem cell transplantation, receiving ECP for acute (n = 4) or chronic (n = 6) GvHD. T-regs were monitored for expression of surface CD4, CD25, GITR, CD45RO, CD62L and intracytoplasmic Foxp3. T-regs were sorted by fluorescence-activated cell sorting to perform functional assays by interferon (IFN)-gamma enzyme-linked immunospot and real-time quantitative polymerase chain reaction (RQ-PCR) to measure Foxp3, transforming growth factor (TGF)-beta, and interleukin (IQ-10 mRNA. Results. ECP was accompanied by a significant increase of CD4+CD25+ T-regs after six procedures, increasing from 8.9% to 29.1% of total CD4 (P

Background and Objectives: Acute lymphoblastic leukaemia (ALL) carrying t(9;22) or t(4;11) genetic abnormalities represents a very high risk subtype of disease (VHR-ALL). Hematopoietic stem cell transplantation (HSCT) remains the best curative option not only for t(4;11) ALL, but also for t(9;22) ALL in the tyrosin-kinase inhibitors era. In the last years, low molecular level of minimal residual disease (MRD) before HSCT was reported as one of the best favourable indexes for survival in ALL. Here we observed that even these patients can show a favourable outcome if submitted to HSCT with very low MRD. Methods: We considered 18 consecutive VHR-ALL patients eligible to HSCT. 16 of them were transplanted in first remission, as soon as possible, employing myelo-ablative conditioning regimens. Molecular MRD has been evaluated before and after HSCT. Results: Immediately before HSCT, MRD revealed: complete molecular remission (MRDneg) for five patients, and a level -3 for seven patients. 100 days after HSCT we had: MRDneg for seven patients and a decrease for all the others after HSCT. After the tapering of immunosuppressive drugs, 13 patients reached the MRDneg in a median time of 8 months (range 3-16). In the intention to treat analysis, 14/18 patients are alive and disease free at the date of analysis. Overall survival and event free survival is of 78% and 66% respectively, with an average follow-up of 45 months (range 6-84) since HSCT. Conclusion: Early transplantation with low MRD level seems to be correlated with a favourable outcome also in VHR-ALL.

BACKGROUND: Imatinib mesylate (IM) was introduced in chronic myeloid leukemia (CML) treatment in the late 1990s and substantially changed the therapeutic approach to the disease, by inducing complete cytogenetic response (CCR) in approximately 60 percent of cases. Nevertheless, some concerns exist about the duration of response to treatment and the onset of resistance to IM. STUDY DESIGN AND METHODS: Twenty-five chronic-phase CML patients in stable CCR (>6 months) treated for at least 1 year with IM at the standard dose (400 mg/day) were mobilized with recombinant human granulocyte–colony-stimulating factor (Filgrastim) at 10 µg per kg for 4 to 6 days, with the aim of collecting at least 2 × 106 CD34+ cells per kg. Standard cytogenetic analysis and first-round and/or nested polymerase chain reaction were performed in basal and postmobilization samples to examine the presence of bcr-abl transcripts. RESULTS: CD34+ cells collection was successful in 16 patients, yielding a median of 3.01 × 106 ± 1.09 × 106 CD34+ cells per kg at the first attempt, and in 4 of the 9 remaining patients who were remobilized after a temporary withdrawal of IM, yielding a median of 2.65 × 106 ± 0.7 × 106 CD34+ cells per kg, with an overall 80 percent success rate. No correlation between mobilization and duration of the disease, length of IM treatment, or previous interferon-α and/or hydroxyurea treatment was found. CONCLUSIONS: Autologous CD34+ cells may be mobilized and collected in most CML patients who achieve CCR after IM treatment, with a view to possible use in the event that resistance to IM occurs in patients not eligible for allogeneic peripheral blood progenitor cell transplantation or those lacking an HLA-matched donor.

According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133+cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 106of CD133+cells (range 2.85 × 106–30.84 × 106), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133+cells of 90,60% (range 81,40%–96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 106cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial).

We analyzed appearance of non transferrin bound iron (NTBI) in 30 transplant eligible patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) during conventional chemotherapy treatment program and evaluated possible relationship with transfusional body iron intake, iron parameters and clinical complications. For each course, serum samples for NTBI detection were taken prior to chemotherapy, during treatment and during subsequent bone marrow myelosuppression: NTBI was assessed by HPLC. Appearance of NTBI was observed from the start of induction treatment and was still detectable during bone marrow myelosuppression; the recovery of the bone marrow function coincided with the disappearance of NTBI. This kinetic was observed in all subsequent high doses chemotherapy courses, independently from confounding variables such as transfusional iron intake and transferrin saturation. NTBI seems to be a consequence of chemotherapy induced lysis of bone marrow cells and, partly, of hepatocytes after cytotoxic injury. The subsequent persistence of NTBI throughout bone marrow myelosuppression is related to the transient suspension of erythropoietic activity. Moreover, NTBI levels >2μM at the beginning of iatrogenic myelosuppression were associated with higher risk of sepsis caused by Gram negative Bacilli (RR 2.571), also compared with other infectious complications (RR 1.954).

To assess the economic outcomes produced when a conventional antibiotic treatment regimen requiring three administrations per day was replaced with a treatment regimen requiring only one daily administration, the efficacy, tolerability and cost of ceftazidime was compared with that of ceftriaxone (both drugs in combination with amikacin) for the empirical treatment of febrile granulocytopenic patients with haematological malignancy. 102 febrile patient-episodes were randomly assigned to receive ceftazidime (6g in three divided doses) or ceftriaxone (2g as a single daily dose), both in combination with amikacin. The response was evaluable in 94 patients (47 in each group). 75 (80%) patients had an absolute granulocyte count lower than 100/mm(3) at the onset of fever or during the first week of antibiotic therapy. 61 (64.9%) were affected by acute leukaemia. Multiple daily ceftazidime plus amikacin was effective in 33 of 47 (70.2%) patients, and single daily ceftriaxone plus amikacin in 31 of 47 (66%) patients (p > 0.2). Among patients successfully treated, median time to defervescence was 3.3 days (range 1 to 11) for ceftazidime plus amikacin and 4.5 days for ceftriaxone plus amikacin (range 1 to 15) [p = 0.14]; study drugs were continued for 12 (range 7 to 26) and 12.3 days (range 7 to 28), respectively. Our study demonstrated that single daily administration of ceftriaxone was as effective and well tolerated as multiple daily administration of ceftazidime when both were administered in combination with amikacin. Cost analysis showed that compared with the thrice daily regimen, administration of single daily doses of ceftriaxone for a 12-day treatment period would result in a net cost saving of $US392 (626 940 Italian lire).

The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133(+) cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133(+) cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133(+) cells. Validation of CD133(+) cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as 'proofs of principle' of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133(+) cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133(+) cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies.

Objectives. Graft-versus-host disease (GVHD) is a major obstacle to safe allogeneic hematopoietic stem-cell transplantation, leading to significant mortality. Recently, T-helper (TH)-17 cells have been shown to play a central role in mediating several autoimmune diseases. The aim of our study was to investigate the relationship between TH-17 cells and GVHD occurring in transplanted patients. Methods. Blood samples were collected from 51 hematopoietic stem-cell transplantation patients and 15 healthy donors. Patients with GVHD were monitored for the presence of TH-17 cells by ELISPOT or flow cytometry in the peripheral blood and by confocal microscopy in GVHD lesions. Cytokine plasma levels were detected by ELISA. Results. An increased TH-17 population (up to 4.8% of peripheral blood CD4+T lymphocytes) was observed in patients with acute GVHD and (up to 2.4%) in patients with active chronic GVHD along with an inflammatory process. In contrast, the percentage of TH-17 cells drastically decreased in patients with inactive chronic GVHD. TH-17 cells consisted of both interleukin (IL)-17(+)/interferon (IFN)-gamma(-) and IL-17(+)/IFN-gamma(+) subsets and expressed IL-23 receptor. Interestingly, IFN-gamma(+)TH-17 cells were able to infiltrate GVHD lesions as observed in liver and skin sections. Moreover, the proportion of TH-17 was inversely correlated with the proportion of regulatory T cells observed in the peripheral blood and tissues affected by GVHD. Finally, we demonstrated a strong correlation between TH-17 levels and the clinical status of patients with GVHD. Conclusions. These findings support the hypothesis that TH-17 are involved in the active phases of GVHD and may represent a novel cellular target for developing new strategies for GVHD treatment.

Despite recent advances, allografting remains the only potential cure for myeloma. From July 1999 to June 2005, 100 newly diagnosed patients younger than 65 years were enrolled in a prospective multicenter study. First-line treatment included vincristin, adriamycin, and dexamethasone (VAD)–based induction chemotherapy, a cytoreductive autograft (melphalan 200 mg/m2) followed by a single dose of nonmyeloablative total body irradiation and allografting from an human leukocyte antigen (HLA)–identical sibling. Primary end points were the overall survival (OS) and event-free survival (EFS) from diagnosis. After a median follow-up of 5 years, OS was not reached, and EFS was 37 months. Incidences of acute and chronic graft-versus-host disease (GVHD) were 38% and 50%, respectively. Complete remission (CR) was achieved in 53% of patients. Profound cytoreduction (CR or very good partial remission) before allografting was associated with achievement of posttransplantation CR (hazard ratio [HR] 2.20, P = .03) and longer EFS (HR 0.33, P < .01). Conversely, development of chronic GVHD was not correlated with CR or response duration. This tandem transplantation approach allows prolonged survival and long-term disease control in patients with reduced tumor burden at the time of allografting. We are currently investigating the role of “new drugs” in intensifying pretransplantation cytoreduction and posttransplantation graft-versus-myeloma effects to further improve clinical outcomes. (http://ClinicalTrials.gov; NCT-00702247.)

The development of nonmyeloablative conditionings has recently reduced the transplant-related mortality (TRM) and extended the eligible age for transplantation up to 65–70 years. From January 2000 to June 2005, 106 newly diagnosed patients younger than 65 years were enrolled in a prospective phase II study at 15 Italian Centers. Fifty-eight were also previously described in a comparison of autografting with allografting based on a genetic randomisation (Bruno et al. N Engl J Med 2007). Here we report on a larger GITMO experience with a longer follow-up. Induction chemotherapy consisted of VAD-based regimens, followed by a cytoreductive autograft with melphalan 200 mg/m2, and by a non-myeloablative 2 Gy TBI-based allograft from an HLA-identical sibling. Graft-vs-host disease (GVHD) prophylaxis included cyclosporin and mycophenolate mofetil. Primary endpoints were overall (OS) and event-free (EFS) survivals. Secondary endpoint was TRM. One-hundred-two (96%) patients, median age 54 (30–65), completed the tandem program whereas 4 withdrew their consent. After a median follow-up of 54 (21–94) months, OS was not reached and median EFS was 35 (31–56) months post-transplant. Incidences of acute grade II-IV GHVD and extensive chronic GVHD were 40% and 50% respectively. Fourteen (13%) patients died from TRM, 14 (13%) from disease progression, 2 from lung cancer (2%) and 1 from lymphoma (1%). Overall response, defined as complete (CR) and partial remission, was 91% (93/102), with 53 patients achieving CR. Overall 39/102 patients relapsed, however only 8/53 of those who reached CR post-transplant. By multivariate-analysis disease response prior to allografting was significantly associated with longer OS (HR 0.27, CI 0.09–0.80, p

Background Allogeneic SCT remains the only curative option in myelodysplastic syndromes (MDS). We prospectively evaluated in a multicenter phase 2 GITMO (Italian Bone Marrow Transplantation Group) trial, the feasibility of allogeneic stem cell transplantation (ASCT, primary protocol end-point) after induction treatment with azacitidine (Vidaza, Celgene). According to the protocol, patients had to perform at least 4 and up to 12 cycles of azacitidine as induction treatment before ASCT. Patients not eligible for ASCT could continue treatment until disease progression. Methods Between November 2010 and September 2014, 102 patients were enrolled by 20 Italian hematology centers. Nine patients never started treatment due to progressive disease (PD, n=2), refusal (n=3), or unknown causes (n=4). Of remaining 93 patients, 70 had with IPSS Int-2/High-risk MDS, 15 WHO-defined acute myeloid leukemia (AML) and 8 chronic myelomonocytic leukemia (CMML). There were 32 females and 61 males with median age 59 yrs (range 21-66.5 yrs). At treatment start, ECOG performance status was: 0 in 66 patients, 1 in 17 and 2 in 10 patients, while MDS HCT-index was: low (0) in 43 patients, intermediate (1-2) in 31 and high (>2) in 18 patients. All 93 patients started azacitidine s.c. at the standard dose of 75 mg/sqm/day, 7 days/month, at a median of 0.8 months (range 0-105 months) from initial MDS diagnosis. Results At response assessment after a median of 4.5 cycles (range 1-11), 25% of patients (n=19) achieved complete remission (CR), 3% marrow-CR (n=2), 14% partial remission (PR, n=11), 9% hematologic improvement (HI, n=7), 35% had stable disease (SD, n=27) and 14% PD (n=11). ASCT was performed in 48 patients (52%), after a median of 4.5 azacitidine cycles (range: 1-11). Data on ASCT are presently available for 45 patients, transplanted with bone marrow (n=10 pts) or peripheral blood stem cells (n=35 pts) from an HLA-identical donor (sibling in 11, and MUD in 34 cases). Forty-five patients did not receive ASCT due to: disease relapse or progression (n=16, 35%), adverse events (n=12, 27%), refusal (n=5, 11%), or other causes (n=12, 27%). At a median follow-up of 18.5 months (range 0.2-31.5), 43 patients are alive (25 received ASCT), and 50 patients died. Causes of death are reported in Table 1. Median overall survival (OS) for the whole patient cohort was 13.4 months (95% CI: 10.9-18.6, Figure 1). Disease status after azacitidine was predictive of significantly improved survival for patients in CR/PR/HI, compared to SD or PD (n=77 patients who completed at least 4 cycles, Figure 2). ASCT considered as time-dependent covariate was associated to a significantly longer survival (p=0.008, HR 0.4, 95% CI: 0.2-0.8). Stratification of MDS according to IPSS and karyotype was not associated to survival. Conclusions Our study shows that allogeneic transplantation following azacitidine is feasible in HR-MDS/CMML and AML, with about 50% of patients being able to undergo HSCT. These data favourably compare with previous studies in MDS on ASCT preceded by conventional chemotherapy. Disease status at the time of HSCT confirms its significant prognostic role for survival, also in the context of hypomethylating treatment, where stabilization of disease also represents a therapeutic target. Disease relapse or progression remain the major causes of death indicating the need for other strategies aimed at improving disease control in MDS. Figure 1. Overall survival (n=93 pts) Figure 1. Overall survival (n=93 pts) Figure 2. Survival according to disease status at response assessment after a median of 4.5 azacitidine cycles (n=77 evaluable pts) Figure 2. Survival according to disease status at response assessment after a median of 4.5 azacitidine cycles (n=77 evaluable pts) Disclosures Voso: Celgene: Honoraria, Research Funding. Off Label Use: Azacitidine used as induction treatment before allogeneic stem cell transplantation in MDS. Finelli:Janssen: Other: Speaker; Novartis: Other: Speaker; Celgene: Other: Speaker, Research Funding.

Goal of work: We compared the efficacy of ceftriaxone (CA regimen) and piperacillin-tazobactam (PTA regimen) in association with amikacin in the treatment of febrile episodes in severely neutropenic hematological patients. Patients and methods: A total of 252 febrile episodes in 224 patients were randomized. Main results: The CA regimen was effective in 62/122 evaluable episodes (50.8%), and the PTA regimen was effective in 64/121 (52.9%; P > 0.2). Median time to failure was 4 and 5 ays (P > 0.1). Further infections developed in 21/122 episodes (17.2%) with the CA regimen and in 12/121 (9.9%) with the PTA regimen (P=0.06). The overall mortality at the end of the febrile episode was 11/243 (4.5%); seven deaths were considered to be related to infection. Conclusions: Patients treated with piperacillin-tazobactam and amikacin tended to become afebrile sooner and to suffer a lower rate of further infections, even though our data did not show any statistically significant differences between the two groups.

This study was performed to evaluate the incidence, risk factors, and outcome of cytomegalovirus (CMV) infection in autologous stem cell transplantation (ASCT), with the aim of performing preemptive therapy in patients with antigenemia. Starting from 2001, 171 consecutive ASCTs were performed in 136 patients; 102 of these patients were seropositive for CMV at the onset of hematological disease. In all these patients, a CMV pp65 antigenemia assay was determined weekly, starting from the day when the absolute neutrophil count went above 500/microL, and until day 60 after ASCT; subsequently, antigenemia was determined only when a CMV infection was suspected. Among the 136 transplanted patients, 40 (29.4%) presented a positive antigenemia; all of them were seropositive for CMV before ASCT; and no cases of primary infection were seen. The incidence of CMV infection in the seropositive population was 40/102 (39.3%); 6 patients (5 with multiple myeloma and 1 with non-Hodgkin's lymphoma) who received 2 ASCTs developed CMV infections after both transplantations, so that positive antigenemia developed after 46/171 (26.9%) transplantations. First positive antigenemia presented a median of 32 days (range 7-57) after stem cell reinfusion. The median antigenemia level at the first appearance was 2/200,000 (range 1-1000). No significant prognostic factors could be shown. Enteritis was present in 5 patients; 2 of them also had fever, and 1 of them also had thrombocytopenia. In 5 patients fever without any other clinical signs or symptoms was present; 30 patients were asymptomatic. Fourteen patients were treated with anti-CMV drugs. CMV reactivation was successfully treated in all patients, and no patient died from CMV disease.

Congenital absence of the inferior vena cava (AIVC) has been reported as a risk factor of deep vein thrombosis (DVT) in young people. DVT is caused by an interaction between congenital coagulation abnormalities and acquired risk factors. We observed an 18-year-old patient with AIVC who developed recurrent deep vein thrombosis at the left leg. Molecular studies showed an etherozigousity for FV Leiden gene (G1691A) and a homozigousity for methylenetetraidrofolate reductase gene (C677T) in absence of folate and vitamin B12 deficiency. After the second DVI episode, the patient has been treated with heparin and oral anticoagulant without discontinuation.

Autoimmune thrombocytopenia (AITP) is a disorder due to specific platelet auto-antibodies directed against platelet surface glycoproteins. AITP in adults is usually chronic, idiopathic and frequently refractory to conventional treatments. Myelo- and immuno- suppressive chemotherapy followed by autologous peripheral blood stem cell (PBSC) transplantation is an experimental approach for severe chronic refractory AITP. We report a case of a woman with AITP, refractory to the conventional therapy, submitted to T-cell-depleted autologous PBSC transplantation, which obtained long term stable response on platelet count. We deem that the positive outcome of our patient depends on T-cells depletion of the graft, which reduces autoreactive T clones.

It is well known that L-asparaginase (L-Ase) treatment may cause thrombotic events in patients with acute lymphoblastic leukemia (ALL). The mechanism of this effect is not well understood although a reduction in plasma antithrombin III (AT III) levels is observed. In our study, a group of patients treated with L-Ase received AT III concentrates as adjuvant treatment. This adjuvant treatment reduced the levels of plasma D-dimer and thrombin-antithrombin complex, which are considered as early markers of a hypercoagulability state. These preliminary data suggest that large randomized trials will have to be conducted to improve our understanding of the role of AT III concentrates in ALL therapy.

Abstract 743 Background: Acute Graft versus host disease (aGvHD) is a severe complication of allogeneic hematopoietic stem cell transplantation (HSCT). Conventional treatment with high dose steroids fails to achieve a complete and sustained response in more than 50% of patients. Several second line treatments have been described but none of these can be considered superior or a standard of care (Paul J. Martin et al, BBMT 2012). Among these treatments, the use of third party mesenchymal stromal cells (MSC) has been proposed (LeBlanc et al, Lancet 2008). In this study, we assessed the safety and efficacy of third party human MSC, in a prospective, multicenter, phase I study (EudraCT 2008–007869-23). Methods: Forty-seven patients with steroid-resistant, acute or chronic grade II-IV GvHD were enrolled into this study. Human MSC were obtained from bone marrow harvests of healthy donors and expanded in vitro using serum free medium supplemented with human platelet lysate (Capelli C et al, BMT, 2007; Capelli C. et al, Cytotherapy 2009). In vitro expanded MSC were produced in two officially authorized Cell Factories and tested in four Italian Hematology Units. The primary endpoint of this study was the safety. Secondary endpoints were the response of GvHD (evaluated 28 days after the last MSC infusion), as well as the overall survival and transplant-related deaths. Blood samples were periodically collected before and after MSC infusion to measure plasma levels of IL2Ralpha by ELISA, as previously described by our group (Dander E et al, Leukemia 2012). Results: Between August 2009, and June 2012, 47 patients (16 children, 31 adults, median age 25.5 years, range 1 to 67) were treated. The median dose of infused MSC was 1.5×106 cells per kg bodyweight. Enrolled patients presented with aGvHD in 37 cases, chronic overlap syndrome in 7 cases, and chronic classic GvHD in 3 cases. Fifteen pts had grade II GvHD, 23 grade III and 9 grade IV, according to NIH criteria. In 17 cases GvHD involved a single organ, in 24 cases 2, and in 6 cases 3 organs. Prior to MSC infusion 22 patients had received only high dose steroids, 12 patients received one cycle of pentostatin (1 mg/kg bodyweight for 3 days, Schmitt T. et al BMT, 2011: 46 580–585), while 13 received other conventional immunosuppressants. Patients received a median of 3 MSC infusions (range 1 to 8). No side effects were registered immediately after MSC infusion and no complications were lately referred as MSC-related. Overall, in 30 patients (63.8%) a clinical response of GvHD was registered. Thirteen of these patients (27.6%) had a complete response and 17 (36.1%) a partial response to treatment. Twenty-two of the 30 responding patients did not require further lines of immunosuppression after MSC infusion. Response was significantly more likely in patients exhibiting grade II GvHD versus those exhibiting more severe gradings (87.5% vs. 51.6%, p = 0.02) and in patients receiving MSC in a time interval of 30 days from the onset of GvHD (75.9% vs. 43.7%, p= 0.05). Current median follow up for this cohort is 200 days (range 30–1066). Responders show a significant lower transplant-related mortality (10.0% vs. 88.2%, p Measurements of plasmatic levels of IL2Ralpha, when comparing responders vs non-responders patients, showed a statistically significant difference in terms of fold decrease of the marker (p=0.027), corroborating clinical results. Similarly, a significant trend of fold decrease change (p=0.058) was observed when comparing responding patients receiving MSC within or after 30 days from the onset of the disease, in line with clinical results. Conclusions: This study confirms that human MSC prepared in academic cell therapy facilities may represent a safe and effective treatment of patients with steroid-refractory GvHD. Plasmatic inflammatory markers may help in evaluating and monitoring of clinical response. The sequential or combined administration of MSC and other immunosuppressants, such as pentostatin, is equally safe and feasible and deserves further investigation. We suggest to consider the use of MSC promptly, as early as possible, after steroid failure. Disclosures: No relevant conflicts of interest to declare.

Abstract 2446 B-Chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of B-lymphocytes expressing CD19, CD20dim and aberrantly expressing the CD5 T-cell marker. Moreover, they over-express the B-cell activation marker CD23. Chimeric Antigen Receptors (CAR) are engineered molecules able to redirect T-cell killing/effector activity towards a selected target in a non MHC-restricted manner. First trials targeting B-CLL were based on both monoclonal antibodies and anti-CD19/anti-CD20.CAR-transduced T cells. However, this approach causes the elimination of normal B-lymphocytes and B-precursors with consequent impairment of humoral immunity. Selective CD23 expression on B-CLL cells renders this molecule an optimal target to design a specific CAR. We have generated a novel CD23-targeting CAR to redirect T cells against CD23+B-CLL. Transduced T cells were tested for cytotoxicity against different CD23+-targets, using a classic 51Chromium release assay, and for specific cytokine release, by multiplex flow cytomix assay. T cells from B-CLL patients were efficiently transduced with the anti-CD23.CAR (average expression 68%, n=10) and redirected specifically toward autologous blasts (average lysis 58%, n=5). On the contrary, anti-CD23 transduced T-cells did not displayed any relevant killing versus normal B cells (average lysis 13%, n=3), differently from anti-CD19.CAR redirected T-cells, which killed tumor and normal B cells in an indistinct manner. Moreover, anti-CD23.CAR redirected T lymphocytes derived from both healthy donors (HD) and B-CLL patients displayed a specific lytic activity against CD23+EBV-LCLs, even in presence of soluble CD23 enriched plasma without being inhibited (average lysis with no plasma 67%; average lysis with 25% of CD23 enriched plasma 79%; average lysis with 50% of CD23 enriched plasma 88%, n=3). We also demonstrated that the expression of the anti-CD23.CAR caused a significant increase in cytokine release from transduced in vitro activated T cells after a 48h stimulation with CD23+ targets. B-CLL derived CD23.CAR-expressing T cells (n=3) secreted 4-fold more INF-gamma, and 1445-fold more TNF-beta, compared to non transduced T cells. Interleukin-2 was also released (average release 2681 pg/mL, n=3) and sustained the antigen-dependent proliferation of CD23.CAR+T cells. To confirm in vivo the in vitro data, we tested NT and CD23.CAR transduced T cells in a recently published xenograft model of B-CLL (Ref biblio, primo nome, giornale, anno). This model is based on the intravenous or subcutaneous injection of the established human B-CLL cell line MEC1 into Rag2−/− gammac−/− mice, which lack not only B and T cells, but also natural killer (NK) cells, thus presenting a profound immunosuppressive environment leading to an high efficiency of both B-CLL and T-cell engraftment. Moreover, this model reproduces the systemic involvement of the disease and it closely resembles the aggressive form human B-CLL, representing an optimal experimental setting to test the efficacy of new therapeutic agents. In the first set of our experiments, 8 weeks-old male mice were subcutaneously injected with 10*106 MEC1 cells in the left flank. Then, mice bearing an established tumor were treated intravenously with a single dose of NT or engineered CD23.CAR T cells (2*106), without any addition of exogenous IL-2. Animals were monitored twice a week for weight and tumor growth (measuring three perpendicular diameters), and sacrificed when the mean tumor volume reached a dimension of 31000 mm3, before presenting clinical signs and symptoms. Compared with NT-treated mice, the infusion of CD23.CAR+T cells resulted in a significant delay in tumor growth, as measured by tumor volume diameter/day (CD23.CAR+ T cells vs NT T cells: p=0.04 at day 12; n=3). In conclusion, our results suggest that CD23.CAR-redirected T cells provide cytotoxic activity against CD23+ B-CLL cells in vitro and in vivo, while sparing normal B lymphocytes, as compared to other available CARs targeting pan-B-cell antigens, such as CD19 and CD20. These results are encouraging and demonstrate the feasibility of generating CD23.CAR+ T lymphocytes for adoptive T-cell therapy of patients with B-CLL. Disclosures: No relevant conflicts of interest to declare.

Thrombotic thrombocytopenic purpura (TTP) is a common illness characterized by platelet thrombi within the microvascularization. In its natural course, this disease has had a mortality rate of 90%. Plasma infusion or exchange achieved a survival rate of 70% to 90%. However, 10% to 30% of patients surviving the initial TTP episode relapse at regular intervals. The treatment of recurrent forms of the disease re mains a challenge; several approaches have been shown to induce medium to long term remissions. We describe a patient with recurrent TTP whose disease remitted after administration of defibrotide.

BACKGROUND: Essential thrombocythemia (ET) is thought to derive from the transformation of a multipotent hematopoietic stem cell, but its molecular pathogenesis remains obscure. An acquired mutation in exon 14 of the Janus tyrosine kinase 2 gene (JAK2) leading to a valine --> phenylalanine change at position 617 (JAK2V617F) in the JH2 pseudokinase domain, has been described in Ph negative chronic myeloproliferative disorders (MPD). It can be found in approximately 95% of polycythemia vera (PV) and in approximately 50–60% of essential thrombocythemia (ET) or primary myelofibrosis. The impact of this mutation on clinical phenotype is still debated. We investigated the frequency of JAK2 mutation and its possible impact and correlation with clinical and laboratory features at diagnosis and during follow-up in ET patients (pts), screened for JAK2 mutational status, referred to our division between 1987 and 2008. METHODS and PATIENTS: JAK2V617F mutation analysis was performed on genomic DNA from bone marrow cells or peripheral blood granulocytes in 115 ET (49 males, 66 females) pts, using allele-specific PCR. Clinical and laboratory features were evaluated at diagnosis and during follow-up, in order to compare ET pts harbouring JAK2 mutation versus (vs) pts harbouring wildtype (WT) gene. For statistical analysis, data were processed using the Graph Pad PRISM 5 DEMO Software. Correlations between clinical variables and JAK2 V617F mutational status were tested for significance (p value < 0,05) by using non-parametric methods, in particular the chi-square test or Fisher’s exact test (two by two table) for categorical nominal variables and the Mann-Whitney rank test for ordinal variables. The log-rank (Mantel-Cox) test applied to Kaplan-Meyer method was employed to estimate thrombotic risk and thrombotic event-free-survival (TEFS) RESULTS: 66 ET pts (57,4%) were JAK2V617 positive. JAK2 mutated pts were older (median age 59 vs 48 years, p = 0,0016) and presented at diagnosis significantly higher hemoglobin levels (Hb 14,3 g/dl vs 13,3 g/dl, p= 0,0027), higher hematocrit (Ht 43% vs 39,8%, p < 0,0001) and lower PLT count (PLT 725 vs 841 ×109/L, p = 0,005) respect to WT group. These PV-like features have also maintained during the course of disease, with statistical significance. No difference was observed in term of gender, white blood count, LDH, progression or entity of splenomegaly, and need of cytoreductive therapy between JAK2 mutated and WT ET pts. A highly significant increase of thrombotic complications was registered for JAK2 positive pts. Considering JAK2 WT ET as reference group, the relative risk (RR) of primary thrombotic event was 2,143 (95% CI: 1.053–4.360, p = 0,035) for JAK2 mutated pts (TEFS at 15 years of 60% in JAK2 mutated group vs 75% in WT group, respectively). Arterial events were more frequent than venous events without statistical difference between two ET groups. A trend of higher risk of recurrent thrombosis (p = 0,056) was also showed for JAK2 mutated respect to WT ET pts. There was no difference about the risk of haemorragic complications, time and incidence of evolution in myelofibrosis between JAK2 mutated or WT ET pts. CONCLUSION: In our population, 57,4% ET pts harbor JAK2V617F mutation. This mutation divides ET pts into two distinct subtypes. JAK2 mutated ET pts present older age and laboratory features at diagnosis and during follow-up, compatible with PV-like phenotype. An increased risk of thrombosis, at diagnosis and during the course of disease, has been showed in mutated ET group, in term of recurrent thrombosis too. These observations seem to confirm a phenotype more aggressive since the diagnosis in JAK2 mutated ET group. A possible biological continuum between JAK2 mutated ET and PV, or more in general between JAK2 positive ET and MPD may be supposed.

Background Chimerism Status (CS) analysis is largely useful for evaluation of donor (D) cells engraftment after allogeneic haematopoietic stem cell transplantation (HSCT). The largest performed method is based on the evaluation of the DNA sequences “Short Tandem Repeats” (STR) and Variable Number Tandem Repeats” (VNTR). Their high polymorphism permits to differentiate donor (D) or recipient (R) origin of the analysed cells. PCR amplification and DNA sequencing permit to quantify D and R amount. Patients and methods CS analysis after HSCT can be conducted on different cell populations in marrow or peripheral blood: whole blood cells, mononucleated cells, or single separated populations. A complete CS (CCS) usually correlates with a favourable outcome of HSCT, conversely a mixed CS may predicts a relapse of disease or a rejection of HSCT, particularly in the condition defined as increasing-mixed CS (IMCS), in which the amount of R origin cells increases along the time. In our study we employed a simple and easily reproducible method to analyse CS on peripheral blood cells at +30, +60, +90 and +120 days after HSCT. CS analysis was conducted separately on granulocytes and mononucleated cells, obtained after double gradient centrifugation. A semi-quantitative method, based on multiplex PCR amplification of 16 STR markers using a commercial kit (PowerPlex® 16 System–Promega), was applied to define the difference between D and R. We assumed the CS in granulocyte population as expression of myeloid engraftment. We also correlated the CS in mononucleated population, with the different leukocyte subpopulations (lymphocytes, monocytes) in peripheral blood count and with the reconstitution of lymphocyte subpopulations (B, T cells and NK cells) at the same time point of analysis, in order to compare the reciprocal course. We evaluated 13 HSCT on 12 patients (pts) (1 pts was submitted to double HSCT for a rejection occurred after the first HSCT), 7 from sibling related donor (SRD) and 6 from matched unrelated donor (MUD). All the pts were affected by neoplastic disease in first (9) or next (3) remission: acute lymphoblastic (5) or myeloblastic leukaemia (4), mielodisplasia (1), chronic myeloid leukaemia (1), multiple myeloma (1). All the conditioning regimens were mieloablative, all the MUD-HSCT and one of the SRD-HSCT received Anti-Thymocyte-Globulin (ATG), 7/8 SRD-HSCT did not received ATG nor other T-depletion procedures. Results: Evaluation on time + 30, +60, +90 and +120 days after HSCT was performed on 13 HSCT, except in two pts who stopped before (+30 and +60) due to their death for relapse or toxicity. A stable CCS in granulocytes was observed in 10/13 HSCT since the first control. 3/13 HSCT presented IMCS followed by relapse (2 pts) or rejection. In mononucleated cells population 10/13 HSCT presented CCS at the first control, 2/10 evolved to CCS in the successive times. The 3 pts who relapsed or rejected presented a IMCS in mononucleated cells too. Considering the different leucocyte populations we observed that the mononucleate cells amount was constituted for only 50% from lymphocytes, the remaining 50% was represented from monocytes. Therefore the analysis of lymphocyte subpopulations showed that T-lymphocytes were virtually absent (either T4 or T8) till to +120 in pts who received ATG, so in these ones CS analysis in mononucleated cells was performed only in monocytes, B lymphocytes and NK cells. Instead pts who did not received ATG presented complete and more rapid reconstitution of all the lymphocyte subpopulations (median at +60 day: T4 200, T8 120, B 60, NK160 × 10^9/l). Conclusions: our method is simple and rapid in analysing CS. Granulocytes CS reflect the engraftment of myeloid lineage. Instead CS in mononucleated cells must be considered in the context of the type of transplantation (MUD or SRD, T-depletion or T repletion) and correlated with the single subpopulations of peripheral blood.

Graft-vs-host disease (GvHD) is a major obstacle to safe allogeneic haematopoietic stem cell transplantation (HSCT), leading to significant morbidity and mortality. Recently, a subset of interleukin (IL)-17-producing cells, named Th17, have been shown to play a central role in the induction of autoimmune-tissue injuries and inflammation. The aim of our study is to analyze the Th17 population in the peripheral blood of transplanted patients experiencing (6) acute, (7) active chronic and (6) chronic GvHD. Both Elispot assay and intracellular staining showed an augmented population of Th17 (both IL-17+/IFN-g− and IL-17+/IFN-g+ producing cells) in patients with both aGVHD and active cGVHD, that can represent up to 2.4% of CD4+ T lymphocytes. In accordance, an increased IL-17 plasma level was detected both in patients with aGVHD (mean IL17A concentration= 14 pg/ml) and active cGVHD (mean IL17A concentration= 12 pg/ml), compared to healthy donors (mean IL17A concentration < 4 pg/ml). Interestingly, the percentage of TH17 cells drastically decreased in patient with quiescent cGVHD. Th17 cells of patients with aGVHD and active cGVHD, were of donor origin, as demonstrated by genotyping, and, in line with what already described, showed high IL23 receptor expression (mean= 40%, range 30%–52%). Noticeably, in the liver and skin GVHD lesions, the solely CD3 cells spontaneously producing cytokines were double positive for INF-γ and IL-17A. Importantly, we demonstrated a strict correlation between the levels of TH17 cells and the clinical status of patients with GVHD. The better understanding of the role played by TH17 in the GVHD pathogenesis could open the way to new highly targeted strategies for the management of GVHD.

Cytokine induced killer cells (CIK) are T/NK cells with demonstrated anti-tumoral activity but lack of GVHD reactivity. They are expanded in vitro after stimulation of PBMC with OKT3, IFN-γ and rhIL-2. This phase I study was designed to test the safety and feasibility of repeated infusions of in vitro expanded donor derived CIK cells given to patients relapsed after allogeneic HSCT. The mean number of starting total nucleated cells was 707 × 106 (range 58–1500 × 106). After a median 22 days of culture, a mean percentage of 51% CD3+CD56+ cells (range 40–71%) was obtained corresponding to an absolute mean number of 1421×106 total CIK cells (range 422–2470 × 106). Eleven patients with AML (n=4), HD (n=3), CMML (n=1), pre-B ALL (n=1) and MDS (n=2), all relapsed after sibling (6) or matched unrelated donor (5) HSCT, entered this study. Before CIK administration, 7 patients had received one or more additional salvage treatments including chemotherapy (5), radiotherapy (1) and unmanipulated DLI (6) without any significant tumor response. The median number of CIK infusions was 2 (range 1–7) and the median number of total CIK cells was 14.5 ×106/kg (7.2–51). The infusions were well tolerated and no acute or late infusion-related reactions were registered. Acute GVHD (grade I and II) was observed in 4 patients 30 days after the last CIK infusion, which progressed into extensive chronic GVHD in 2 cases. In 6 patients, no significant clinical response could be registered so that disease progression and death occurred rapidly. In contrast, 5 patients achieved measurable responses: a patient with MDS, who had been treated with CIK cells alone, showed a hematologic improvement but subsequently progressed and died. One patient with HD received local radiotherapy and 7 CIK infusions (total of 51×106/kg CIK cells) which allowed the achievement of a good PR. After almost 1 year (300 days), he progressed and chemotherapy was given with achievement of a very good PR. A second patient with HD received one DLI at day 516 and 1 CIK infusion (12.2×106/kg) at day 537. At day 572, chemotherapy was initiated due to the persistence of disease. At the end of chemotherapy he received 3 additional CIK infusions for a total of 34×106/kg from days 711–752, without signs of aGVHD and is presently in CR at more than 780 days. One patient with CMML had been treated with DLI on day 102 and with a total of 38×106/kg CIK cells, given in four infusions from days 137–530, because of the appearance of mixed chimerism. Full chimerism was achieved 42 days after the first CIK infusion. The patient remains in CR at day 576. A second MDS patient, who had not achieved any significant response after five DLI (days 411–559), obtained a complete hematologic, cytogenetic and molecular remission after a single CIK infusion (7.6 ×106/kg) given at day 603 and remains in CR at day 680. This study shows that the production of allogeneic CIK cells is feasible, their infusion is generally safe and may induce clinical remission in patients relapsing after HSCT.

High-dose therapy supported by autologous stem cell transplantation (ASCT) has became a widely applied therapy in many haematological malignancies during two decades. ASCT may be curative and even more commonly leads to prolonged survival. Along with the use of mobilised blood progenitor cells and improved supportive care after ASCT, the early treatment-related mortality (100 d) non-relapse mortality (NRM) occurs. We have analysed a cohort of 191 patients (108 male and 83 female) who received ASCT (247 transplant) in 1994–2005 in Bone Marrow Transplant Unit of San Gerardo Hospital. The median age was 49 years (16–70). The most common diagnoses included lymphoma (88), multiple myeloma (67), acute leukemia (32), chronic lymphocitic leukemia (2) and myelodysplastic syndrome (2). At the time of transplant (247 transplant) 93 patients was in complete remission, 67 patients in partial remission and 86 patients in persistent/progression malignancies. The most common conditioning treatment ASCT included: Melphalan (200 mg/m2 or 140 mg/m2) in 55% of case, BEAM in 30%, Busulfan-Cyclophosphamide in 4%, others in 11% (Thiotepa- Cyclophosphamide, Busulfan-melpalan, Mitoxantrone-melphalan, TBI- Cyclophosphamide). The median dose of CD34+ reinfused was 5.21 × 10^6/kg (1.2–15 × 10^6/kg). The median time to achieve an absolute neutrophil count >0.5 × 10^9/l was 12 days (range 0–49) and to achieve a platelet count > 20 × 10^9/l was 19 (range 0–36). The median time of hospitalisation was 22 days (range 10–93). During hospitalisation 158 patients (63%) developed oral mucositis: 100 (53%) of them developed mucosites of grade 1–2, 58 (37%) of grade 3–4. High dose chemotherapy with BEAM was more toxic on the oral mucose than high dose of Melphalan alone. In 41% of patients total parenteral nutrition support was necessary. During hospitalisation 180 patients (73%) developed sepsis: possible infection 61%, Gram + 20%, Gram − 12%, miscellaneous 3%, fungi 2%, viral 1%, Pneumocistis Carinii 1%. Regimens including cyclophosphamide was responsible for most sepsis. 44 patients presented a positive antigenemia of Cytomegalovirus; all of them were seropositive for CMV before ASCT; only one case of primary infection was seen and this patient died after 55 days after ASCT. At 100 days from ASCT 173 patients were in complete remission, 53 of them had a relapse of hematologic malignancies in a median time of 1 year (range 51 days-6 year), 32 patients in partial remission and 35 patients in persistent/progression malignancies. The late complications were: cardiac (30%), hepatic (16%), renal (3%), gastrointestinal (12%), CNS (8%), other (MGUS, cataract, aseptic necrosis of bone, diabete)(31%). The most important cardiac complications were: atrial fibrillation, sinusal tachycardia, arrhythmia, pericarditis. 61 patients (32%) died after ASCT. Death was due to complications related to disease progression in 62% of patients. 25% of patients died from infections. Secondary malignancies (acute myeloid leukemia) were the cause of late non relapse mortality in 2 patients. Fatal cardiovascular complications were observed in 4 patients (acute myocardial infarction or cardiomyopatia most probably due to previous anthracycline therapy).