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BACKGROUND The rate of major arterial and venous thrombosis in primary myelofibrosis (PMF) and post-ET (PET) and post-PV (PPV) secondary myelofibrosis has been evaluated in a limited number of studies. In the present paper we describe the clinical epidemiology of thrombosis in a large series of patients with overt PMF and PPV/PET MF looking at the rate and risk factors. Moreover, we report findings on thrombosis rate in two cohorts of patients treated with Hydroxyurea (HU) or Ruxolitinib (Ruxo). METHODS Patients were registered in the European Registry for Myeloproliferative Neoplasms (ERNEST). This project, promoted by the European LeukemiaNet, is coordinated by FROM - Foundation for Research, Papa Giovanni XXIII Hospital, Bergamo (Italy) and supported by Novartis through a research collaboration . Patients were diagnosed in 6 Centers from Italy, Spain and Sweden, between Jan, 2001 and Dec, 2012, with the required follow-up information. Patients (n= 1010) with PMF (n=584, 59%), PET-MF (n=207, 20%) and PPV-MF (n=219, 21%) were evaluated for incident thrombosis as primary endpoint. Considering death as a competitive event, uni-and multivariate analyses were performed by applying Fine & Gray competing-risk regression models. RESULTS After a median follow-up of 3.8 years (IQR: 1.8-7.1) from diagnosis, 108 thromboses (10.7%) occurred, for an overall incidence rate of 2.0% pts-yr (95% CI: 1.7-2.5). Arterial thromboses were found in 50 patients (46.3%) including cerebral (n=21, 19.4%), myocardial infarction (n=13, 12.0%) and peripheral events (n=9, 8.3%). Venous thromboses were 58 (53.7%), of which 25 (23.0%) were DVT ± PE and 11 (10.2%) were splanchnic. Thrombosis rate was 1.91, 1.60 and 2.79% pts-yr in PMF, PET-MF and PPV-MF, respectively. In univariate analysis, factors significantly associated with an increased thrombotic risk in PMF were age (p=0.013) and the presence of the JAK2 mutation (p=0.003); in addition, a significant higher proportion of PMF patients at low and intermediate-1 vs intermediate-2 or high risk IPSS score, had thrombosis during the follow-up (p=0.008). In multivariate analysis, only JAK2 mutation retained statistical significance (SHR=3.12, 95% CI: 1.40-6.94, p=0.005). Conversely, neither in univariate nor in multivariable analysis, significant risk factors were not found.To investigate the possible interaction of IPSS score and JAK2 mutation we created a model whose results are presented in Fig. 1A: the cumulative incidence function (CIF) of thrombosis was significantly lower in patients with JAK2 wild-type and intermediate-2 or high IPSS score (CIF: 4% projected at 10 years; SHR=1 [reference category]), while patients at the highest risk for thrombosis harbored JAK2 mutation and were categorized at low or intermediate-1 by IPSS score (CIF: 20% projected at 10 years, SHR=7.13, p=0.008). Of note, thrombosis had a significant impact on mortality. After adjusting for sex, age, year of diagnosis, type of MF and IPPS, HR was 1.51, (95% CI. 1.15-1.98, p=0.003).The influence of drug exposure to incident thrombosis was investigated in two cohorts of 559 consecutive patients exposed to HU (n=470) or to Ruxo (n=89), median treatment 2.6 and 3.0 years, respectively. HU- compared to Ruxo-treated patients were older (median age 67 vs. 63 years, p=0.001), more frequently triple negatives (12% vs. 2%, p=0.036), less splenomegalic (spleen length >10 cm: 30% vs. 88%, p CONCLUSION IPSS score, in addition to the survival risk assessment, may be useful, if associated with the JAK2 mutation, to recognize patients at vascular risk and to suggest appropriate anti-thrombotic prophylaxis. The trend towards a benefit of Ruxo, compared to HU, warrants a study in larger case series. Figure 1 Figure 1. Disclosures Barbui: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Passamonti: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Speakers Bureau. Vannucchi: Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Lectures.

Background: A myeloproliferative neoplasm (MPN) is frequent underlying cause of splanchnic vein thrombosis (SVT). We reported that ruxolitinib, a JAK1/2 inhibitor, was safe in patients (pts) with MPN-associated SVT and effective in reducing spleen size at the planned primary analysis at 24 weeks (w) in an investigator-initiated phase II clinical trial (Pieri L, AJH 2016). Herein we present final long term follow up (FU) data. Methods. A total of 21 MPN-SVT entered the trial in 2012. Pts who completed the 24w core study and well tolerated the drug and had evidence of clinically-significant improvement were allowed to enter an extension phase up to w72, then entered a long term FU. The drug was provided by Novartis, that had no role in trial design nor in data analysis. Safety data were reported as cumulative incidence of adverse events (CTCAE v4.03). Clinical responses were defined according to ELN and IWG-MRT criteria. Results. Patients disposition. Eighteen patients were alive at last follow-up at a median of 5.5y, range 3.7-6.5y. Two pts died before w72 (hepatocarcinoma; unknown cause), 1 pt died at w252 (sepsis). Diagnosis of the 18 pts was: PMF 8 (44%), PV 5 (28%), ET 4 (22%), PET-MF 1 (6%). The DIPSS risk score of the 8 pts with PMF was low in 4 and intermediate-1 in 4. At last FU, 14 patients are still on ruxolitinib (78%), 10 pts still on clinical trial, 4 pts shifted to commercial drug. Four pts (22%) withdrawn treatment after w72 because of inefficacy, withdrawal of consent, unknown reasons, shift to observation only (1 each). Safety. Thirteen pts (72.2%) had adverse events; the median number of events per patient was 5 (range, 0-37). Five pts had G2 thrombocytopenia plus 1 pt G3 (28%), 9 had G2 anemia (50%), 1 had G2 neutropenia (6%). There were 4 non-hematologic G3 adverse events (22%), one drug-related (CPK increase) that resolved with dose reduction. Six pts developed G1-2 infections (33%), 4 had Herpes Zoster reactivation (22%), 1 developed second cancer (pheochromocytoma at w96; 6%). Efficacy. Spleen response (defined as percentage reduction of spleen length from left costal margin (LCM) from baseline value as by IWG-MRT criteria) was available in 16 of 18 pts on treatment. Of 8 pts who obtained spleen response at w72, 4 pts had a complete response, while the remaining 4 pts had a spleen reduction >50%. At last FU, 6 of 8 pts (75%) adjudicated as spleen responders at w72 maintained the response. None of the 8 pts who had not obtained a spleen response at w72 acquired it at the last FU. According to ELN/IWG-MRT criteria of disease response, 7 pts had partial response, 11 had stable disease. At abdominal vessel eco scan, thrombosis improved in 3 of 12 evaluated pts (25%) and remained stable in the remaining 9. Of the 16 pts with endoscopic evaluation at last FU, complete resolution of oesophageal varices was documented in 3/12 with baseline varices (25%), worsening in 4 (33%), stable in 5 (42%), while no de-novo formation of F1 varices was documented in the remaining 4 (33%). No pt had gastrointestinal bleeding episodes. Exploratory endpoints. Included changes in the level of JAK2V617F variant allele burden (VAF) at last FU, available in 13 of 15 JAK2V617F mutated pts. VAF remained stable in 2 pts (13%), increased by 34% (range 25-40) in 3 pts, while 8 pts (62%) had a reduction >10% (range 18-96), of whom 6 (46%) had a reduction >50% (median 82%, range 70-96). By correlative analysis of JAK2 VAF changes and spleen response, 3 of 6 pts with VAF reduction of >50% had complete spleen response, as compared to none of the 3 pts who had an increase of VAF. Conclusions. After a median follow-up of 5.5 years, ruxolitinib continues to be safe in pts with MPN-associated SVT and maintains efficacy against splenomegaly in 33% of the pts. Of note, the large majority of pts (67%) showed stabilization or improvement of oesophageal varices, supporting the hypothesis that sustained reduction of enlarged spleen might contribute to decrease the upstream venous system pressure. Significant reduction of JAK2V617F VAF>50% was documented in 40% of the pts, although it was not clearly correlated with clinical improvement. Disclosures Rumi: novartis: Honoraria, Research Funding. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: Ruxolitinib use in essential thrombocythemia

Background Treatment of patients developing steroid-refractory acute GvHD (SR-aGvHD) after allogeneic hematopoietic cell transplantation (HCT) remains an unmet clinical need and a major therapeutic challenge. Mesenchymal stromal cells (MSC) induce immunosuppression and reduce the inflammatory status initiated by aGvHD. We performed an interim analysis of a multicenter, prospective, non-randomized, phase I/II study, planned to test the safety and activity of human umbilical cord (UC), third-party MSC given sequentially after Pentatostatin for the treatment of SR-aGvHD grade III-IV as well as the overlap syndrome (EUDRACT n. 2012-000582-21), NCT02032446). Patients and Methods Third-party UC MSC were produced under GMP conditions as previously described (Capelli C et al Cytotherapy, 2011, 13, 786-801). The treatment schedule was based on the sequential administration of Pentostatin, given iv at dose of 1 mg/m^2 for 3 consecutive days, followed by 3 MSC infusions given at weekly intervals. Between October 2013 and May 2018 (range 4.6 years) we enrolled 27 allogeneic HCT adult recipients (22 male) with SR-aGvHD (n 25) or overlap syndrome (n 2), median age 47 years (range 19-62) transplanted from HLA-identical sibling donors (6; 22%), haploidentical donors (2; 7%), unrelated donors (18; 67%) or cord blood (1; 4%). Target organ involvement included skin in 11 cases (44%), gastrointestinal tract (GI) in 23 (92%) and liver in 12 (48%). Nine patients had grade III (33%), 16 patients grade IV (60%) and 2 patients had severe overlap syndrome (7%). Most of patients exhibited multiple organ involvement (n 16, 64%). Patients started MSC therapy at median of 22 days (10-2101) after the onset of aGvHD or overlap syndrome. Results The infusions of UC MSC-infusion were always well tolerated without any immediate or late toxic event. Response to MSC therapy, by day 30 after the final MSC dose, was evaluable in 16 of 27 patients: 7 achieved CR (26%), 6 PR (22%) and 3 NR (11%). Ten patients died before reaching the evaluation time of response: 8 patients for aGvHD and 2 for haematological disease. One patient was lost to follow up after 9 days from the last UC-MSC infusion. Remarkably, the subgroup of patients with single organ involvement (8 GI, 1 liver) were all evaluable showing 6 patients in overall response (67%, 4 RC and 2 PR). No response was observed in 2 cases of severe overlap syndrome. Patients achieving CR had improved longer term OS vs patients in PR/NR (80% vs 50% at 1 year). For patients with grade III and IV SR-aGvHD, CR rates at day 30 were 67% and 31% respectively. During tapering of steroid therapy 4 out of 7 patients that obtained CR had aGvHD recurrence, at median time of 87 days. We observed relapse of underlying malignancy in 5 patients and all died with active disease. At last follow-up 8 out of 27 patients were alive with no GVHD in 5 of them; 14 patients died from TRM: aGvHD or overlap syndrome itself (n=10), infections (n= 2), cGvHD (n= 2). Two patients with uncontrolled GvHD had clinical signs of Transplant Associated Microangiopathy (TAM). A total of 17 reported AE and 12 SAE were registered. In 23 out of 27 patients infectious complications were observed, in most cases virus related (n.25; CMV followed by BK virus and EBV), and fungal infections (3 aspergillus pneumoniae). Blood-stream documented infections (5) and pneumoniae (5) were also observed. Conclusions The infusion of cord blood derived MSC proved safe in all cases. As expected in patients with steroid refractory aGvHD, a high rate of infectious complications were observed. When considering that most patients (60 %) had grade IV aGvHD, the response rate and the OS are promising and this warrants not only the completion of this Phase I/II study but also planning a future pivotal trial. Disclosures No relevant conflicts of interest to declare.

Introduction: Patients with disease relapse after allogeneic transplantation (alloHSCT) have a poor prognosis. Donor lymphocyte infusion (DLI) is one of the main clinical options to salvage patients in relapse after transplant. Cytokine Induced Killer (CIK) cells are in vitro activated and expanded T lymphocytes which have acquired NK like cytotoxicity as well as CD56 expression. CIK cells have shown Graft versus Leukemia (GvL) activity with little GvHD and therefore may represent an ideal candidate to treat post-transplant relapse. We report the final results of a phase II multicenter pilot study, whose objective was to evaluate the safety and efficacy of sequential administration of donor derived unmanipulated DLI and CIK cells in patients with recurrent hematologic cancers after alloHSCT. Methods Seventy-four patients relapsed after alloHSCT, performed using either a matched related (N=42) or unrelated donor (n= 32), were enrolled in the study. This phase II multicenter study was authorized by Istituto Superiore di Sanità, as for Advanced Therapeutic Medicinal Product (ATMP) regulations, and approved by the Agenzia Italiana del Farmaco (AIFA). The trial was registered as (EUDRACT number 2008-003185-26, ClinicalTrial.gov : NCT01186809). Results Among the 74 patients (including 16 children and 58 adults) enrolled into this study (median age 45, range 1-67), 20 had a diagnosis of ALL (27%), 41 of AML (55%), 4 of MM (5%), 3 of HD (4%), 2 of NHL (3%) and 4 of MPN (5%). All patients relapsed after matched allogeneic transplants (32 unrelated and 42 sibling), of whom 44 (59%) suffered from a hematological, 4 (5%) from a cytogenetic and 26 (35%) from a molecular relapse. The therapeutic strategy consisted of two infusions of unmanipulated DLI (each of 1 x 106/kg cells) at 3 weeks interval, followed by three infusions of donor derived CIK cells given at 3 weeks interval. The first 12 patients were treated with increasing numbers of CIK cells, in groups of three patients per dose level. Since dose limiting toxicity (DLT) was never observed (acute GVHD of grade III or more), the highest dose planned (5 x 106/kg, 5 x 106/kg and 10 x 106/kg) was then administered to all patients. Ten patients died for disease progression, 1 patient developed aGVHD (grade I, skin only) and 1 withdrawn for medical decision before or during the DLI treatment and could not proceed to the planned subsequent CIK administration. Sixty-two patients received at least one infusion of CIK cells, of whom 43 patients (61%) completed the cell therapy program, while 3 patients are still under treatment. The study flow is outlined in Figure 1. As per protocol, clinical response was determined 100 days after the last CIK administration and the study was analyzed on an intent to treat basis. An early death occurred in 24 (32%) patients (4 during the DLI), no response was observed in 18 (24%) patients, a stable disease in 1 patient (1%), a complete remission in 21 (28%) and a partial remission in 6 (8%), for an overall response rate of 36%. In 4 patients clinical response could not be evaluated (3 patients still in treatment and 1 withdrawn from the protocol). Acute GVHD was observed in a total of 11 patients (15%): grade 1 (n=4), 2 (n=2) and 3-4 (n=5). During follow up, chronic GVHD was observed in 8 patients (11 %) (3 mild, 4 moderate and 1 severe). By univariate analysis, progression free survival (PFS) and overall survival (OS) were significantly associated (p Conclusion Our study shows that administration of CIK cells is feasible in patients with recurrent hematologic cancer after alloHSCT with a relatively low toxicity in terms of GvHD. Particularly in the setting of the molecularly relapsed patients, long-term survival can be achieved. In future studies, we are planning to test CIK cells in preventing post-transplantation relapse in high risk AML. Finally, CIK cells may represent an innovative platform to transduce chimeric antigen receptors in allogeneic T cells with a reduced risk to induce GvHD. Disclosures Biondi: Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board.

Introduction Graft-versus-Host Disease (GvHD) is one of the leading causes of mortality and morbidity following allogeneic stem cell transplant. In vivo T cell depletion by alemtuzumab as part of the transplant conditioning is an effective strategy to reduce the risk of GvHD. While it is recognised that the overall incidence of GvHD is reduced by alemtuzumab, the incidence of chronic GvHD as defined by the National Institute of Health (NIH) consensus criteria, the impact on outcome, and the pattern of organ involvement have not been defined yet in this transplant setting. Methods Consecutive patients (n = 323) undergoing allogeneic stem cell transplantation at the Queen Elizabeth Hospital, Birmingham, between January 1 2008 and June 30 2012 were reviewed in this retrospective, single centre study. Medical records were examined and data regarding the development of GvHD were collected; NIH consensus criteria for diagnosis and staging of chronic GvHD were stringently applied. Clinical characteristics of GvHD occurring in patients transplanted following T cell depletion by alemtuzumab administration (n=248) were compared with those of patients transplanted with a T cell replete graft (n=75). Patients receiving alemtuzumab were mainly treated with reduced-intensity conditioning protocols, while patients in the no-T-cell depletion group were mainly treated with a myeloablative, sibling transplant. Results After a median follow up of 38.4 months, the cumulative incidence (CI) of grade II-IV classic acute GvHD was 35% and 48% for patients transplanted respectively with or without T cell depletion by alemtuzumab (p= 0.041, Figure 1); with a CI of grade III-IV classic acute GvHD of 13% and 27% (p=0.007). The 2-years CI of grade II-IV late acute GvHD was not significantly different in the two groups (20% and 23% for patients respectively treated with or without alemtuzumab, p=0.589, Figure 2). T cell depletion by alemtuzumab significantly reduces the 3 years cumulative incidence of classic chronic GvHD (5% versus 31%, p Conclusion This retrospective analysis provides for the first time data on the incidence rates of NIH-defined GvHD categories in patients transplanted after T cell depletion by alemtuzumab. Patients transplanted with alemtuzumab experienced a lower incidence of classic acute and classic chronic GvHD compared to patients not receiving T cell depletion. In contrast, alemtuzumab conditioning appeared to have no effect on the incidence of late acute GvHD or overlap syndrome, suggesting that these two entities of GvHD are driven by different immunological mechanisms as compared to classic acute and classic chronic GvHD. We also confirmed the utility of the NIH classification of GvHD and of the NIH global severity score to predict survival in alemtuzumab-conditioned allogeneic stem cell transplant. Disclosures No relevant conflicts of interest to declare.

Background Patients with Philadelphia-negative myeloproliferative neoplasms (MPN) can develop venous thrombosis and MPN are the leading cause of splanchnic vein thrombosis. Cerebral vein thrombosis (CVT) is a rare life-threatening disease that in approximately 3% of cases encounters MPN among risk factors and tends to recur in 2-4% of patients as CVT and in 4-7% as venous thrombosis at other sites. Little or no information is available on patients with MPN who develop CVT. Objective and design To investigate the characteristics and clinical course of CVT in patients with MPN we carried out a multicenter (n=11), observational, retrospective cohort study. Patients Centers were asked to provide information on index patients with MPN who developed CVT (group MPN-CVT). For each of them, 2 patients with MPN and venous thrombosis other than CVT (group MPN-VT) and 4 patients with MPN and no venous thrombosis (group MPN-NoVT) were provided, matched by sex, age at diagnosis of MPN (+/-5 years) and type of MPN (polycytemia vera, essential thrombocytemia, myelofibrosis) with index patients. Results From January 1982 to June 2013, 48 MPN-CVT, 87 MPN-VT and 178 MPN-NoVT patients were identified in a population of 5,500 patients with MPN. Diagnosis of MPN and thrombosis coincided in 46% of MPN-CVT and 29% of MPN-VT patients (p=0.046). Compared to MPN-NoVT, MPN-CVT and MPN-VT patients had a higher prevalence of thrombophilia abnormalities (40% and 35% vs 21%, p=0.015) and, among those with essential thrombocytemia, of the JAK2 V617F mutation (76% and 78% vs 55%, p=0.059). Compared to MPN-VT, MPN-CVT patients had a higher rate of recurrent thrombosis (42% vs 25%, p=0.049) that in two-third of patients in both groups was venous, with a similar site distribution. This difference occurred despite a shorter median follow-up period (6.1 vs 10.3 years, p=0.019), a higher proportion of patients on long-term antithrombotic treatment (94% vs 84%, p=0.099) and a similar proportion of patients on cytoreductive treatment (75% vs 72%, p=0.745) among MPN-CVT than MPN-VT patients. The incidence of recurrent thrombosis was 8.8% patients/year in MPN-CVT and 4.2% patients/year in MPN-VT patients (log-rank test, p=0.022) and CVT was the only variable in a multivariate model including blood counts, thrombophilia, cytoreductive and antithrombotic treatment, that was predictive of recurrent thrombosis (HR 1.86, 95%CI 1.00-3.58). Conclusions Patients with MPN develop recurrent thrombosis in a much higher proportion than those without, particularly if they had a CVT. Patients with MPN and CVT have an approximately 2-fold increased probability to develop recurrent thrombosis than those with MPN and venous thrombosis at other sites, independently of other risk factors. Disclosures: No relevant conflicts of interest to declare.

Background The combined use of chemotherapy and monoclonal antibodies has proved highly effective for the treatment of CLL but often results in severe life threatening immunosuppression. The development of adoptive therapy with autologous T cells could be clinically relevant to overcome these problems. Methods We have devised a novel, simple and efficient method for ex vivo expansion of normal autologous T cells from the peripheral blood of CLL patients for adoptive therapy, using blinatumomab (CD3xCD19) and rhIL-2 in serum-free medium. The complete phenotype of in vitro expanded T cells was analyzed by flow cytometry and their cytotoxic activity by calcein release assays. Results We performed 18 expansions of T cells, starting from a very small volume of peripheral blood from untreated CLL patients (mean 10.3 ml, range 2-30 ml) that contained a mean of 9.2x106 T cells (range 0.4 to 51x106)(Fig.1). This method allowed reproducible expansion in about 21 days of a mean 410x106 CD3+ T cells (range 71 to 2184x106). The mean fold expansion of T cells in about 3 weeks of in vitro culture was 224 (range 4.4-1326). The only significant contaminant in final Blinatumomab Expanded T cell cultures (BET) were NK cells (mean 18.5%). Indeed addition of blinatumomab and rhIL-2 to the cultures led to a rapid decrease in CLL B cells, which took place from days 7 to 14 onwards and resulted in their complete depletion within 3 weeks (mean 0.2% CLL B cells at days 18-25). Only in one case, a significant percentage of CLL B cells could be observed at the end of culture, but this was due to the particularly high percentage neoplastic cells in the starting population in this patient (98%), resulting in relatively late depletion of these cells, which took place between days 14 and 21, and therefore remained detectable in this case at day 24 (3.8% CLL B cells at day 24). Despite the very low percentage of starting T cells in this specific patient (1.2%), 152x106 T cells could be obtained, equivalent to a 42 fold expansion. In the 18 expansions performed, the resulting BET cells contained both CD4+ and CD8+ cells in varying proportions (median 46.2% and 44.4% respectively). Only in two cases the final product was composed predominantly of CD4+ cells (95%). Expanded T cells were polyclonal, as shown by TCR Vβ expression which was within the normal range by flow cytometry. Indeed CMV specific clones, detected by CMV peptide (pp65495-503)-loaded HLA-A*0201 tetramer, were expanded using this method and detected in equivalent proportion before and after expansion. Final T cells were composed predominantly of the effector and central memory subsets. Th1 were slightly prevalent over Th2 cells (means 20% and 10%, respectively), whereas Th17 and Treg were less than 1%. Since CLL derived T cells have been shown previously to have enhanced expression of the synapse regulators CD272 and CD279 compared to normal T cells, leading to impaired immunological synapse formation, we have analyzed these markers in both starting and BET cells from 4 patients. We observed that CD272 and CD279 diminished in BET compared to the starting CLL T populations (from 73% to 19% and 61% to 18%, respectively). These data suggest that stimulation and expansion with blinatumomab and rhIL-2 has normalized expression of these regulators on CLL T cells. Indeed BET were highly cytotoxic against CD19+ targets cell lines or primary CLL cells, with 70-90% lysis at a 3:1 effector target ratio in presence of blinatumomab. Finally BET were compared to Xcellerated cells expanded using anti-CD3/CD28 Dynabeads and rhIL-2. The expansion protocols using either blinatumomab or anti-CD3/CD28 Dynabeads showed equivalent efficiency and comparable cell composition at the end of culture. Further comparison of the T cell subsets present in BET or CD3/CD28 cultures is in progress. Conclusions These data altogether suggest that the use of blinatumomab and rhIL-2 provides a reproducible, simple and GMP-compliant protocol, allowing expansion of large numbers of autologous polyclonal T cells depleted of CLL cells, from relatively small volumes of peripheral blood from CLL patients. This approach is an attractive option for adoptive therapy in these patients after immunosuppressive treatments. Disclosures: No relevant conflicts of interest to declare.

Abstract 314 Background: Akt/mTOR activation has been found in association with dysregulated JAK2/STAT5 signaling due to JAK2V617F mutation. Increased phosphorylation of STAT5 and Akt was detected in patients (pts) with myeloproliferative neoplasms (MPN) (Grimwade LF, BJH 2009). We reported (Bogani C, ASH 2009) that RAD001, an oral inhibitor of mTOR, inhibited the proliferation of human and murine JAK2V617F-mutated cells and impaired colony formation by MPN progenitor cells, suggesting potential clinical activity. Methods: We will report final data from an investigator-initiated, multicenter phase I/II trial with RAD001 in myelofibrosis, primary (PMF) and post-polycythemia vera/essential thrombocythemia (PPV/PET MF). Inclusion criteria were intermediate/high risk score (Lille criteria) or need of treatment because of progressing splenomegaly. Phase I involved a 3+3 pts scheme in 3 sequential cohorts at 5.0, 7.5, and 10 mg daily for 3 mo to establish maximum tolerated dose (MTD). Phase II was a two-stage Simon design involving 16+14 pts at MTD for 4 mo. The protocol was approved by IRBs and pts provided informed consent. The study was supported by Agenzia Italiana per il Farmaco (AIFA) and AIRC-Gruppo Italiano Malattie Mieloproliferative (AGIMM). Results: Phase I: there was no DLT at 10 mg daily, considered as MTD; there were 2 major (MR), 3 moderate (MO) and 1 minor (MI) and 3 no (NR) responses. In part 1 of Phase II, >1/16 pts achieved MR; according to study design, 14 additional pts were enrolled in part 2 (n=30). 20 patients had low and 10 intermediate Lille score; according to IWG-MRT criteria, 5, 10, 8 and 7 were low, int-I, int-II and high risk score, respectively. There were 16 PMF, 8 PPV- and 6 PET-MF. 20 pts were JAK2V617Fpos, 3 were MPLW515pos. Patients evaluable for intention-to-treat (ITT) analysis as of aug 1st were 26; the remaining 4 pts will be updated at meeting. Therapy was discontinued in 5 pts: 1 pt's decision at d60 without evidence of any >grade-2 toxicity, 2 at d60 and d90 for physician's decision due to grade-2 toxicity, 1 death due to respiratory failure at d60, 1 at d30 for grade-3 acute renal failure. 21/26 pts (81%) were available for per-protocol analysis. Therapy was generally well tolerated; commonest toxicities were grade-2 mouth ulcers and grade-1/2 hypertrigliceridemia. Hematological toxicities: 3 grade-2 and 4 grade-3 reversible anemia, one grade-2 neuthropenia, one grade-2 and 1 grade-3 reversible thrombocytopenia. A reduction of spleen size consistent with CR, PR or NR was obtained in 8%, 46%, and 46% of the pts, respectively. 11 of 21 pts (52%) had complete resolution of systemic symptoms, and 14 of 19 pts (74%) reported disappearance of pruritus. Two pts achieved PR in anemia with decrease in transfusion requirement >50%, while in 3 pts Hb increased of 2g/dL. A CR in platelets was obtained in 3 of 19 pts and CR in leucocytes in 2 of 18 pts with abnormal blood count. Overall, according to ITT analysis: 6 major (23%), 6 moderate (23%), 2 minor (8%) and 12 NR (46%) (EUMNET criteria). According to IWG-MRT criteria, there were 6 (23%) clinical improvement. Per-protocol analysis: 33% major, 19% moderate, 5% minor, 43% NR; 24% clinical improvement. No disease progression. Changes induced by RAD001 in some biological parameters were preliminary evaluated and will be finally updated at meeting. Blood levels of CCDN1 mRNA, a target of mTOR, significantly decreased in responders (MR+MO) versus non-responders (P=0.02). In the 13 JAK2-mutated pts who completed the treatment, the V617F allele burden was 61.6+/−15.8 versus 66.6+/−19.6% at baseline. There was a trend (P=0.07) towards a reduction of granulocyte WT1mRNA copy number in responders versus non-responders. We found no correlation of circulating CD34+ cells or CXCR4 expression with clinical response. A set of 46 inflammatory protein markers and cytokines were quantified before and at d30 of treatment. Some, including IL-10 and MIP-1beta, showed significant decrease while others increased, including Factor VII, IL-8 and MMP-2. Levels of MIP-1 beta were significantly lower in responders (P=0.006). Conclusions: These data indicate that mTOR pathway targeting with RAD001 in MF pts induces an appreciable rate of response in splenomegaly, constitutional symptoms, and pruritus, with low-grade toxicity. However, effects on JAK2V617F mutational load were minimal, and should be better evaluated after longer trial duration. Disclosures: Vannucchi: Novartis: Membership on an entity's Board of Directors or advisory committees. Off Label Use: This is an investigator-initiated, non-sponsored, clinical trial with RAD001 in myelofibrosis. Paratore:Novartis: Employment.