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This geoepidemiological study, performed in Italy and France, shows that Erdheim-Chester disease is increasingly diagnosed and cases cluster in specific geographic areas, namely southern Italy and central France. Disease frequency inversely correlates with the Human Development Index.

F095Allogeneic, donor-derived CD19-CAR T cells can be readily generated for the treatment of pediatric patients with relapsed/refractory BCP-ALLF095Allogeneic CD19-CAR T cells are effective and, compared to autologous CAR, they neither increase toxicity, nor induce high GvHD incidence

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies, but is absent on normal tissues, including hematopoietic progenitor cells, and may therefore be an appropriate candidate for T cell–mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity, PRAME-specific cytotoxic T lymphocytes (CTLs), we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal, PRAME-specific CTL lines and elicited high-avidity CTLs, with a high proportion of cells recognizing a previously uninvestigated HLA-A*02–restricted epitope, P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME+hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts, but also against leukemic progenitor cells as assessed by colony-forming–inhibition assays, which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors, indicating that this approach may be of value for immunotherapy of PRAME+hematologic malignancies.

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies, but is absent on normal tissues, including hematopoietic progenitor cells, and may therefore be an appropriate candidate for T cell–mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity, PRAME-specific cytotoxic T lymphocytes (CTLs), we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal, PRAME-specific CTL lines and elicited high-avidity CTLs, with a high proportion of cells recognizing a previously uninvestigated HLA-A*02–restricted epitope, P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME+ hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts, but also against leukemic progenitor cells as assessed by colony-forming–inhibition assays, which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors, indicating that this approach may be of value for immunotherapy of PRAME+ hematologic malignancies.

Adoptive transfer of autologous Epstein-Barr virus–specific cytotoxic T lymphocytes (EBV-CTLs) to solid organ transplant (SOT) recipients has been shown safe and effective for the treatment of EBV-associated posttransplantation lymphoproliferative disorders (PTLDs). SOT recipients, however, require the continuous administration of immunosuppressive drugs to prevent graft rejection, and these agents may significantly limit the long-term persistence of transferred EBV-CTLs, precluding their use as prophylaxis. Tacrolimus (FK506) is one of the most widely used immunosuppressive agents in SOT recipients, and its immunosuppressive effects are largely dependent on its interaction with the 12-kDa FK506-binding protein (FKBP12). We have knocked down the expression of FKBP12 in EBV-CTLs using a specific small interfering RNA (siRNA) stably expressed from a retroviral vector and found that FKBP12-silenced EBV-CTLs are FK506 resistant. These cells continue to expand in the presence of the drug without measurable impairment of their antigen specificity or cytotoxic activity. We confirmed their FK506 resistance and anti-PTLD activity in vivo using a xenogenic mouse model, suggesting that the proposed strategy may be of value to enhance EBV-specific immune surveillance in patients at high risk of PTLD after transplantation.

Adoptive transfer of autologous Epstein-Barr virus–specific cytotoxic T lymphocytes (EBV-CTLs) to solid organ transplant (SOT) recipients has been shown safe and effective for the treatment of EBV-associated posttransplantation lymphoproliferative disorders (PTLDs). SOT recipients, however, require the continuous administration of immunosuppressive drugs to prevent graft rejection, and these agents may significantly limit the long-term persistence of transferred EBV-CTLs, precluding their use as prophylaxis. Tacrolimus (FK506) is one of the most widely used immunosuppressive agents in SOT recipients, and its immunosuppressive effects are largely dependent on its interaction with the 12-kDa FK506-binding protein (FKBP12). We have knocked down the expression of FKBP12 in EBV-CTLs using a specific small interfering RNA (siRNA) stably expressed from a retroviral vector and found that FKBP12-silenced EBV-CTLs are FK506 resistant. These cells continue to expand in the presence of the drug without measurable impairment of their antigen specificity or cytotoxic activity. We confirmed their FK506 resistance and anti-PTLD activity in vivo using a xenogenic mouse model, suggesting that the proposed strategy may be of value to enhance EBV-specific immune surveillance in patients at high risk of PTLD after transplantation.

For the adoptive transfer of tumor-directed T lymphocytes to prove effective, there will probably need to be a match between the chemokines the tumor produces and the chemokine receptors the effector T cells express. The Reed-Stemberg cells of Hodgkin lymphoma (HL) predominantly produce thymus- and activation-regulated chemokine/CC chemokine ligand 17 (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), which preferentially attract type 2 T helper (Th2) cells and regulatory T cells (Tregs) that express the TARC/MDC-specific chemokine receptor CCR4, thus generating an immunosuppressed tumor environment. By contrast, effector CD8+T cells lack CCR4, are nonresponsive to these chemokines and are rarely detected at the tumor site. We now show that forced expression of CCR4 by effector T cells enhances their migration to HL cells. Furthermore, T lymphocytes expressing both CCR4 and a chimeric antigen receptor directed to the HL associated antigen CD30 sustain their cytotoxic function and cytokine secretion in vitro, and produce enhanced tumor control when infused intravenously in mice engrafted with human HL. This approach may be of value in patients affected by HL.

For the adoptive transfer of tumor-directed T lymphocytes to prove effective, there will probably need to be a match between the chemokines the tumor produces and the chemokine receptors the effector T cells express. The Reed-Stemberg cells of Hodgkin lymphoma (HL) predominantly produce thymus- and activation-regulated chemokine/CC chemokine ligand 17 (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), which preferentially attract type 2 T helper (Th2) cells and regulatory T cells (Tregs) that express the TARC/MDC-specific chemokine receptor CCR4, thus generating an immunosuppressed tumor environment. By contrast, effector CD8+ T cells lack CCR4, are nonresponsive to these chemokines and are rarely detected at the tumor site. We now show that forced expression of CCR4 by effector T cells enhances their migration to HL cells. Furthermore, T lymphocytes expressing both CCR4 and a chimeric antigen receptor directed to the HL associated antigen CD30 sustain their cytotoxic function and cytokine secretion in vitro, and produce enhanced tumor control when infused intravenously in mice engrafted with human HL. This approach may be of value in patients affected by HL.

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed in many hematologic malignancies, including chronic myeloid leukemia (CML). The sensitivity of CML to donor lymphocyte infusion after allogeneic stem cell transplantation suggests this tumor can be highly susceptible to cellular immunotherapy targeted to tumor associated antigens. We therefore tested whether functional PRAME-specific cytotoxic T lymphocytes (PRAME CTLs) could be generated and expanded from healthy donors and CML patients, or whether the limited immunogenicity of this CTA coupled with tumor-associated anergy would preclude this approach. Using optimized culture conditions and HLA-A*02–restricted PRAME-peptides, we have consistently generated PRAME CTLs from 8/9 healthy donors and 5/6 CML patients. These CTLs released IFNγ in response to PRAME peptides (between 113 ± 8 and 795 ± 23 spot forming cells/105 T cells) and lysed PRAME peptide–loaded cells (45 ± 19% at an effector:target [E:T] ratio of 20:1) in a MHC-restricted fashion. Importantly, these CTLs recognized and had cytotoxic activity against HLA-A*02+/PRAME+ tumor cell lines, and could recognize and respond to primary CML cells. PRAME CTLs were generated almost exclusively from the naive T-cell compartment, and clonal analysis showed these cells could have high αβTCR-peptide avidity. PRAME CTLs or vaccines may thus be of value for patients with CML.

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed in many hematologic malignancies, including chronic myeloid leukemia (CML). The sensitivity of CML to donor lymphocyte infusion after allogeneic stem cell transplantation suggests this tumor can be highly susceptible to cellular immunotherapy targeted to tumor associated antigens. We therefore tested whether functional PRAME-specific cytotoxic T lymphocytes (PRAME CTLs) could be generated and expanded from healthy donors and CML patients, or whether the limited immunogenicity of this CTA coupled with tumor-associated anergy would preclude this approach. Using optimized culture conditions and HLA-A*02–restricted PRAME-peptides, we have consistently generated PRAME CTLs from 8/9 healthy donors and 5/6 CML patients. These CTLs released IFNγ in response to PRAME peptides (between 113 ± 8 and 795 ± 23 spot forming cells/105T cells) and lysed PRAME peptide–loaded cells (45 ± 19% at an effector:target [E:T] ratio of 20:1) in a MHC-restricted fashion. Importantly, these CTLs recognized and had cytotoxic activity against HLA-A*02+/PRAME+tumor cell lines, and could recognize and respond to primary CML cells. PRAME CTLs were generated almost exclusively from the naive T-cell compartment, and clonal analysis showed these cells could have high αβTCR-peptide avidity. PRAME CTLs or vaccines may thus be of value for patients with CML.

Bone marrow–derived mesenchymal stem cells (BM-MSCs) are stromal cells with the ability to proliferate and differentiate into many tissues. Although they represent powerful tools for several therapeutic settings, mechanisms regulating their migration to peripheral tissues are still unknown. Here, we report chemokine receptor expression on human BM-MSCs and their role in mediating migration to tissues. A minority of BM-MSCs (2% to 25%) expressed a restricted set of chemokine receptors (CXC receptor 4 [CXCR4], CX3C receptor 1 [CX3CR1], CXCR6, CC chemokine receptor 1 [CCR1], CCR7) and, accordingly, showed appreciable chemotactic migration in response to the chemokines CXC ligand 12 (CXCL12), CX3CL1, CXCL16, CC chemokine ligand 3 (CCL3), and CCL19. Using human pancreatic islets as an in vitro model of peripheral tissue, we showed that islet supernatants released factors able to attract BM-MSCs in vitro, and this attraction was principally mediated by CX3CL1 and CXCL12. Moreover, cells with features of BM-MSCs were detected within the pancreatic islets of mice injected with green fluorescent protein (GFP)–positive BM. A population of bona fide MSCs that also expressed CXCR4, CXCR6, CCR1, and CCR7 could be isolated from normal adult human pancreas. This study defines the chemokine receptor repertoire of human BM-MSCs that determines their migratory activity. Modulation of homing capacity may be instrumental for harnessing the therapeutic potential of BM-MSCs.

Bone marrow–derived mesenchymal stem cells (BM-MSCs) are stromal cells with the ability to proliferate and differentiate into many tissues. Although they represent powerful tools for several therapeutic settings, mechanisms regulating their migration to peripheral tissues are still unknown. Here, we report chemokine receptor expression on human BM-MSCs and their role in mediating migration to tissues. A minority of BM-MSCs (2% to 25%) expressed a restricted set of chemokine receptors (CXC receptor 4 [CXCR4], CX3C receptor 1 [CX3CR1], CXCR6, CC chemokine receptor 1 [CCR1], CCR7) and, accordingly, showed appreciable chemotactic migration in response to the chemokines CXC ligand 12 (CXCL12), CX3CL1, CXCL16, CC chemokine ligand 3 (CCL3), and CCL19. Using human pancreatic islets as an in vitro model of peripheral tissue, we showed that islet supernatants released factors able to attract BM-MSCs in vitro, and this attraction was principally mediated by CX3CL1 and CXCL12. Moreover, cells with features of BM-MSCs were detected within the pancreatic islets of mice injected with green fluorescent protein (GFP)–positive BM. A population of bona fide MSCs that also expressed CXCR4, CXCR6, CCR1, and CCR7 could be isolated from normal adult human pancreas. This study defines the chemokine receptor repertoire of human BM-MSCs that determines their migratory activity. Modulation of homing capacity may be instrumental for harnessing the therapeutic potential of BM-MSCs.

Prognosis of a significant proportion of patients with chemotherapy-refractory or multiply-relapsed CD30+ Non-Hodgkin's Lymphoma (NHL) or Hodgkin lymphoma (HL) still remain poor. Targeting CD30 with monoclonal antibodies in HL and anaplastic large cell lymphoma was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. Immunotherapeutic approaches targeting CD30 by chimeric antigen receptor (CAR) has been demonstrated to be of value in two independent clinical trials, although clinical benefit was sub-optimal. We designed a new CAR construct characterized by an anti-CD30 single-chain variable-fragment cassette (AC10), linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. The inducible Caspase-9 (iCasp9) safety switch was included in both constructs with the goal of promptly controlling undue toxicity. As a selectable marker, we added in frame the CD34 antigen. The in vitroanti-tumor efficacy was evaluated by using either the NHL cell line: Karpas299, or the HL cell lines: L428, in both short-term cytotoxic assay (51Cr release assays) and long-term co-cultures for 6 days. Supernatant from co-culture experiments was analyzed by Elisa. We assessed the antitumor effect of CAR.CD30 T cells in a in vivoNSG mouse model engrafted i.v. with lymphoma FF-luciferase cell lines Karpas299 or L428, and monitored tumor growth by IVIS Imaging system. For tumor re-challenging, mice of the NHL model surviving until day +140, were i.v.infused with 0.2x106 Karpas299 cells, and subsequently followed for additional 110 days. Persistence of CAR.CD30 T cells was evaluated, together with a deep characterization of memory profile of T cells.

Survival rates of children with relapsed/refractory (r/r) BCP-ALL remain unsatisfactory and little progress has been made in the past 2 decades. Similarly, relapse of childhood B-NHL is usually associated with an aggressive disease and poor outcomes. Targeted immunotherapy with T-cells genetically modified to express a CD19-directed CAR showed an unprecedented antitumor efficacy, leading to the recent FDA and EMA approval of two CD19-CAR products for treatment of BCP-ALL and B-NHL. Relevant toxicities have, however, been reported, mainly related to the development of severe Cytokine Release Syndrome (CRS) and/or of neurotoxicity.

Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Merli: Amgen: Honoraria; Novartis: Honoraria; Sobi: Consultancy; Bellicum: Consultancy. Algeri:Bluebird bio: Consultancy, Honoraria; Atara Biotherapeutics: Consultancy, Honoraria; Miltenyi: Honoraria. Locatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

In view of the exciting results reported in patients with CD19+ malignancies given CAR T cells, it is expected that a continuously growing number of patients will be offered this treatment and, thus, will be exposed to gene-modified products. Since the techniques of gene manipulation are relatively new, some of the risks associated to CAR T therapy may be unpredictable.

Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Based on the clinical success observed in acute lymphoblastic leukemia (ALL) with chimeric antigen receptor engineered T (CAR T), we hypothesized that combining the specificity of a CAR with the innate allo-reactivity of KIR-mismatched NK cells might provide a powerful tool for adoptive cell therapy. The use of a third-party bank of CAR-NK cells offers the advantage of an immediate availability to be exploited in the allogenic setting and could be associated with a lower toxicity profile than CAR-T cells.

No relevant conflicts of interest to declare.

Background:Allogeneic HSCT from either an HLA-identical sibling or an unrelated donor is a potentially curative treatment for patients with hemoglobinopathies and erythroid disorders (ED), such as Thalassemia Major (TM), Sickle Cell Disease (SCD) and Diamond-Blackfan Anemia (DBA). The limited historical experience with HLA-haploidentical HSCT in this setting has reported a disease-free survival probability lower than that reported using HLA-matched donors. In the last few years, we have developed a novel method of graft manipulation, based on the selective depletion of α/β+ T-cells and CD19+ B-cells (ClinicalTrial.gov identifier: NCT01810120), which was shown to be safe and effective in children with multiple types of non-malignant disorders (Bertaina el al, Blood 2014). To further optimize this approach through the acceleration of the recovery of adaptive immunity, we designed an ongoing phase I/II trial aimed to test the safety and efficacy of post-transplant infusion of donor T-cells transduced with the iC9 suicide gene (BPX-501 cells) in children with either malignant or non-malignant disorders (ClinicalTrials.gov identifier: NCT02065869). As the transduced gene contains sequences for the CD19 marker, BPX-501 cells (CD3+/CD19+) can be easily tracked in peripheral blood. We report on 10 children with hemoglobinopathies and ED who were enrolled in the phase II portion of the study.

A new method of graft manipulation based on physical removal of αβ+T cells and CD19+B cells, leaving mature NK cells and γδ T cells in the graft, has been recently developed for HLA-haploidentical HSCT. We demonstrated that γδ T cells collected from transplanted patients are endowed with capacity of killing leukemia cells after ex vivotreatment with zoledronic acid (ZOL). Thus, we hypothesized that infusion of ZOL in patients receiving this type of graft, may boost γδ T cell cytotoxic activity against leukemia cells.

French: Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

Introduction

Background:Among patients with beta-thalassemia early detection of transfusion-induced myocardial iron loading and its intervention with aggressive chelation therapy may delay or reverse heart failure. Three dimensional speckle tracking echocardiography (3D-STE) is a novel tool that may early detect myocardial affection in these patients.

Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Galimberti:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Saglio: NOVARTIS: Consultancy. Pane:NOVARTIS: Consultancy, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

No relevant conflicts of interest to declare.

Introduction.

E-AVARE were observational studies in Austria and Switzerland investigating febrile neutropenia (FN) risk assessment and granulocyte colony-stimulating factor (G-CSF) guideline implementation in real life Non-Hodgkin-lymphoma (NHL) treatment. A combined analysis of both studies was performed to better understand the characteristics of NHL patients who receive pegfilgrastim and the outcomes of these patients in everyday clinical practice.Eligible patients had NHL, were eligible to receive G-CSF, and were planned to receive ≥3 chemotherapy (CT) cycles. The Austrian study included pegfilgrastim treated patients only, while the Swiss study included all G-CSF formulations used. To evaluate pegfilgrastim-related outcomes, only data from patients who completed ≥3 CT cycles and who received pegfilgrastim in ≥1 cycle were included (primary analysis set). Primary outcome: proportion of patients with investigator-assessed high (≥20%) overall FN risk in cycle 1 receiving pegfilgrastim as primary prophylaxis (PP). Other outcomes: investigator-assessed CT-related FN risk, overall incidence of FN and unplanned hospitalizations. Exploratory analysis: description of age cohorts (<65 or ≥65 years [yrs]).277 patients fulfilled the inclusion criteria for the primary analysis set. Table 1 shows patient characteristics and outcomes. In cycle 1, 237 patients had an investigator-assessed ≥20% overall FN risk and of these 219 (92%) received pegfilgrastim as PP. Among all 277 patients, pegfilgrastim was administered as PP in 236 (85%); pegfilgrastim was first administered in the second or later cycle in 41 patients, of these 8 (3%) received daily G-CSF and 33 (12%) received no G-CSF in cycle 1. Older patients were less likely to receive chemotherapy with curative intent or with high FN risk; FN incidence was similar between age groups but older patients had a greater duration of hospitalization for FN. No serious adverse drug reactions were reported.In patients with NHL pegfilgrastim was used predominantly as PP in patients assessed at an overall high risk of FN, in accordance with guidelines. Older patients who received CT with intermediate FN risk were more likely than younger patients to be assessed as high overall FN risk. FN risk seemed well managed with pegfilgrastim use in both older and younger patients, but FN may carry a greater burden in patients aged ≥65 yrs, although less intensive therapies were used in this subset of patients.Medical Writer: Margit Hemetsberger (Amgen-sponsored)Willenbacher: Amgen: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Jaeger:Amgen: Employment, Equity Ownership. Bast:Amgen: Employment, Equity Ownership. Renner:Amgen: Consultancy, Honoraria, Speakers Bureau.

E-AVARE were observational studies in Austria and Switzerland investigating febrile neutropenia (FN) risk assessment and granulocyte colony-stimulating factor (G-CSF) guideline implementation in real life Non-Hodgkin-lymphoma (NHL) treatment. A combined analysis of both studies was performed to better understand the characteristics of NHL patients who receive pegfilgrastim and the outcomes of these patients in everyday clinical practice.

Interferon-alpha 2 (IFN) is able to induce hematological response in about 70-80% of ET patients but some of them could be defined as bad responders.IFN binding its receptor results in tyrosine cross-phosphorylation and auto-phosphorylation of the JAKs proteins (Tyk2 and Jak1). These phosthyrosines recruit and activate STAT family member such as STAT1 and STAT3. These proteins induce the transcription of SOCSs, whose role is to extinguish cytokine signaling by inhibition of JAK kinase-activity directly through the KIR-domain, and indirectly promoting the proteasomal degradation of Jak2, by SOCS-box-motif.In summary, IFN induces the expression of SOCSs, which inhibit TPO mediated signaling through Jak2 double inhibition. This allows IFN-α and TPO pathway to cross-talks by means of the JAK-STAT-SOCS cascade.To identify molecular markers that identify those patients who respond to IFN, we analyzed bone marrow cells transcript levels of specific genes involved in the IFN receptor pathway, whose signal cross-talks with the TPO dependent JAK-STAT pathway. In particular we investigated the mRNA expression of JAK1, TYK2, STAT1, STAT3, SOCS1 and SOCS3.We analyzed 60 ET patients treated with 3 million units of IFN-α-2b 5 times a week as induction (3 months), and 3 times a week as maintenance. Responses were classified as follow: Good-Responders(R) (n=44), those who achieved complete response according to European Leukemia Net criteria, and Bad-Responders(NR) (n=17) who didn’t reach the criteria.The mRNA expression of genes of interest was measured in bone marrow samples from ET patients by RTq-PCR and tested for their predictive value using receiver operating characteristics (ROC) curves.Data were normalized as following: [mRNA normalized copy number (NCN)=mRNA target gene/mRNA GUSB].An IFN score was calculated as an average in log2 of mRNA levels of genes differently expressed between Good-R and Bad-R.Main clinical characteristics were similar between the two groups of response. JAK2 V617F mutation was detected in 56,8% of Good-R and 58,8% of Bad-R (p=0,81) and no difference was found in JAK2V617F allele burden (p=0,17) and mRNA expression (p=0,2). Patients showed a median spleen volume of 500 ml in Good-R and 250 ml in Bad-R group (p=0.01).Bad-R compared with Good-R showed higher mRNA expression of JAK1 (13.4 vs 4.7; p<0.00001), STAT3 (2.7 vs 2.4; p=0.0002) and SOCS3 (1.1 vs 2; p=0,002). The AUC, using the normalized gene expression values, was 0.88 for JAK1, 0.81 for STAT3 and 0.7 for SOCS3.Average expression in log2 of these three genes was calculated and used as IFN score. The ROC curve AUC analysis for IFN-score revealed an AUC of 0.9 (95% CI:0.8-1.0). The score value with highest combined sensitivity (94.1%, 95% CI: 71.3-99.8) and specificity (88.6%, 95% CI: 75.4-96.2) was 4.74, with a likelihood ratio of 8.28. In our cohort, all Bad-R patients but one, showed IFN-score higher than the established cut off, and this further support the accuracy of our IFN-responsive score for ET patients.We identified a set of three genes whose expression could be compounded into IFN score that showed a significant correlation with response in ET patients.The IFN score could represent a predictive biomarker for responsiveness to IFN and may likely become a substantial aid to the physician.No relevant conflicts of interest to declare.

Interferon-alpha 2 (IFN) is able to induce hematological response in about 70-80% of ET patients but some of them could be defined as bad responders.

Paratore: Novartsi: Employment. Galimberti:Novartis: Employment. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Abstract 2795

No relevant conflicts of interest to declare.

Abstract 4088

The cancer testis antigen PRAME is a potential target for adoptive T-cell or vaccine therapy of many hematologic malignancies and solid tumors. PRAME-specific cytotoxic T lymphocytes (PRAME-CTLs) can be detected in patients with hematologic malignancies and we have shown that they can be generated and expanded ex-vivo, using an optimized combination of artificial antigen presenting cells (aAPC) (K562 cell line genetically modified to express the HLA-A*02, CD80, CD40L and OX40L molecules) and cytokines (IL12, IL7 and IL15). Four HLA-A*02 PRAME-derived epitopes (P100, P142, P300 and P425) have previously been identified using a proteosome-mediated digestion analysis. However, this strategy, since relying only on the major cleavage site targeted by the immune-proteosome machinery for epitopes generation, may limit the potential clinical value of the identified peptides. We have now adopted an alternative method that uses a peptide-library consisting of 125 synthetic pentadecapeptides, overlapping by 11 aminoacids, spanning the entire PRAME protein. We evaluated whether novel HLA-A*02 restricted CD8+ T-cell responses to multiple immunogenic epitopes can be identified and used to consistently generate polyclonal PRAME-CTL lines from healthy donors and patients with hematologic malignancies. CD8+ T lymphocytes from 14 HLA-A*02 healthy donors and 3 patients with chronic myelogenenous leukemia (CML) were primed with autologous CD40L-activated B blasts loaded with the PRAME-peptide library in the presence of low doses of IL12, IL7 and IL15, and then expanded by weekly re-stimulation with peptide loaded aAPC and IL-2. The frequency and specificity of PRAME-CTLs were evaluated using IFNg Elispot and 51Cr release assays against PHA-blasts loaded with the PRAME-library. Using this approach we consistently generated PRAME-CTLs in 12 of the 14 HLA-A*02 healthy donors (526±101 SFC/105 cells as assessed by IFNg Elispot assay) compared to an irrelevant peptide-library (7±2 SFC/105). Similarly, PRAME-CTLs were generated from all 3 CML patients (441±250 SFC/105 cells vs 22±10 SFC/105 against an irrelevant peptide-library). These PRAME-CTLs were also able to target autologous tumor blasts (57±6 IFNg SFC/105), demonstrating that the same peptides were processed and presented physiologically. A Cr51 release assay confirmed that the PRAME-reactive T cells were cytotoxic, lysing autologous-PHA blasts loaded with the peptides derived from the PRAME-library (63±14% at a 20:1 E: T ratio), but not with irrelevant peptides (<15%). MHC class-I blocking experiments using specific antibodies showed that both IFNg release and cytotoxic activity were HLA-restricted. Using pentadecapeptides sub-pools, we found that the responses of our expanded PRAME-CTLs were polyclonal, since they consistently released IFNg in response to 1 to 6 pentadecapeptides pools (59% were specific for 1 or 2 pools, 25% to 3 pools, and 16% to 6 pools). Moreover, the approach we describe has allowed us to identify 6 potential new immunogenic 15-mer peptides that are processed and presented by tumor cells, and should facilitate expansion of polyclonal PRAME-CTLs for adoptive transfer or after vaccine administration to patients with PRAME+ hematological malignancy.

The cancer testis antigen PRAME is a potential target for adoptive T-cell or vaccine therapy of many hematologic malignancies and solid tumors. PRAME-specific cytotoxic T lymphocytes (PRAME-CTLs) can be detected in patients with hematologic malignancies and we have shown that they can be generated and expanded ex-vivo, using an optimized combination of artificial antigen presenting cells (aAPC) (K562 cell line genetically modified to express the HLA-A*02, CD80, CD40L and OX40L molecules) and cytokines (IL12, IL7 and IL15).

Adoptive transfer of autologous EBV-CTLs to hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) recipients is a safe and often effective means for prevention and treatment of EBV-associated post transplant lymphoproliferative disorders (PTLD). Although immunosuppressive drugs can be tapered in patients developing PTLD, they often cannot be completely withdrawn because of the risk of graft rejection, a particular concern in lung, heart or liver transplant recipients. These immunosuppressive drugs may limit the expansion, persistence and efficacy of transferred EBV-CTLs. One of the most widely used immunosuppressive agents is FK506 whose effects are highly dependent on binding FKBP12 proteins, since T cells generated from FKBP12 knockout mice are completely resistant to the inhibitory effects of FK506. We have generated EBV-CTLs resistant to FK506 by knocking down FKBP12 using a small interfering RNA (siRNA) stably expressed from a retroviral vector. After extensively screening potential target sequences, we identified one, designed as siRNA4, that knocks down >90% of FKBP12 protein expression in T cell lines and also in EBV-CTLs, as assessed by Western blotting. We then generated two retroviral vector encoding for siRNA4/eGFP and irrelevant siRNA/eGFP, respectively. These vectors were used to transduce established EBV-CTL lines generated from 7 EBV-seropositive donors. Transduction efficiency was 46.3±22.5% and 55.4±27.5% for siRNA4 and irrelevant-siRNA, respectively. We measured the proliferation of transduced CTLs in the presence of FK506, in short term and long term cultures. Using a thymidine uptake assay, we found that the inhibiton of proliferation by increasing concentrations of FK506 was significantly diminished in siRNA4+ CTLs compared to control CTLs (41±4% inhibition for siRNA4+ CTLs vs 74± 2% for control CTLs). To evaluate the effects of knocking down FKBP12 in long-term cultures, control and siRNA4+ CTLs were stimulated weekly with the antigen (autologous EBV-LCL) with or without the addition of FK506 (5ng/ml) and low dose IL-2 (20U/mL). We found that the proportion of siRNA4+ CTLs increased over time not only as a percentage of GFP+ cells (from 46±22% to 89.4±5.3% after 5 stimulations) but also numerically (median fold expansion: 34.3, range 5–60). In contrast, control EBV-CTLs did not show any selection in culture, since the percentage of GFP+ cells remained unchanged (from 56±27% to 57±23.1%) and CTLs ceased to proliferate (median fold expansion: 2, range 0–5). Finally, we found that siRNA4+ CTLs retained their antigen specificity, having MHC-restricted cytotoxic activity against EBV-targets (66±22% vs 16±12% for autologous and allogenic LCL, respectively at an E:T ratio of 20:1 for siRNA4+ CTLs; 61±12% vs 15±12% for autologous and allogenic LCL, respectively, for control CTLs). Modified CTLs also maintained their production of IFNγ in response to specific EBV-peptides, as assessed by ELIspot assays. We are currently evaluating the in vivo expansion of genetically modified EBV-CTLs in the presence of FK506. In conclusion, we have developed a strategy that produces EBV-CTLs resistant to FK506. This strategy may be beneficial to improve EBV immune reconstitution in patients at high risk of developing post transplant lymphoma.

Adoptive transfer of autologous EBV-CTLs to hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) recipients is a safe and often effective means for prevention and treatment of EBV-associated post transplant lymphoproliferative disorders (PTLD). Although immunosuppressive drugs can be tapered in patients developing PTLD, they often cannot be completely withdrawn because of the risk of graft rejection, a particular concern in lung, heart or liver transplant recipients. These immunosuppressive drugs may limit the expansion, persistence and efficacy of transferred EBV-CTLs. One of the most widely used immunosuppressive agents is FK506 whose effects are highly dependent on binding FKBP12 proteins, since T cells generated from FKBP12 knockout mice are completely resistant to the inhibitory effects of FK506. We have generated EBV-CTLs resistant to FK506 by knocking down FKBP12 using a small interfering RNA (siRNA) stably expressed from a retroviral vector. After extensively screening potential target sequences, we identified one, designed as siRNA4, that knocks down >90% of FKBP12 protein expression in T cell lines and also in EBV-CTLs, as assessed by Western blotting. We then generated two retroviral vector encoding for siRNA4/eGFP and irrelevant siRNA/eGFP, respectively. These vectors were used to transduce established EBV-CTL lines generated from 7 EBV-seropositive donors. Transduction efficiency was 46.3±22.5% and 55.4±27.5% for siRNA4 and irrelevant-siRNA, respectively. We measured the proliferation of transduced CTLs in the presence of FK506, in short term and long term cultures. Using a thymidine uptake assay, we found that the inhibiton of proliferation by increasing concentrations of FK506 was significantly diminished in siRNA4+ CTLs compared to control CTLs (41±4% inhibition for siRNA4+ CTLs vs 74± 2% for control CTLs). To evaluate the effects of knocking down FKBP12 in long-term cultures, control and siRNA4+ CTLs were stimulated weekly with the antigen (autologous EBV-LCL) with or without the addition of FK506 (5ng/ml) and low dose IL-2 (20U/mL). We found that the proportion of siRNA4+ CTLs increased over time not only as a percentage of GFP+ cells (from 46±22% to 89.4±5.3% after 5 stimulations) but also numerically (median fold expansion: 34.3, range 5–60). In contrast, control EBV-CTLs did not show any selection in culture, since the percentage of GFP+ cells remained unchanged (from 56±27% to 57±23.1%) and CTLs ceased to proliferate (median fold expansion: 2, range 0–5). Finally, we found that siRNA4+ CTLs retained their antigen specificity, having MHC-restricted cytotoxic activity against EBV-targets (66±22% vs 16±12% for autologous and allogenic LCL, respectively at an E:T ratio of 20:1 for siRNA4+ CTLs; 61±12% vs 15±12% for autologous and allogenic LCL, respectively, for control CTLs). Modified CTLs also maintained their production of IFNγ in response to specific EBV-peptides, as assessed by ELIspot assays. We are currently evaluating the in vivo expansion of genetically modified EBV-CTLs in the presence of FK506. In conclusion, we have developed a strategy that produces EBV-CTLs resistant to FK506. This strategy may be beneficial to improve EBV immune reconstitution in patients at high risk of developing post transplant lymphoma.

Imatinib mesylate was given to 26 Philadelphia positive (Ph+) chronic myeloid leukemia (CML) patients who were in late chronic phase (CP) and in stable complete cytogenetic response (CCR) after interferon-alfa (IFN-α), but showed persistent positive residual disease at PCR analysis under this treatment. At diagnosis median age was 40 years (range 21–64) and according to Sokal’s score, 18 patients were low risk and 8 were intermediate risk. Median IFN treatment was 88 mo.s (range 15–202) and median CCR duration was 73 mo.s (range 10–148). Imatinib was administered at the standard dose of 400 mg/die, after stopping IFN for 1 week. Residual disease was measured on bone marrow (BM) cells at baseline, before starting Imatinib, at 3, 6, 12, 18 mo.s and at the last follow-up (median 32 mo.s, range 21–49), by assaying BCR-ABL transcripts using quantitative PCR (RQ-PCR). The copy number (CN) of BCR/ABL and ABL transcript were derived by the interpolation of CT values to the appropriate standard curve, and the result, for each sample, was expressed as ratio of BCR/ABL mRNA copies to ABL mRNA x 100 (normalized copy number - NCN). Imatinib treatment resulted in a progressive and consistent decline of residual disease in all but one patient, from a median of 0.89 at baseline to 0.01 at the end of follow-up. Major molecular response (BCR/ABL levels <0.1) was reached in 20 patients (77%) and BCR/ABL transcripts were undetectable in 13 (50%). Achievement of molecular response was significantly correlated with post-IFN baseline transcript level (mean 1.194 for patients achieving complete molecular response vs 18,97 for those who did not; p<0.001), but not with other clinical/biological patient characteristics. In all patients, imatinib was well tolerated with no side effects requiring drug dose reduction or dose discontinuation. Albeit obtained from an unusual subset of selected patients with favourable prognosis, and likely particularly sensitive to imatinib, present results confirm the efficacy of combining Imatinib and IFN-α and further support investigating treatment approaches employing these two drugs.

Imatinib mesylate was given to 26 Philadelphia positive (Ph+) chronic myeloid leukemia (CML) patients who were in late chronic phase (CP) and in stable complete cytogenetic response (CCR) after interferon-alfa (IFN-α), but showed persistent positive residual disease at PCR analysis under this treatment. At diagnosis median age was 40 years (range 21–64) and according to Sokal's score, 18 patients were low risk and 8 were intermediate risk. Median IFN treatment was 88 mo.s (range 15–202) and median CCR duration was 73 mo.s (range 10–148). Imatinib was administered at the standard dose of 400 mg/die, after stopping IFN for 1 week. Residual disease was measured on bone marrow (BM) cells at baseline, before starting Imatinib, at 3, 6, 12, 18 mo.s and at the last follow-up (median 32 mo.s, range 21–49), by assaying BCR-ABL transcripts using quantitative PCR (RQ-PCR). The copy number (CN) of BCR/ABL and ABL transcript were derived by the interpolation of CT values to the appropriate standard curve, and the result, for each sample, was expressed as ratio of BCR/ABL mRNA copies to ABL mRNA x 100 (normalized copy number - NCN).

We created a relational database of patients with cutaneous T-cell lymphoma (CTCL) to collect in a standardised fashion, anagraphic variables, clinical history, clinical, histological, haematological, and immunological information on CTCL patients hospitalised at IDI-IRCCS, Rome, Italy. At present, there are data on 424 patients, hospitalised from 1983 to July 2005. Active follow-up is performed yearly to ensure a standardised ascertainment of survival time. For deceased patients, the actual date of death (for all causes) is recorded, while surviving patients are censored at the date of last contact.

We created a relational database of patients with cutaneous T-cell lymphoma (CTCL) to collect in a standardised fashion, anagraphic variables, clinical history, clinical, histological, haematological, and immunological information on CTCL patients hospitalised at IDI-IRCCS, Rome, Italy. At present, there are data on 424 patients, hospitalised from 1983 to July 2005. Active follow-up is performed yearly to ensure a standardised ascertainment of survival time. For deceased patients, the actual date of death (for all causes) is recorded, while surviving patients are censored at the date of last contact. The database includes 29 patients with Sezary syndrome (SS). Follow-up times ranged from 0 to 105 months. At first hospitalisation the median values of cells/mL were: white blood cells (WBC) 8750, neutrophils 4250, eosinophils 140, basophils 120, lymphocytes 2760, monocytes 500, CD3+ 2780, CD4+ 2431, CD8+ 192, CD19+ 96. Seventeen patients were deceased. We included in the Kaplan-Meier survival analysis only patients who were diagnosed before July 2004 (n=26). Median survival time from diagnosis was 52 months. No significant differences were observed in mortality for WBC (cutoff 9000 cells/uL), neutrophils (cutoff 4500 cells/uL), basophils (cutoff 200 cells/uL), lymphocytes (cutoff 3000 cells/uL), monocytes (cutoff 500 cells/uL), CD3+ (cutoff 2000 cells/uL), CD4+ (cutoff 2000 cells/uL), CD8+ (cutoff 200 cells/uL), CD19+ (cutoff 70 cells/uL). A lower survival was observed for patients with eosinophils <200 cells/uL (p=.08). Survival in patients with SS does not seem to be influenced by haemathologic parameters. However, patients with long-term survival (>90 months) are observed, and their characteristics should be further investigated. Survival analysis of 26 patients with Sézary syndrome, Rome, Italy, 1991–2004. Survival analysis of 26 patients with Sézary syndrome, Rome, Italy, 1991–2004.