ALBERT

Description: Abstract 3987 Multiple Myeloma (MM) is a clonal plasma cell disorder whose growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type 1 (IGF-1). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma and has been suggested to induce apoptotsis by reducing NF-κB signalling. Other cytotoxic mechanisms have been suggested, including increased reticulum stress leading to an unfolded protein response. We analyzed the impact of recombinant IGF-1 combined with the proteasome inhibitor bortezomib on human plasma cell lines in vitro and in vivo and on fresh human myeloma cells ex vivo. Using an MTT assay, we found that IGF-1 enhanced the cytotoxic activity of bortezomib in vitro against the LP1, RPMI8226, U266 and MM1.S lines, at a concentration of IGF-1 of 100 ng/mL. This potentiating effect was confirmed on MM1.S cells using a flow cytometric analysis of annexin V staining, and showed that the enhanced toxicity could be inhibited by the presence of a monoclonal antibody directed against the IGF-1 receptor (IGF1-R). IGF-1 was also found to enhance the cytotoxic activity of other proteasome inhibitors against MM1.S cells, including MG115, MG132, PSI and epoximicin. In vivo studies were performed in SCID mice bearing MM1.S xenografts. Mice received weekly administrations of bortezomib (0.5 mg/kg, i.p.) with or without recombinant IGF-1 (0.03 mg/kg, i.p.). The co-administration of IGF-1 with bortezomib significantly delayed tumor growth in comparison to that observed in mice treated with bortezomib alone. Fresh human myeloma cells exposed to bortezomib ex vivo displayed a larger annexin V positive fraction when they were co-incubated with IGF-1 then when they were exposed to bortezomib alone. This effect, which could be observed in subpopulations of CD45 hi and CD45 lo cells, could be reversed by an antibody directed against IGF-1R. Thus in each of these situations, IGF-1 increased the sensitivity of multiple myeloma cells to the cytotoxic effect of bortezomib. Analysis of pro- and anti-apoptotic proteins in MM1.S cells by immunoblotting showed that the addition of IGF-1 to bortezomib significantly enhanced the content o Bax, Bad and Bak and significantly reduced the content of Bcl2, BclX-L and Bfl-1. Exploration the NFkB pathway showed that exposure to IGF-1 and bortezomib induced a reduction of IkBalpha, an increase in phosphor-IKBalpha as well as a decrease in NFkB p65. Other observations made with the IGF-1/bortezomib combination include an increase in the content of cleaved caspase 3 and in P21 protein. Cell cycle distributions of cells exposed to bortezomib alone or the IGF-1/bortezomib combination were similar. Preliminary data showed an increased content of CHOP protein, suggesting that the IGF-1/bortezomib combination might enhance reticulum stress in MM1.S cells, thus leading to an Unfolded Protein Response (UPR) and to cell death. These results suggest that IGF-1 sensitizes myeloma cells to proteasome inhibitors by contributing to the enhancement of the reticulum stress. Overall these results suggest that exposure of myeloma cells to one of their key growth factors, IGF-1, significantly enhanced their sensitivity to bortezomib as well as to other proteasome inhibitors. This phenomenon appears to involve several pathways and may be dependent on the high baseline level of reticulum stress present in myeloma cells. Disclosures: No relevant conflicts of interest to declare.

Description: Allopurinol for tumor lysis syndrome (TLS) prophylaxis is mostly given at 300 mg daily, but the exact dose is variable and depends on physician’s choice. In order to assess which baseline factors mostly drive the choice, we performed an analysis of the FLORENCE study database. FLORENCE is a double-blinded randomized trial, the largest one in TLS prophylaxis, conducted in 11 European countries (Croatia, Czech Republic, Germany, Hungary, Italy, Poland, Romania, Russia, Serbia, Spain, Ukraine) and Brazil, with this latter country accounting only for 1 patient. Patients with hematologic malignancies (HM) were stratified according to TLS risk (intermediate or high; Cairo et al, British Journal of Haematology, 2010) and serum uric acid (sUA) level (≤ or 〉 7.5 mg/dl) and randomized at one-to-one ratio to receive TLS prophylaxis with febuxostat or allopurinol. At randomization, 341 patients were blindly assigned by the physician to receive standard or high daily dose level of study drug, containing either allopurinol 300/600 mg or fixed febuxostat 120 mg, respectively. A total of 286 (84%) patients were assigned to receive standard dose level, 253 (88%) and 33 (12%) of which were at intermediate and high TLS risk, respectively. By contrast, in a total of 55 (16%) patients the high dose level of treatment was chosen, 28 (51%) and 27 (49%) of which were at intermediate and high TLS risk, respectively. In order to identify which factors might have influenced the physician’s choice, an analysis of variance (ANOVA) model was used considering the dose level as dependent variable and each of the following variables as covariates (one at a time): TLS risk grade (intermediate or high), sUA level (≤ or 〉 7.5 mg/dL), type of HM (acute leukemia or chronic lymphocytic leukemia / non Hodgkin lymphoma), presence/absence of high disease burden (defined as bulky disease for lymphomas or white blood cells count ≥ 100 x 109/L for leukemias), lactate dehydrogenase level (〈 or ≥ 2 x upper limit of normal) and country. Since the standard dose level was chosen mainly for patients at intermediate TLS risk while the high one was equally distributed between TLS risk groups, ANOVA models were performed in the two TLS risk subgroups with the dependent and independent variables described above. In the intermediate TLS risk subgroup (n=281), all the covariates were statistically significant excluding high disease burden. Conversely, in the high TLS risk subgroup (n= 60) only country reached statistical significance (p= 0.053) despite the small sample size. Our study confirms that 300 mg/day is the preferred dose of allopurinol for patients at intermediate TLS risk; in this patient group objective disease-related factors as well as country drove the investigator’s choice. On the other hand, the 300 mg and 600 mg daily dose levels were almost equally distributed in patients at high TLS risk and only country impacted on the physician’s choice for this subgroup. The availability of a fixed posology scheme, as febuxostat offers, may overcome the subjective nature of variability in the clinical practice of TLS prophylaxis, in particular for patients at high TLS risk. Disclosures Baldini: MENARINI RICERCHE: Employment. Off Label Use: Febuxostat in Tumor Lysis Syndrome prophylaxis. Rossi:MENARINI RICERCHE: Employment. Spina:MENARINI RICERCHE: Membership on an entity's Board of Directors or advisory committees. Federico:MENARINI RICERCHE: Membership on an entity's Board of Directors or advisory committees. Nagy:MENARINI RICERCHE: Consultancy. Jordan:MENARINI RICERCHE: Consultancy. Aurer:MENARINI RICERCHE: Consultancy. Ribera:MENARINI RICERCHE: Consultancy. Borsaru:MENARINI RICERCHE: Consultancy. Glushko:MENARINI RICERCHE: Consultancy. Grosicki:MENARINI RICERCHE: Consultancy. Rego:MENARINI RICERCHE: Consultancy. Nizzardo:MENARINI RICERCHE: Employment. Scartoni:MENARINI RICERCHE: Employment. Scordari:MENARINI RICERCHE: Employment. Matera:MENARINI RICERCHE: Employment. Maggi:MENARINI RICERCHE: Employment. Capriati:MENARINI RICERCHE: Employment. Simonelli:MENARINI RICERCHE: Employment.

Description: The capacity to generate lymphokine-activated killer (LAK) cells and the susceptibility of the neoplastic cells to both allogeneic and autologous LAK effectors were studied in B and T chronic lymphoproliferative disorders. While in B-cell chronic lymphocytic leukemia (B-CLL) the depressed natural killer function could be restored after a 7-day incubation with recombinant interleukin (IL-2), B-CLL mononuclear cells showed a reduced LAK activity compared with normal LAK cells. Furthermore, in all but 1 of the 20 B-CLL samples tested the leukemic cells were totally resistant to autologous LAK effectors. In most cases the leukemic cells were also resistant to normal allogeneic LAK cells. Competition experiments demonstrated that the patients' LAK cells, as well as normal LAK effectors, were capable of recognizing B-CLL cells, pointing, therefore, to a postbinding cytolytic defect. In hairy cell leukemia (HCL) an overall reduced LAK activity against allogeneic targets was documented, but, at variance from B-CLL, hairy cells were often susceptible to the lytic effect of normal LAK cells, and in half of the cases tested the neoplastic population was also sensitive in an autologous system. Similarly to B- CLL, in the great majority of T chronic lymphoproliferative disorders studied, the pathologic cells were resistant to normal and autologous LAK effectors and a defective LAK generation was found. These results demonstrate that in most B and T chronic leukemias the LAK function is defective and, when inducible, does not appear directed against the leukemic population. The possibility of exploiting an immunotherapeutic approach with IL-2/LAK cells in the management of chronic lymphoproliferative disorders does not gain support by these findings.

Description: Background: Acute lymphoblastic leukemia (ALL) is a malignant disorder with a long-term remission of less than 50% of adult patients and of nearly 80% of children. Relapsed and refractory (r/r) adult and childhood B-ALL patients, have significant unmet medical needs. Adoptive transfer of patient-derived T cells engineered to express a chimeric antigen receptor (CAR) by viral vectors has achieved complete remission and durable response in highly refractory populations (June CH et al. Science 2018). In addition, unmodified Cytokine Induced Killer (CIK) cells (CD3+, CD56+ T cells) have clearly demonstrated a high profile of safety in ALL patients (Introna M et al. Biol Blood Marrow Transplant. 2017). Here, we demonstrate the feasibility and reproducibility of a GMP-compliant clinical-grade culture and gene-modification protocol of allogeneic CIK cells using the non-viral Sleeping Beauty (SB) transposon system (Singh H et al, Plos One 2013) to obtain CD19CAR expressing CIK cells (Magnani CF et al, Oncotarget 2016, Magnani CF et al, Hum Gene Ther. 2018, Biondi A et al. J Autoimmun. 2017) starting from a limited amount of an easily available material such as peripheral blood (PB). Methods: Fifty mL of PB were centrifuged on Ficoll gradient to obtain mononuclear cells (PBMCs). PBMCs were then simultaneously electro-transferred with the SB GMP-grade DNA transfer CD19.CAR/pTMNDU3 plasmid (human 3rd generation anti-CD19CD28OX40z CAR under the pTMNDU3 promoter), and transposase pCMV-SB11 plasmid (kindly provided by L. Cooper, MDACC, Houston, TX, USA). CIK populations (Introna M et al, Haematologica 2007) were then generated according to the method enclosed in the filed patent EP20140192371 (Magnani CF et al, Oncotarget 2016). The manufacturing process and the quality control tests were performed in a good manufacturing practices (GMP) academic cell factory authorized by Agenzia Italiana del Farmaco (AIFA) in the context of an ongoing phase I clinical trial (NCT03389035) for children and adults with relapsed/refractory B-cell precursor ALL post hematopoietic stem cell transplantation (HSCT). Results: We manufactured nine batches by seeding a mean of 102.52x106 allogeneicPBMCs derived from 50 ml of peripheral blood (range 46.1 - 193.17x106). After 21-22 days of culture the mean harvesting was 5.0x109 nucleated cells (range 1.15 - 16.00x109) with a mean viability of 97.56% (min. 95.24% - max 99.43%). These cells were mostly CD3+ lymphocytes (mean 98.54%, min. 94.85% - max 99.68%) which had a median fold increase of 87.3. Expanded CD3+ cells expressed CD56+ and surface CAR at variable levels among the batches (mean 44.79% and 43.78%, respectively) and had a median vector copy number (VCN) of 2.56 VCN/cells, according to pre-clinical data (Magnani CF et al, Hum Gene Ther. 2018). In all the nine batches, CARCIK-CD19 cells demonstrated potent and specific in vitro cytotoxicity towards the CD19+ REH target cell line (mean 82.96%, min. 61.89% - max 97.72%). Cell products appear to be highly polyclonal and no signs of genotoxicity by transposon insertions could be observed by integration site (IS) analysis performed by Sonication Linker Mediated (SLiM)-PCR and Illumina MiSeq sequencing. The GMP batches were released after about 10 days after the end of production. Quality control release specifications and results are reported in Table 1. Conclusions: Overall, these results demonstrate that clinical-grade SB transduction of allogeneic CIK cells with CD19 CAR is feasible and allows rapid and efficient expansion of highly potent CARCIK-CD19 cells starting from easily available small amounts of PB, with important implications for non-viral technology. In summary our data represent a solid ground for the future development of further SB-based platforms. A clinical trial investigating allogeneic CARCIK-CD19 in r/r pediatric and adult ALL post HSCT is currently ongoing (NCT03389035). Disclosures Gritti: Autolus: Consultancy. Rambaldi:Celgene: Consultancy; Omeros: Consultancy; Novartis: Consultancy; Italfarmaco: Consultancy; Pfizer: Consultancy; Amgen Inc.: Consultancy; Roche: Consultancy.

Description: Cooperation between in vitro exogenous prolactin (PRL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) at an early step of in vitro erythroid differentiation has been shown in a previous study. To gain more insight into the role of PRL in in vivo hematopoiesis, we have now addressed the involvement of endogenous PRL in the growth of hematopoietic progenitors in a bone marrow (BM) stroma environment. The possible modulation of local PRL production by the inflammatory mediator platelet-activating factor (PAF), which is known to be produced by BM cells and to regulate pituitary PRL release, has also been evaluated. Development of burst-forming unit-erythroid (BFU-E) colonies from CD34+ hematopoietic progenitors cultured on a BM stroma cells (BMSC) layer was slightly, but significantly, reduced in the presence of an antihuman PRL antibody. Pretreatment of BMSC with PAF increased the BFU-E colony efficiency of cocultured CD34+ cells, and this effect was completely abrogated by the antiserum. PAF-modulated release of PRL by BMSC was confirmed by an enzyme-linked-immunospot (Elispot) technique. In addition, immunoprecipitation and Western blotting experiments showed two immunoreactive products in the BMSC culture medium. These corresponded to the nonglycosylated (23 kD) and glycosylated (25.5 kD) forms of pituitary PRL that are also expressed by the B-lymphoblastoid cell line IM9-P3. Specific increase of the nonglycosylated form and decrease of the glycosylated form was observed after PAF treatment. Polymerase chain reaction (PCR) amplification of reverse transcribed RNA using PRL-specific primers showed the presence of PRL message in BMSC and IM9-P3 cells. In situ hybridization experiments with a rat PRL cDNA probe cross-reacting with human PRL mRNA confirmed its presence in a small fraction of unstimulated BMSC and in the majority of PAF-stimulated BMSC. The enhancing effect of PAF on PRL-mediated colony formation, PRL release, and mRNA activation was counteracted by pretreating BMSC with the PAF-receptor (R) antagonist WEB 2170. Lastly, responsiveness of BMSC to PAF was substantiated by the presence of the PAF-R mRNA on these cells.

Description: Cooperation between in vitro exogenous prolactin (PRL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) at an early step of in vitro erythroid differentiation has been shown in a previous study. To gain more insight into the role of PRL in in vivo hematopoiesis, we have now addressed the involvement of endogenous PRL in the growth of hematopoietic progenitors in a bone marrow (BM) stroma environment. The possible modulation of local PRL production by the inflammatory mediator platelet-activating factor (PAF), which is known to be produced by BM cells and to regulate pituitary PRL release, has also been evaluated. Development of burst-forming unit-erythroid (BFU-E) colonies from CD34+ hematopoietic progenitors cultured on a BM stroma cells (BMSC) layer was slightly, but significantly, reduced in the presence of an antihuman PRL antibody. Pretreatment of BMSC with PAF increased the BFU-E colony efficiency of cocultured CD34+ cells, and this effect was completely abrogated by the antiserum. PAF-modulated release of PRL by BMSC was confirmed by an enzyme-linked-immunospot (Elispot) technique. In addition, immunoprecipitation and Western blotting experiments showed two immunoreactive products in the BMSC culture medium. These corresponded to the nonglycosylated (23 kD) and glycosylated (25.5 kD) forms of pituitary PRL that are also expressed by the B-lymphoblastoid cell line IM9-P3. Specific increase of the nonglycosylated form and decrease of the glycosylated form was observed after PAF treatment. Polymerase chain reaction (PCR) amplification of reverse transcribed RNA using PRL-specific primers showed the presence of PRL message in BMSC and IM9-P3 cells. In situ hybridization experiments with a rat PRL cDNA probe cross-reacting with human PRL mRNA confirmed its presence in a small fraction of unstimulated BMSC and in the majority of PAF-stimulated BMSC. The enhancing effect of PAF on PRL-mediated colony formation, PRL release, and mRNA activation was counteracted by pretreating BMSC with the PAF-receptor (R) antagonist WEB 2170. Lastly, responsiveness of BMSC to PAF was substantiated by the presence of the PAF-R mRNA on these cells.

Description: Background and aim: In the last decade HDT-ASCT has significantly improved progression-free and overall survival of patients with MM. Achievement of CR or good partial response and the tumor reduction attained with the induction pretransplant chemotherapy have been shown to be the most relevant prognostic factors for long-term survival. In recent years, novel drugs such as thalidomide and bortezomib have been introduced in the treatment of MM. Bortezomib (B) (VelcadeÒ) as a single agent, gives a response rate ranging from 35% to 50%, including up to a 9% CR rate in relapsed/refractory patients, and of 40%, with a 10% CR rate in the up-front setting. Thalidomide (T) has been identified as the first independently active agent in MM since the introduction of melphalan and prednisone and currently represent the milestone of initial treatment in elderly patients. Since B and T target different molecular pathways, we started a phase II trial in order to assess efficacy, toxicity and rate of PBSC collection after VTD regimen delivered as induction pretransplant chemotherapy in patients with newly diagnosed MM. Patients and Methods: from June 2007 to July 2008 14 pts (M/F: 11/3) with a median age of 56 years (range: 42–71) were enrolled in the study; six pts (43%) were more than 60 yo and 7 pts had a previous history of M-GUS lasting in median 54 months. At time of treatment there were 71%, 22% and 7% having Durie and Salmon staging III, II and I respectively, while ISS was 1 in 22%, 2 in 50% and 3 in 28% of cases. One pt had renal impairment, extensive bone disease was documented in 78% of cases with 2 pts showing extramedullary disease. Sixty-five percent of pts (9/14) had IgG MM, 14% IgA, 7% light chain, and 14% non secretory myeloma. Unfavourable cytogenetic was recorded in 36% (5/14) of cases. Bortezomib was administered at 1.3 mg/m2 on days 1,4, 8, 11 as short IV infusion, thalidomide at daily dose of 100 mg PO and Dexamethasone (40 mg/die PO) the day of bortezomib infusion and the day after (640 mg for each cycle) every 4 weeks for 3–4 courses. All patients received deep venous thrombosis prophylaxis consisting of aspirin 100 mg daily (44%), low molecular weight heparin (28%) and low dose warfarin (28%). Following VTD regimen patients underwent to high-dose cyclophosphamide (4 g/m2) with G-CSF support and peripheral stem cell harvest. MEL 200 was given as conditioning regimen. All patients received standard dose of zoledronic acid monthly. Results: At present time all patients are evaluable for VTD and PBSC collection while 10 for response after transplant. After 3 courses of VTD 93% of pts achieved more than a partial response including 57% of CR and 36% of VGPR. One pt achieved a PR. VTD regimen resulted well tolerated with main toxicity consisting of WHO grade III peripheral neuropathy recorded in 37 % of pts. There were no pts with relevant hematologic toxicity or other non-hematologic toxicities and there were no chemotherapy reduction or delay because of toxicity. None of pts developed DVT. A sufficient amount of CD34+ cells (median 6.5 × 106/kg; range: 2.7–11.6) was collected in 13 of 14 evaluable pts after a median of 1.4 aphaeresis (range:1–2). One pt failed to collect after CTX and was treated with HD-Ara-C obtaining an adequate number of CD34+ cells for transplant. The median CD 34+ cells infused in the 10 transplanted pts was 3.3 ×106/kg (range: 2.0 – 4.7). Times to platelet (20×109/L) and granulocyte (500×109/L) recovery were 13 and 10 days respectively. After HDC and ASCT 7 of 10 patients presented CR (70%) and 2 (20%) a VGPR with an ORR of 90%. One pt presented progression disease after 6 months from transplant. Conclusion: these very preliminary data suggest that VTD regimen given as pretransplant chemotherapy is effective and well tolerated regimen; the capacity to give high response rate in a short time without to compromise PBSC collection makes VTD regimen a good option for initial treatment of newly diagnosed MM patients although more pts and longer follow-up are needed to assess the real impact on survival of this regimen.

Description: Introduction . BEAM (carmustine, etoposide, aracytin, melphalan) is a conditioning regimen used in autologous hemopoietic stem cell transplantation for Hodgkin Lymphoma and non Hodgkin Lymphoma, with acceptable toxicity and high efficacy. In our study we replaced the carmustine with fotemustine, an analogous chloroethylnitrosourea. Primary end-point was to valuate the feasibility of this modified conditioning regimen. Patients and Methods . 86 patients were consecutively conditioned in seven BM-Units with FEAM before receiving aHSCT. 44 patients (63%) were male, 32 (37%) female. Median age was 51 years (range, 18–77). 23 patients (27%) had HL, 60 (70%) had NHL (2 SLL, 1 LPL, 1 MZL, 2 FL, 6 MCL, 37 DLBCL, 3 BL, 1 B-LBL, 1 T-LBL, 4 u-PTCL, 2 AIL), 1 patient had a B-CLL, 1 a B-ALL FAB L3 and 1 an aggressive NK-cell leukemia. In the lymphoma group, 22 patients (27%) were in stage II (10 with bulky disease), 20 (24%) in stage III and 41 (49%) in stage IV. Among lymphoma patients, 19 (23%) had a bone marrow involvement, 5 (6%) a central nervous system involvement, including 2 primary central nervous system lymphoma. At the time of transplantation, 41 patients (48%) were in CR, 3 (3%) in VGPR, 32 (37%) in PR, 10 (12%) had a resistant-progressive disease (R/PD); 30 patients (35%) were at first line of therapy, 56 (65%) had received more than one line of therapy. Patients received fotemustine 150 mg/m2 on days -7, -6, etoposide 200 mg/m2 and aracytin 400 mg/m2 on day -5, -4, -3, -2, and melphalan 140 mg/m2 on day -1. The median number of CD34+ cells infused was 3.8 × 106/Kg recipient body weight (range, 1–21.8). Results . Only 2 patients were not evaluated for engraftment and toxicity. Among evaluable patients, all engrafted. The median time to neutrophil (N 〉0.5 × 106/L) and platelet (PLT 〉20 × 109/L) recovery was 11 (range, 9–19) and 13 days (range, 6–105) respectively. 55 patients (65%) received trasfusions of red blood cell units, with a median of 2 units (range, 1–8). All patients received platelet trasfusions with a median of 2 units (range, 1–15). Toxicity . No chemotherapy-induced nausea and vomiting (CINV) was observed in 17 patients (20%), 53 patients (63%) had CINV grade I–II, 14 patients (17%) grade III, no grade IV was observed. No mucositis was observed in 16 patients (19%), 45 patients (54%) had mucositis grade I–II, 17 patients (20%) grade III, 6 patients (7%) grade IV. No diarrhea was observed in 50 patients (60%), 29 patients (34%) had diarrhea grade I–II, 5 patients (6%) grade III, no grade IV was observed. No epatic toxicity was observed in 80 pts (95%), 1 patient had epatic toxicity grade I, 1 patient grade II and 2 patients grade III, no grade IV was reported. Only one patient had a transient renal toxicity grade II. No pulmonary toxicity was observed. Fever 〉38, 5°C was documented in 68 patients (81%) with a median duration of 4 days (range 1–25). GRAM-were identified in 15 patients, GRAM+ in 14 patients, yeasts were isolated in 3 patients and there was only one infection by Pneumocystis Carinii. In the other 35 patients (51%) no organism was identified as the source of the fever which was classified as FUO. Outcome . At a median follow-up of 5 months (range, 1–16), 79 patients (92%) are alive. On 75 evaluated patients, fifty-nine (79%) are alive and free from disease. Among the seven deceased patients, three died for PD at day +111, +110 and +75 from transplantation respectively, one died for bacterial meningitis at day +45 from transplantation, after a complete hematologic recovery, one in PR died for gastric haemmorrhage from tumor site, and two (1 CR, 1 VGPR) died for comorbidity, respectively at day +150 and +240 from transplantation. TRM at 100 days was 1%. Among the 75 patients who were evaluated after aHSCT, thirty-five patients, out of thirty-six who were in CR before aHSCT, maintained the CR after, for the other thirty-nine, twenty-seven (69%) achieved the CR (three of these had a CNS involvement before aHSCT), one achieved a VGPR, two a PR (ORR 77%), four had a stable disease and five progressed. Considering as treatment failure the relapse, the progression or the death for any cause, in our study the treatment failure was assessed on 16%. Conclusions . Our study demonstrated the feasibility and the safety of the FEAM, regard its toxicity it was not superior with respect to BEAM. However, a longer follow-up is needed to valuate the efficacy of this modified conditioning regimen in term of clinical response.

Description: Background Febuxostat, a potent and selective xanthine oxidase inhibitor, showed a significantly superior serum uric acid (sUA) control during chemotherapy (CT) in comparison to Allopurinol (Spina M. et al, ASCO 2014 and Annals of Oncology in press) when given as 120 mg oral fixed daily dosed and has been recently approved in the European Union (EU) for the prevention and treatment of hyperuricemia in adult patients undergoing CT for hematologic malignancies (HM) at intermediate- high Tumor Lysis Syndrome (TLS) risk, with recognition of the high relevance of its clinical benefit. Some pediatric HM are highly prone to develop TLS; an age-appropriate drug formulation for TLS prophylaxis is lacking in this population, resulting in an unmet medical need that Febuxostat is going to address. The similarities between adult and pediatric patients in terms of TLS pathogenesis, clinical manifestations and current management allow using pharmacokinetic (PK) information to extrapolate clinical efficacy and safety from adult to pediatric patients, as well as within the different pediatric ages (EMEA/CHMP/EWP/147013/2004). Methods A phase I/II study (Floret) has been designed to explore the PK/pharmacodynamic (PK/PD) profile of Febuxostat in the pediatric population in comparison to adults. Febuxostat daily dose will be 120 mg for adolescents and adults with a lower 80 mg dose to be administered to adolescents only, to obtain confirmatory data on the optimal exposure in terms of safety and effectiveness compared to adults. Considering the difference in body size and organ maturation, the selected daily doses of Febuxostat to be tested in children aged 6 to 11 years are 40 and 60 mg. As the only approved strengths of Febuxostat in the EU are 80 and 120 mg, an age-appropriate 20 mg tablet formulation of Febuxostat is developed to ensure the adequate dosing in children. In accordance to the European Medicines Agency Guidelines CHMP/EWP/147013/2004 and CHMP/EWP/18599/2006, the choice of population PK analysis, using non-linear mixed effect models, is considered appropriate to obtain the necessary precision of PK parameters estimates for the comparability assessment between the groups of patients. The PD/efficacy measurement, namely the sUA area under curve from baseline to the evaluation visit, corresponding to the primary efficacy measure explored during the Florence study, a Phase III comparative trial in adults vs Allopurinol (Spina M et al, ASCO 2014), together with the exposure data will abridge the efficacy results from the Florence to the Floret study and will confirm that potential PK deviation, if any, is of no clinical relevance. Conclusion: The present PK/PD approach allows reducing the number of blood samples from each patient, in particular the lowest aged pediatric patients, while replacing a conventionally designed PK study and a robust sized Phase III study in pediatrics. By bridging PK/PD data, the Floret study not only will allow Febuxostat to rapidly address an unmet medical need in a vulnerable population, but it offers a model - whenever applicable - to be adopted to accelerate new drugs availability to the pediatric population while fully complying with regulatory requirements and minimize the number of patients in clinical trials. Disclosures Spina: Menarini Ricerche SpA: Consultancy. Off Label Use: Febuxostat in pediatric Tumor lysis syndrome. Mazzei:Menarini Ricerche SpA: Employment. Baldini:Menarini Ricerche SpA: Employment. Pedretti:Menarini Ricerche SpA: Employment. Mezzalana:Menarini Ricerche SpA: Employment. Matera:Menarini Ricerche SpA: Employment. Manunta:Menarini Ricerche SpA: Employment. Calamai:Menarini Ricerche SpA: Employment. Scordari:Menarini Ricerche SpA: Employment. Scartoni:Menarini Ricerche SpA: Employment. Capriati:Menarini Ricerche SpA: Employment. Simonelli:Menarini Ricerche SpA: Employment.

Description: Background Immunotherapy using patient-derived CAR T cells has achieved complete remission and durable response in highly refractory populations. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. Allogeneic Cytokine Induced Killer (CIK) cells, a T-cell population characterized by the enrichment of CD3+CD56+ cells, have demonstrated a high profile of safety in acute lymphoblastic leukemia (ALL) patients (Introna M et al. Biol Blood Marrow Transplant. 2017). CIK cells could be easily engineered by the non-viral Sleeping Beauty (SB) transposon for the clinical application (Magnani CF et al, Hum Gene Ther. 2018, Biondi A et al. J Autoimmun. 2017). Methods CIK cells were generated from 50 ml of donor-derived peripheral blood (PB) by electroporation with the GMP-grade CD19.CAR/pTMNDU3 and pCMV-SB11 plasmids according to the method enclosed in the filed patent EP20140192371. After lymphodepletion with Fludarabine (30 mg/m2/day) x 4 days and Cyclophosphamide (300 mg/m2/day) x 2 days, CARCIK-CD19 were infused in pediatric and adult B-cell ALL (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT). The clinical trial follows a four-dose escalation scheme (1x106, 3x106, 7.5x106 and 15x106 transduced CAR+ T cells/kg) using the novel Bayesian Optimal Interval Design (BOIN). During the cell manufacturing period, bridging anti leukemic therapy from patient registration to the beginning of the lymphodepletion, was allowed. The primary endpoint was to define the Maximum Tolerated Dose (MTD) and a safety assessment. Key secondary endpoints included the assessment of complete hematologic response (CR), defined as 〈 5% bone marrow (BM) blasts, circulating blasts 〈 1%, no clinical evidence of extramedullary disease, as well as the characterization of CARCIK-CD19 persistence in PB and BM (NCT03389035). Results We manufactured eighteen batches by seeding a median of 126.8x106 allogeneicPBMCs. At the end of expansion, the mean harvesting was 6.46x109 nucleated cells (range 1.39 - 16.00x109). Manufactured cells were mostly CD3+ lymphocytes (mean 98.90% ±SE 0.30%). Of these, 43.57%±3.73% were CAR+, 47.07%±2.74% were CD56+, 80.44%±2.53% were CD8+. The quality requirements for batch release were met in 17 productions. As of the data cut-off date (July 19, 2019), 4 pediatric and 7 adult patients were infused with a single dose of CARCIK-CD19 (n=2 HLA identical sibling, n=4 MUD, n=5 haploidentical donor). The leukemic burden in the BM post lymphodepletion/pre-infusion ranged from 0% to 96%. CARCIK-CD19 were characterized by a high profile of safety in all treated patients. Toxicities reported were a grade I cytokine release syndrome and an infusion-related DMSO-associated seizure, with absence of dose-limiting toxicities, neurotoxicity and graft-versus-host disease (GvHD) in any of the treated patients. Four out of 5 patients, receiving the highest doses, achieved CR and CRi at day 28. The 3 patients in CR were also MRD- (by flow cytometry and RT-PCR) while the CRi was MRD+ and relapsed at day+49. Robust expansion was achieved in the majority of the patients as defined by detectable CAR T-cell detection (vector copy number VCN, range 4645-977992 transgene copies/ug) and flow (range 0.5-30%) in PB. The median time to peak engraftment was 14 days. The magnitude of expansion was correlated with the CD19+ burden in the BM at the time of the infusion (P value = 0.0006, R square 0.7469). CD8+ T cells represented the predominant CARCIK-CD19 T-cell subset (78.88%±5.33% d14 n=6) along with CD3+CD56+ CIK cells and CD4+ T cells to a lesser extent. The majority of CAR T cells had a central and effector memory phenotype. CAR T cells were measurable by VCN up to 6 months with a mean persistence of 70.5 ± 14.85 days (follow up ranging from 28 days to 1 year). No major difference was observed by integration analyses of the patients' PB and the cell products. The vector integration sites reflected the classical random distribution of SB without any tendency for gene dense regions. Conclusions Our ongoing phase I/II trial demonstrates that SB-engineered CARCIK-CD19 cells are able to expand and persist in pediatric and adult B-ALL patients relapsed after HSCT, with important implications for a non-viral technology. These encouraging results prompted us to expand our study. Disclosures Gritti: Autolus Ltd: Honoraria; Roche: Other: Not stated; Abbvie: Other: Not stated; Becton Dickinson: Other: Not stated. Rambaldi:Celgene: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, travel support; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.