ALBERT

Description: Abstract 317 Introduction: The novel agents bortezomib, thalidomide and lenalidomide have been successfully incorporated into autologous stem-cell transplantation (ASCT) as up-front therapy for newly diagnosed MM. However, several reports have raised concerns about the impact of novel agent-based induction regimens on PBSC collection. Furthermore, the ability to successfully collect PBSCs following initial therapy with two of these newer drugs needs to be confirmed in large phase III clinical trials. Methods: The GIMEMA Italian Myeloma Network designed a phase III study to compare VTD with thalidomide-dexamethasone (TD) as induction therapy prior to double ASCT. Primary study endpoint was the rate of complete or near complete response to each of these two induction regimens, while their toxicity profile – including the impact on PBSC mobilization and collection - was a secondary study endpoint. To address this latter issue, we performed a post-hoc analysis to compare the effect of the triplet VTD induction regimen versus the doublet TD combination on CD34+ cell collection. After three 21-day cycles of VTD or TD induction therapy, patients received intermediate dose cyclophosphamide (CTX 4 g/m2) followed by G-CSF (10 mcg/Kg/die) to mobilize and collect PBSCs. The target threshold to safely perform double ASCT was 4 × 106 CD34+ cells/Kg. Results: Patients evaluable for PBSC collection were 435 out of the 474 who received induction therapy. Of these, 223 were initially randomized to VTD and 212 to TD induction therapy. The median number of collected CD34+ cells was 9.7 × 106/Kg in the VTD arm and 10.7 × 106/Kg in the TD arm (p= n.s.). The planned yield of 4 × 106 CD34+ cells/Kg was achieved with a single harvest in more than 90% of patients in both treatment groups (96% in VTD and 92% in TD, p= n.s.). A yield of CD34+ cells 〉10 × 106 /Kg was reported in 51% and 56% of patients treated with VTD and TD, respectively (p= n.s.). Only 5 patients (2%) in VTD group and 2 patients (1%) in the TD arm failed to collect more than 2 × 106 CD34+ cells/Kg (p= n.s.). The majority of patients (86% in VTD and 82% in TD, p=n.s.) received CTX as an in-patient procedure, the median time of hospitalization being 4 days. Less than 5% of patients developed grade 3–4 infectious complications (2% in the VTD group vs 3% in TD, p=n.s.) which required hospitalization in only 2 patients. Following ASCT, no significant difference was observed between the two treatment arms in terms of hematologic recovery and non hematological toxicity. Kaplan-Meier curves of TTP and PFS were almost superimposable for patients with a CD34+ yield 〉10 × 106/Kg and in the range between 4 and 10 × 106/Kg (group 1). These curves were very similar also for patients who collected between 2 and 4 × 106/Kg CD34+ cells or 10 × 106/Kg CD34+ cells was in the 90% range, a value significantly better than what was seen in the remaining subgroups. In a multivariate Cox regression analysis, yields of CD34+ cells 〉10 × 106/Kg and in the range of 4 to 10 × 106/Kg were independently associated with prolonged PFS (p=0.001 and =0.027, respectively), while CD34+ cells 〉10 × 106/Kg predicted for extended OS (p=0.002). Absence of t(4;14) and/or del(17q), and ISS stage 1 or 2 were additional favorable prognostic factors for both PFS and OS, while randomization to VTD independently predicted for longer PFS. Conclusions: Results of the present analysis showed that both TD and VTD shared the advantage of no adverse impact on PBSC collection and the engraftment potential of collected PBSCs. The target for CD34+ cell collection (〉4 × 106/Kg) was achieved with a single harvest in more than 90% of patients in both treated groups and a collection failure was reported in 1% to 2% of patients. These favorable results are due to early PBSC collection, which was performed after 3 cycles of TD and VTD, and use of CTX plus G-CSF which allows better stem cell collection and less likelihood of a collection failure. Of particular note, both VTD and TD were associated with a 50% to 59% probability to collect 〉10 × 106 CD34+ cells/Kg, a variable independently associated with extended PFS and OS. Disclosures: Off Label Use: bortezomib and thalidomide used as induction therapy for newly diagnosed multiple myeloma patients. Baccarani:Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Cavo:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Genzyme: Honoraria.

Description: A new hyper-branched water-soluble polymer was synthesized by reacting β-cyclodextrin with pyromellitic dianhydride beyond the critical conditions that allow the phenomenon of gelation to occur. The molar ratio between the monomers is a crucial parameter that rules the gelation process. Nevertheless, the concentration of monomers in the solvent phase plays a key role as well. Hyper-branched β-cyclodextrin-based polymers were obtained performing the syntheses with excess of solvent and cross-linking agent, and the conditions for critical dilution were determined experimentally. A hyper-branched polymer with very high water solubility was obtained and fully characterized both as for its chemical structure and for its capability to encapsulate substances. Fluorescein was used as probe molecule to test the complexation properties of the new material. Beilstein J. Org. Chem. 2014, 10, 2586–2593. doi:10.3762/bjoc.10.271

Description: Ibuprofen sodium salt (IP) was encapsulated in cyclodextrin nanosponges (CDNS) obtained by cross-linking of β-cyclodextrin with ethylenediaminetetraacetic acid dianhydride (EDTAn) in two different preparations: CDNSEDTA 1:4 and 1:8, where the 1: n notation indicates the CD to EDTAn molar ratio. The entrapment of IP was achieved by swelling the two polymers with a 0.27 M solution of IP in D 2 O, leading to colourless, homogeneous hydrogels loaded with IP. The molecular environment and the transport properties of IP in the hydrogels were studied by high resolution magic angle spinning (HRMAS) NMR spectroscopy. The mean square displacement (MSD) of IP in the gels was obtained by a pulsed field gradient spin echo (PGSE) NMR pulse sequence at different observation times t d . The MSD is proportional to the observation time elevated to a scaling factor α. The α values define the normal Gaussian random motion (α = 1), or the anomalous diffusion (α 〈 1, subdiffusion, α 〉 1 superdiffusion). The experimental data here reported point out that IP undergoes subdiffusive regime in CDNSEDTA 1:4, while a slightly superdiffusive behaviour is observed in CDNSEDTA 1:8. The transition between the two dynamic regimes is triggered by the polymer structure. CDNSEDTA 1:4 is characterized by a nanoporous structure able to induce confinement effects on IP, thus causing subdiffusive random motion. CDNSEDTA 1:8 is characterized not only by nanopores, but also by dangling EDTA groups ending with ionized COO − groups. The negative potential provided by such groups to the polymer backbone is responsible for the acceleration effects on the IP anion thus leading to the superdiffusive behaviour observed. These results point out that HRMAS NMR spectroscopy is a powerful direct method for the assessment of the transport properties of a drug encapsulated in polymeric scaffolds. The diffusion properties of IP in CDNS can be modulated by suitable polymer synthesis; this finding opens the possibility to design suitable systems for drug delivery with predictable and desired drug release properties. Beilstein J. Org. Chem. 2014, 10, 2715–2723. doi:10.3762/bjoc.10.286

Description: A new hyper-branched water-soluble polymer was synthesized by reacting β-cyclodextrin with pyromellitic dianhydride beyond the critical conditions that allow the phenomenon of gelation to occur. The molar ratio between the monomers is a crucial parameter that rules the gelation process. Nevertheless, the concentration of monomers in the solvent phase plays a key role as well. Hyper-branched β-cyclodextrin-based polymers were obtained performing the syntheses with excess of solvent and cross-linking agent, and the conditions for critical dilution were determined experimentally. A hyper-branched polymer with very high water solubility was obtained and fully characterized both as for its chemical structure and for its capability to encapsulate substances. Fluorescein was used as probe molecule to test the complexation properties of the new material.

Description: Ibuprofen sodium salt (IP) was encapsulated in cyclodextrin nanosponges (CDNS) obtained by cross-linking of β-cyclodextrin with ethylenediaminetetraacetic acid dianhydride (EDTAn) in two different preparations: CDNSEDTA 1:4 and 1:8, where the 1:n notation indicates the CD to EDTAn molar ratio. The entrapment of IP was achieved by swelling the two polymers with a 0.27 M solution of IP in D2O, leading to colourless, homogeneous hydrogels loaded with IP. The molecular environment and the transport properties of IP in the hydrogels were studied by high resolution magic angle spinning (HRMAS) NMR spectroscopy. The mean square displacement (MSD) of IP in the gels was obtained by a pulsed field gradient spin echo (PGSE) NMR pulse sequence at different observation times t d. The MSD is proportional to the observation time elevated to a scaling factor α. The α values define the normal Gaussian random motion (α = 1), or the anomalous diffusion (α 〈 1, subdiffusion, α 〉 1 superdiffusion). The experimental data here reported point out that IP undergoes subdiffusive regime in CDNSEDTA 1:4, while a slightly superdiffusive behaviour is observed in CDNSEDTA 1:8. The transition between the two dynamic regimes is triggered by the polymer structure. CDNSEDTA 1:4 is characterized by a nanoporous structure able to induce confinement effects on IP, thus causing subdiffusive random motion. CDNSEDTA 1:8 is characterized not only by nanopores, but also by dangling EDTA groups ending with ionized COO− groups. The negative potential provided by such groups to the polymer backbone is responsible for the acceleration effects on the IP anion thus leading to the superdiffusive behaviour observed. These results point out that HRMAS NMR spectroscopy is a powerful direct method for the assessment of the transport properties of a drug encapsulated in polymeric scaffolds. The diffusion properties of IP in CDNS can be modulated by suitable polymer synthesis; this finding opens the possibility to design suitable systems for drug delivery with predictable and desired drug release properties.

Description: Enantiodifferentiating geometrical photoisomerizations of (Z)-cyclooctene and (Z,Z)-1,3-cyclooctadiene were performed by using the pyromellitate-linked cyclodextrin network polymer, termed “cyclodextrin nanosponge (CDNS)”, as a supramolecular sensitizing host. The photochirogenic behavior of the nanosponges incorporating β- or γ-cyclodextrin was significantly different from that reported for the conventional sensitizer-appended monomeric cyclodextrins, affording chiral (E)-cyclooctene and (E,Z)-cyclooctadiene in enantiomeric excesses critically dependent on the solution pH and solvent composition employed, revealing the active roles of chiral void spaces of CDNS in the photochirogenic reaction.

Description: Abstract 4289 The aim of this study was to generate cytotoxic T-lymphocytes (CTL) clones directed against AML cells and to identify new immunogenic antigens with the following properties: i) to be overexpressed by leukemic cells and not by normal tissues; ii) to be shared among different AML subtypes; iii) to play a role in leukemic growth/survival; iv) to be expressed by leukemic stem cells. To this end, we loaded normal dendritic cells (DC) from a healthy donor with apoptotic bodies from primary AML cells and used loaded DC to stimulate autologous lymphocytes (i.e. lymphocytes from the donor). Donor was selected to be partially matched with the leukemic patient for MHC-class I, in that he shared two MHC-class I alleles at the HLA-supertype level (HLA-B7 and HLA-B44). With this strategy, a CD8+ T-cell line was generated that recognized both loaded DC and AML cells used for loading, but not DC loaded with normal cells derived from the patient (PHA-blasts). This CTL line was cloned by limiting dilutions and 180 clones were screened by IFN-g elispot against 2 different AML samples that were expressing both HLA-B7 and HLA-B44; one additional mismatched AML sample was used as negative control. Two clones were selected that recognized HLA-B7+/HLA-B44+ AML cells but not the negative control (namely, clone 31D3 and 8E12). To determine the HLA-restriction element and to verify shared antigen expression among AML subtypes, we tested both clones against a panel of 18 HLA-typed primary AML samples. We found that clone 31D3 was restricted by HLA-B7 and clone 8E12 by HLA-B44. Each clone recognized 5/5 HLA-matched AML samples of different subtype. In addition, the clones did not recognize both resting and activated normal myeloid, nor lymphoid and CD34+ cells expressing the proper HLA-restriction allele. In addition to elispot activity, both clones showed killing of AML cells in a CFSE-based cytotoxic assay. To confirm the lack of reactivity against normal tissues, we analyzed the activity of both clones against HLA matched or mismatched fibroblasts and we found that clone 8E12 displayed a low reactivity activity against normal matched fibroblasts, while clone 31D3 was not reactive. We then focused the analysis on clone 31D3 and tested whether its activity was restricted to leukemia or also directed against a panel of HLA-B7 positive (N=3) or negative (N=5) solid tumors. Interestingly, we found that the clone 31D3 could recognize one colon carcinoma and one melanoma cell line expressing the HLA-B7 supertype. In particular, the melanoma cell line (G4-mel) was HLA-B35+ (HLA-B35 belongs to the HLA-B7 supertype) and was recognized at very high levels. The availability of a tumor cell line expressing adequate amounts of antigen will make the generation of a cDNA library for antigen identification more feasible than with primary leukemic cells. Finally, to further confirm that HLA-B35 is the proper MHC restriction element and to determine to what extent the antigen is shared among different tumors, we transduced 8 tumor cell lines, 3 normal B-lymphoblastoid and 1 fibroblast cell line with the HLA-B35 allele. Clone 31D3 efficiently recognized 6/8 HLA-B35 transduced tumor cell lines (1 AML, 2 melanomas, 2 colon and 1 breast carcinoma) but none of the control cell lines. In conclusion, clone 31D3 is restricted by the HLA-B7 supertype, it recognizes an antigen that is expressed by leukemic cells and not by normal tissues and, most importantly, is shared among tumors of different histology. Since the vast majority of malignant cells tested (both primary cells and in vitro established cell lines) were targeted by this clone, the putative antigen might be a protein playing an important role in tumor growth or survival. We are now testing the activity of the clone against purified leukemic stem cells and attempting to identify this shared tumor antigen. Disclosures: No relevant conflicts of interest to declare.

Description: Over the past 2 decades considerable evidence has accumulated on the association between hepatitis C virus (HCV) and hepatitis B virus (HBV) and several hematologic malignancies, most notably B-cell non-Hodgkin lymphoma (NHL). In this review we summarize this evidence, address possible mechanisms whereby hepatitis viruses may contribute to lymphomagenesis, and discuss the therapeutic fallouts from this knowledge. Most of this evidence is on HCV, and this is the main focus of the review. Moreover, we mainly address the association with NHL, the most prevalent hematologic malignancy, and the most extensively investigated with regard to an association with hepatitis viruses. Available evidence on the association with other hematologic malignancies is also addressed briefly.

Description: Background/Aims Transient monoclonal gammopathy is a common alteration of laboratory test after allogeneic stem cells transplantation (alloBMT). However, until now, only scattered works have been published about it. The main paper reported on PubMed, regarding transient monoclonal gammopathy, was presented by the Dana Farber's group at the end of the eighties. The author of that paper showed an apparently strong correlation between development of graft versus host disease (GvHD) and appearance of a monoclonal gammopathy. Starting from that observation, we decided to evaluate among our allogeneic transplanted patients the incidence of M-component and its possible relationship with GvHD. Patient and Method 67 patients undergoing alloBMT at the Haematology Unit of Alessandria (Italy) between 2006 and 2010 were evaluated: 52% of patients were male and 48% were females. Pre-transplantation diagnosis included: 34 acute myeloid leukaemia (50.7%), 8 acute lymphoblastic leukaemia (11.9%), 7 non-Hodgkin Lymphoma (10.4%), 6 chronic leukaemia (9%), 4 myelodysplastic syndrome (6%), 2 Hodgkin lymphoma (3%) and 6 other less common malignancies (9%). All patients had, at least, two pre-transplantation serum electrophoresis with no evidence of pre-existing monoclonal component; for the analysis, we haven’t considered patients submitted to alloBMT for myeloma. Controls of serum electrophoresis were performed at 90, 180 and 360 days after transplantation. In our survey, 17 patients relapsed after alloBMT, 27 patients developed GvHD and 26 patients died. Post-transplantation follow up ranged from 81 to 2514 days with a median of 496 days. Results As a whole, 35/67 (52%) of the patients developed monoclonal gammopathy after transplantation. Comparing patients with or not monoclonal gammopathy after alloBMT, an increased GvHD development (54% vs 34%) and a decreased relapse incidence (19% vs 32%) was observed. Otherwise, analysing the appearance of monoclonal gammopathy at defined time-points, we have not detected any difference in overall survival, GvHD development, relapse incidence and post-transplantation mortality at +90 and +180 days post transplant. Vice versa, an increased GvHD development (50% vs 21% at median +378 days) was observed in patients with an appearance of monoclonal gammopathy at +360 days; so it seems that the presence of a M-component is associated only lately after 360 days to the possibility of GvHD development. Conclusion Evidence for monoclonal B-cell proliferation is common within the first year after alloBMT. The few papers published in the past found this proliferation more frequently associated with GVHD but without any long term adverse effect. Our data would seem to confirm a correlation between appearance of monoclonal gammopathy post-transplantation and GvHD development. In the past, the explanation for the evidence of a monoclonal gammopathy was associated to an aberrant immune reconstitution after alloBMT. Nevertheless in the last year it has been shown that B cells are involved in the pathogenesis of chronic GVHD (cGVHD) and anti-B-cell therapy can be used for the treatment of cGVHD. A prospective study with a larger population should be considered, in order to confirm our results and assay post-transplantation monoclonal gammopathy as an early marker for GvHD development. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3020 Background. In patients with newly diagnosed MM, three/four-drug combinations seem to be more effective compared with two-drug associations in terms of both rate and duration of remission. Moreover, there is an emergent body of evidences that consolidation/maintenance therapy improves quality of response and remission duration. However, the impact of these strategies in relapsed/refractory MM (r-rMM) are still unknown. Methods. This is a multicenter, phase II study including patients with r-rMM having measurable disease, no more than 4 prior lines of therapy, adequate performance status, cardiac and liver function. As induction therapy patients received 6 28-day cycles of oral thalidomide 100 mg/day continuously at bedtime, oral dexamethasone 20 mg on day 1–2, 4–5, 8–9, 11–12, pegylated Liposomal Doxorubicin (pLD) 30 mg/m2 iv on day 4 and bortezomib 1.3 mg/m2 iv on day 1, 4, 8, 11 (ThaDD-V). As consolidation patients underwent 6 28-day cycles of rotating bortezomib 1.3 mg/m2 iv on day 1, 4, 8, 11 plus oral dexamethasone 20 mg on day 1–2, 4–5, 8–9 (3 courses) or thalidomide 100 mg/day continuously at bedtime plus oral dexamethasone 20 mg on day 1–4 (3 courses). Patients eligible and having suitable stem cell storage underwent ASCT instead of standard consolidation at the discretion of attending physician. Maintenance therapy included thalidomide 100 mg/day until relapse or intolerable toxicity. Since in the first 20 patients we recognized an excess of peripheral neuropathy, protocol was amended as follow: bortezomib 1.3 mg/m2 on day 1, 4, 11 and thalidomide 50 mg/day in all therapeutic phases. The primary end-points of this study were best response and toxicity of the planned therapy. Results. Forty-six patients were enrolled. Median age was 63.5 years (range 31–80 years) and the median number of prior regimens was 1 (range 1–4). Twenty-four patients (52%) had undergone autologous stem cell transplantation, 30 (65%) had received anthracyclines, 27 (59%) thalidomide, 8 (17.5%) bortezomib and 16 (35%) were refractory to the last regimen. After induction 16 patients (34.5%) achieved CR (6=13% sCR), 15 (32.5%) VGPR and 4 (8.5%) PR with a ORR of 76.5%. Seven patients (15%) progressed. Out of 46 patients undergone induction, 26 (20 standard, 6 ASCT) received consolidation therapy since 1 patients died during induction, 8 had progressive disease, 1 had second neoplasm, 7 had severe toxicities and 3 undergone allogeneic stem cell transplantation. Excluding 6 patients who have obtained sCR before, 5 (25%) out of 20 patients had further improvement in response. Therefore, best response after induction and consolidation were: 25 CR (54%; 8 sCR=17.5%), 16 VGPR (34.5%) and 2 PR (4.5%). Maintenance therapy did not further improve response. Patients receiving ≤ 2 prior regimens had a CR rate significantly higher than those heavily treated (41% vs 0%; p= 0.010) whereas prior ASCT, thalidomide or bortezomib, refractory disease and bortezomib dose-intensity did not affect quality of response. After a median follow-up of 31 months (range 12–53), 28 patients relapsed and 20 died. Median TTP was 18.5 months, median PFS was 17.5 months and median survival was 40 months. Median TTP of patients achieving PR-VGPR was 16 months (3 years= 10%) whereas in those obtaining CR it was 32.5 months (3 years= 45%; p=0.032) vs not reached (3 years= 85%) in patients achieving sCR (p=0.003). Main toxicity was peripheral neuropathy (PN). Indeed, in the first 20 patients we observed 6 (30%) grade 2 and 3 (15%) grade 3 PN. After amendment, grade 2 and 3 PN occurred in 3 (11.5%) and 2 patients (7.5%), respectively. DVT occurred in 2 patients (4.5%) and severe infection in 7 (15%). Grade 3–4 neutropenia, anemia and thrombocytopenia occurred in 4 (8.5%), 2 (4%) and 7 patients (15%), respectively. Finally, only one patients died of myocardial infarction during induction. During consolidation therapy other 2 patients developed grade 3 peripheral neuropathy. During maintenance with thalidomide no patients developed severe neuropathy requiring discontinuation. Conclusions. Multi-drug combination namely ThaDD-V as induction followed by consolidation-maintenance therapy seems to be very effective in patients with r-rMM provided that this procedure is used early on relapse when very deep responses are still possible. A reduced dose-intensity of bortezomib significantly decreases PN without jeopardizing outcome. Disclosures: Offidani: Celcene, Janssen Cilag: Honoraria. Polloni:Celgene: Honoraria. Corvatta:Celgene: Honoraria. Gentili:Celgene: Honoraria. Brunori:Celgene, Janssen-Cilag: Honoraria. Catarini:Cerlgene, Janssen-Cilag: Honoraria. Malerba:celgene, Janssen-Cilag: Honoraria. Leoni:Celgene, Janssen-Cilag: Honoraria.