ALBERT

Description: Abstract 2926 Introduction Angiogenesis plays a central role in the progression of both solid and hematological tumors. In particular, in multiple myeloma (MM) the critical role of bone marrow (BM) microenvironment and angiogenesis has been well documented. The past decade has witnessed a dramatic improvement in the therapeutic options in MM. However, the disease remains incurable, underscoring the need for continued efforts towards understanding MM biology and exploitation of novel therapeutic approaches. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cells. The Eph receptors, a large family of receptor tyrosine kinases (RTKs) activated by ephrins binding, have been implicated in many processes involved in malignancy, including alteration of the tumor microenvironment and in angiogenesis, in both of which EpHA3 likely plays an active role. Aberrant expression of EpHA3 is seen in many types of hematolologic malignancies (some leukemic cell lines, T-cell lymphoma, acute lymphoblastic leukemia, myeloproliferative neoplasms) although it is not expressed ubiquitously. Finally, the over-expression of Eph is believed to be sufficient to confer tumorigenic potential although probably further mechanisms can occur to abnormally activate the receptor. Basing on the role of EpHA3 in haematological malignancies, a first-in-class engineered IgG1 antibody targeting the EpHA (KB004) was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EpHA3 overexpressing hematological myeloid malignancies refractory to conventional treatment. We investigated the EpHA3 role and its preferential membrane–bound by GPI linker ligand EFNA5, in MM patients in order to define EpHA3 as new molecular target for a novel therapeutic approach with a specific anti EpHA3 monoclonal antibody. The EpHA3 expression has been studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with monoclonal gammopathy of undetermined significance (MGECs), of control subjects (normal ECs) and in MM cell lines. Methods After written informed consent, BM aspirates have been collected from 20 MM and 4 MGUS patients. Normal ECs were derived from 3 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed the expression levels of EpHA3 in normal ECs, MGECs and MMECs and MM cell lines evaluating the mRNA and protein levels by RT-qPCR and by WB coupled to ImmunoFluorescence analysis. The biological effects of EpHA3 targeting in MMECs have been studied silencing the EpHA3 mRNA in MMECs and testing them at 72h after silencing in series of functinal assays including viability assay by trypan blue exclusion staining and by in vitro angiogenesis assay followed by measurement of mesh areas and vessel length. Moreover, we studied EFNA5 mRNA expression levels in Normal ECs, MGECs and MMECs and in MM cell lines by PCR. Results Our data showed that EpHA3 mRNA levels are progressively increased from ECs to MGECs reaching the highest values in MMECs. Subsequent analysis by WB and immunofluorescence confirmed EpHA3 protein upregulation among the different EC types. The MMECs in which EpHA3 has been silenced revealed a protein level reduction of approximately 60% when compared to the control. We could not detect major viability defects. Furthermore, in vitro angiogenesis inhibition was marginal when compared to the not silenced counterpart. To know whether EpHA3 may impact not only MM angiogenesis but also plasma cells, three MM cell lines were studied for the EpHA3 expression. We found the plasma cell lines gave constant over expression of EpHA3. Finally, the preliminary data regarding EFNA5 mRNA expression level showed it is expressed in either MMECs and MM plasma cell lines. The evaluation of KB004 effect on MMECs in term of apoptosis induction and in vitro tube formation inhibition, as well as the analysis of EpHA3 levels in primary MM plasma cells are in progress. Conclusions From this study we expect to characterize the role of the EpHA3in MM patients and to provide experimental evidences supporting the possibility of using EpHA3 as a new molecular target for MM by proving the in vitro efficacy of a monoclonal antibody to target the angiogenesis of MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 729 Introduction: The prognosis of primary plasma cell leukemia (PPCL) remains poor. The “novel agents” have recently shown promising results in PPCL patients in case reports or small retrospective series. Here we describe the first prospective, multicenter, phase II clinical trial of PPCL, where lenalidomide in combination with low dose dexamethasone (Ld) was tested as initial therapy in newly diagnosed patients fulfilling the IMWG diagnostic criteria of PPCL. Patients and methods: Ld regimen consisted of lenalidomide 25 mg/d for 21 days and oral dexamethasone 40 mg on days 1, 8, 15, and 22 for each 28-day cycle. After 4 cycles, responding patients not eligible for stem cell transplantation (SCT) continued until 8 cycles of full-dose Ld, followed by a maintenance dose of single agent lenalidomide equal to 10 mg/d on days 1–21 of each 28-day cycle. Patients responding after 4 cycles and eligible for SCT proceeded according to the treatment centre's transplant policy. Patients not responding after 4 cycles or progressing during this treatment were considered off-study. Appropriate dose reductions (in particular for patients with reduced renal function at baseline), double contraception methods and anti-thrombotic/anti-infective prophylaxis were recommended. The primary end-point was early response rate according to IMWG uniform response criteria. Secondary end-points were PFS, OS, feasibility and efficacy of SCT, safety. Results: According to the Simon optimal two-stage adaptive design, twenty-three patients were enrolled in between March 2009 and May 2011. The male/female ratio was 1.1, and median age was 60 years (range 44–80). The median absolute number and percentage of circulating plasma cells were 4.280/μl (range 1.500–114.660) and 34% (range 21–90) respectively. Fifteen patients (65.2%) had abnormal renal function at presentation. Twenty-one patients (91.3%) were tested by FISH analysis and cytogenetic abnormalities were detected in all of them, del13q being the most frequently found (16 patients). Seventeen patients showed multiple chromosomal lesions. Involvement of chromosome 14 was observed in 18 patients, three of whom showed t(4;14). Chromosome 1q gain and del17p were detected in 10 and 7 patients, respectively. In the intention-to-treat (ITT) population, overall response rate (ORR) after at least one Ld cycle was 73.9% (17/23), with 8 patients (34.7%) achieving partial remission (PR), 5 (21.7%) very good PR (VGPR), 3 (13%) complete response (CR), and 1(4.3%) near CR (nCR) (VGPR or better: 39%). In the efficacy-evaluable (EE) population, 14 out of 15 patients who received the initially planned 4 Ld cycles (65.2% of the ITT population) responded (ORR 93.3%), achieving 5 PR (33.3%), 5 VGPR (33.3%), 1 near-CR (6.6%) and 3 CR (20%) (VGPR or better: 59.9%). The maintenance phase was reached and safely performed in 4 responding patients not eligible for SCT, 3 of whom relapsed after 2, 8 and 22 months, respectively. After Ld induction therapy, 8 patients received single (n. 4) or double (n. 4) autologous SCT (ASCT); another patient underwent a sequence of ASCT followed by non-myeloablative allogeneic SCT (AlloSCT). Six eligible patients did not receive ASCT frontline, due to initial Ld failure or adverse events; three of them underwent single ASCT (n. 2) or tandem ASCT/non-myeloablative AlloSCT (n. 1) after a bortezomib-based salvage therapy, achieving 2 CR and 1 PR. After a median follow-up of 23 months, median OS and PFS in ITT population were not reached and 22 months, respectively. All transplanted patients remained alive, although three of them relapsed and started salvage treatments; OS was 12 months in the 11 patients who did not receive ASCT (p 〈 0.001). The correspondent figures for PFS were 29 and 16 months, respectively (p 〈 0.01). Considering ITT population, multivariate analyses showed that SCT was positively correlated to both OS and PFS. There were 17 episodes of grade 3/4 non hematological toxicity, which occurred in 13 patients (5 infections, 3 renal, 3 metabolic, 2 gastro-intestinal, 2 skin, 1 fatigue, 1 thromboembolic), causing early interruption of Ld treatment in 4 patients. Grade 3/4 hematological toxicity (mainly neutropenia) occurred in 11 patients (47.8%). Conclusions: Ld may be a feasible and effective initial therapeutic option for PPCL, particularly in patients who receive ASCT after a short course of induction treatment. Disclosures: Musto: Celgene: Honoraria, Research Funding. Off Label Use: Lenalidomide as first line therapy of plasma cell leukemia. Petrucci:Celgene: Honoraria. Cascavilla:Celgene: Honoraria. Di Raimondo:Celgene: Honoraria. Caravita:Celgene: Honoraria. Morabito:Celgene: Honoraria. Offidani:Celgene: Honoraria. Bringhen:Celgene: Honoraria. Boccadoro:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Palumbo:Celgene: Consultancy, Honoraria.