ALBERT

Description: Abstract 4438 BACKGROUND: Graft Failure (GF) occurs in 5–27% of patients (pts) after allogeneic hamatopoietic stem cell transplant (HSCT) and is associated with high morbidity and mortality related to infections and hemorrhagic events. Graft function may be poor as result of graft rejection, primary disease relapse or Poor Graft Function (PGF). The incomplete recovery of blood counts is defined primary PGF and the decreasing blood counts after successful engraftment secondary PGF. Several factors may determine GF: disease risk and status, conditioning regimen, HSC source, HLA compatibility, T cell content, immunosuppression, GvHD, viral infections, drugs. GCSF and Rhu-EPO are readily available and effective in PGF but with no effects on platelets. Second transplantation from the same donor, with or without conditioning therapy, can boost the haematopoietic recovery in pts with GF. Unfortunately, both a second peripheral CD34+ mononuclear cells (MNC) mobilization and a marrow harvest in the operating room may be contraindicated early after the first donation as not safe for donors. Intrabone SCT can overcome the risk of graft failure even with a low number of CD34+ MNC, as it has been demonstrated in cord blood transplant. Here we investigate in three adult pts with GF a bone-to-bone boost (BBB) with a small marrow harvest from respective donors, unfit for a second conventional donation. AIM: to evaluate the feasibility of the BBB technique in 3 pts with graft failure. METHODS: pts were 2 males (57, 53 y) with PGF with a diagnosis of AML and CMML, respectively, and a female (44 y) with graft rejection and AML relapse. In the first two patients prolonged pancytopenia and hypoplastic marrow were documented, with diagnosis of primary PGF and secondary PGF, respectively, donor chimerism ranging from 80–100% (STR and HLA), without evidence of leukemia. In the third patient, after prolonged pancytopenia an AML relapse was documented with 89% blasts on bone marrow aspirate. In PGF patients no conditioning regimen was administered before the boost at day 30 and 72 after SCT, respectively. In the patient with AML relapse Melphalan 200 mg/mq was given 48 h before the infusion, at day 35 after SCT. The 3 donors were related, haploidentical. For the BBB procedure small quantities of bone marrow (〈 200ml) were collected from the posterior iliac crest bilaterally of the donors, at the bedside, during deep sedation and analgesya. Shortly after the unmanipulated marrow harvested was infused in superior-posterior iliac crest mono- or bilaterally, depending on the volume, during deep sedation and analgesya. In pt 1 Mononucleated cell (MNC) dose was 0.9 × 10^8/Kg for a volume of 166 ml. In pt 2 MNC dose was 0.4 × 10^8/Kg for a volume of 88 ml. In pt 3 MNC dose was 0.3 × 10^8/Kg for a volume of 140 ml. RESULTS AND CONCLUSION: In this cases the BBB technique proved feasible and safe for both the donor and the patient. Patient 1 received a second PBSC boost, without conditioning, 3 months after the BBB, and he's now alive, in CR, 13 months after the first transplant. Patient 2 died 3 months after the first transplant for pneumonia and sepsis. Patient 3 is alive, in CR, 4 months after the first HSCT. This practice can give the chance of HSC boost to patients with GF without the need of a GCSF mobilization for donors, with a minimal invasive operation. This can give the option to overcome and resolve infectious and hemorrhagic complications, bridging patients to further therapies and procedures. The intra-bone SCT may be a facilitating tool for microenvironment reconstitution, seeding and subsequent differentiation and may as well have a tolerogenic effect, through the mesenchymal stromal cells infused with the harvest. Further studies are necessary to assess the efficacy of this procedure. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2861 Introduction: The AKT inhibitor Perifosine (Æterna Zentaris GmBH, Germany) has been shown in phase II studies to induce partial responses in a variety of solid tumors. Sorafenib (Nexavar, Bayer) is an oral multikinase inhibitor exerting in vitro and in vivo antiproliferative, antiangiogenic, and proapoptotic effects in a variety of hematological and nonhematological tumors. Our preclinical data demonstrating that the combination of Perifosine and Sorafenib induces gene expression profiling and signaling changes associated with a synergistic cytotoxic activity against lymphoma cell lines in vitro and in vivo, established the rationale for this ongoing phase II study aimed to determine safety and activity of Perifosine/Sorafenib combination therapy in relapsed/refractory non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). Methods: Between July 2008 and July 2010, 26 out of 36 planned patients (18 males and 8 females; median age, 42 years; range, 19–73 years) with relapsed/refractory diffuse large B cell lymphoma (DLBCL, n = 3), follicular lymphoma (FL, n = 3), Waldenstrom macroglobulinemia (WM, n = 1), chronic lymphocytic leukemia (CLL, n = 4), and HL (n = 15) who have failed second- or subsequent-line salvage chemo-radiotherapy were enrolled in this trial. Prior to study entry, patients received a median of 5 (range 2 – 11) lines of treatment with autologous SCT performed in 19 (73%) and an additional allogeneic SCT in 10 (38%) patients. At study entry, 7 patients had relapsed and 19 refractory lymphoma. Perifosine (50 mg BID, per os) was initially administered as single agent for 4 weeks to assess tolerability and tumor response. Patients achieving less than partial remission (PR) were given the combination therapy, i.e., Perifosine (50 mg BID, per os) plus Sorafenib (400 mg BID, per os) until disease progression or clinical significant toxicity. Patients achieving ≥PR went off-study and continued with Perifosine (50 mg BID, per os) alone until disease progression or clinical significant toxicity. Tumor responses were assessed according to the revised response criteria for malignant lymphoma of the International Working Group. NCI CTCAE v3.0 was used for toxicity assessment. The study was approved by the Institutional Ethical Committee. Results: After a 4-week treatment with Perifosine alone, 22 out of 26 patients achieved

Description: Abstract 1647 Background and Aims: Mantle cell lymphoma (MCL) is the aggressive subtype of non Hodgkin's lymphoma with the poorest long-term survival probability. There is no consensus for its treatment: in most cases, R-CHOP immuno-chemotherapy, with its 34% complete remission (CR) rate, is still considered the standard of care. Very encouraging results have been reported using more aggressive induction treatments as first-line therapy but these have a role only in the initial management of clinically fit patients less than 60–65 years of age. Since the median age at diagnosis is 63 years and intensive treatment tend to be poorly tolerated by elderly patients, a frontline therapy that can be administered to this category of patients is needed. The main objective of the present study was to design a well tolerated regimen, applicable to the vast majority of patients over 60 years with MCL, while improving the limited efficacy of R-CHOP immunochemotherapy. To achieve this goal, we have used two novel drugs, i.e. ofatumumab and bendamustine. Ofatumumab is a fully human monoclonal antibody targeting a unique epitope on the CD20 molecule: its superior complement-dependent cytotoxicity compared to rituximab may translate into longer duration of treatment response. Bendamustine is a unique agent with both nitrogen mustard group and a benzimidazole ring: when given to relapsed or refractory MCL patients in combination with rituximab, it has shown a very favourable toxicity profile, as well as considerable activity. Methods: Previously untreated patients with MCL, any stage (patients with disease limited only to the bone marrow were excluded), aged ≥ 60 years, were enrolled into a therapeutic protocol consisting of six cycles of immunochemotherapy as follows: ofatumumab 1000 mg i.v. on day 1 (300 mg only for the first cycle); bendamustine 120 mg/sm i.v. on day 2 and 3; dexamethasone 40 mg i.v. on day 1 to 4. Each cycle of therapy was administered every 21 days. Hematopoietic growth factors were used in case of neutropenia. Results: As of July 31, 2011, 36 of the 50 planned patients have been enrolled and 19 have received the whole treatment. Cyclin D1 immunohistochemistry was positive in 18 patients (95%), thus confirming the diagnosis, while in the patient found negative at cyclin D1 analysis, Sox11 was used to confirm MCL; 1 patient (5%) had blastoid histology. Median age was 69 years (range 60–81), stage at diagnosis was IV in 95% of patients; MIPI score was low in 8 (42%), intermediate in 8 (42%) and high in 3 patients (16%); there were 15 male (79%) and 4 female patients (21%). Bone marrow (BM) involvement was detected by immunohistochemistry in 15 (79%) patients and by polymerase chain reaction (PCR) in all patients. Involvement of peripheral blood was detected by flow-cytometry in 12 cases (63%), by PCR in 18 cases (94%), while no data were available in the remaining case. A molecular probe was available for all patients: bcl1/IGH rearrangement was detected by PCR in 11 patients (58%), and an IGH allele-specific oriented primer was designed for the remaining 8 patients (42%). Most adverse events (AEs) were mild or moderate in severity. The most common grade ≥ 3 treatment-related AE were neutropenia (10.5%), febrile neutropenia (10.5%), anemia (5.2%) and thrombocytopenia (5.2%). Two patients (10.5%) experienced tumor lysis syndrome: one was complicated by acute renal failure and the other by disseminated intravascular coagulation; both recovered with adequate therapy. Infectious complications consisted of one episode of pneumonia and one herpes zoster reactivation: both cases were resolved with antibiotic and antiviral therapy, respectively. Of note, 10 patients (52%) experienced cytomegalovirus reactivation, detected as pp65 antigen immunofluorescence, without evidence of infection: all these patients received anti-CMV therapy with negativization of the assay. Among the 19 patients evaluable for response, overall response rate was 94%, with CR in 17 patients, partial remission in one patient and progressive disease after the second cycle in one patient; 15 out of 15 patients evaluable for minimal residual disease in the BM showed molecular remission. Conclusions: Chemotherapy with bendamustine and ofatumumab appears generally safe and well tolerated to date in MCL patients aged ≥ 65 years requiring treatment. Preliminary data about efficacy are encouraging: accrual is ongoing for further evaluation. Disclosures: Off Label Use: ofatumumab, in label for CLL.

Description: Introduction: HL patients with refractory disease or relapsing after autologous stem cell transplantation (SCT) have very poor prognosis with currently available salvage chemotherapy. ITF2357 (Italfarmaco S.p.A., Milano, Italy) is an orally bioavailable hydroxamate inhibitor of class I and II histone deacetylases (HDACs) with preclinical and clinical activity as single agent in hematopoietic cancers. Our preclinical data demonstrating a synergistic activity of ITF2357 with the alkylating agent Meclorethamine in HL cell lines, established the rationale for this currently ongoing phase II study aimed to determine activity and safety of the sequential ITF2357 and Meclorethamine treatment. Methods: Patients with relapsed/refractory HL who have failed second- or subsequent-line salvage chemo-radiotherapy were enrolled. Eligibility criteria included prior treatment with autologous and/or allogeneic SCT, prior treatment with single agent Meclorethamine, at least one target lesion ≥2 cm, ECOG performance status of 0–1, and platelet ≥75,000/μL. ITF2357 (50 mg QID, per os, days 1–3) followed by Meclorethamine (6 mg/sqm, intravenously, day 4) was dosed in 3-week cycles until disease progression or appearance of clinical significant toxicity, but for a maximum of 12 cycles. Tumor responses were determined after cycles 2, 6, 9 and 12 by computed tomography (CT) and positron emission tomography (PET) scan. Serum levels of thymus- and activation-regulated chemokine (TARC) were assessed by ELISA prior to each cycle of therapy. Results: To date, 19 patients have been enrolled (16 males and 3 females; median age, 33 years; range, 21–61 years), including 8 patients enrolled in a preliminary compassionate use trial, and 11 patients of a planned 23 enrolled in this ongoing phase II trial. Prior to study entry, patients received a median of 5 (range 2–7) lines of treatment with autologous SCT performed in 15 (79%) and an additional allogeneic SCT in 5 (26%) patients. At study entry, 6 patients had relapsed and 13 refractory HL. Seventeen of 19 patients received a median of 3 cycles (range, 1–10) of ITF2357/Meclorethamine and are evaluable for response by CT and PET scans. Best response to therapy included 2 (12%) complete remissions (CR) and 3 (18%) partial remissions (PR), for an overall response rate (ORR) of 30%. In addition, 5 (29%) patients had stable disease (SD) with 4 (23%) patients achieving SD for ≥4 months, while 7 (41%) patients progressed. After the first cycle of therapy, serum TARC levels were decreased by 70±16% (mean±SEM, P ≤0.05) in 5 patients who achieved major clinical responses (PR+CR), and by 16±14% (P = ns) in patients who achieved SD. Overall, therapy was well tolerated without significant adverse events, and no patient required dose reductions for management of toxicities. The most common drug-related non-hematological toxicities were grade 1–2 nausea (12/17) and fatigue (14/17). Four patients experienced infections [pneumonia (n = 1), oral herpes simplex (n = 2), oral candidiasis (n = 1)]. No prolongation of QT/QTc interval has been detected over 70 therapy cycles. Hematological toxicities included grade 1–2 anemia (13/17), neutropenia (7/17), and thrombocytopenia (12/17). Grade 3–4 neutropenia and thrombocytopenia were observed in 7 and 8 patients, respectively. RBC and platelet transfusions were required by 4 and 5 patients, respectively. Conclusions: Preliminary results from this ongoing trial suggest that ITF2357, in combination with Meclorethamine, demonstrates significant anti-tumor activity in heavily pretreated relapsed/refractory HL and is well tolerated. Preliminary data also suggest that early decrease in serum TARC levels may predict response to therapy.

Description: Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs.HSCs are maintained in their niche by binding to cellular determinants through adhesion molecules and diverse strategies are currently used to promote their egress from BM to PB. Traditionally, the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, other compounds have been recently tested. One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the blood. The use of this drug is currently approved by FDA and EMA in combination with G-CSF, in patients affected by lymphoma or multiple myeloma whose cells mobilize poorly with G-CSF alone. Clinical trials demonstrated that Plerixafor alone safely and rapidly mobilizes HSCs also in healthy donors, beta-thalassemia patients and pediatric patients affected by malignancies. Previous characterization studies on non-human primates and human samples of Plerixafor mobilized cells in comparison to cells mobilized by G-CSF alone or in combination with Plerixafor showed a different expression profile, cell composition and engrafting potential in a xenotransplant model. From these studies remains unsolved whether Plerixafor, G-CSF, or their combination mobilizes different primitive HSC populations, defined both by multimarker immunophenotype and in vivo functional analysis. In the present study we investigated by controlled comparative analysis the functional and molecular hallmarks of human HSCs collected from BM, G-CSF and/or Plerixafor mobilized peripheral blood. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+CD38/lowCD90+CD45RA-CD49f+ cells and primitive populations of HSCs. These cells possess higher ability to home to hematopoietic niches and engraft in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The higher content of CXCR4+ and CD49f+ cells correlates with this feature. Furthermore, global gene expression profiling highlights the superior in vivo reconstitution activity of Plerixafor mobilized cells. The "stemness" signature of cells dislodged from their niche by the drug is attenuated by the combined use with G-CSF, which emphasizes the gene expression profile induced by G-CSF treatment. These data indicate that a qualitative advantage accounts for the superior performance of Plerixafor mobilized cells. These findings provide the rationale for using a suboptimal dose of more primitive HSCs when target cell number for transplantation is limited, or when G-CSF mobilization is too risky like in sickle cell anemia patients. Moreover, CD34+ cells mobilized by Plerixafor alone or with the combination of G-CSF are efficiently transduced by a lentiviral vector encoding for human ß-globin gene (GLOBE LV) and are able to engraft and differentiate in vivo, supporting their use for gene therapy applications. Disclosures Ciceri: MolMed SpA: Consultancy.

Description: Abstract 586 Gene therapy of inherited blood diseases requires harvest of hematopoietic stem cells (HSCs) from patients and autologous transplantation of genetically modified cells. In order to achieve correction of the disease, high number of HSCs and previous conditioning of the host bone marrow (BM) are necessary. In the clinical application of gene therapy for thalassemic patients the choice of the HSC source is a crucial issue. On one side, the minimal target dose poses a challenge for the use of steady state BM since reinfusion of high numbers of beta globin gene modified CD34+ cells is probably necessary to gain sufficient correction of the genetic defect in order to achieve transfusion independency; on the other side, the disease related features and complications of thalassemic patients (i.e. splenomegaly and thrombophilia) dictate caution in the use of G-CSF as mobilizing agent. In April 2011 a clinical protocol exploring the use of Plerixafor (AMD3100) as single agent was started (“Plerixafor mobilized stem cells as source for gene therapy of beta-thalassemia”, acronym AMD-THAL, EudraCT2011-000973-30). Aims of the trial were to explore the ability of Plerixafor in inducing safe and effective stem cells mobilization in adult patients affected by beta-thalassemia, to characterize stem/progenitor cells mobilized from the BM and peripheral blood of treated subjects and to achieve gene transfer efficiency of mobilized CD34+ cells at a level comparable to that obtained using steady state BM. Four patients (01, 02, 03 and 04) were enrolled and already mobilized to date (August 2012). All patients are affected by transfusion dependent beta-thalassemia and aged 28 (01), 41 (02), 39 (03), 33 (04). Two are splenectomized (02 and 03); all subjects are regularly iron chelated with adequate organ function. Administration of Plerixafor subcutaneously as single agent and at the single dose of 0.24 mg/kg resulted in mobilization of CD34+ cells/mcl with a peak of 78 cells at 9 hrs (01), 70 cells at 7 hrs (02) and 69 cells at 8 hrs (03); suboptimal mobilization was observed in patient 04 (peak 18 at 8 hrs). Patient 03 received a second dose at 0.40 mg/kg 24 hrs after the first dose and underwent a second leucoapheretic procedure. Harvest by leukoapharesis resulted in procurement of the following CD34+ cells/kg: 1.84 × 106 (01) and 4.43 × 106 (02) with a unique leukoapheretic procedure, and 3.57 × 106 (03) with two leukoapheresis. No apheresis was performed for patient 04 because the minimum target of 20 CD34+ cells/mcl in peripheral blood was not reached. CD34+ cells selection through Clinimacs Miltenyi resulted in the following yield: 1.2 × 106 CD34+ cells/Kg, 65% recovery (01), 2.66 × 106 CD34+ cells/Kg, 60% recovery (02), 1.78 × 106 CD34+ cells/Kg, 50% recovery (03). No severe adverse event occurred. Recorded side effects were: grade 3 hypotension related to the apheretic procedure (01), mild grade 1 facial disestesia (02 and 04) and hyperleukocytosis (02: WBC from 13.6 to 42.6 × 103/mcl). In addition, steady state and Plerixafor primed BM aspirates were performed to analyze any modification in CD34+ concentration in the BM following Plerixafor administration. In fact, Plerixafor administration resulted in enrichment of CD34+ cells concentration in the BM. Purified CD34+ cells from leukoapheresis of the 4 treated patients were analyzed for their biological and functional properties, subpopulations composition and expression profile. In vivo reconstitution potential and lymphomyeloid differentiation of CD34+ cells were tested following transplantation in NSG mice. Experiments are ongoing but preliminary results indicate that cells mobilized by Plerixafor have a primitive phenotype with a high reconstitution potential and are efficiently transduced with a lentiviral based vector, named GLOBE, encoding for the human beta-globin (Roselli et al., 2010), thus being a suitable source of target cells for gene therapy. Disclosures: No relevant conflicts of interest to declare.