ALBERT

Description: Abstract 2249 Poster Board II-226 BACKGROUND. Allogeneic hematopoietic stem cell transplantation (HSCT) is being used to treat a range malignant and nonmalignant conditions. However, it often causes potentially lethal Graft-versus-Host Disease (GVHD). Several studies revealed that mesenchymal stromal cells (MSCs) from human bone marrow can down regulate GVHD after HSCT. METHODS. MSCs were obtained from BM, expanded and characterized by their morphology, immunophenotype, immunosuppressive potential for autologous, partially and fully mismatched lymphocytes. Twenty five patients (pts) got 39 (range, 1 to 4) MSCs infusions for 27 episodes of acute GVHD, which was defined as steroid resistant grade IV aGVHD in all but one patient. RESULTS. GvHD characteristics. Acute GVHD started from day +6 to d +46 from HSCT (median d+18) excluding single pt in whom it occurred on d+150. In 2 pts disease manifested in a hyper acute form before the engraftment (on d+6 and d+8, respectively). In all but one pt GI GVHD was defined as grade IV with full clinical picture of it. Skin GVHD accompanied GI symptoms in 17 pts and 13 was 〉 grade II, in 4 grade IV. Liver symptoms presented in 13 pts, one pt was determinate as VOD. In 3 pts liver involvement was defined as grade IV. In all 24 of 25 pts had GVHD grade IV 4 and one grade III. Previous conventional anti GVHD therapies included: MP in all pts, MTX, various calcineurin inhibitors, MMF, ECP, serotherapy. Only 3 pts showed temporary limited partial response. Treatment with MSCs. The 1st infusion with MSC was given on day +32.5 (range, 8 to 74); d+50 (range, +28 to +180) post diagnosis of aGVHD and HSCT HSCT, respectively. In 22 evaluable pts we treated 24 separate episodes of GVHD: 22 first episodes and in 2 pts relapse of GVHD. In 24 of 39 cases treatment was performed with frozen MSC and in 15 with fresh cultured cells. In 37/39 cases passages 1 to 3 were used. Median number of infused cells was 1 (range, 0.3 to 3.1) x10 6 per kg of pt body weight in each treatment. Initial response was seen in 17/24 episodes of aGVHD (70.8%), in 8 partial (PR) and in 9 complete (CR). Two pts experienced GVHD relapse after achieving CR and very good PR: one case was treated successfully while another was resistant to MSCs. The latest pt with grade IV aggressive liver GVHD received MSCs injections intra hepatic arteries under radiological control following IRB approval with no anti GVHD effect. The procedure was uneventful with no evidence of microembolisation, no changes of blood flow or deterioration of liver tests (cytolysis). We observed a trend for better effect with higher MSC cell dose: total and per first infusion MSCs dose (1.93±1.28 vs. 1.23±0.50, p=0.078; 1.28±0.79 vs. 0.88±0.28, p=0.086). In all cases effect was seen after the first procedure. No difference was noted in the anti GVHD activity between fresh vs. frozen MSCs. There were no immediate or late toxicity or side effects. Overall survival. 14/25 patients are alive up to 20 months follow up. 8/11 pts, died from GVHD (4 within 1-10 d from MSCs infusion), and 3 from other unrelated causes including TTP-1, DAH-1 and disease progression-1. CONCLUSION. Treatment with MSCs seems to be a new novel type of therapy for steroids resistant GVHD with promising preliminary results and low toxicity. Obviously multicenter 2 arm randomized study is in need to confirm these encouraging. Disclosures: No relevant conflicts of interest to declare.

Description: G-CSF induced hematopoietic stem/progenitor cell (HSC/HPC) mobilization is regulated by a complex interplay between extracellular matrix (ECM), cytokines/chemokines, adhesion molecules, and proteases, which affect stem cell retention and proliferation in the bone marrow microenvironment. Several studies suggest that membrane type 1-matrix metalloproteinase (MT1-MMP), expressed on the surface of various cell types, is a key enzyme for normal cell motility and tumor cell migration and invasion. We found that enriched human CD34+ cells express various surface MT1-MMP levels, depending on the cell source and G-CSF treatment. CD34+ cells obtained from BM of healthy donors treated with G-CSF were found to have the highest mean fluorescence intensity (〉900 arbitrary units), while level of expression was lower in CD34+ cells derived from G-CSF mobilized peripheral blood from healthy donors (159±40), human steady-state (SS) BM (80±19), and human cord blood (41±4). Following 48 hr incubation of human SS-BM CD34+ cells with G-CSF, the expression of MT1-MMP increased 2-fold compared to untreated cells, whereas treatment with other cytokines, such as SCF, SDF-1 and IL-6, had only a minimal impact. Immunocytochemical analysis of human cord blood CD34+ enriched cells plated on fibronectin or hyaluronate-coated cover slips revealed that in response to SDF-1, MT1-MMP changes its localization in the polarized and spreading cells, suggesting a role in the process of HPCs directional migration. Indeed, incubation with neutralizing antihuman MT1-MMP Ab, targeting its catalytic site, significantly reduced human progenitor migration towards a gradient of SDF-1 in transwells. Interestingly, low concentrations of tissue inhibitor of the metalloproteinase-2 (TIMP-2) enhanced SDF-1 induced transwell migration, while higher concentrations hampered this process. More importantly, 5 daily injections of G-CSF to NOD/SCID mice previously engrafted with human cells, up-regulated MT1-MMP expression on CD45+ and CD34+ human cells in the BM and peripheral blood of the mobilized chimeric mice compared to untreated, control chimeras. Treatment of chimera mice with antihuman MT1-MMP Ab on days 3–5 of G-CSF induced mobilization, significantly reduced the number of CD45+ and CD34+ human cells in peripheral blood. In summary, based on our data we suggest that following G-CSF treatment, increased levels of MT1-MMP on the surface of human progenitors in the BM facilitates their mobilization most probably due to pericellular ECM degradation and/or activation of other regulatory molecules, pointing to the essential role of MT1-MMP in G-CSF induced mobilization.

Description: Abstract 1143 Poster Board I-165 Introduction Cyclosporine (CSA) is the backbone of graft vs. host disease (GVHD) prophylaxis in the last decades. It is established that CSA levels in the 1st weeks after transplant are critical for the rate and severity of GVHD. Initially, we gave CSA starting on day -1 in all our protocols. However, 8 years ago, we have changed CSA initiation in most of our protocols to day -4 in order to have stable, controlled therapeutic blood levels of CSA prior to transplant. Since nowadays the use of CSA is the most widespread prophylaxis of GVHD, we found it essential to compare the initiation of CSA on day -4 to day -1, consecutively determining the preferred initiating timing of CSA for patients who undergo allogeneic transplantations. Methods Out of 1716 patients that underwent allogeneic transplantation, we identified 2 groups of patients that received T-cell repleted grafts in which only CSA was used for GVHD prevention, starting on days -1 or -4 (n=219 and 261 respectively). The guidelines for CSA cessation and DLI were uniform in both groups. Both groups were compared for age, sex, donor type and matching, disease, disease status upon transplant, graft type, engraftment, GVHD (both acute and chronic), GVHD associated death and overall survival. Results The groups were found to be equal for age (p=0.83), sex (p=0.58), donor type (p=0.54), matching (p=0.98), disease type (malignant or non-malignant; p=0.25), graft type (PBSC or BM; p=0.45) and disease status (remission or active; p=0.42). The median time to ANC〉500 was 16 and 15 days in the CSA -1 and -4 groups respectively with a trend toward better engraftment with initiation of CSA on day -4 (figure 1A, P=0.07). However, platelet engraftment was significantly better with CSA -4, with a median of 14 and 12 days in the CSA -1 and -4 groups respectively (figure 1B, p=0.0005). 112 and 138 patients developed acute GVHD (aGVHD) of any grade, respectively. Out of them 54% and 44% had severe (grade 3-4) aGVHD (p=0.45). The median time to aGVHD was similar, with a median of 29 and 28.5 days in the CSA -1 and -4 groups respectively (p=0.54). However, 64 patients developed cGVHD in the CSA -1 group, while 102 did so in the CSA -4 group (figure 2A, p=0.0002. Hazard ratio 0.59, 95% CI 0.37 to 0.73). Of these patients, 46.8% and 40.2% of the patients had extensive cGVHD (p=0.70), respectively. Additionally, despite lower GVHD rate in the CSA -1 group, GVHD associated death occurrence was more frequent then in the CSA -4 group (41/148 and 17/132 patients, p=0.02). Kaplan Meier analysis of all cause mortality showed higher mortality in the CSA -1 group (67.6% and 50.5%, figure 2B, p=0.074. Hazard ratio 1.24, 95% CI 0.98 to 1.58). Conclusions The initiation of CSA on day -4 improves engraftment, conversely increases the risk for cGVHD of any grade (possibly through prevention of tolerance), but reduces the risk of GVHD associated death and improves overall survival. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2223 Poster Board II-200 Introduction: In general it is expected that strong immune suppression that alleviates GVHD, will increase the risk of relapse. Alefacept (Amevive®) is an immunosuppressive dimeric fusion protein that is used for psoriasis control. We showed its remarkable effect in acute steroid resistant/dependent and chronic extensive GVHD. Methods: To date, 42 patients with a median age of 31.5 years (range 3-66) were treated by us with alefacept due to acute (n=28) or chronic (n=14) steroid resistant/dependent GVHD (27 males, 15 females). Twenty were transplanted from HLA matched family members, 14 from HLA matched unrelated donors, 8 from mismatched donors and 2 from unrelated cord blood units. Pretreatment acute GVHD grade ranged 2-4 (median 3) and involved the skin (30), gut (19) and liver (7). All the patients with chronic GVHD had extensive involvement prior to therapy. Results: The median time from transplantation to alefacept was 42.5 days and 13 months in acute and chronic GVHD respectively (range 18-110d and 3-47.5m) and a median of 9 (range 1-25) injections that were given per patient. Thirty-four out of the 41 evaluable patients (83%) responded to the treatment (23/28 and 11/13 in the acute and chronic group respectively). Despite this high response rate, demonstrating the deep immunosuppressive and immunomodulative effect of alefacept, only 5/41 evaluable patients (figure 1) have relapsed (with a median follow-up of 30.8 months on the 17 survivors). Other than the 5 patients that relapsed (all treated with calcineurin inhibitor and steroids), full-donor peripheral blood chimerism (100% donor cells and no residual host-type DNA) was stable throughout the treatment period and later in all but one patient, that developed mixed chimerism under alefacept treatment. His chimerism returned to full-donor chimerism with taper-down of immune suppression. Currently, 17/42 (40.5%) patients are alive (figure 1), most with improved or stable GVHD. Twenty-six patients died due to either: GVHD progression (n=14), progression of the basic disease (n=4), infections (n=5) or other causes (n=3). Conclusions: Alefacept is effective and safe for the treatment of acute or chronic steroid resistant/dependent GVHD and may discriminate between GVHD and GVL. Disclosures: No relevant conflicts of interest to declare.

Description: After engraftment of allogeneic stem cell transplantation (allo-SCT) patients are severely immuno-suppressed and are susceptible for recurrent opportunistic infections on one hand; on the other hand, particularly without immunosuppressive treatment over-activity of the graft can lead to uncontrolled acute or chronic graft-versus-host disease (GvHD); optimal immunological activity of the graft is necessary to control the underlying malignant disease and to prevent relapse. Therefore, accurate immune monitoring is crucial for the assessment and appropriate management of the immune-compromised patients after allo-SCT. Since October 2005 up to 6 consecutive blood samples were collected from 51 patients undergoing allo-SCT in Hadassah Medical Center following engraftment. Immune function was analyzed by Cylex Immuknow™ assay, an FDA approved immune cell function test for the assessment of cell-mediated immunity. ATP activity from magnetically separated and lysed CD4 lymphocytes is measured by light intensity, thereby reflecting the current immune function of the patient. From other clinical applications of this test, the following stratification of the immune response was established according to the following levels of ATP activity: Low (0–225 ng/mL), moderate (225–525 ng/mL) and high activity (〉525 ng/mL) of immune response. Our preliminary results from allo-SCT patients are in keeping with their clinical course and the known stratification of the ATP level:The gradual increase to moderate ATP levels over several months after allo-SCT are in keeping with normal immune reconstitution with an uneventful post transplant clinical course;high ATP levels were observed in patients prior to the clinical presentation of acute GVHD;low ATP levels were associated with recurrent infections and relapse. Remarkably, there was no correlation with the recovery of the white cell count after allo-SCT. Our results with consecutive tests are the first study of Cylex Immuknow™ assay in patients after allo-SCT and are in keeping with other currently known clinical applications of this assay for other indications. Our preliminary observations indicate a promising contribution of Cylex Immuknow™ assay as a simple and fast monitoring technique for patients undergoing allo-SCT, which might also predict clinical complications. Our future follow-up studies aim to confirm these preliminary results in order to gain confidence in the assay’s clinical contribution as a possible standard test for patients after allo-SCT.

Description: The aim of allogeneic stem cell transplantation (SCT) is to combine tumor cytoreduction with optimal doses of chemoradiotherapy and to replace host with donor immunohematopoietic cells. Induction of host-vs.-graft tolerance by engraftment of donor stem cells, enables durable engraftment of donor lymphocytes, with subsequent induction of graft vs. leukemia effect (GVL) expected to eliminate all residual chemoradio-resistant malignant cells of host origin by alloreactive donor cells. Relapse shortly post SCT indicates tumor refractoriness and carries poor prognosis. Multi-donor stem cell transplantation (MDT) is an innovative transplantation strategy aiming at both increasing the number of donor stem cells infused to the recipient to improve the chance of engraftment of at least one of the donors, thus avoiding the need for re-transplantation in case of rejection, while in parallel improving the displacement of residual host cells and GVL effect by stimulation of alloreactive donor T cells as bystander effect to the rejection of 1 donor by the other donor in order. We describe 4 patients that were treated with MDT following early post allogeneic transplant relapse (n=3, resistant AML) or rejection (n=1). In order to reduce the risk for the patients, and with the assumption that MDT will stimulate anti host immune response leading to engraftment and GVL, we chose to combine transplantation from 2 donors (including the 1st donor in the patients transplanted due to relapse and both parents in the patient transplanted due rejection). The patients received a non myeloablative conditioning treatment. All patients engrafted (2 with the 2nd donor, 1 with 1st donor and 1 from his mismatched mother) with 100% donor chimera. Time to engraftment was short - median 11d (range 10–12). CR was achieved with the appearance of GVHD probably due to stimulation of donor vs. donor (and therefore against host alloantigens). No relapse was documented during follow up. Two of the recipients are currently alive with follow-up ranging 1.5–25m. Conclusions: The use of MDT induced engraftment and GVL in patients with chemo-immuno-resistant leukemia. In view of the profound anti host activating effect of MDT, we plan to assess its therapeutic potential on engraftment facilitation when reduced intensity conditioning is used in patients in need for SCT lacking an HLA matched related or unrelated donor.

Description: Most of the hematological malignancies where allogeneic stem cell transplantation (SCT) is a therapeutic option occur in elderly patients. Additionally, in this age group, the probability of finding a matched sibling donor decreases due to mortality and co-morbidities of patient’s siblings which may exclude them from donation. In such case, SCT from an unrelated donor (UD) can offer a therapeutic solution for elderly patients in need although such a procedure is considered more hazardous than SCT from a matched sibling (mostly due to procedure and GVHD related morbidity and mortality). We present our data on transplantation, in 68 elderly (age〉/= 55 years) patients that underwent SCT from a matched sibling (n=51) or UD (n=17) mostly using fludarabine-based regimen (table 1). Median recipient age was 58.3y in the sibling group and 60.8y in the UD group (NS) (range 55–69.5y and 55–75y, respectively). With a median follow-up of 40 months of the survivors after sibling SCT, the 1-year OS was 32%. Similarly, in the UD group the 1-year OS was 35% (survivors’ median follow-up 48.5m) (NS). Transplant related mortality occurred in 22/49 (45%) and 9/17 (53%) of the patients transplanted from sibling or UD respectively (NS). Occurrence of acute GVHD was noted in 26/43 and 7/11 (figure 1) evaluable patients transplanted from matched sibling or UD respectively (NS). Diagnosis of chronic GVHD was made in 50% (14/28) and 63% (5/8) of the patients in the sibling and UD group correspondingly. GVHD related mortality occurred in 11 and 2 patients, respectively. We conclude that using fludarabine based regimen, allogeneic SCT from an UD is equally effective as in patients undergoing SCT from matched family member. Age per-se should not be currently used as an exclusion criterion for either initiation of donor search or SCT from UD. Table 1- Patients’ characteristics Figure Figure

Description: Reduced-intensity or nonmyeloablative stem cell transplantation (NST) is designed to induce host-versus-graft tolerance by engraftment of donor stem cells. The rationale behind NST is to induce optimal graft-versus-leukemia (GVL) effects for elimination of all malignant cells by donor alloreactive immunocompetent cells as an alternative to standard high-dose myeloablative chemoradiotherapy. NST based on the use of fludarabine, low-dose busulfan, and anti–T-lymphocyte globulin (ATG) was employed in 24 patients aged 3 to 63 years with chronic myeloid leukemia (CML) in first chronic phase (CP). Graft-versus-host disease (GVHD) prophylaxis consisted of low-dose cyclosporine (CSP), in some cases with low-dose methotrexate. Early discontinuation of CSP was attempted in cases of mixed chimerism in an attempt to amplify GVL effects. All 24 patients showed rapid 3-lineage engraftment, mostly without complete aplasia; 6 patients did not require transfusion of any blood products. NST was associated with minimal procedure-related toxicity. The incidence of acute GVHD (grade I or higher) was 54%; however, this incidence increased following CSP withdrawal. After a follow-up of up to 70 months (median, 42 months), 21 of 24 patients remained alive and disease free. The GVL effects induced by donor immunocompetent lymphocytes eradicated all host hematopoietic cells, as evidenced by molecular testing. The Kaplan-Meier probability of survival and disease-free survival at 5 years is 85% ± 8% (95% confidence interval, 70%-100%). NST may successfully replace myeloablative stem cell transplantation, providing a safer, well-tolerated therapeutic option for all patients with CML in first CP with a matched donor. However, this conclusion must be tested in a prospective randomized clinical trial.