ALBERT

Beschreibung: Abstract 3308 Poster Board III-196 The use of lineage-specific chimerism studies following allotransplantation has shown that low levels of donor T-cell chimerism can be seen following some preparative regimens despite good engraftment of donor myeloid-origin cells. Furthermore, low levels of early T-cell chimerism have been associated with eventual graft rejection, malignant relapse and poorer event-free survival [Maris et al Blood 102: 2021-2030 (2003); Saito et al Biol Blood Marrow Transplant 14: 1148-1155 (2008)]. There is no consensus regarding the management of such differential engraftment. Withdrawal of immunosuppression, DLI, and second transplant with repeat conditioning have all been used. We utilized a strategy of low dose DLI (initial dose 0.1-1 × 10e7 CD3+ cells/kg) without additional conditioning or withdrawal of immunosuppression in patients with donor T-cell chimerism 〈 50% following allotransplant who had no evidence of relapse/progression of malignancy. Nineteen consecutive patients treated this way between Nov 2005 and April 2009 were included in this analysis - median age 59 (range 36-63);15M, 4F; NHL 5, AML 5, MDS 3, CLL2, CML 2, MPS 2; donor =MRD 3, MUD 16; Preparative regimen for transplant- reduced intensity 18, myeloablative 1, and consisted of fludarabine/busulfan (11), fludarabine/cyclophosphamide (6), other (2). Alemtuzumab was used in the preparative regimen in 15 and rabbit antithymocyte globulin in 2. The first DLI was administered a median of 66 days post BMT (range 36-237). Median CD3+ and CD34+ cell dose was 1.0 ×10e7/kg (range 0.1-1) and 0.24 × 10e6 (0.02-0.74) respectively. Cryopreserved G-CSF mobilized cells collected at the time of BMT were used for the DLI in all patients. Median (range) donor chimerism in blood CD3+ cells and CD33+ cells prior to DLI was 14% (0-34%) and 100% (10-100%) respectively. Pre and post DLI donor T-cell chimerism is shown in the Figure below. Full donor CD3 chimerism (FDC) (〉90% donor-derived CD3+ cells) was achieved after one DLI in 9 evaluable patients (50%) at a median 90 d post infusion (range 30-180 d). Eight patients received additional DLI (median 1 range 1-3) at a CD3+ cell dose up to 4 × 10e7/kg and 7 of these patients achieved FDC in CD3+ cells [total success rate in achieving FDC = 89%]. FDC was durable in all cases. Acute GVHD (none 〉 gd 2 overall) developed in three evaluable patients following DLI (17% - median 43d [35-57d]) and chronic GVHD (extensive mild 1, moderate 8, severe 1) developed in ten patients (55% -median 119d [53-166d]). One patient died of chronic GVHD. With a median follow-up of 728d (135-1240d) estimated 2yr survival from DLI is 74%. Conclusion This strategy is highly effective at correcting poor donor T-cell chimerism without inducing severe GVHD. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Introduction Extramedullary leukemia (EML) will develop in approximately 3% of the patients with acute leukemia. Only a few retrospective studies, and no prospective or randomized studies, have assessed the effectiveness and toxicity of radiation for EML. Here we review the EML patients treated with radiation therapy at the Oregon Health and Sciences University (OHSU) Radiation Oncology department. Methods and Materials From 1987 to 2005, 17 patients with EML underwent 20 radiation courses at the OHSU Radiation Oncology department. All patients had either biopsy-proven EML or had pre-established diagnoses of leukemia and were treated for EML as presumptive relapse. Patient data and disease history were either extracted from the patient chart or obtained from the cancer tumor registry. Variables used for analysis included patient age, gender, histological diagnosis, tumor location, radiation dose, fraction size, acute toxicities, last follow up or date of death, disease recurrence site after radiation therapy, initial symptom with presenting EML, effect of radiation on symptom(s), and time to EML. Univariate and multivariate analyses were done. Kaplan-Meier survival curves and Cox regression analyses were generated. Results The mean age of our patients was 37.5 years, with a range from 7.4 to 78.5 years. Males made up 76% of the patients population. The most common location for an EML was soft tissue (25%), followed by central nervous system (20%), and mucosal (15%). Surgical intervention was performed in only 5 of the 17 EML patients. The 17 patients received 20 treatment courses. The most frequently used radiation energy was 6 Megavoltage photons (55% of the cases), while the next most common was cobalt 60 (10%) and a mixed energy beam (10%). Radiation therapy was quite effective at relieving symptoms with a 94% response rate and 61% having a complete response. Pain was palliated in 88% of patients, while mass effect was decreased in 100% of patients. The mean radiation dose given was 21.8 Gray (range 10–39.6 Gy). We did not observe a radiation dose response to symptom palliation (Table 1). Leukemia recurrence of any type occurred at a median of 5.8 months from the last day of radiation treatment. There was a low incidence of acute grade 1 or 2 toxicities (39%) and no acute grade 3 or 4 toxicities or late toxicities. Our 17 patients had a mean and median overall survival of 20.7 months and 5.6 months, ranging from less than 1 month to 149 months. Regression and correlation models failed to show any significant prognostic factor (age, gender, quality of radiation, total radiation dose, initial presenting EML symptom, or time from diagnosis of leukemia to diagnosis to EML) influencing overall survival. Conclusions The role of radiation in EML is for symptom relief. Low dose radiation provides excellent palliation with minimal toxicity. A radiation dose response was not seen in our small patient population. Table 1 Radiation Dose response in Extramedullary Leukemia Dose # Patients # Symptoms # symptoms with a response # symptoms with a complete response 10–19.9 Gy 6 7 7 (100%) 4 (57%) 20–29.9 Gy 4 5 5 (100%) 3 (60%) 30 Gy or more 4 6 5 (83%) 4 (80%)

Beschreibung: Recent advances in understanding the role of JAK2 V617F mutation in Bcr-Abl negative myeloproliferative (MPD) diseases pathogenesis opened up a possibility to develop highly targeted therapies against these debilitating ailments. We used a Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F-dependent proliferation. Further extensive SAR of initial hits resulted in identification of R723, a potent and selective JAK2 inhibitor. This molecule is strongly antiproliferative (IC50 130–200 nM) against mouse BaF3 cells used for initial screening as well as against human UKE1 and SET2 cell lines harboring the same mutation. On the other hand, R723 has only weak activity in IL2-dependent (i.e. JAK1/JAK3-dependent) proliferation assays performed with human primary T (IC50 1300 nM) and mouse T-cell leukaemia CTLL2 cells (IC50 600 nM). A 10 to 20 fold cell-based selectivity of R723 was further confirmed by measuring inhibition of constitutive STAT5 phosphorylation in SET2 and BaF3 cells versus inhibition of IL-2 inducible STAT5 phosphorylation in human primary T and mouse CTLL2 cells using FACS-based approach. Compound R723 has low nonspecific antiproliferative activity against JAK2-independent MOLT4, A549 and H1299 cell lines with an IC50 ranging from 4 to 6 uM. The molecule has been also proven to be potent (IC50 of 2 nM against JAK2 in biochemical assay) and highly selective (window of more than 500 fold over JAK1 and 10 fold over JAK3) inhibitor of JAK2 kinase in vitro. Moreover, when tested in biochemical assay against a panel of more than 200 kinases at a concentration of 20 nM (IC90 for JAK2), R723 inhibited none of them. The selectivity of R723 was further confirmed using a variety of cell-based assays probing T-, B- and mast cell activation. Compound R723 was further evaluated in a stress-induced erythropoiesis mouse model, where kinetics of EPO-dependent hematocrit recovery from phenylhydrazine-induced anemia was assessed. Significant delay in recovery was observed at doses of 75 and 100 mg/kg bid indicating strong compound effect on EPOR signaling in vivo. The result could not be attributed to general toxicity effects as 14 day toxicology study did not identify any abnormalities at doses tested. As a result, R723 could become the basis for next generation of potent and selective compounds targeting JAK2-dependent myeloproliferative diseases.

Beschreibung: Neo-vascularization has been implicated in a number of inflammatory diseases as well as tumor growth. Both angiogenesis, the sprouting of resident tissue endothelial cells (ECs), and vasculogenesis, the recruitment of bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs), are thought to participate in neo-vascularization. EPCs have been implicated in tumor growth, however, the biologic significance of EPCs during inflammation is unclear. We studied neo-vascularization and the role of EPCs during inflammation in well-characterized murine models of graft-versus-host disease (GVHD). We found a significantly increased number of donor BM-derived EPCs in peripheral blood and BM in allogeneic bone marrow transplantation (allo-BMT) recipients with GVHD at different time points after BMT. We next quantified neo-vascularization during inflammation in GVHD target organs by immunofluorescence microscopy and by flow cytometry. We found significantly increased numbers of donor-derived ECs in the liver as well as a significantly higher vessel density in the liver, illeum and colon. We adoptively transferred selected GFP+ EPCs and observed incorporation into the neo-vasculature of the inflamed intestines (Fig. 1A) and liver during GVHD. Taken together, these data suggest that neovascularization during GVHD is due to vasculogenesis from donor EPCs. Next we used an antibody (E4G10) against the vascular endothelial adhesion molecule VE-cadherin, which recognizes a terminal epitope that is exposed on circulating EPCs, but is masked in the established vasculature, and found a significant reduction of EPCs in the peripheral blood and BM. We observed that depletion of EPCs was associated with a significant inhibition of donor BM-derived neo-vascularization in the liver, illeum and colon during GVHD. E4G10 treated recipients had significantly better survival and lower clinical GVHD scores in all tested models (B6BALB/c [1×106 T], B6B6D2F1 [1×106 T], B6B6D2F1 [2×106 T], B6B6D2F1 [3×106 T]). We found significantly reduced numbers of allo-reactive donor T cells in secondary lymphoid organs during GVHD, but no changes in the expression of activation markers and homing molecules, as a consequence of E4G10 administration. In blinded histopathological analyses we found significantly less GVHD and reduced numbers of tissue-infiltrating CD3+ T cells in the liver, illeum and colon in E4G10-treated allo-BMT recipients. To better emulate the clinical setting, we first assessed the role of EPCs in tumor growth in allo-BMT recipients. We transferred sorted GFP+ EPCs as well as renal carcinoma (RENCA) cells to BALB/c recipients and found that GFP+ EPCs were recruited to the neo-vasculature of lung metastases. We detected a significant inhibitory effect of E4G10 administration on tumor growth, as determined by in vivo bioluminescence imaging, in both tumors tested (RENCA, A20 lymphoma) as well as a significant survival prolongation in tumor-bearing mice that were treated with E4G10 in the RENCA and C1498 (AML) model. Finally, we performed experiments in which tumor-bearing allo-BMT recipients received allogeneic T cells, which mediate the favorable graft-versus-tumor (GVT) effect but also cause inflammation in GVHD target organs. We found that administration of E4G10 led to a significantly higher rate of tumor-free survival in all models (B6'BALB/c [1×105 B6 T and 2×105 RENCA], B10BR'B6 [1×105 B10BR T and 2×105 C1498], B6'BALB/c [2×105 B6 T and 5×105 A20]), which was due to both attenuation of GVHD as well as inhibition of tumor growth (Fig. 1B). We conclude that depletion of EPCs is a strategy to simultaneously ameliorate inflammatory disease and tumors, providing a new approach to improve therapeutic outcome of allogeneic hematopoietic stem cell transplantation. This study demonstrates the biological significance of EPCs for neo-vascularization during inflammation and identifies the specific targeting of EPCs to disrupt neo-vascularization as a novel therapeutic concept to decrease inflammation. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group.

Beschreibung: The majority of clinical laboratories use the one stage factor assay for plasma factor measurements because it is simple and easy to automate. However, quality assessment scheme surveys on factors show that inter-laboratory agreement needs to be improved and the one stage assay needs to be standardized better. In this study, one stage factor assays were improved by optimizing assay calibrations. A serious drawback of current factor assay calibrations is that they can’t cover the clinically relevant range (from zero to high activities) by a reliable single calibration. To have measured activities in the calibrated range, high and low samples must be re-run, using higher and lower sample pre-dilutions, respectively. However, measuring samples diluted less than 1:10 should be avoided, because that violates the basic assumption of the one stage method that the deficient plasma reagent alone provides the other factors, and that the effect of other factors in the sample is negligible. Here, factor assays were improved by the introduction of a zero calibration point and the establishment of calibration curves with an optimization approach. In each of the deficient (

Beschreibung: The International Prognostic Scoring System (IPSS) requires cytogenetic data to evaluate risk in MDS. Cytogenetic data were not available for all patients from the CALGB trial 9221 (Silverman et al, JCO2002; 20:2429). Therefore, we developed an alternative prognostic model that identified a homogeneous subgroup of high-risk MDS patients based on the following risk factors: percent marrow blasts, number of cytopenias, age, gender, FAB classification, and time since diagnosis (IPSS used data at time of diagnosis; CALGB 9221 used data at time of randomization). The model was validated using 2,318 patients from the MDS Registry, Düsseldorf. Using these baseline prognostic factors, we predicted survival for each of the 191 CALGB patients, identified a high-risk subgroup with a survival prognosis of ≤ 1.2 years (the IPSS INT-2 survival median), and compared azacitidine to supportive care. Patients were analyzed as randomized (azacitidine or supportive care) according to the intention-to-treat (ITT) principle. The two groups had similar demographic and disease characteristics. All 70 high-risk patients were followed until death. There was a statistically significant difference in overall survival curves between the azacitidine and supportive care groups (p=0.03). The one-year survival rate was 63% (95% CI: 47 to 78%) in the azacitidine group and 37% (95% CI: 19 to 54%) in the supportive care group (p=0.03; 26% difference with a 95% CI of 3 to 49%). The two-year survival rate was 35% (95% CI: 20 to 50%) in the azacitidine group and 13% (95% CI: 1 to 25%) in the supportive care group (p=0.03; 22% difference with a 95% CI of 3 to 41%). Similarly, statistically significant differences in time to AML transformation, and death or AML transformation, were observed. Transfusion independence was defined as free from transfusions for at least 2 months. Among patients who were RBC transfusion dependent at baseline, a significantly greater number of patients from the azacitidine group (11/25, 44%) compared with patients from the supportive care group (1/14, 7%) achieved transfusion independence (p=0.03; 37% difference with a 95% CI of 7 to 59%). Additionally, patients in the azacitidine group with baseline transfusion independence experienced significantly prolonged duration of RBC (p

Beschreibung: Busulfan based ASCT protocols are effective therapy for patients (pt.s) with poor risk NHL and HD. This therapy historically required pt.s to be hospitalized to manage treatment related toxicities until neutrophil engraftment with a median stay in most institutions of 21 days. We have developed a comprehensive outpatient approach for the management of pt.s undergoing ASCT. While potential benefits include decreased hospital utilization and increased patient satisfaction, there is paucity of data regarding its feasibility and safety. We report on treatment related mortality (TRM) and outcome results using a comprehensive outpatient care approach in 169 consecutive patients with NHL and HD transplanted at a single institution between February 1998 and December 2006. Excluding the planned admission for stem cell infusion on Day 0, all other aspects of their management (chemotherapy, supportive care) were to be performed in the outpatient facility. Exceptions to this model occurred for pt.s with circumstances that might compromise the safety of the outpatient management, otherwise this was standard practice. Pt.s were seen daily in the clinic during the first 30 days, unless they were hospitalized. The most common reasons for admission included the development of neutropenic fever or mucositis. One hundred sixty-nine consecutive pt.s with a median age of 54 yr.s (22–73) underwent ASCT for NHL (57 diffuse B-cell large cell (DLC), 53 HD, 17 Mantle Cell (MC), 24 Follicular NHL (FL), 6 ALCL, 3 Burkitts, and 9 other subtypes). The conditioning regimen consisted of dose targeted oral BU (1 mg/kg every 6 hours) on days -8 thru -5, CY 60 mg/kg on days -3 and -2 and VP-16 10 mg/kg days -4, -3 and -2. Autologous peripheral blood stem cells were used for 151 pt.s, bone marrow for 8 pt.s and 10 pt.s received both cell sources. The median CD34+ cell dose was 4.9 × 106/kg. Median time to neutrophil and platelet engraftment was 11 (range 9–34) and 17 (range 0–85) days, respectively. There were no (0%) treatment related deaths in the 169 pt.s during either the first 100 days or 1 year post-transplant. Eight high risk patients (3 DLC, 2 ALCL, 1 Burkitts NHL, 1 HD and 1 FL) died of progressive disease by day 100 (days 52 - 85). Stratifying for disease risk per the CIBMTR criteria, the 3-year Kaplan-Meier overall survival (OS) and progressive-free survival (PFS) for pt.s with DLC NHL in all pt.s (n=57), intermediate risk pt.s (n=24) and low risk pt.s (CR1, n= 9) is 67%, 86% and 100%; and 46%; 60% and 100%, respectively. The 3-year OS and PFS for pt.s with HD with low/intermediate risk (n=24) and high risk (n=29) is 78% and 72%; and 51% and 45%, respectively. The 3-year OS and PFS for patients with MC NHL (low risk n=9, intermediate risk n=6) is 68% and 65%, respectively. The 3-year OS for pt.s with FL is 75%. In summary, outpatient ASCT with expectant inpatient management for treatment related toxicities can be conducted safely in the setting of a comprehensive outpatient treatment program with treatment related mortality outcomes that is at least as comparable, if not superior, to those reported in the literature for conventional inpatient ASCT for similar patient cohorts.

Beschreibung: Relapse/progression of malignancy (RM) is an important cause of treatment failure and death following allogeneic hematopoietic stem cell transplantation (allo-HCT). CTLA-4 is an important negative regulator of effector T-cell activation. CTLA-4 inhibition has demonstrated potent anti-cancer effects in animal models, and in patients with some solid tumors. CTLA-4 blockade following allo-HCT may potentially augment graft-versus-malignancy but GVHD may also possibly be increased. We report the safety and preliminary efficacy of a dose-escalation study of a neutralizing human monoclonal antibody targeting CTLA-4 (ipilimumab) in patients with RM following allo-HCT. Eligibility criteria included allo-HCT ≥90 days previously, 〉 50% donor T-cell chimerism, no prior grade 3/4 GVHD, no prophylaxis/therapy for GVHD for ≥ 6 weeks. Patients received a single dose of ipilimumab over 90 min. DLI at a dose of 5 × 10e6 CD3 cells/kg was allowed 8 weeks following ipilimumab if no GVHD occurred and RM was present. A total of 29 patients were treated at four centers (23M, 6F; median age 43 (21–65); Hodgkins disease [HD] =14 Myeloma [MM]=6, CML=2, CLL=2, AML=2, NHL=1, Renal Ca =1, Breast Ca=1; 19 related donors, 10 unrelated; 6 myeloablative, 23 RICT).(4 at dose-level 1 [DL1] 0.1 mg/kg, 3 at 0.33 mg/kg [DL2], 4 at 0.66 mg/kg [DL3], 3 at 1.0 mg/kg [DL4] and 15 at 3.0 mg/kg [DL5]). Eight patients had failed prior DLI. Median time between BMT and ipilimumab was 366 d (125–2368). Dose-limiting toxicity was not encountered. No patient developed clinically significant GVHD within 90 days following ipilimumab alone. Three possible immune adverse events were documented: grade 3 polyarthropathy 14 weeks following ipilimumab, and 6 weeks post DLI, which resolved with corticosteroid therapy, (AML, DL1); grade 1 hyperthyroidism with thyroid-stimulating antibody 6 weeks post ipilimumab (CLL, DL3). Grade 2 pneumonitis responsive to corticosteroids (HD, DL5). Ten patients received DLI after ipilimumab. Three patients developed objective evidence of disease response after ipilimumab alone: PR in a patient with mantle cell NHL lasting 3m [DL4]; ongoing CR in a two patients with HD [DL5]. Two of these patients had failed prior DLI. Three additional patients demonstrated possible anti-cancer effects (MR in a HD patient, reduction of PB and BM blasts in AML, DL1; maintenance of molecular remission in a CML patient given ipilimumab alone for 3.5 yrs despite stopping imatinib, DL1). This study shows that doses of ipilimumab (up to 3.0 mg/kg) can safely be administered to patients with RM following allo-HCT without inducing/exacerbating GVHD while inducing regressions of malignancy including durable CR in some patients.

Beschreibung: Abstract 1335 Poster Board I-357 Alloreactive T cells are crucial for graft-versus-host-disease (GVHD) pathophysiology, and we hypothesized that controlling their trafficking can ameliorate GVHD. P-selectin is a dimeric glycoprotein found on most inflamed endothelium, which interacts with multiple lectin-type molecules on leukocytes, including T cells. We used murine allogenienc BMT models to study GVHD and found that P-selectin−/− recipients exhibited significantly less GVHD mortality and morbidity, as well as decreased GVHD of the skin, liver and small bowels. However, WT and P-selectin−/− allo-BMT recipients had comparable large bowel GVHD. This decrease in target organ and systemic GVHD was associated with diminished infiltration of alloactivated T cells into the Peyer's Patches and small bowels, coupled with increased numbers of donor T cells in the spleen and secondary lymphoid organs (SLO) on day 14 and day 35 post-transplant. However, donor alloreactive T cells in WT and P-selectin−/− allo-BMT recipients had similar alloactivation and apoptosis, and donor alloactivated T cells from WT and P-selectin−/− allo-BMT recipients with GVHD showed similar proliferation in vitro in a mixed leukocyte reaction, suggesting that the inflammatory environment in WT and P-selectin−/− recipients was comparable. Finally, non-transplanted P-selectin−/− mice, and P-selectin−/− mice which had received the allo-BMT conditioning regimen but not a donor graft, had similar cellularity in the majority of tissues examined as corresponding WT controls. This suggests that the differential cellularity of donor alloactivated T cells in WT and P-selectin−/− allo-BMT recipients with GVHD is probably largely dependent on trafficking and tissue infiltration during inflammation. Since P-selectin glycoprotein ligand 1 (PSGL1) is the best-described P-selectin ligand, and all leukocytes constitutively bear high levels of membrane PSGL1, we next hypothesized that PSGL1−/− donor alloreactive T cells would be defective in trafficking into GVHD target organs, and that PSGL1−/− donor T cells would cause decreased target organ damage, systemic GVHD, and mortality. However, allo-BMT recipients of WT and PSGL1−/− donor T cells had comparable survival and clinical GVHD scores, and further analyses on day 14 post-transplant revealed that recipients of WT and PSGL1−/− donor T cells also had similar numbers of donor alloactivated T cells in the spleen, liver, mesenteric and peripheral lymph nodes, and Peyer's Patches. Additionally, WT and PSGL1−/− donor T cells had comparable proliferation as measured by CFSE dilution, and comparable alloactivation in vivo as determined by levels of CD25, CD44, and CD62L, suggesting similar T cell function. As PSGL1−/− and WT donor T cells appeared to have equal functionality and accumulated in GVHD target tissues and lymphoid tissues in a similar fashion, we asked whether PSGL1−/− T cells might display other P-selectin ligands. Flow cytometric analyses of T cells from non-transplanted PSGL1−/− mice, and analyses of PSGL1−/− alloactivated T cells on day 14 after allo-BMT, revealed that these cells displayed substantial levels of cell-surface P-selectin ligands as defined by positive staining with recombinant P-selectin-IgG-Fc fusion protein at levels similar to those found on WT T cells, suggesting that although absence of P-selectin on host tissues may ameliorate GVHD, multiple donor leukocyte P-selectin ligands interact meaningfully with P-selectin. Our studies suggest that P-selectin may be required for trafficking into inflamed tissues but not SLO, and that donor T cells may utilize multiple P-selectin ligands apart from PSGL1 to interact with P-selectin and traffic into inflamed tissues during GVHD. We conclude that targeting P-selectin may be a viable target for GVHD prophylaxis or treatment. Disclosures No relevant conflicts of interest to declare.

Beschreibung: AGVHD is a major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Primary treatment of aGVHD with corticosteroids achieves a complete response (CR) rate of 20–40%. Mesenchymal stem cells (MSC) derived from adult bone marrow have immunomodulatory effects. Preclinical and clinical data suggest that MSC inhibit T-cell alloreactivity. Thus, we initiated a trial to investigate the use of Prochymal, ex-vivo cultured adult human MSC derived from unrelated donors, added to conventional steroid therapy for the primary treatment of aGVHD. Patients (pts) and Methods: Pts must have had newly clinically diagnosed aGVHD, grades II-IV using standard grading criteria, after undergoing a related or unrelated allogeneic SCT, or donor lymphocyte infusion (DLI). Endpoints of the study included safety of administration of Prochymal and response rates of aGVHD by day 28 after Prochymal infusion. Treatment with steroids (2mg/kg) and Prochymal, randomized to 2 (low) or 8 million (high) cells/kg, was initiated at the time of GVHD diagnosis. Pts were kept at therapeutic levels of tacrolimus, cyclosporine, or MMF for GVHD prophylaxis. 2 doses of Prochymal were given 3–5 days apart, with the first given within 72 hrs of steroid initiation. Corticosteroids were maintained at 2 mg/kg for at least 1 week. Pts were monitored for response and toxicity weekly for 4 weeks, and then followed for safety on a 2 year long-term follow-up study. Results: 32 pts were enrolled with interim data available for 30 pts (21 males, 9 females) with median age 52 (range 34–67). AGVHD developed following matched sibling (n=15), matched unrelated (n=11), or DLI (n=4) infusions. Overall aGVHD was noted to be grade II (n=20), grade III (n=7), and grade IV (n=3), and involved GI (n=13), skin (n=11), GI and skin (n=4), and GI and liver (n=2). Prochymal dose was low in 17 pts and high in 13 pts. All pts received both Prochymal infusions. 26 of 29 evaluable pts (90%) initially responded to treatment of their aGVHD: 19 achieved CR with no evidence for GVHD, and 7 achieved PR as documented by a reduction in 1 organ stage. Nine pts (31%) eventually required a second line agent to control aGVHD. No infusional toxicities were noted. One pt developed atrial fibrillation 1 day following the second Prochymal infusion. No unexpected ectopic tissue formation was noted in any pt by CT scans at day 28. Currently, 8 pts have died from progression of aGVHD (n=2), intracranial bleed following fall (n=1), relapse (n=1), and infection (n=4) at a median of 44 days (range 13–58) following Prochymal infusion. Conclusions: The addition of Prochymal to corticosteroids resulted in a high response rate with minimal added toxicity when used for the primary treatment of aGVHD, and suggests that MSC have unique biological properties that may be effective for treatment of GVHD. Longer follow-up is necessary to determine if Prochymal has any impact on survival. Phase III studies are warranted, including studies in steroid refractory GVHD.