ALBERT

Description: Because of its potent immunosuppressive yet stem cell–sparing activity, high-dose cyclophosphamide was tested as sole prophylaxis of graft-versus-host disease (GVHD) after myeloablative allogeneic bone marrow transplantation (alloBMT). We treated 117 patients (median age, 50 years; range, 21-66 years) with advanced hematologic malignancies; 78 had human leukocyte antigen (HLA)–matched related donors and 39 had HLA-matched unrelated donors. All patients received conventional myeloablation with busulfan/cyclophosphamide (BuCy) and T cell–replete bone marrow followed by 50 mg/kg/d of cyclophosphamide on days 3 and 4 after transplantation. The incidences of acute grades II through IV and grades III through IV GVHD for all patients were 43% and 10%, respectively. The nonrelapse mortality at day 100 and 2 years after transplantation were 9% and 17%, respectively. The actuarial overall survival and event-free survivals at 2 years after transplantation were 55% and 39%, respectively, for all patients and 63% and 54%, respectively, for patients who underwent transplantation while in remission. With a median follow-up of 26.3 months among surviving patients, the cumulative incidence of chronic GVHD is 10%. These results suggest that high-dose posttransplantation cyclophosphamide is an effective single-agent prophylaxis of acute and chronic GVHD after BuCy conditioning and HLA-matched BMT (clinicaltrials.gov no. NCT00134017).

Description: Key Points Posttransplantation cyclophosphamide is effective as sole GVHD prophylaxis for myeloablative HLA-matched–related or –unrelated BMT. Despite low chronic GVHD with PTCy, relapse and survival are comparable with outcomes reported using other GVHD prophylactic approaches.

Description: Background: The results of NMA, related HLA-haploidentical (haplo) BMT with PTCy are comparable to matched BMT (Blood 2015;125;3024). However, patients (pts) may lack related donors, often because of familial disease susceptibility or cytotoxic antibodies against donor HLA molecules. Although partially HLA-mMUD BMT could be considered if a MUD is unavailable, mismatches 〉 1 HLA antigen have historically been associated with unacceptable rates of GVHD, graft failure, and nonrelapse mortality (NRM). We hypothesized that PTCy may overcome this HLA barrier. Methods: We developed a prospective, regimen-finding study of NMA BMT for hematologic malignancies pts without an acceptable matched or first-degree related haplo donor. The objective was to identify a regimen with ≤ 25% severe acute GVHD and ≤ 20% NRM by day 100, using mMUD's or non-first-degree relatives. Results of mMUD BMT with our standard conditioning are presented. Pt eligibility included age 0.5-75 years, no suitable family donor, ECOG PS ≤ 2, LVEF ≥ 35%, FEV1 and FVC ≥ 40% predicted, and adequate hepatic function. The mismatched donor shared ≥ 5/10 HLA alleles (based on molecular typing of HLA-A, -B, -Cw, -DRB1, and -DQB1) with ≥ 1 allele matched for a HLA class I gene and ≥ 1 allele matched for a class II gene. Donors matched at ≥ 1 allele at each locus were prioritized, followed by donors having the fewest number of mismatched loci. All pts received fludarabine (30 mg/m2 IV days -6 to -2), Cy (14.5 mg/kg IV days -6 and -5), TBI (200 cGy day -1) and T-cell replete bone marrow. GVHD prophylaxis consisted of high-dose PTCy (50 mg/kg IV days 3 and 4), mycophenolate mofetil on days 5-35, and either sirolimus (15 pts) or tacrolimus (1 pt) on days 5-180. GCSF was given from day 5 until neutrophil recovery. Safety was continuously monitored. Results: Sixteen pts (median age 57, range 37-66) received mMUD BMT on this study from 7/2011-6/2014 (9 AML cases, 4 MDS or MPN, 1 CML, 2 PTCL). Major reasons for utilizing a mMUD included the absence of an adult haplo first-degree relative and the presence of prohibitive donor-specific anti-HLA antibodies (DSA's). The unrelated grafts had a median of 2 HLA mismatches and included three 7/10 matches, one 6/10 match, and one 5/10 match. HLA-C mismatch was present in 10 grafts (63%) and complete DP mismatch in 6 (38%).By revised Disease Risk Index, 25% of pts were high risk, 69% intermediate risk, and 6% low risk; 69% were in CR at BMT. The median graft doses were 3.36 x 108 total nucleated cells/kg and 3.87 x 107 CD3+ cells/kg. All pts engrafted, and there were no non-relapse deaths or prohibitive toxicities. By competing-risk analysis, the estimated cumulative incidence (CuI) of neutrophil recovery was 100% by day 29, with a median of 20 (range 14-29) days. The CuI of platelet recovery ≥ 20,000/µL was 100% by day 46, with a median of 32 (range 12-46) days. By day 60, 14/15 (93%) evaluable pts achieved full donor chimerism in either CD3+ or unsorted cells. Notably, there were no cases of acute grade 3-4 GVHD. Acute grade ≥ 2 GVHD was limited to 2 cases of skin-only grade 2 GVHD and 1 case of grade 2 skin and ungradable visceral GVHD. By competing-risk analysis, the estimated CuI of acute grade 2 GVHD was 12% at day 100 (90% CI, 0-27%) and 19% at day 180 (90% CI, 2-37%)(Fig A). The CuI of any chronic GVHD at 1 and 2 years was 7% (90% CI, 0-18%)(Fig A). Critical illness prior to relapse was limited to 1 case of sepsis which resolved. With a 3-year median follow-up, the probability of PFS was 56% at 1 year and 50% at 3 years (Fig B). The estimated CuI of relapse was 44% at 1 year. Median OS has not been reached, with an estimated 1 year and 3 year OS of 68% (Fig B). Conclusion: These results suggest that virtually no pt should be denied allogeneic BMT because of lack of an HLA-matched or haplo donor. mMUD searches were successful in pts with DSA's that were too high for desensitization. Disclosures Off Label Use: posttransplantation cyclophosphamide for GVHD prophylaxis.

Description: Tissue-derived adenosine, acting via the adenosine A2A receptor (A2AR), is emerging as an important negative regulator of T-cell function. In this report, we demonstrate that A2AR stimulation not only inhibits the generation of adaptive effector T cells but also promotes the induction of adaptive regulatory T cells. In vitro, antigen recognition in the setting of A2AR engagement induces T-cell anergy, even in the presence of costimulation. T cells initially stimulated in the presence of an A2AR agonist fail to proliferate and produce interleukin-2 and interferon (IFN)-γ when rechallenged in the absence of A2AR stimulation. Likewise, in an in vivo model of autoimmunity, tissue-derived adenosine promotes anergy and abrogates tissue destruction. Indeed, A2AR stimulation inhibits interleukin-6 expression while enhancing the production of transforming growth factor-β. Accordingly, treating mice with A2AR agonists not only inhibits Th1 and Th17 effector cell generation but also promotes the generation of Foxp3+ and LAG-3+ regulatory T cells. In this regard, A2AR agonists fail to prevent autoimmunity by LAG-3−/− clonotypic T cells, implicating an important role for LAG-3 in adenosine-mediated peripheral tolerance. Overall, our findings demonstrate that extracellular adenosine stimulates the A2AR to promote long-term T-cell anergy and the generation of adaptive regulatory T cells.

Description: Allogeneic transplantation of hematopoietic stem cells remains the only curative approach for patients with sickle cell disease (SCD), yet procedural toxicities and graft-versus-host disease limit this approach to children. We chose a low-dose radiation approach utilizing rapamycin based upon its unique ability to promote T cell tolerance even when T cells are stimulated in the presence of costimulation (Powell, et al, J Immunol. 1999). We confirmed this approach in vivo in a murine bone marrow transplantation model comparing a short course of conventional immunosuppression with cyclosporine to that with rapamycin, with long-term, high-level chimerism attained only in mice treated with rapamycin (Powell et al., manuscript submitted). We have now begun accrual to an IRB approved clinical trial testing this approach in adults with SCD and herein report the results in our first two subjects. Protocol entry criteria include those previously reported (Walters, et al, NEJM, 1996) with the addition of pulmonary hypertension defined as a tricuspid-regurgitant jet velocity (TRV) 〉 2.5 m/s (Gladwin et al, NEJM, 2004). Additionally, patients must have failed a 6-month course of hydroxyurea. The conditioning regimen consists of a single radiation dose of 300cGy, alemtuzumab (1mg/kg total), and oral rapamycin targeting trough levels between 10–20 ng/ml. The graft consists of unmanipulated mobilized peripheral blood progenitors obtained from an HLA-matched sibling. The first patient is a 24-year-old female referred due to recurrent transient ischemic attacks and strokes despite chronic exchange transfusions. Her initial evaluation was notable for evidence of significant hemolysis with a total bilirubin of 9.8 mg/dl, an LDH 1,172 units/L (range 113–226), an absolute reticulocyte count of 318,000/uL and an undetectable haptoglobin. Cardiac doppler-echocardiogram revealed a TRV of 3.7 m/s, consistent with severe pulmonary hypertension. The conditioning was well tolerated and the patient did not require parenteral antibiotics or nutritional support. Assessment of donor chimerism, measured by microsatellite PCR and hemoglobin electrophoresis, revealed an early peak of myeloid chimerism which has stabilized at approximately 60%, with lower levels of lymphoid chimerism at approximately 5–10% now more than 300 days post-transplant. Hemoglobin levels stabilized at 11–12 g/dL, without detectable sickle hemoglobin, now allowing for therapeutic phlebotomy. Remarkably, the LDH and TRV fell concurrently toward the normal range. Patient 2 is a 26 year old male with frequent crises requiring hospitalization twice monthly. At one month post-transplant, myeloid chimerism is 97%, with lymphoid chimerism currently unmeasurable due to lymphopenia, and similar to that seen in patient 1 at the same time point. Patient 2 also required neither parenteral antibiotics nor nutritional support. Finally, neither patient to date has developed any symptoms or signs of either acute or chronic GVHD. Thus with two patients accrued to date, we have demonstrated the ability of allogeneic HSC transplantation to achieve mixed hematopoietic chimerism without the development of GVHD and for the first time, established the reversibility of the associated pulmonary hypertension.

Description: Murine models of bone marrow transplantation were used to study the mechanisms governing the activation of donor lymphocyte infusions (DLIs) manifesting as lymphohematopoietic graft-versus-host (LH-GVH) and graft-versus-leukemia (GVL) reactivities. We demonstrate here that established mixed chimerism influences the potency of DLI-mediated alloreactivity only in the MHC-mismatched but not MHC-matched setting. In the MHC-matched setting, high levels (≥ 40%) of residual host chimerism correlated negatively with DLI-mediated alloreactivity irrespective of the timing of their administration, the donor's previous sensitization to host antigens, or the level of residual host APCs. In vivo administration of Toll-like receptor (TLR) ligands was required to maximize DLI-mediated LH-GVH and GVL reactivities in chimeras with low levels (≤ 15%) of residual host chimerism. In contrast, coadministration of DLI with antigen-presenting cell (APC) activators was insufficient to augment their LH-GVH response in the presence of high levels of host chimerism unless the host's T cells were transiently depleted. Together, these results show the cardinal influence of donor-host incompatibility on DLI-mediated GVH responses and suggest that in MHC-matched chimeras, the induction of optimal alloreactivity requires not only donor T cells and host APCs but also TLR ligands and in the presence of high levels of host chimerism depletion of host T cells.