Sirolimus For Graft-versus-host Disease Prophylaxis Research Articles

Anti-thymocyte globulin with CsA and MMF as GVHD prophylaxis in nonmyeloablative HLA-mismatched allogeneic HCT.

Nonmyeloablative regimens are used for allogeneic hematopoietic cell transplantation (HCT) of older or medically unfit patients, but successful outcome is still hindered by graft-versus-host disease (GVHD), especially in the setting of HLA-mismatched HCT. New GVHD prophylaxis strategies are emerging, including the triple drug strategy, that improve the GVHD-free and relapse-free survival (GRFS). Because the impact of ATG in HLA-mismatched Flu-TBI-based nonmyeloablative HCT has not been investigated, we did a retrospective analysis in three Dutch centers. 67 patients were evaluable, with a median age of 56 years. Overall survival, relapse-free survival and GRFS at 4 years were 52%, 43%, and 38%, respectively. NRM findings and cumulative incidence of relapse at 4 years were 26% and 31%, respectively. At 1-year grade II-IV had occurred in 40% of the patients, and the incidence of moderate-severe chronic GVHD incidence was 16%. Acknowledging the limitations of retrospective analyses, we conclude that the use of ATG for HLA-mismatched truly nonmyeloablative Flu-TBI HCT is feasible and results in acceptable long term outcomes, especially with regards to GRFS. We consider ATG in combination with cyclosporin and mycophenolate mofetil as an alternative for the triple drug strategy that uses sirolimus for GVHD prophylaxis in this particular setting.

Dynamics of Immune Cell Reconstitution in Allogeneic Hematopoietic Cell Transplant Patients Receiving Post-Transplant Cyclophosphamide (PTCy)

In the setting of haploidentical hematopoietic cell transplantation (haplo-HCT), post-transplant cyclophosphamide (PTCy) selectively eliminates alloreactive T cells in-vivo, resulting in favorable graft versus host disease (GVHD), non-relapse mortality (NRM) and relapse outcomes. However, few studies have examined the impact of PTCy on immune reconstitution (IR). We quantified IR in 63 patients after haplo-HCT with PTCy, mofetil mycophenolate and tacrolimus (TAC) and compared results to 93 patients with 8/8 HLA matched related or unrelated donors (MD) receiving TAC, methotrexate and sirolimus for GVHD prophylaxis. Both groups received reduced intensity conditioning for hematologic malignancies. The median age of the Haplo-PTCy and MD cohorts was 55 and 57 years. Patient samples were analyzed using multi-color flow cytometry panels to characterize distinct lymphocyte populations. All IR values are expressed as median absolute cell count per μL. One month after HCT, recovery of all T cell subsets (CD3, CD4Tcon, Treg, CD8) was significantly reduced in the PTCy cohort compared to MD (Figure A, B, C). Recovery of CD4Tcon was also reduced at 2 and 3 months after PTCy (p NK cells were lower 1 month after PTCy (52.7 vs 91.1, p=0.08), but were significantly higher at 2, 3 and 6 months (153.4 vs 94.8, p=0.001, 153.7 vs 87.5, p=0.008, 180 vs 102, p=0.01, respectively, Figure D) compared to the MD cohort. Delayed NK cell recovery at 1 month after PTCy was due entirely to reduced numbers of CD56dim NK cells (Figure E). Subsequently recovery of CD56dim NK cells was similar in both cohorts. Recovery of CD56bright NK cells was significantly increased in the PTCy cohort (p Consistent with prior reports, 1 year cumulative incidence of extensive cGvHD was lower in the PTCy cohort compared to the MD cohort, 13% (5-26%, 95% CI) and 40% (30-50%, 95% CI) respectively, p=0.003, without increased NRM (p=0.28) or relapse (p=0.17). In summary, the effect of PTCy on IR was most pronounced 1 month after transplant with significantly delayed recovery of CD3, CD4Tcon, Treg, CD8 and CD56dim NK cells. Slow recovery of CD4Tcon persisted for 3 months and delayed recovery of Treg persisted for 1 year. Beginning 2 months after HCT, recovery of both CD56dim and CD56bright NK cells was more rapid in the PTCy cohort. Further studies will examine the effects of these differences in IR on clinical outcomes such as relapse, infections and GVHD.

A Phase I Trial of Total Marrow and Lymphoid Irradiation (TMLI)-Based Transplant Conditioning in Patients (Pts) with Relapsed/Refractory Acute Leukemia

Background: Current hematopoietic cell transplant (HCT) regimens for patients with relapsed refractory acute leukemia have 3-year overall survival (OS) rates for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) of 19% and 16% respectively. Previous studies demonstrated that intensification of total body irradiation (TBI) is not possible due to excessive regimen-related toxicity. Because image-guided targeted radiation therapy (e.g., total marrow and lymphoid irradiation (TMLI)) allows for the precise delivery of radiation through the sculpting of radiation to areas of high risk and disease burden, intensification of radiation dose to target structures as part of a HCT preparative may be possible without increased radiation-related toxicities or non-relapse mortality. Herein we report the results of a phase I trial that combines TMLI (1200-2000 cGy) with fixed doses of etoposide (VP16) and cyclophosphamide (CY); the primary objective is to determine the maximum tolerated dose/recommended phase II dose of TMLI.Methods: TMLI together with VP16 and CY before allogeneic HCT was assessed for patients with relapsed/refractory AML and ALL. TMLI was administered on days -10 to -6, VP16 60 mg/kg (adj bw) on day -5, and CY 100 mg/kg (ideal bw) on day -3. The initial radiation dose was 1200 cGy, delivered in 150 cGy fractions twice daily. The radiation dose was escalated in increments of 150 cGy, up to 1500 cGy, by use of a standard 3x3 design. At this point, the dose was raised in 100 cGy increments to a 2000 cGy maximum with a rolling 6 design. Bone marrow (n=3) or peripheral blood stem cells (n=48) were given on day 0. Tacrolimus and sirolimus were administered for graft versus host disease (GVHD) prophylaxis. Dose limiting toxicity (DLT) was defined according to the Bearman and CTCAE 3.0 scales, the latter for hematologic toxicity. The radiation dose delivered to the liver and brain was kept at 1200 cGy. Median normal organs received 16-60% of the dose (oral cavity 28%, lung 44%, esophagus 33%).Results: From 3/14/2008 to 1/30/2014, 51 patients underwent transplantation on this trial. (See table.)Our phase I trial/safety studies found the TMLI/CY/VP16 conditioning regimen to be well tolerated at TMLI doses up to 2000 cGy; 1-year estimates of non-relapse mortality and overall survival were 8.3% (95% CI: 2.6-18.4) and 54.4% (95% CI: 39.3-67.3) respectively (median follow-up: 23.5 months).Relapsed, progressed, or persistent disease after transplant occurred in 33 patients (bone marrow, 26; extramedullary disease, 6; concurrent bone marrow/extramedullary, 1). Of the 18 patients who were treated with a dose of 1700 cGy or higher, 17 achieved a complete remission at the day +30 evaluation. No radiological-based maximum tolerated dose (MTD) was defined. We determined that the median organ dose at 2000 cGy would be lower than that seen for total body irradiation (TBI), but a higher dose may result in reaching or exceeding TBI organ dose levels. We therefore stopped at 2000 cGy, above which non-targeted organs may no longer be protected.Acute GVHD (aGVHD) developed in 28 (55%) of patients; of those 7 (14%) developed grades 3-4. The most common toxicities across the tested dose levels were grade 1 GI toxicity and grade 2 stomatitis. One patient (treated at 1500cGy) developed grade 3 stomatitis. No additional DLTs were experienced across all dose levels.Conclusion: A dose of 2000 cGy targeted to lymph nodes and marrow in combination with CY and VP16 can be safely administered in the context of related and unrelated HCT, using tacrolimus and sirolimus for GVHD prophylaxis. We did not see increased incidence of aGVHD, and the day +100 NRM rate was <5%. A phase II trial is currently being conducted.TablePatient characteristicsVariableMedian (range) or NAge at transplant (yrs)34 (16-57)Disease diagnosis AML ALL Ph- ALL Ph+ biphenotypic undifferentiated3313221Disease status at HSCT 1 RL 2 RL IF14334Cytogenetic risk (SWOG criteria) favorable intermediate unfavorable unknown significance122199KPS at HSCT80 (60-100)Donor source sibling HLA matched unrelated mismatched (1 allele) unrelated25521WBC at HSCT1.4 (0.1-14.9)% Blasts in blood at transplant*4 (0-93)% Blasts in marrow at transplant*52 (8-98)Extramedullary disease at time of HSCT11*Excludes patients with solely extramedullary disease, n=4 DisclosuresStein:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Research Funding. Snyder:Incyte: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Forman:Mustang: Research Funding; Amgen: Consultancy.

Aprepitant (Emend®) Significantly Increases Sirolimus Levels In Patients Undergoing Allogeneic Hematopoietic Stem Cell transplantation.

AbstractAbstract 1311Drug interactions are common with immunosuppressive agents used during the process of hematopoietic stem cell transplant (HCT). Aprepitant (Emend®) (APR), a selective antagonist of substance P/neurokinin-1 (NK1) receptors, is a novel antiemetic agent that is increasingly used for patients undergoing HCT. APR is both a substrate and a moderate inhibitor of human cytochrome P450 (CYP450), specifically CYP3A4. Use of APR presents a potential for interactions with immunosuppressive medications. Tacrolimus and sirolimus are immunosuppressive agents commonly used for prevention and treatment of graft versus host disease (GVHD) in patients undergoing allogeneic HCT. These two drugs are substrates for CYP3A4 isoenzyme and hence agents that are known to inhibit CYP3A4 may increase bioavailability of these immunosuppressants. We retrospectively examined the effect of oral APR on serum tacrolimus/sirolimus drug levels in 14 consecutive patients (APR treated group) and compared these data to 71 consecutive control patients (control group) who did not receive APR during conditioning for allogeneic stem cell transplant. Patients for both groups were identified and selected for analysis from a prospective observational research database. All patients underwent reduced-intensity Allo HCT using fludarabine and melphalan conditioning regimen from May 2008 to July 2009 at City of Hope National Medical Center.Oral APR was administered as125 mg on day -4 and 80 mg on days -3 and -2 of HCT process. The first dose of APR was given on the same day as melphalan (day -4). Tacrolimus (0.02 mg/kg/day) and sirolimus (12 mg loading dose, followed by 4 mg/day) were started on day -3; doses were subsequently adjusted to maintain drug levels of 5–10 ng/ml. Baseline characteristics were similar for patients in both groups in terms of age, gender, donor type, stem cell source and GVHD prophylaxis regimen. Drug levels were monitored twice a week; the median number of drug levels tested over the first 30 days post-HCT were 8 for APR group and 8.5 for the control group. The post-loading sirolimus levels (drawn 1 to 3 days after loading dose) were significantly higher in APR group (29.2 vs. 13.5 ng/ml, p=0.003, Table), while the levels were not different for tacrolimus (9.2 vs. 8.6 ng/ml, p=0.13). There was a trend for higher median tacrolimus levels over the first 30 days for the patients in the Control group (8.2 vs. 7.3 ng/ml, p=0.05). In response to elevated post-loading sirolimus levels, clinicians drastically reduced the sirolimus dose which led to sub-therapeutic sirolimus levels (< 5 ng/ml) in 6/14 patients in the APR group, however, there were no obvious differences in the incidence of early non-relapse related mortality or acute GVHD. Thrombotic microangiopathy was not observed in any of the patients in the APR group. Our data demonstrate that concomitant administration of APR with sirolimus leads to a two fold increase in sirolimus levels. Close monitoring and dose-adjustment of sirolimus is warranted for patients receiving these two drugs concomitantly in the context of allogeneic HCT. [Display omitted] Disclosures:Off Label Use: tacrolimus and sirolimus for GVHD prophylaxis (off-label use).

Prevention of Acute GvHD During MHC Haploidentical BMT: Evaluating the Efficacy of T-Cell Costimulation Blockade Using a Novel Rhesus Macaque Transplant Model.

Abstract 2455Poster Board II-432 Introduction:While hematopoietic stem cell transplantation (HSCT) offers a cure for many hematologic diseases, it remains plagued by often fatal graft-versus-host disease (GvHD). Despite the inadequacy of current GvHD prevention strategies, especially for MHC-mismatched HSCT, the pace of the clinical introduction of novel therapeutics has been slow, likely due to the lack of a suitable translational model to rigorously test the immunologic and clinical impact of novel biologic therapies. Among the most promising of these therapies include those that block T cell costimulation blockade. While they have been used for both autoimmune disease and to prevent rejection of solid organ transplants, costimulation blockade reagents have not yet been evaluated for efficacy in preventing clinical GvHD. Here we describe a novel primate model of MHC-mismatched GvHD, that has allowed us, for the first time, to evaluate the mechanisms controlling GvHD in a primate translational system, and to evaluate the efficacy of costimulation blockade for the prevention of primate GvHD, even across haplo-MHC barriers. Methods:Using DNA microsatellite-based pedigree analysis and MHC haplotype determination, we have developed the first MHC-defined Rhesus macaque HSCT system. MHC haplo-identical transplant pairs were chosen, and recipients prepared for transplant with TBI (8 Gy, as a single dose, with lung shielding to 6 Gy). Animals were either treated with no immunosuppression post-transplant (controls) or with a costimulation blockade-based regimen which included CD28/B7 blockade with abatacept (20mg/kg every 7 days), CD40/CD154 blockade with the 3A8 anti-CD40 monoclonal antibody (maintenance dosing at 5mg/kg twice weekly) as well as sirolimus to maintain serum trough levels between 5-10 ng/mL. Either leukopheresis-derived peripheral blood stem cells or bone marrow was used for transplant (average total nucleated cell dose = 9.3 +/-2.7×108/kg; average CD3+ cell dose = 1.1 +/- 0.88 ×108/kg) Donor engraftment was measured by microsatellite analysis, and GvHD was graded clinically using standard scales. The immune phenotype after transplant was determined by multicolor cell- and serum-based flow cytometric analyses. Results:Seven haploidentical transplants have been completed. Three controls received no immunosuppression. These animals demonstrated rapid and complete donor engraftment, with donor T cell activation and proliferation occurring within one week of transplant, coincident with the onset of severe clinical GvHD, which predominantly targeted the GI tract. Flow cytometric analysis showed loss of CD127 expression on both CD4+ and CD8+ T cells, consistent with their rapid clonal expansion and differentiation. Multiplexed luminex cytokine analysis demonstrated high-level secretion of the inflammatory cytokines IFNγ, and IL18, as well as the counter-regulatory cytokine IL-1RA. Importantly, no rise in TNF, IL-1b, nor IL17 was measured despite severe GvHD.In contrast, four treated animals received a haplo-identical BMT in the setting of abatacept/anti-CD40 and sirolimus for GvHD prophylaxis. All of these recipients demonstrated rapid donor engraftment, but, unlike the controls, they were protected against clinical GvHD—they displayed neither the skin rash nor the profuse diarrhea noted in the control animals. Flow cytometric analysis demonstrated maintenance of CD127 expression on both CD4+ and CD8+ T cells. Furthermore, luminex analysis revealed that expression of IFNγ, IL18 and IL-1RA were all normal in the setting of GvHD prophylaxis with costimulation blockade and sirolimus. Conclusions:We have established a robust model of haplo-identical HSCT and GvHD using an MHC-defined Rhesus macaque colony. This model has allowed us to begin to determine the mechanisms underlying GvHD during primate haplo-identical BMT and to assess the efficacy of novel regimens to prevent this disease. We find that unprotected primate GvHD is characterized by rapid T cell proliferation, with concomitant loss of expression of CD127 on both CD4+ and CD8+ T cells. In addition, it is associated with a cytokine storm, including high level secretion of IFNγ, IL18 and IL-1RA into the serum. Finally, we find that CD28/CD40-directed costimulation blockade in combination with sirolimus can effectively inhibit both the clinical and cellular hallmarks of GvHD during haplo-identical BMT, and thus may deserve close clinical scrutiny as a possible prophylaxis strategy during these high risk transplants. Disclosures:No relevant conflicts of interest to declare.

Posterior reversible encephalopathy syndrome due to sirolimus

Posterior reversible encephalopathy syndrome (PRES) was first described in 15 patients by Hinchey et al. in 1996.1 The most common clinical symptoms and signs included headache, altered alertness and confusion, vomiting, seizures, and visual disturbance ranging from blurred vision to cortical blindness. Neuroimaging typically showed white matter edema in the posterior portions of the cerebral hemispheres, particularly in the parieto-occipital regions. The presumed etiologies for the 15 patients included hypertensive encephalopathy (n=4), preeclampsia (n=3), cyclosporine (n=4), tacrolimus (n=3), and interferon- (n=1). With control of blood pressure and withdrawal or reduction of the immunosuppressive agent, all of the patients had resolution of symptoms within 2 weeks and neuroimaging normalized. Since the initial description of PRES, there have been a number of reports of its incidence following allogeneic bone marrow transplant in patients receiving cyclosporine or tacrolimus for graft-versus-host disease (GVHD) prophylaxis.2, 3, 4 Toxic levels of the immunosuppressive medications do not appear to be required for the development of PRES.5 In a previous report of a lung transplant recipient patient on sirolimus that developed a clinical syndrome similar to PRES, the neurotoxicity was attributed to dialysis disequilibrium syndrome.6 The patient improved with adjustment in the dialysis regimen and continued on sirolimus without recurrent neurological complaints. An association between treatment with sirolimus and PRES has otherwise never been reported. Here we present a case of PRES occurring in the setting of treatment with sirolimus for GVHD prophylaxis.

Overcoming T cell–mediated rejection of bone marrow allografts by T-regulatory cells: Synergism with veto cells and rapamycin

Recently, we have shown that anti-third-party cytotoxic T lymphocytes (CTLs) depleted of alloreactivity against the host are endowed with marked veto activity and can facilitate bone marrow (BM) allografting without graft-versus-host disease. We also demonstrated synergism between rapamycin (RAPA) and the veto cells. CD4(+)CD25(+) T-regulatory (Treg) cells are suppressor cells that can enhance alloengraftment. We investigated whether donor Tregs would be synergistic with veto CTLs and RAPA in augmenting alloengraftment or, conversely, would suppress veto CTL effects. Lethally irradiated C3H mice were transplanted at day 2 after irradiation with Balb-nude BM. Graft rejection was induced by purified host-type T cells infused 1 day prior to BMT. The addition of Tregs led to moderate enhancement of engraftment. RAPA at different doses was synergistic with Tregs. The addition of veto CTLs to Tregs enabled reducing the effective RAPA dose fourfold. Combining all three agents was necessary to overcome rejection at low-dose RAPA. Chimerism analysis at 5 to 9 months revealed a significant presence of host-type cells coexisting with the predominant donor T cells, suggesting that tolerance had been attained. The synergistic effects between Tregs, veto CTLs, and RAPA offer an attractive approach for facilitating alloengraftment.