Combination Of Sirolimus Research Articles

Significant Activity Of The mTOR Inhibitor Sirolimus and HDAC Inhibitor Vorinostat In Heavily Pretreated Refractory Hodgkin Lymphoma Patients

BackgroundPreclinical models have suggested that HDAC and mTOR inhibitors have synergistic antineoplastic activity in Hodgkin lymphoma and other cancers by reducing the activity of AKT, mTOR and HDAC. MethodsWe designed a phase I study to determine the safety of the mTOR inhibitor sirolimus (1mg-5mg PO daily q 28 days) and HDAC inhibitor vorinostat (100mg-400mg PO daily q 28 days) in advanced cancers with an expansion cohort at the recommended phase 2 dose (RP2D) of sirolimus 4mg and vorinostat 300mg for patients with refractory classical Hodgkin lymphoma. The expansion cohort included optional pre- and post-treatment tumor biopsies, peripheral blood mononuclear cells (PBMCs), plasma/serum collections for pharmacodynamic (PD) and pharmacokinetic (PK) endpoints. ResultsA total of 16 patients (men, n=8; women, n=8), median age 33 years, median of 6.5 prior therapies (including autologous SCT [n=15], autologous and allogeneic SCT [n=4]) were enrolled in dose escalation (n=1) or RP2D (n=15) cohorts. At the median follow-up of 3.2 months, 15 (94%) patients demonstrated decreased FDG uptake including 2 CRs (13%) and 5 PRs (31%) using CHESON criteria, with a total CR+PR rate of 44%. The median reduction of tumor was -27% (-100% to +20%, Figure). The estimated median time-to-treatment failure has not been reached and 63% of the patients continued on therapy with further improvement in their level of disease response. Major grade 3-4 treatment-related toxicities included grade 3 thrombocytopenia (4 patients, 25%), grade 4 thrombocytopenia (6, 38%), grade 3 anemia (1, 6%), febrile neutropenia (1, 6%) and led to treatment interruptions and/or dose modifications in 10 (63%) patients. Five (31%) patients had archival tissue available for targeted next-generation sequencing and one patient had a loss of TSC2, an abnormality that putatively activates mTOR. This patient has had a continuing response to therapy (-56%) for 8.4+ months. PD studies in pre- and post-treatment tumor biopsies, PBMCs and plasma as well as PK analysis continue. ConclusionThe combination of sirolimus and vorinostat is well tolerated with encouraging activity in very heavily pretreated patients with Hodgkin lymphoma refractory to standard therapies. Enrollment continues. [Display omitted] Disclosures:Off Label Use: sirolimus - mTOR inhibitor vorinostat - HDAC inhibitor.

Clinicopathologic features of post‐transplant lymphoproliferative disorders arising after pediatric small bowel transplant

Few studies examined the clinicopathologic features of PTLD arising in pediatric SBT patients. Particularly, the association between ATG and PTLD in this population has not been described. Retrospective review of 81 pediatric patient charts with SBT--isolated or in combination with other organs--showed a PTLD incidence of 11%, occurring more frequently in females (median age of four yr) and with clinically advanced disease. Monomorphic PTLD was the most common histological subtype. There was a significant difference in the use of ATG between patients who developed PTLD and those who did not (p < 0.01); a similar difference was seen with the use of sirolimus (p < 0.001). These results suggested a link between the combination of ATG and sirolimus and development of more clinically and histologically advanced PTLD; however, the risk of ATG by itself was not clear. EBV viral loads were higher in patients with PTLD, and median time between detection of EBV to PTLD diagnosis was three months. However, viral loads at the time of PTLD diagnosis were most often lower than at EBV detection, thereby raising questions on the correlation between decreasing viral genomes and risk of PTLD.

Additive effect of sirolimus and anti‐death receptor 5 agonistic antibody against hepatocellular carcinoma

Despite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10-20% of cases after liver transplantation, and the use of potent adjuvant anticancer drugs would be welcome. The aim of this study was to evaluate the efficiency of a combined therapy of rapamycin (sirolimus) and anti-death receptor (DR)5 monoclonal antibody (mAb) on HCC. We first assessed the side effects of anti-DR5 mAb administration in vivo by giving various doses of anti-DR5 mAb. Cell proliferation assays were then performed using mouse Hepa1-6 cells or human Huh7 cells to quantify the relative cell viability under various concentrations of sirolimus, anti-DR5 mAb or a combination. Next, one million Hepa1-6 cells were transplanted into C.B17-SCID-beige mice subcutaneously, and four groups were created: (1) untreated, (2) anti-DR5 mAb alone, (3) sirolimus alone and (4) anti-DR5 mAb+sirolimus. Anti-DR5 mAb (200 and 300μg/day) induced liver dysfunction with partial necrosis of the liver, but 100μg/day was well tolerated with transaminitis, but normal bilirubin and only minor histological liver damage. In vitro, anti-DR5 mAb lysed Hepa1-6 and Huh7 cells in a dose-dependent manner, and combinations of sirolimus and anti-DR5 mAb demonstrated an additive effect. In vivo studies demonstrated that tumour sizes were significantly smaller in the combined therapy group than in the monotherapy groups. Combining sirolimus and low-dose anti-DR5 mAb has a significant effect against HCC. This strategy represents a potential novel approach for the management of HCC.

Belatacept and Sirolimus Prolong Nonhuman Primate Islet Allograft Survival: Adverse Consequences of Concomitant Alefacept Therapy

Calcineurin inhibitors (CNI) and steroids are known to promote insulin resistance, and their avoidance after islet transplantation is preferred from a metabolic standpoint. Belatacept, a B7-specific mediator of costimulation blockade (CoB), is clinically indicated as a CNI alternative in renal transplantation, and we have endeavored to develop a clinically translatable, belatacept-based regimen that could obviate the need for both CNIs and steroids. Based on the known synergy between CoB and mTOR inhibition, we studied rhesus monkeys undergoing MHC-mismatched islet allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on CoB-resistant rejection, some animals also received CD2 blockade with alefacept (LFA3-Ig). Nine rhesus macaques were rendered diabetic with streptozotocin and underwent islet allotransplantation. All received belatacept and sirolimus; six also received alefacept. Belatacept and sirolimus significantly prolonged rejection-free graft survival (median 225 days compared to 8 days in controls receiving basiliximab and sirolimus; p = 0.022). The addition of alefacept provided no additional survival benefit, but was associated with Cytomegalovirus reactivation in four of six animals. No recipients produced donor-specific alloantibodies. The combination of belatacept and sirolimus successfully prevents islet allograft survival in rhesus monkeys, but induction with alefacept provides no survival benefit and increases the risk of viral reactivation.

Effectiveness of Sirolimus in Combination with Cyclosporine against Chronic Rejection in a Pediatric Liver Transplant Patient

The patient is a 3-year-old boy who received living-donor liver transplantation (LDLT) for hepatoblastoma, with his mother as the donor. Oral tacrolimus was started at a dose of 0.3 mg every 12 h from day 1, with the dosage adjusted on the basis of trough concentrations. The levels of aspartate aminotransferase (AST), alanine transferase (ALT), and total bilirubin (T-bil) were 110 U/L, 182 U/L, and 12.6 mg/dL, respectively, when chronic rejection (CR) was pathologically diagnosed. Then, sirolimus at a dose of 1.0 mg/d was added to the tacrolimus-based regimen. The T-bil level rapidly decreased to 5.4 mg/dL, without changes in AST and ALT. Because the intracellular receptor of sirolimus and tacrolimus is FK506-binding protein 12, we switched tacrolimus to cyclosporine at a dose of 60 mg/d to avoid competitive inhibition between these 2 drugs. The target trough concentration of sirolimus and cyclosporine was set to around 15 ng/mL and 180 ng/mL, respectively. The concentration/dose ratio of sirolimus was significantly correlated with the blood cyclosporine level (r=0.5293, p<0.05), suggesting the pharmacokinetic interaction between these 2 drugs. Thereafter, the levels of AST and ALT as well as the T-bil were successfully decreased to 73 U/L, 83 U/L, and 3.0 mg/dL, respectively. These results suggest that sirolimus therapy in combination with cyclosporine may be an effective treatment against CR after liver transplantation.

Combination Therapy of Sirolimus and Sorafenibfor Recurrent Hepatocellular Carcinoma after Liver Transplantation

Backgrounds: Sirolimus and sorafenib both have been used in recurrent Hepatocellular Carcinoma (HCC) patients after Liver Transplantation (LT). In the present study, we evaluated the side effects and efficacy of acombination therapy consisting of sirolimus and sorafenib. Methods: We retrospectively reviewed patients who had recurrent HCC after LT between 2005 and 2012. Toxicity was evaluated by reviewing medical records for each follow-up visit. Efficacy was evaluated according to the modified RECIST guidelines. Results: A total of 24 patients who received combination therapy were reviewed to evaluate drug toxicity. Side effects included hand-foot syndrome (n=12, 50%), diarrhea (n=7, 29.2%), fatigue (n=2, 8.3%), and alopecia (n=1, 4.2%). Among the 24 patients enrolled in this study, 19 were evaluated for efficacy. A complete response was observed in only 1 case (5.3%), while a partial response was observed in 2 cases (10.5%). Five cases (26.3%) showed disease stabilization. The median overall survival after initiation of the combination therapy was 21.6 months. In comparison, 26 recipients with recurrent HCC received non-combination therapy. The median survival of patients receiving a noncombination therapy was 12.0 months. However, there was no statistically significant difference in patient survival rate between the combination and non-combination therapy groups (P=0.101). Conclusion: Combination therapy of sorafenib and sirolimus for recurrent HCC LT recipients may be useful for disease management. However, controlled prospective study is needed to further evaluate the safety and efficacy of combined sorafenib and sirolimus therapy.

Synergistic Control of Acute GvHD: Effectively Down-Regulating T Cell Proliferation and Cytotoxicity with Combined mTOR Inhibition and CD28:CD80/86 Costimulation Blockade.

AbstractAbstract 2998 Introduction:GvHD remains the most deadly complication of HSCT despite current prevention strategies. To address the unmet need for better GvHD control, we have created a non-human primate (NHP) model with which to rigorously test mechanism and efficacy of novel therapeutics. In this study, we determined whether a novel combination of mTOR inhibition (with sirolimus) and CD28:CD80/86 costimulation blockade (with belatacept) could control GvHD. Here we show for the first time that these two agents combine synergistically to prevent both the clinical and immunologic manifestations of primate aGvHD. Methods:Rhesus macaque recipients were irradiated (9.6 Gy in 2 fractions at 7cGy/min), and then transplanted with G-CSF-mobilized PBSC from a haplo-identical donor (1–5×108 TNC/kg). Recipients were treated with either sirolimus alone (n = 4, troughs targeted at 5–10 ng/mL), belatacept alone (receiving weekly doses of 20 mg/kg), or combination therapy. Clinical GvHD was monitored using our previously described NHP grading scale (Miller et al., Blood 2010), and multiparameter flow cytometric analysis was performed. Results:Untreated controls (n = 5) developed rapid, severe histopathologically-proven aGvHD and succumbed rapidly (MST = 7 days). Recipients treated with either sirolimus or belatacept alone were partially protected from the clinical manifestations of GvHD. Sirolimus-treated recipients (n = 6) developed predominantly GI disease (with diarrhea but no elevation of bilirubin) and had an MST of 14 days (Figure 1). Recipients treated with belatacept alone (n = 3) developed primarily liver aGvHD (bilirubin rapidly rising to 6–30 × normal with histologically-confirmed lymphocytic infiltration) and an MST of 11 days. In striking contrast, recipients treated with combined sirolimus + belatacept (n = 5) demonstrated neither uncontrolled diarrhea nor hyperbilirubinemia at the timed terminal analysis (1 month post-transplant).We employed multiparameter flow cytometry to determine the immunologic consequences of sirolimus and belatacept on T cell proliferation (using Ki-67 expression) and cytotoxity (using granzyme B expression). We found that the clinical synergy observed with combined therapy was recapitulated immunologically. Thus, while untreated aGvHD was associated with rampant CD8+ proliferation (with 83 +/− 14% Ki-67+ CD8+ vs 4.7 +/− 0.6% pre-transplant), sirolimus or belatacept as monotherapy both partially controlled proliferation (35 +/− 3% and 65 +/− 23% Ki-67+ CD8+ with sirolimus or belatacept, respectively). Combined sirolimus + belatacept dramatically reduced proliferation (to 8 +/− 3%, favorably comparing with 13% Ki-67+ CD8+ T cells using standard Calcineurin Inhibitor/Methotrexate (CNI/MTX) prophylaxis).Sirolimus and belatacept both also partially controlled GvHD-related T cell cytotoxicity. Thus, while untreated aGvHD was associated with excessive granzyme B expression in CD8+ T cells (82 +/− 2% granzyme Bvery high CD8+ cells vs 0.3 +/− 0.2% pre-transplant) sirolimus or belatacept monotherapy also partially controlled cytotoxicity (8 +/− 1% and 35 +/− 1% granzyme Bvery high with sirolimus or belatacept, respectively). Combination therapy dramatically reduced the proportion of these cells, to 1.5 +/− 0.8 % granzyme Bvery high, favorably comparing with 4% granzyme Bvery high using CNI/MTX.The ability of sirolimus, belatacept, or the combination to control Ki-67 and Granzyme B expression closely correlated with survival (Figure 2A, B) supporting a pathogenic role for these highly proliferative and cytotoxic cells in aGvHD pathology. Moreover, significant co-expression of granzyme B in the Ki-67+ cells was observed (Figure 2C) suggesting that dual-positive Ki-67/Granzyme B cells may mark a pathogenic population, amenable to tracking in the peripheral blood. Implications:These results reveal a previously undiscovered synergy between sirolimus and belatacept in the control of primate aGvHD, and provide support for future clinical investigation of this novel prevention strategy. They also identify CD8+/Ki-67+/Granzyme Bvery high dual-positive T cells as a potentially sensitive biomarker of GvHD pathogenesis, amenable to monitoring in either the blood or in GvHD target organs. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Sirolimus, Tacrolimus and Low-Dose Methotrexate Based Graft-Versus-Host Disease (GVHD) Prophylaxis After Non-Ablative or Reduced Intensity Conditioning in Related and Unrelated Donor Allogeneic Stem Cell Transplantation: Decreased Severe Early aGVHD but Persistence of Late Acute Gvhd

AbstractAbstract 4194 Background:The morbidity and mortality associated with acute and chronic graft-versus-host disease (GVHD) remain significant after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Encouraging results have been reported with the combination of sirolimus, tacrolimus, and low-dose methotrexate after non-myeloablative allogeneic peripheral blood stem cell transplantation (PBSCT) (Alyea EP et al, Biol Blood Marrow Transplant 2008). We conducted a retrospective analysis of 74 patients with hematologic malignancies who underwent a non-myeloablative (NMA) or reduced intensity (RIC) allo-HSCT using sirolimus, tacrolimus, and low-dose methotrexate for GVHD prophylaxis. Methods:Between April 2008 and June 2011, 74 patients (M/F: 45/29), median age 51 years (range: 23–69) were transplanted for non-Hodgkin’s lymphoma (63%), chronic lymphocytic leukemia/small lymphocytic lymphoma (20%), Hodgkin lymphoma (12%) and acute lymphoblastic leukemia or myelodysplasia (3%). At transplantation, 36 patients (49%) were in complete remission and 38 patients (51%) had chemotherapy sensitive active disease. Conditioning regimen was NMA in 59 patients and RIC in 15 patients. Donors were HLA-matched related (MRD, n = 30, 41%), matched unrelated (MUD, n = 37, 50%) or 9/10 HLA-mismatched unrelated (MMUD, n = 7). All patients received peripheral blood mobilized grafts. GVHD prophylaxis consisted of sirolimus and tacrolimus that were started on day -3 and doses were adjusted to maintain target serum trough levels of 3–12 ng/mL and 5–10 ng/mL, respectively, followed by methotrexate 5 mg/m2 on days +1, 3, 6. Tapering of sirolimus and tacrolimus began at day 60 in the absence of GVHD or relapse, with the goal of complete discontinuation of immunosuppressants by 6 months or earlier if patients were not in CR at transplant. Recipients of MUD (31 of 37) or MMUD (7 of 7) grafts were given 2 doses of anti-thymocyte globulin (ATG) on days −3, and −2. Forty-eight patients with CD20+ malignancies (65%) received rituximab at day -8 and +21, 28, 35, 42. Acute GVHD (aGVHD) was scored by IBMT Severity Index and chronic GVHD (cGVHD) based on NIH consensus criteria. Results:The cumulative incidence of Grade B-D and C-D classic aGVHD at day +100 were 14.9% and 2.7%, respectively. The cumulative incidence of aGVHD at 1-year was 31.1 % for grade B-D and 5.4 % for grade C-D, due to late-onset Grade B-D and Grade C-D aGVHD rates of 16.2% and 2.7%, respectively. The incidence of cGVHD at 1 and 2 years was 15% and 28%, respectively. The use of peri-transplant rituximab did not affect the incidence of Grade B-D aGVHD (p=0.55) or cGVHD (p=0.94). There was no difference in the incidence of Grade B-D aGVHD at day 100 between ATG (13.9%) and no ATG (15.8%) cohorts. There was a trend towards a modestly higher 2-year incidence of cGVHD in patients who did not receive ATG, despite the fact that these were predominantly recipients of MRD (37% vs. 21% in ATG cohort, p=0.12). Two patients (2.7%) developed thrombotic microangiopathy and 4 patients (5.4%) had renal toxicity, requiring modification of the immunosuppressant treatment. No sinusoidal obstructive sydrome was reported. To date, 15 patients have died; causes of death include disease recurrence (n=9) and GVHD (n=6). The cumulative incidence of non-relapse mortality and relapse at 1 year were 4% and 27%, respectively. The 1-year relapse rate in the ATG and no ATG cohorts was 11% and 28%, respectively (p=0.10). With a median follow up 2.6 (range: 0.8–3.9) years, progression-free and overall survival at 2 years were 64 % and 77 %, respectively. Conclusions:As previously reported this GVHD prophylaxis regimen is safe and effective, providing lower rates of acute GVHD compared to historical data. The incidence of chronic GVHD by NIH criteria was low due to an increase in late-onset acute GVHD. Rituximab therapy did not affect the incidence of acute and chronic GVHD. The addition of ATG in MUD and MMUD recipients resulted in a trend toward lower rates of chronic GVHD with no increase in the rate of relapse. Future strategies will need to focus on decreasing the incidence of late onset acute and chronic GVHD without increasing the risk of relapse. Disclosures:No relevant conflicts of interest to declare.

Tacrolimus/Sirolimus Vs. Tacrolimus/Methotrexate for Graft-Vs.-Host Disease Prophylaxis After HLA-Matched, Related Donor Hematopoietic Stem Cell Transplantation: Results of Blood and Marrow Transplant Clinical Trials Network Trial 0402

AbstractAbstract ▪739▪This icon denotes a clinically relevant abstractThe combination of a calcineurin inhibitor and methotrexate has been the standard of care in graft-vs.-host disease (GVHD) prophylaxis for over 25 years, with resultant rates of grade II-IV acute GVHD between 30–50%. The mTOR inhibitor, sirolimus, has demonstrated promise in a number of Phase II trials as an immunosuppressant used for GVHD prophylaxis. The BMT CTN, sponsored by the NHLBI and NCI, conducted a multicenter, randomized controlled trial comparing the combination of tacrolimus and sirolimus (Tac/Sir) with tacrolimus and methotrexate (Tac/Mtx) as GVHD prophylaxis after matched, related donor (MRD) hematopoietic stem cell transplantation (HSCT). Methods:Eligible patients were between ages 2 – 60 years, and had acute leukemia in remission, myelodysplasia or chronic myeloid leukemia in chronic or accelerated phase. All had adequate organ function, and a 6/6 HLA-A, B, DRB1 matched sibling donor. 304 patients were randomly assigned to either Tac/Sir (n = 151) or Tac/Mtx (n = 153) as GVHD prophylaxis after TBI-based conditioning and MRD HSCT. An intent-to-treat analysis was performed on the primary endpoint of Grade II-IV GVHD-free survival 114 days from randomization. Ten subjects who received busulfan-based conditioning and were previously reported were excluded from analysis. Three subjects who did not undergo HSCT are included in the primary analysis, but not secondary analyses. Results:Treatment groups were well balanced. The median age of participants was 44 years (range 13 – 59) and 83% had acute leukemia. Neutrophil and platelet engraftment were both faster in the Tac/Sir group (14 vs. 16 days, p < 0.001; 16 vs. 19 days, p = 0.03, respectively), but this did not affect the time to first hospital discharge (20 vs. 21 days, p = 0.37). The incidence of grade II-IV and grade III-IV acute GVHD at 100 days were lower in the Tac/Sir group (26 vs. 34%, p = 0.17; 8 vs. 15%, p = 0.05). Day 100 treatment-related mortality was no different between groups (7 vs. 7%, p = 0.43). The primary endpoint of 114-day acute GVHD-free survival was not statistically different between groups (67 vs. 62%, p = 0.38, Figure). The cumulative incidence of relapse at 2 years from transplantation was not different between groups (27 vs. 30%, p = 0.81). The competing-risk cumulative incidence of chronic GVHD was higher in the Tac/Sir arm (54 vs. 43%, p =0.044). Overall toxicities were not different between groups, with two notable exceptions. The peak and average OMAS oral mucositis scores were lower in the Tac/Sir arm (peak 0.70 vs. 0.96, p < 0.001; average 0.31 vs. 0.47, p < 0.001), however, there was an increased rate of the endothelial injury syndromes, veno-occlusive disease (11 vs. 4%, p = 0.03), and thrombotic microangiopathy (5 vs. 1%, p = 0.05) in the Tac/Sir arm. Causes of death were not different between groups. At 2 years from transplantation, disease-free (DFS) and overall survival (OS) were not different between study arms (DFS 53 vs. 53%, p = 0.76; OS 60 vs. 61%, p = 0.44). Conclusions:No difference in 114-day acute GVHD-free survival was noted between treatment arms. Compared with Tac/Mtx in MRD HSCT, Tac/Sir is associated with more rapid engraftment, less severe acute GVHD and oral mucositis, excess chronic GVHD and endothelial injury syndromes, and similar long-term outcomes. Understanding the trade-offs between regimens, Tac/Sir can be used as an alternative to Tac/Mtx in MRD HSCT. [Display omitted] Disclosures:Cutler:Pfizer, inc: Research Funding; Astellas, Inc: Consultancy, Research Funding. Off Label Use: Sirolimus - Prevention of GVHD Tacrolimus - Prevention of GVHD. Waller:Outsuka: Research Funding.

Everolimus and Sirolimus in Combination with Cyclosporine Have Different Effects on Renal Metabolism in the Rat

Enhancement of calcineurin inhibitor nephrotoxicity by sirolimus (SRL) is limiting the clinical use of this drug combination. We compared the dose-dependent effects of the structurally related everolimus (EVL) and sirolimus (SRL) alone, and in combination with cyclosporine (CsA), on the rat kidney. Lewis rats were treated by oral gavage for 28 days using a checkerboard dosing format (0, 3.0, 6.0 and 10.0 CsA and 0, 0.5, 1.5 and 3.0 mg/kg/day SRL or EVL, n = 4/dose combination). After 28 days, oxidative stress, energy charge, kidney histologies, glomerular filtration rates, and concentrations of the immunosuppressants were measured along with 1H-magnetic resonance spectroscopy (MRS) and gas chromatography- mass spectrometry profiles of cellular metabolites in urine. The combination of CsA with SRL led to higher urinary glucose concentrations and decreased levels of urinary Krebs cycle metabolites when compared to controls, suggesting that CsA+SRL negatively impacted proximal tubule metabolism. Unsupervised principal component analysis of MRS spectra distinguished unique urine metabolite patterns of rats treated with CsA+SRL from those treated with CsA+EVL and the controls. SRL, but not EVL blood concentrations were inversely correlated with urine Krebs cycle metabolite concentrations. Interestingly, the higher the EVL concentration, the closer urine metabolite patterns resembled those of controls, while in contrast, the combination of the highest doses of CsA+SRL showed the most significant differences in metabolite patterns. Surprisingly in this rat model, EVL and SRL in combination with CsA had different effects on kidney biochemistry, suggesting that further exploration of EVL in combination with low dose calcineurin inhibitors may be of potential benefit.

The combination of sirolimus plus tacrolimus improves outcome after reduced-intensity conditioning, unrelated donor hematopoietic stem cell transplantation compared with cyclosporine plus mycofenolate

Different types of graft-versus-host disease prophylaxis have been proposed in the setting of reduced intensity and non-myeloablative allogeneic stem cell transplantation. An alternative combination with sirolimus and tacrolimus has recently been tested although comparative studies against the classical combination of a calcineurin inhibitor and mycophenolate mofetil or methotrexate are lacking. We describe the results of a prospective, multicenter trial using sirolimus + tacrolimus as immunoprophylaxis, and compare this approach with our previous experience using cyclosporine + mycophenolate in the setting of unrelated donor transplantation setting after reduced-intensity conditioning. Forty-five patients received cyclosporine + mycophenolate between 2002 and mid-2007, while the subsequent 50 patients, who were transplanted from late 2007, were given sirolimus + tacrolimus. No significant differences were observed in terms of hematopoietic recovery or acute graft-versus-host disease overall, although gastrointestinal acute graft-versus-host disease grade ≥ 2 was more common in the cyclosporine + mycophenolate group (55% versus 21%, respectively, P=0.003). The 1-year cumulative incidence of chronic graft-versus-host disease was 50% versus 90% for the patients treated with the sirolimus- versus cyclosporine-based regimen, respectively (P<0.001), while the incidence of extensive chronic disease was 27% versus 49%, respectively (P=0.043). The 2-year non-relapse mortality rate was 18% versus 38% for patients receiving the sirolimus- versus the cyclosporine-based regimen, respectively (P=0.02). The event-free survival and overall survival at 2 years were 53% versus 29% (P=0.028) and 70% versus 45% (P=0.018) among patients receiving the sirolimus- versus the cyclosporine-based regimen, respectively. In conclusion, in the setting of reduced intensity transplantation from an unrelated donor, promising results can be achieved with the combination of sirolimus + tacrolimus, due to a lower risk of chronic graft-versus-host disease and non-relapse mortality, which translates into better event-free and overall survival rates, in comparison with those achieved with cyclosporine + mycophenolate.

Clinical activity of mTOR inhibition in combination with cyclophosphamide in the treatment of recurrent unresectable chondrosarcomas

Chondrosarcomas (CS) represent a heterogeneous group of rare sarcomas, poorly responsive to chemotherapy or radiotherapy. When local therapies in recurrent or metastatic disease are exhausted, chemotherapy plays a marginal role. Different molecular pathways have been shown to be activated in CS. In this retrospective study, we summarize our experience in treating a cohort of patients with recurrent unresectable CS with a combination of sirolimus (SIR) and cyclophosphamide (CTX). Ten consecutive patients with unresectable CS were offered off-label treatment with SIR and CTX between 2007 and 2012. Tumor response, progression-free survival (PFS), adverse events, and other relevant clinical data were analyzed. The median patients' age was 49 (range 28-68). Median disease-free interval since the primary diagnosis was 22.5 months. Median time from the disease recurrence to initiation of SIR and CTX treatment was 21.7 months due to additional local surgical treatments, excision of metastases, or slow asymptomatic progression. One (10 %) objective response was observed, and six (60 %) patients had stabilization of disease for at least 6 months. Three patients had progressive disease. Median PFS was 13.4 months (range 3-30.3). No significant adverse events were observed. Although advanced CS remains an incurable disease, our experience suggests that a combination of SIR and CTX is well tolerated and may have meaningful clinical activity with disease control rate of 70 %. Further prospective studies are warranted.

A dose escalation trial for the combination of erlotinib and sirolimus for recurrent malignant gliomas

In order to achieve higher dosages than previously used in clinical trials, we conducted a phase I trial to determine the maximum tolerated dose (MTD) for the combination of erlotinib and sirolimus for the treatments of recurrent malignant gliomas. Patients with pathologically proven World Health Organization (WHO) grade III glioma and grade IV glioblastoma and radiographically proven tumor recurrence were eligible for this study. Treatments included once daily erlotinib, which was given alone for the first 7 days of treatments, then in combination with once daily sirolimus. Sirolimus was given with a loading dose on day 8 followed by a maintenance dose starting on day 9. Dose-limiting toxicity (DLT) was determined over the first 28 days of treatments, and the MTD was determined in a 3 + 3 classic study design. 19 patients were enrolled, and 13 patients were eligible for MTD determination. The MTD was determined to be 150 mg daily for erlotinib and 5 mg daily (after a 15 mg loading dose) for sirolimus. The DLTs included rash and mucositis (despite maximal medical managements), hypophosphatemia, altered mental status, and neutropenia. The combination of erlotinib and sirolimus is difficult to tolerate at dosages higher than previously reported in phase II trials.

Anti-Thymocyte Globulin (Thymoglobulin), Tacrolimus, and Sirolimus as Acute Graft-versus-Host Disease Prophylaxis for Unrelated Hematopoietic Stem Cell Transplantation

Acute graft-versus-host disease (aGVHD) is a major cause of morbidity and mortality in patients undergoing unrelated hematopoietic stem cell transplantation. We prospectively evaluated the efficacy of intermediate-dose rabbit anti-thymocyte globulin (Thymoglobulin® a total of 4.5 mg/kg given over days-3, -2, and -1) in combination with tacrolimus and sirolimus for the prevention of aGVHD. We enrolled 47 recipients who underwent unrelated hematopoietic stem cell transplantation. Patients received daily granulocyte colony-stimulating factor starting on day +6 until neutrophil engraftment (median duration, 11 days; range, 9-15 days). Twenty-two patients received HLA 8/8 and 25 received 7/8 matched grafts, respectively. The median follow-up duration was 23.6 months (range, 18.8-27.9 months). The cumulative incidence of grade II to IV aGVHD was 23.4% (95% confidence interval, 12.4-36.3). At 2-year follow-up, the cumulative incidence of nonrelapse mortality was 31.9%, cumulative incidence of relapse was 24.6%, and cumulative incidence of chronic GVHD was 33%. Progression-free survival at 1 year was 54%, with a median of 17.7 months. Overall survival at 1 year was 65%, with no median reached. These results suggest that the combination of Thymoglobulin, tacrolimus, and sirolimus in patients undergoing unrelated hematopoietic stem cell transplantation is well tolerated and associated with a low incidence and severity of aGVHD and chronic graft-versus-host disease.

Phase I study of sirolimus plus gemcitabine in patients with advanced solid tumors: A Spanish Group for Research on Sarcomas (GEIS) study.

3096 Background: In preclinical studies, combination of sirolimus with gemcitabine enhances apoptosis in vitro and increases anti-tumor efficacy in vivo. Methods: Patients with advanced solid tumors, age 18-70 years, no prior mTOR inhibitor or gemcitabine, ECOG PS 0-1, and adequate hematological, renal and hepatic function, were enrolled in this phase I study to assess safety, tolerability, pharmacokinetics (PK), and to identify the dose limiting toxicity (DLT), maximum tolerated dose (MTD) and recommended dose (RD) of the combination of sirolimus and gemcitabine. A 3+3 dose escalation design with cohorts of 3-6 patients was used. Sirolimus was given po continuously. Gemcitabine was given iv 10mg/m2/minute on days 1 and 8 every 3 weeks. Dose levels 1, 2 and 3 corresponded to sirolimus 2, 2 and 5mg/24h plus gemcitabine 800, 1000 and 1000mg/m2 respectively. After observing DLTs at higher dose level and poorer mTOR signaling inhibition at lower doses, a new cohort of sirolimus 5mg/24h plus gemcitabine 800 mg/m2 was added. Skin biopsies pre and post treatment were performed to assess the inhibition of mTOR pathway. Results: 19 patients were enrolled: median age 51 years (36-70); gender 12M, 7F. Median number of cycles was 4. Patients were treated at 4 dose levels, the MTD was reached at level 3 and the RD was: sirolimus 5mg/24h and gemcitabine 800mg/m2. 3 DLTs were observed, 1 at dose level 2 and 2 at dose level 3: transaminitis grade 3, thrombocytopenia grade 3 and thrombocytopenia grade 4. Other toxicities grade 1-2 included anemia, neutropenia, asthenia, mucositis and high cholesterol levels. 2 patients achieved partial response (1 uterine cervix cancer and 1 colon cancer). Immunohistochemistry of pS6 in skin biopsies showed significative inhibition of mTOR pathway at RD. PK parameters estimated were in agreement with those previously reported in the literature. No influence of sirolimus administration on gemcitabine clearance was found. Conclusions: Combination of sirolimus and gemcitabine is feasible and safe, allowing administration of active doses of both agents and achieving mTOR signaling inhibition. A phase II study to assess the activity of this combination in sarcomas is ongoing.

Clinical activity of mTOR inhibition in combination with cyclophosphamide in the treatment of recurrent nonresectable chondrosarcomas.

10043 Background: Chondrosarcomas (CS) represent a heterogeneous group of rare sarcomas, poorly responsive to chemotherapy or radiotherapy. When local therapies in recurrent or metastatic disease are exhausted, chemotherapy plays a marginal role. Different molecular pathways have been shown to be activated in CS. In this retrospective study we summarize our experience in treating a cohort of patients with recurrent nonresectable CS with a combination of sirolimus and cyclophosphamide. Methods: Nine consecutive patients with nonresectable CS were offered off-label treatment with sirolimus and cyclophosphamide between 2007-2011. Tumor response, progression-free survival (PFS), adverse events and other relevant clinical data were analyzed. Results: The median patients’ age was 52 (range, 35-68). Median disease-free interval (DFI) since the primary diagnosis was 24 months. Median time from the disease recurrence to initiation of sirolimus and cyclophosphamide treatment was 20.9 months due to additional local surgical treatments/excision of metastases/slow and asymptomatic progression. One (11%) objective response was observed and five (56%) patients had stabilization of disease for at least 6 months. One patient has been treated for only 3.5 months and reported symptomatic improvement at this stage, two patients had progressive disease. Median time to treatment failure (TTF) was 15 months (range, 2.8-30.3). No significant adverse events were observed. Conclusions: Although advanced CS remains an incurable disease, our experience suggests that a combination of sirolimus and cyclophosphamide is well-tolerated and has meaningful clinical activity with clinical benefit rate of 67%. Further prospective studies are warranted.

A preclinical study on the combined treatment of nimotuzumab and sirolimus in glioblastoma.

2066 Background: The human epidermal growth factor receptor (EGFR) is an ideal therapeutic target for inhibiting glioblastoma (GBM) growth as the signaling pathway is highly dysregulated in GBM. However, trials so far with EGFR inhibitors have not shown clinically meaningful improvement in response rates and survival. One of the postulated mechanisms of resistance is the constitutive activation of the downstream, PI3K/AKT/mTOR pathway, independent of the upstream EGFR activation status. Hence, the dual blockage of the pathways with an anti-EGFR agent and mTOR inhibitor is postulated to have synergistic anti-tumour effects. We aim to investigate the anti-tumour effect of combined nimotuzumab (anti-EGFR monoclonal antibody) and sirolimus (mTOR inhibitor) in a GBM preclinical model. Methods: Primary human GBM cells were derived from surgical GBM specimens. The endogenous expressions of glial-fibrillary acidic proteins and EGFR were determined by IHC staining and Western Blot analysis. Cell viability assays were then carried out in these GBM cells and immortalized human normal astrocytes (iHNA) using a range of concentrations of nimotuzumab alone, sirolimus alone or combined treatment. Results: GBM cells treated with nimotuzumab showed a dose- dependent cell kill and the optimal concentration was determined to be 2 µg/ml. Treatment of the GBM cells with sirolimus showed that the drug was capable of inducing cell death at varying levels and the optimal level was determined to be 0.1 mM. Combined treatment with nimotuzumab (2µg/ml) and rapamycin (0.1 mM) showed a dose dependent cell kill in GBM cells which was not observed in iNHA cells. The combined treatment resulted in only 10% of residual gliomas at 24 h post treatment. Single treatment with rapamycin has no cytotoxic effect whereas treatment with nimotuzumab alone exerts a cytotoxicity effect of 33%. Taken together, we observed an additive effect of cell kill when rapamycin is used together with nimotuzumab in human glioma cells. Conclusions: In this study, combined treatment of nimotuzumab and sirolimus resulted in a greater cytocidal effects in GBM cells than either agent alone. We will further examine this combination regimen in a subsequent phase I clinical trial.

The novel combination of sirolimus and bortezomib prevents graft-versus-host disease but maintains the graft-versus-leukemia effect after allogeneic transplantation

We have previously shown that bortezomib induces a depletion of alloreactive T cells and allows the expansion of T cells with suppressive properties. In the current study, we analyzed the potential synergistic effect of bortezomib in conjunction with sirolimus in order to reduce-graft-versus-host disease without hampering graft-versus-leukemia effect in the allogeneic transplant setting. We evaluated the effect of sirolimus, bortezomib or the combination of both in the proliferation and activation of in vitro stimulated T lymphocytes. Pathways involved in this synergy were also analyzed using Western blot assays. Finally, BALB/c mice receiving C57BL/6 allogeneic donor bone marrow with splenocytes were used to measure in vivo the effect of this novel combination on the risk of graft-versus-host disease. The combination of both drugs synergistically inhibited both activation and proliferation of stimulated T cells. Also, the production of Th1 cytokines (IFN γ, IL-2 and TNF) was significantly inhibited. This effect was due, at least in part, to the inhibition of Erk and Akt phosphorylation. In vivo, the combination reduced the risk of graft-versus-host disease without hampering graft-versus-leukemia effect, as shown in mice receiving graft-versus-host disease prophylaxis with sirolimus plus bortezomib being infused with tumor WEHI cells plus C57BL/6 donor BM and splenocytes. The current study reveals a synergistic effect of the combination sirolimus and bortezomib to prevent graft-versus-host disease while maintaining the graft-versus-leukemia effect.

Results of a phase II study of sirolimus and cyclophosphamide in patients with advanced sarcoma

BackgroundActivation of the mammalian target of rapamycin (mTOR) pathway has been demonstrated in sarcoma. Trials using mTOR inhibitor in sarcoma have shown low objective response rates but progression-free survival (PFS) rates suggest cytostatic effects. The combination of sirolimus and cyclophosphamide demonstrated synergistic anti-sarcoma activity in preclinical models; therefore, we conducted a phase II trial of sirolimus and cyclophosphamide in patients with advanced sarcoma. MethodsPatients received 4mg sirolimus daily and 200mg cyclophosphamide d1-7 and 15-21 every 28days. The primary objective was to estimate the 24-week PFS rate with a target of ⩾25%. Patients were followed for World Health Organisation (WHO) criteria tumour response by imaging every 8weeks. Serum levels of sirolimus, lipids and vascular endothelial growth factor were measured. Tumour tissue was analysed for mTOR, S6 ribosomal protein and cytochrome P450 3A4/5 by quantitative immunofluorescence. ResultsForty-nine eligible patients were enrolled from September 2008 to December 2009. Patients received a median of four cycles of therapy. Starting doses of drugs were tolerated in 79%. One patient achieved partial tumour response, 10 were progression-free for ⩾24weeks and two completed 12 cycles of treatment. Median PFS and overall survival (OS) were 3.4 and 9.9months, respectively. Serious adverse events attributed to therapy occurred in 11% and included infection, pneumonitis and thrombosis. Hypertriglyceridaemia from treatment and lower tumour phosphorylated-mTOR are associated with longer survival. ConclusionsSirolimus and cyclophosphamide were tolerated by the majority of patients. About 20% of patients had stable sarcoma for at least 6months but objective tumour response was infrequent.

A Prospective Phase II Trial: The Combination of Tacrolimus, Sirolimus, and Rabbit Anti-Thymocyte Globulin (Thymoglobulin® Thymo) to Prevent Acute Graft Vs. Host Disease (aGVHD) in Patients Receiving Unrelated Hematopoietic Stem Cell Transplantation (UHSCT)

Abstract 4551 Background:Acute graft versus host disease continues to affect approximately 60% of patients undergoing UHSCT, with significant mortality and morbidity. Methods:We prospectively evaluated the efficacy of combining Thymo (4.5 mg/kg divided doses on days -1,-2, and -3), tacrolimus and sirolimus in preventing aGVHD. The cumulative incidence (CI) rate of grade II-IV aGVHD was calculated using death without grade II-IV aGVHD and relapse as competing risks (Cr). The Cr: for non relapse mortality (NRM) CI was death due to relapse, for relapse CI was death without relapse, for chronic GVHD CI was relapse & non relapse mortality without cGVHD. Kaplan-Meier method was used to calculate overall (OS) and progression free survival (PFS). The incidence of infections and other complications were reported with the Wilson’s 95% Confidence Interval (in table below). Results:Between August 2008 and November of 2010, we enrolled 47 patients (pts) with median age of 50(20–70) years. The Median follow-up time is 23.6 months (18.8–27.9). There were 21 AML, 10 MDS, 4 ALL, 2 CML, 2 CMML, 1 CLL, 3 Myelofibrosis, 2 multiple myeloma, 2 NHL. Preparative regimens included Bu/Flu 30, Bu/Flu-TBI 10, VP16/TBI 3, R-BEAM 1, and Flu/MEL-TBI 3. All pts received peripheral blood stem cells mobilized with G-CSF. Median CD34+ dose was 7.31×10 6/kg (1.9–18.6). Median donor age was 32.6 (19.0–61.0) years. All patients received daily G-CSF starting day +6 till engraftment. Twenty two pts received 8/8 and 25 received 7/8 HLA matched grafts respectively. All patients’ engrafted, with median day of 11 (9–15). Twenty deaths occurred throughout the whole follow up period, due to: relapse 6, aGVHD 4, cGVHD 2, sinusoidal obstruction syndrome (SOS) 2, bleeding 1, multi organ failure 2, sepsis 2 and pneumonia 1. Twelve patients experienced disease relapse. Fourteen patients had non-relapse mortality.Twelve pts developed Grade II-IV aGVHD, 5 grade II, 4 grade III, and 3 grade IV. The CI rate for grade II-IV aGVHD at 200 days post transplant is 0.23.4% (12.4, 36.3); CI of NRM at day 1057 is 31.9% (18.4, 46.2). CI of relapse at day 1057 is 30.4% (15.2,47.1). CI of cGVHD at day 890 is 40.2(21.5, 58.2), with total of 16 cases: 8 mild, 7 moderate and one sever based on NIH consensus criteria. Median PFS is 17.7 months. PFS at 6 months is 63% and 54% at 1 year. Median OS has not been reached. OS at 6 months is 73%, and 65% at one year. The incidence of infections and other transplant related morbidities are shown in the table below. There were 2 cases of thrombotic thrombocytopenic purpura (TTP) before day 100. 16 CMV by PCR, 10 EBV by PCR, 9 HSV, 10 BK cystitis, 31 bacterial infections, 4 oral candidiasis, and 3 SOS. Conclusion:These early results suggest that the combination of Thymo, tacrolimus and sirolimus in pts undergoing UHSCT is well tolerated and is associated with a low rate and severity of acute GVHD.Clinical OutcomeIncidenceConfidence IntervalCI Grade II-IV aGVHD23.4(12.4, 36.3)CI Non-relapse mortality31.9(18.4, 46.2)CI cGVHD40.2(21.5, 58.2)CI Relapse30.4(15.2, 47.1)TTP before day 1004.26(1.17, 14.25)CMV PCR34.04(22.17, 48.33)EBV PCR21.28(11.99, 34.90)HSV19.15(10.42, 32.54)BK cystitis21.28(11.99, 34.90)Bacterial Infections65.96(51.67, 77.83)Oral Candidiasis8.51(3.36, 19.93)SOS6.38(2.19, 17.16) Disclosures:Al-Kadhimi:Genzyme Pharmaceuticals: Research Funding. Off Label Use: The use of thymoglobulin, Sirolimus and Tacrolimus in blood and marrow transplant. Lum:Transtarget Inc: Equity Ownership, Founder of Transtarget.