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Background The median age of AML is approximately 70 years, and the survival of older adults age ≥60 years remains poor with standard therapy. Clofarabine (CLO) has significant single agent activity in AML, and in vitro studies demonstrate synergy with Ara-C. We therefore performed a phase I study to determine the maximum tolerated dose (MTD) of CLO with standard dose Ara-C (100mg/m2/day D1-7 by 24hr continuous infusion) as AML induction therapy. CLO was administered daily × 5 days beginning D2 to allow for pharmacokinetic (PK) & pharmacodynamic (PD) studies. Methods Enrollment was restricted to newly-diagnosed de novo AML patients age ≥60 years considered candidates for intensive therapy; those with prior MDS or hypoplastic AML (<20% bone marrow cellularity) were excluded. Pts were treated in cohorts of 3–6 to determine MTD of CLO with standard dose infusional Ara-C (100mg/m2 × 7). DLT was defined as grade III/IV non-hematologic toxicity occurring in >1 pt per cohort. The CLO starting dose was 30mg/m2/day ×5 (dose level 1), with the intention to increase to CLO 40mg/m2 if tolerated. However, CLO dose reductions of 25% (CLO 22.5 mg/m2/day × 5, dose level -1) & 50% (CLO 15mg/m2/day ×5, dose level -2) were allowed in the event of dose-limiting toxicity (DLT). Pts achieving complete remission (CR) received 2 cycles consolidation with CLO ×5 & Ara-C 100mg/m2 × 5. Results A total of 13 pts (median age 69 yrs, range 61–77; 10 male) have been treated. DLT was observed at dose level 1 (Table 1), and the protocol was amended to mandate additional hydration with CLO, and antibiotic/antifungal prophylaxis. At dose level -1, 1 pt died from PE on D27 after hematologic recovery, considered treatment-related per protocol, mandating dose de-escalation to dose level -2. Pt 13 is in active induction therapy, and not yet evaluable for response, although DLT has not been encountered. Febrile neutropenia occurred in 11/13 pts. The MTD in this study is CLO 15mg/m2/day × 5 with standard infusional Ara-C (dose level -2). Significant clinical activity was observed, particularly at higher CLO doses, including CR in 2 pts with complex karyotype. In contrast, only 1/5 evaluable pts at dose level-2 achieved CR. PK & PD studies are in progress. Conclusions In contrast to prior reports using intermediate dose bolus Ara-C (1g/m2), the MTD of CLO combined with standard dose infusional Ara-C in older adults with AML is lower, and the toxicity profile appears different. Significant clinical activity was noted at higher CLO dose levels & with complex karyotype. Based on this observation an escalation to CLO 20mg/m2 is now planned. CLO & Standard Dose Infusional Ara-C: Toxicity & Efficacy Patient CLO Dose Level Age Cytogenetics Response (CR Duration) DLT Fail - failure to achieve CR 1 1 (30mg) 66 −7 Death Infection/Renal 2 1 61 −7, complex CR (15 mths) none 3 1 69 intermediate CR (2 mths) none 4 1 77 5q-, complex Death Infection/Renal/Vasc Leak 5 −1 (22.5mg) 75 +8 Death Pneumonia 6 −1 67 diploid CR (5 mths) none 7 −1 71 9q- Death Pulm Embolism Day 27 8 −2 (15mg) 74 5q-, complex Fail none 9 −2 64 diploid Fail none 10 −2 63 diploid Fail none 11 −2 73 complex CR (5 mths) none 12 −2 72 diploid Fail none 13 −2 63 diploid - -

NST provides graft-versus-leukemia (GVL) activity with reduced regimen related toxicity. We used a conditioning regimen consisting of Pentostatin 4 mg/m2 daily × 3 days and 200 cGy TBI 24 hrs prior to stem cell infusion in patients (pts) with high-risk/relapsed/refractory hematologic malignancies. The importance of the timing of stem cell infusion relative to Pentostatin administration was examined in a comparison of two sequential protocols. In cohort 1 (n=39) Pentostatin was given on days -21, -20, and -19. In cohort 2 (n=39) Pentostatin was given on days -10, -9, and -8. The median age of the day -21 cohort was 52 years (range 22–70) and the day -10 cohort was 59.5 years (range 36–70). The median number of prior therapies in the day -21 cohort was 4 (range 0–8), including prior autologous SCT in 22 pts, while in the day -10 cohort, the median number of prior therapies was 6 (range 0–18), including prior autologous SCT in 9 pts. Post-grafting immunosuppression was cyclosporine (CsA)/mycophenolate mofetil (MMF). In both protocols, CsA 2.0 mg/kg IV q12 hrs was administered on days -1, 0, and +1, and then converted to oral 5 mg/kg PO BID with a taper beginning on day +70. In the second protocol, pts with unrelated donor transplants tapered CsA starting at day +100. MMF at 15 mg/kg PO BID was administered on days 0–27 for related donor transplants in both protocols and in the second protocol this was extended until day +40 for the unrelated donor transplants. In the day -21 protocol, SCTs were obtained from matched related (n=14) or unrelated (n=25) donors. The cumulative incidence of all grades of acute GVHD at day 100 was 40% and was more common in unrelated donor (60%) vs. related donors (15%) transplants. In the day -10 protocol, SCT was performed with products from matched related (n=15) or unrelated (n=21) donors. The cumulative incidence of grade II–IV acute GVHD was approximately 35% (30% in related versus 40% in unrelated donors). In both protocols, the median chimerism values for CD3+ cells and white blood cells at day 28 were >80% and 90% donor cells, respectively. T, B and natural killer (NK) lymphocyte frequency and numbers were identical at protocol entry. One day following TBI conditioning, there was a 57% depression in CD3+ cells in the day -21 protocol that was retained on day 28 post transplant (55%). In the day -10 protocol, there was a significant depression (47%) in the frequency of CD3+ cells one day following TBI, which was depressed further (68%) on day 28 post transplant. The dendritic cells (DCs), both CD123+ and CD11c+, were not suppressed by the conditioning regimen. Separation of pts on day 28 into cohorts with an absolute number of CD3+ cells >0.25×106/ml revealed that those pts with a higher CD3+ cell count had a median OS time of 36.7 weeks, which was significantly longer than pts whose absolute number of CD3+ cells <0.25×106 (median OS of 21 weeks). This differential effect on OS was not observed when pts were separated on the basis of either CD4+ or CD8+ cells. In conclusion, there is a lower incidence of acute GVHD in the day -10 protocol, which can be associated with a significant reduction in the frequency and absolute number of circulating T cells. The significant reduction in T cells, observed one day following TBI and 28 days post transplant, is associated with the timing of transplant relative to Pentostatin conditioning and a significant prolongation of OS.

NST is increasingly being used to introduce a graft-versus-malignancy (GVM) effect with reduced regimen related toxicity. The first NST protocol, which accrued patients (pts) between 9/01 and 7/04, 39 pts with high risk/relapsed/refractory hematopoietic malignancies who were not candidates for full intensity allogeneic SCT underwent NST using Pentostatin/TBI. Their median age was 52 years (range 22–70). The median number of prior therapies was 4 (range 0–8) including prior autologous SCT in 22 pts. Conditioning consisted of Pentostatin 4 mg/m2 daily on day -21, -20, and -19, followed by 200 cGy TBI on day -1. The second protocol, between 10/04 and 6/06, 24 pts received the same conditioning regime on days -10, -9, and -8, and TBI on day -1. The median age of these pts was 59.5 years (range 36–70) and the median number of prior therapies was 2 (range 0–6). Post-grafting immunosuppression was cyclosporine (CsA)/mycophenolate mofetil (MMF). In the both protocols CsA 2.0 mg/kg IV q12 hrs was administered on days -1, 0, and +1 and then converted to oral 5 mg/kg PO BID with a taper beginning on day +70. In the second protocol pts with unrelated donor transplants remain on CsA until day + 100. MMF at 15 mg/kg PO BID was administered on days 0–27 for related donor transplants in both protocols and in the second protocol this was extended until day +40 for the unrelated donor transplants. Results: In the day -21 protocol SCTs were from matched related (n=14) or unrelated (n=25) donors. The cumulative incidence of all grades of acute graft-versus-host disease (GVHD) at day 100 was 40% and was more common in unrelated donor transplants (60% vs. 15%). In the day -10 protocol, SCT was performed with products from matched related (n=8) or unrelated (n=16) donors. The cumulative incidence of grade II–IV acute GVHD was approximately XX% (XX% in related versus XX% in unrelated donors). In both protocols, the median chimerism values for CD3+ cells and WBC at day 28 were >80% and 90% donor cells, respectively and T, B and NK lymphocyte frequency and numbers were identical at protocol entry. One day following TBI conditioning there was a 32% depression in CD3+ cells in the day -21 protocol that was partially recovered on days 28 and 70 post transplant (27% and 16% respectively). In the day -10 protocol there was a 45% depression in the frequency of CD3+ cells (p<0.05) on day following TBI, which was depressed further (72%) on day 28 post transplant with partial recovery on day +70 (24% depression). In both protocols the CD8+ T cells were depressed at these time points; however, the CD4+ cells were only depressed in the second protocol. Similarly, by day 28 post transplant the frequency of monocytes, granulocytes and NK cells had recovered to pre-transplant levels. The DCs, both CD123+ and CD11c+ were not suppressed by the conditioning regime, although 70 days following transplant in the day -10 protocol the CD123+ cell frequency was significantly. Conclusions: There appears to be a trend for a lower incidence of acute GVHD in the day -10 protocol, which can be associated with a reduction in the frequency of circulating T cells. This significantly greater reduction in T cell frequency, observed one day following TBI and 28 days post transplant, is directly associated with the timing of transplant relative to pentostatin conditioning as the post-grafting immunosuppression was similar between the two protocols at these time points.

To determine the role of rATG to modify the development of acute GvHD we conducted a randomized, prospective, open-label, multi-center pilot study in patients with ALL or AML in first or second remission. CsA was the only agent used for GvHD prophylaxis. Patients (N=22) were randomized (1:1) to receive either 4.5 mg/kg or 8.5 mg/kg of ATG during the preparative regimen. The rATG infusions were completed on Day -1, within 18 hours prior to SCT on Day 0. The low (N=11) and high dose groups (N=11) were comparable for age, diagnosis and remission status. The mean age was 41.4 and 39.6 years in the low and high dose groups respectively. In both groups 6/11 patients had AML and 5/11 had ALL. All patients received a fully ablative preparative regimen from a HLA matched related donor. Results: Mean time to neutrophil engraftment was 6.5 and 7.3 days for the low and high dose arms, respectively. The incidence of Grade > II acute GvHD was 0/11 for the low dose arm and 3/11 for the high dose arm. Infections were seen in 9/11 patients in both groups. The cumulative number of hospitalization days was 30.4 and 47.6 days (p=n.s.) in the low and high dose groups respectively. The overall survival at 180 days was 63.6 % in the low dose group and 54.5 % in the high dose group (p=n.s.). The disease free survival (DFS) at day 180 was 54.5% in both groups. The treatment related mortality (TRM) was 18.2 % in the low dose and 45.5 % in the high dose (p=n.s.) The relapse rate was 18.2 % in the low dose group and 0% in the high dose group (p=n.s). Conclusion: This pilot study suggests that rATG may modify the development of serious acute GvHD in patients who received a fully ablative SCT. The low, combined, 3/22, incidence of acute GvHD (13.6%) in patients who received rATG(Thymoglobulin) and post transplant CsA suggests that this combination is an acceptable regiment for GvHD prophylaxis. The lower dose of rATG, 4.5 mg/kg, was as effective and was associated with a lower TRM as compared to the higher, 8.5mg/kg dose. The use of rATG administered during the preparative regimen was well tolerated and may provide an effective alternative to methotrexate when used as part of an ablative conditioning regimen in HLA identical related donor SCT.

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.

Despite significant advances in the treatment of acute myeloid leukemia (AML) in younger adults, there has been little progress in the treatment of older patients (age≥60 years), who comprise the majority of those with the disease. Clofarabine is a purine nucleoside analog with single agent activity in patients with relapsed AML. In addition, clofarabine potentiates Ara-C cytotoxicity in vitro through increased intracellular Ara-CTP accumulation, making this an attractive combination. A phase I/II study has therefore been initiated combining clofarabine with standard dose cytarabine (100mg/m2/day x 7) in pts age ≥60 years with de novo AML. The starting dose of clofarabine was 30mg/m2/day x 5 beginning on day 2 (Dose level I). Patients were accrued in cohorts of 3–6 to establish dose limiting toxicity (DLT); cohort expansion at the maximum tolerated dose (MTD) is planned in phase II using a Simon 2-stage design. Detailed plasma and intracellular pharmacokinetics were performed during induction therapy with Ara-C alone (day 1), and following the addition of clofarabine (day 2) to determine the effect of clofarabine on intracellular Ara-CTP accumulation. Pts with residual AML on d14–21 restaging bone marrow (BM) biopsy were eligible to receive Re-Induction with 5 days of clofarabine & Ara-C. Those achieving complete remission were also eligible to receive 1–2 cycles of consolidation with Ara-C (d1–5) & clofarabine (total 3 cycles of planned therapy). Dose limiting toxicity was encountered at dose level I (see Table 1). 2/4 pts achieved CR, in 1 case with residual cytogenetic abnormality, and there were 2 treatment-related deaths from infxn (culture neg sepsis, n=1; Candida tropicalis, n=1). In the latter case (pt 4), BM aplasia was achieved, but the pt died on d25 prior to hematologic recovery. In view of the DLT, the protocol has therefore been amended to allow 25% dose de-escalation of clofarabine to Dose Level -I (22.5mg/m2/day x 5), and to limit eligibilty to pts age 60–75 yrs inclusive. Routine use of aggressive pre-hydration and antibiotic and antifungal prophylaxis is now mandated. Clofarabine & cytarabine is a highly active induction regimen in older adults age ≥60 yrs with de novo AML, but has significant myelosuppressive and infectious toxicity. The study is proceeding in phase I at Dose Level -I to establish the MTD. Phase I, Dose Level I PT AGE, GENDER FAB CYTOGENETICS F/U BM TOXICITY (Gr.III/IV) OUTCOME MLD - multilineage dysplasia; F&N - fever & neutropenia; CR - complete remission 1 66M M2 Diploid D21: residual AML renal, infxn Death 2 61M M2 Complex D14: aplastic F&N CR 3 69M M2 with MLD Intermediate Risk D21: recovering F&N CR 4 77F M2 Diploid D14: aplastic renal, infxn, capillary leak Death

Background: Activating mutations of FLT3 occur in roughly 30% of adults with AML and are associated with an increased relapse rate and reduced survival. Lestaurtinib (CEP-701), an oral FLT3 kinase inhibitor, has activity as monotherapy in relapsed/refractory AML patients with FLT3 mutations. In vitro studies have demonstrated synergistic cytotoxic effects when FLT3 mutant AML cells were exposed to chemotherapy followed by lestaurtinib. Methods: Patients with AML in first relapse were randomized 1:1 to receive chemotherapy alone or chemotherapy followed by treatment with lestaurtinib. Eligible patients were required to have a FLT3 activating mutation (ITD or Asp835) at first relapse. Chemotherapy was determined by duration of initial remission: 1–6 months, MEC (mitoxantrone, etoposide, cytarabine); 6–24 months, HiDAC (high-dose cytarabine). Both regimens consisted of 5 days of chemotherapy, and, for those patients randomized to receive lestaurtinib (80 mg bid), treatment was started two days after the final dose of chemotherapy. The primary endpoint was complete remission (CR), determined within 42 days of study entry. Prior to initiation of chemotherapy, leukemia cells of the patients randomized to lestaurtinib were tested for in vitro sensitivity to lestaurtinib using a cytotoxicity assay. Plasma samples were obtained at baseline and Days 15 and 42 to determine FLT3 inhibitory activity. Results: To date, 42 patients have been enrolled and 34 have completed the primary endpoint assessment (12 male and 22 female, median age 58, range 26–72). In general, lestaurtinib was well tolerated, with mild to moderate gastrointestinal symptoms and fatigue attributed to the drug. Of the pretreatment leukemia samples available for cytotoxicity analysis, 78% were sensitive to lestaurtinib in vitro. Thirteen of 17 (76%) patients achieved a plasma FLT3 inhibitory activity of greater than 85%. All patients who achieved this degree of plasma FLT3 inhibitory activity and whose pretreatment leukemia cells were sensitive in vitro to lestaurtinib achieved a clinical response. Conversely, patients with insensitive cells or low drug plasma levels did not respond. Ten of 17 patients randomized to lestaurtinib showed evidence of response (CR or partial response [PR]), with 5 achieving CR, 3 CR with incomplete blood count recovery (CRi), and 2 PR. Four of 17 patients randomized to receive chemotherapy alone had achieved a response (2 CR, 2 CRi). Accrual is ongoing, and updated clinical and correlative data will be presented. Conclusions: These results demonstrate that real-time correlative studies are able to accurately predict response to lestaurtinib, and the clinical benefit achieved for these patients provides important evidence that targeting FLT3 remains a valid therapeutic approach for AML with FLT3 activating mutations.