ALBERT

Description: Key PointsFirst-line CPX-351 vs 7+3 control in newly diagnosed AML improves 60-day mortality, remission rate, and OS (HR = 0.46, P = .01) in sAML subset.

Description: Aplastic anemia (AA), a potentially fatal disease, may be cured with marrow transplantation. Survival in pediatric patients has been excellent early after transplantation, but only limited data are available regarding late effects. This study evaluates late effects among 152 patients followed 1-38 years (median, 21.8 years). Transplantation-preparative regimes were mostly cyclophosphamide with or without antithymocyte globulin. Survival at 30 years for the acquired AA patients is 82%, and for the Fanconi anemia patients it is 58% (P = .01). Multivariate analysis demonstrated that chronic GVHD (P = .02) and Fanconi anemia (P = .03) negatively impacted survival. Two Fanconi patients and 18 acquired AA patients developed a malignancy that was fatal for 4. There was an increased incidence of thyroid function test abnormalities among those who received total body irradiation. Cyclophosphamide recipients demonstrated normal growth, basically normal development, and pregnancies with mostly normal offspring. Quality-of-life studies in adult survivors of this pediatric transplantation cohort indicated that patients were comparable with control patients except for difficulty with health and life insurance. These data indicate that the majority of long-term survivors after transplantation for AA during childhood can have a normal productive life.

Description: Abstract 3759 For genetic blood diseases, such as primary immunodeficiencies, gene therapy targeted to hematopoietic stem cells (HSCs) is a feasible and now proven effective therapeutic option for patients who lack a histocompatible HSC. However, the risk of adverse outcomes resulting from insertional oncogenesis is a major concern. We are investigating whether inclusion of the herpes simplex virus thymidine kinase (HSVtk) gene into integrating vectors into rhesus macaque HSCs confers ganciclovir (GCV) sensitivity allowing ablation of vector-containing cells from the blood and other hematopoietic compartments, as an approach to increasing safety of gene therapy procedures. HSVtk suicide genes have been studied in detail in transduced mature T cells, but never in stem and progenitor cells. We infused autologous CD34+ cells transduced ex vivo with gammaretrovirus vectors encoding the HSVtk as suicide gene along with marker genes into 4 rhesus macaques, following myeloablative irradiation. In the first animal, a vector consisting of the MND backbone driving the sr39 high affinity tk mutant, and IRES and a truncated NGFR marker gene was used. A stable marking level of 5% NGFR+ circulating cells was observed for 6 months following transplantation, confirmed by q-PCR. The drug GCV was infused at 5 mg/Kg BID for 21 days. This animal had complete elimination of vector-containing cells in all peripheral blood lineages as assessed by flow cytometry and qPCR, and remains negative now 4 months after GCV discontinuation. Three additional animals were transplanted with autologous CD34+ cells transduced with a vector containing a standard HSVtk gene and GFP as a marker. These animals had lower stable marking levels of approximately 1% at 4 months post-transplant, and after 21 days of GCV, had a clear decrease in the level of GFP+ cells, but not complete ablation, likely due to lower drug-sensitivity of the tk protein expressed by this vector. Cells with a lower level of GFP expression were not eliminated, supporting this hypothesis. Additional animals receiving cells transduced with the sr39 tk retroviral vector and with a lentiviral vector containing a codon-optimized HSVtk are in progress. These data suggest that inclusion of a suicide gene in integrating vectors may be an effective way to address genotoxicity concerns, should clonal outgrowth occur, and increase safety of HSC-targeted gene therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Risk factors for grades 2-4 acute graft-versus-host disease (GVHD) and for chronic GVHD as defined by National Institutes of Health consensus criteria were evaluated and compared in 2941 recipients of first allogeneic hematopoietic cell transplantation at our center. In multivariate analyses, the profiles of risk factors for acute and chronic GVHD were similar, with some notable differences. Recipient human leukocyte antigen (HLA) mismatching and the use of unrelated donors had a greater effect on the risk of acute GVHD than on chronic GVHD, whereas the use of female donors for male recipients had a greater effect on the risk of chronic GVHD than on acute GVHD. Total body irradiation was strongly associated with acute GVHD, but had no statistically significant association with chronic GVHD, whereas grafting with mobilized blood cells was strongly associated with chronic GVHD but not with acute GVHD. Older patient age was associated with chronic GVHD, but had no effect on acute GVHD. For all risk factors associated with chronic GVHD, point estimates and confidence intervals were not significantly changed after adjustment for prior acute GVHD. These results suggest that the mechanisms involved in acute and chronic GVHD are not entirely congruent and that chronic GVHD is not simply the end stage of acute GVHD.

Description: Abstract 3626 Background: CPX-351 is a liposomal formulation of cytarabine (Ara-C) and daunorubicin (DNR) in a 5:1 molar ratio designed to maximize anti-tumor synergy. CPX-351 accumulates within bone marrow with preferential uptake of liposomes by leukemia cells followed by intracellular release of encapsulated drug. A Phase 2b study randomized untreated older patients with AML 2:1 to CPX-351 or standard 7+3. At entry patients were stratified by age, cytogenetics, and presence or absence of antecedent hematologic disorder (AHD)/prior cytotoxic treatment that evolved into AML (sAML) into a high-risk group (age ≥70 or adverse cytogenetics or sAML) or standard-risk group (age 60–69 and non-adverse cytogenetics and de novo AML). The standard-risk group was relatively homogeneous, while the high-risk group gathered together patients with one, two or all three risk factors. Response after CPX-351 was increased in all patients (66.7% vs. 51.2%). Patients with secondary AML had coherent improvement in survival (HR=0.40, p=0.01), EFS (HR=0.51, p=0.04), and CR + CRi rate (57.6% vs. 31.6%). This report presents the results of uni/multivariate analyses for survival and event free survival. Methods: Untreated de novo or sAML patients, aged 60–75, PS= 0–2, serum creatinine 〈 2.0 mg/dL, total bilirubin 〈 2.0 mg/dL, ALT/AST 〈 3 × ULN, and LVEF ≥50% were eligible. Patients with history of treatment for their AHD were eligible. Patients received up to 2 induction and 2 consolidation courses of CPX-351 (100 u/m2; D 1, 3, 5) or 7+3 (Ara-C= 100 mg/m2/d and DNR= 60 mg/m2). Consolidation with hematopoietic stem cell transplantation (HSCT) was permitted. The primary endpoint was CR + CRi rate. Event free survival was calculated from randomization to the date of documentation of persistent leukemia after induction, relapse after achievement of response, or death, whichever occurred first. Allogeneic transplants were permitted for post remission treatment. The association between baseline characteristics and survival was assessed by univariate and multivariate Cox regression analyses. Response was included as a time-dependent variable. The multivariate model used stepwise selection to identify prognostic factors after accounting for potential treatment effects. Results: Significant factors affecting overall survival (OS) in the univariate analysis included: response (p=0.01), ≥2 risk factors (p=0.013), secondary AML (p=0.021), and adverse cytogenetics (p=0.038). After accounting for treatment, response (p=0.003) and ≥2 risk factors (p=0.005) were strongly associated with OS in the multivariate model. Response and 60-day deaths were similar in both study arms for patients with 0 or 1 risk factor. Patients with 2 or 3 risk factors treated with CPX-351 had rates of response and 60-day death rates that were similar to those with only 0 or 1 risk factor. Control arm patients with multiple risk factors did much worse, with fewer CRs, no CRi's, and a substantially higher rate of early deaths. Conclusions: CPX-351 is highly active in every subgroup of older patients with newly diagnosed AML. This analysis demonstrates that CPX-351 minimizes the loss of response and increase in 60-day mortality that occurs with 7+3 treatment among patients with secondary AML and adverse cytogenetics. The relative benefit of CPX-351 is greatest among patients with ≥ 2 risk factors. Disclosures: Lancet: Celator Pharmaceuticals: Research Funding. Cortes:Celator Pharmaceuticals: Research Funding. Kovacsovics:Celator Pharmaceuticals: Research Funding. Hogge:Celator Pharmaceuticals: Research Funding. Kolitz:Celator Pharmaceuticals: Research Funding. Hoering:Celator Pharmaceuticals: Consultancy. Chiarella:Celator Pharmaceuticals: Employment. Louie:Celator Pharmaceuticals: Employment. Feldman:Celator Pharmaceuticals: Research Funding.

Description: Natural killer (NK) cells are cytotoxic leukocytes defined in humans and rhesus macaques by the expression of CD56 and/or CD16, and the absence of T, B, and myeloid markers. Functionally, they are defined by lysis of tumor targets in a major histocompatibility complex (MHC)–unrestricted fashion, and production of cytokines critical to innate immunity. Ex vivo expanded NK cells are being developed as adoptive transfer therapeutics for a variety of malignancies. However, the ontogeny and hematopoietic lineage relationships of human NK cells have been difficult to study, given differences in phenotype and function between human and murine NK cells, poor NK engraftment and development in human xenografts, and restricted development of NK cells from hematopoietic stem and progenitor cells (HSPC) in vitro. In addition, several phenotypic subsets of NK cells have been described, and their relationships and relative locations within the hematopoietic hierarchy are supported by limited in vivo data. We “barcoded” individual CD34+ HSPC from 3 macaques using lentiviral vectors carrying highly diverse oligonucleotides, allowing precise quantitation of clonal contributions via low cycle PCR amplification followed by high-throughput sequencing and computational analysis (Lu et al, Nat Biotech). We quantitated barcode levels, and thus individual HSPC clonal contributions, over time and between lineages, following TBI and infusion of autologous barcoded CD34+ cells. Reconstitution of the NK compartment was clonally distinct from T, B and myeloid (My) lineages. Following reconstitution of NK, T, B and My from uni-lineage progenitors at 1-2 months(m), by 3-4 m, as expected from murine and in vitro models, multi-lineage clones began to contribute and dominate, first B/My, then B/T/My. However, NK cells remained clonally distinct through 9 m, despite overall clonal diversity and marking levels in NK lineage similar to B/T and My. Even the most abundant clones in the NK lineage were not found contributing in any significant way to B/T or My lineages. Shared B/T/My/NK clones finally began to contribute more extensively at 9-14 m. We fractionated peripheral blood NK cells into 3 NK subsets and compared them to overall PB and lymph node NK cells at 4.5-6.5 m. The majority (75-90%) of PB rhesus NK cells are found in the CD16+/CD56- PB subset (corresponding to human CD16+/CD56dim cytotoxic NK), and this subset accounted for the clonal pattern in overall NK cells (Pearson r=0.98 vs overall NK cells), and the disparity from B/T/My, with r values all

Description: Abstract 655 Introduction: CPX-351 is a liposomal formulation encapsulating CYT and DNR at a 5:1 molar ratio that maximizes synergy. CPX-351 was well tolerated and demonstrated markedly prolonged plasma half-life for CYT (t1/2=31.1 hours) and DNR (t1/2=21.9 hours) in Phase 1. Responses (CR + CRp) were noted in patients with prior 7+3 exposure including some with multiple relapses and primary induction failure. A Phase 2 study using 2:1 randomization to demonstrate efficacy and safety of CPX-351 versus conventional 7+3 regimen is summarized here. Methods: Subjects with de novo or 2o AML (based on history of MDS, MPD, or prior chemotherapy exposure), ECOG PS of 0–2, SCr 70, and 2o AML. Cytopenia-associated adverse event rates were higher in the CPX-351 treatment arm: febrile neutropenia (64.7% vs. 51.2%), infections of all grades (84.7% vs. 68.3%), bacteremia (40% vs. 22%), petechiae (32.9% vs. 12.2%) and ecchymoses (11.8% vs. 2.4%). Median time to count recovery after induction was longer after CPX-351, 38 vs. 34 days for ANC〉1000 and 44 vs. 33 days for Plt〉100K. CPX-351 was associated with reduced early mortality compared to 7+3 (4.7% vs. 14.6% at 60 days, p= 0.053, Chi Square). Complete analysis of adverse events will be presented. Kaplan Meier analysis reveals a hazard ratio for CPX-351 for overall survival after a minimum of 6 months follow-up to be 0.83 (95%CI: 0.62–1.13; p=0.24). The estimated mean difference within the first year was 2.2 months. Response (CR + CRp) was highly predictive of prolonged survival with the hazard ratio of responders vs. non responders of 0.55 (95% CI: 0.41–0.73; p

Description: The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027–induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027–induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo.

Description: Abstract 2061 The impact of graft versus host disease (GvHD) on readmission rates following allogeneic stem cell transplantation (SCT) has not been reported. We hypothesised that patients with GvHD would have a higher readmission rate than patients without GvHD. To test this hypothesis a retrospective review was undertaken of 187 consecutive patients who underwent allogeneic SCT from 1/1/2006 to 30/4/2009 at the Royal Marsden Hospital, UK. Data were collected from the electronic patient record. The study was approved by the hospital audit committee. Patient characteristics: male 113(60%), median age 47 yrs (17–70), diagnosis: acute leukaemia 123 (66%), chronic leukaemia 22 (12%), lymphoma 23 (12%), myeloma 12 (6%), other 7 (4%), donor: sibling 60 (32%), unrelated 115 (61.5%), cord 12 (6.5%), stem cell source: PBSC: 158 (84.5%), BM 17 (9%), cord 12 (6.5%); donor sex: M 111(59.5%), F 64 (34%), cord 12 (6.5%); match: full 130 (69.5%), mismatch 45 (24%), cord 12 (6.5%), conditioning intensity: full 73 (39%), reduced 114 (61%); use of alemtuzumab 113 (60%); GvHD prophylaxis: cyclosporine 116 (62%), mycophenolate 32 (17%), cyclosporine and methotrexate 26 (14%), other 13 (7%). Median follow up was 3.8 (2.2–5.5) yrs from transplant. 118/187 (63%) of patients developed GvHD. There was no significant difference between patients with or without GvHD except for increased use of alemtuzumab in the non-GvHD group (71% v 54%, p=0.024). GvHD was diagnosed clinically at the transplant centre. Biopsies were undertaken if the diagnosis was unclear. Glucksberg criteria were used to stage acute GvHD (aGvHD). NIH criteria were used to diagnose chronic GvHD (cGvHD). 45/118 (38%) had biopsy-proven GvHD. 88/187 (47%) had aGvHD (grades: 1 – 17 (9%), 2 – 43 (23%), 3 – 15 (8%), 4 –13 (7%)). 58/187 (31%) of patients had cGvHD. 36/187 (19%) had both acute and chronic GvHD (52 (29%) had aGvHD alone, 22 (12%) had cGvHD alone). 8 developed GvHD following donor lymphocyte infusion. 104/118 (88%) required steroid treatment. 61/118 (52%) commenced steroids within 100 days of transplant. 61/118 (52%) were steroid refractory. 5 patients had received a second allogeneic transplant for relapse to induce GvHD and graft versus malignancy effect. The median duration of initial transplant admission was 31 days (20–138) in GvHD group compared to 32 days (16–103) in non GvHD group (p=NS). 14 patients died during initial admission (5 in GvHD group, 9 in non-GvHD group) and were excluded from readmission analysis. The overall readmission rate was higher in GvHD patients (89% (101/113) v 41/60 (68%), p=0.001). In the first 100 days post transplant, 42/56 (75%) of patients who had started steroids were readmitted compared to 60/117 (51%) in patients who started steroids after day 100 or had no GvHD (p=0.003).Critical care unit admission was higher in the GvHD group (38/113 (34%) v 7/60 (12%),p=0.002). The mean total number of admission days was higher in the GvHD group (42 days v 18 days, p

Description: Background: Genetic polymorphisms in the TPMT gene cause inter-individual variability in 6MP metabolism and tolerability. In the context of ALL maintenance therapy (SJCRH Total XII) that was heavily reliant upon 6MP and methotrexate (MTX) and without individualizing 6MP based on TPMT genetics, dose-limiting toxicity due to TPMT defects resulted in interruptions of 6MP and MTX administration, which have been associated with worse ALL outcomes in some studies (McLeod, Br J Haematol 1999; Welch, Cancer Chemother Pharmacol 1996). Overall 6MP dose intensity (i.e. prescribed dose /protocol dose) was 83% (Relling, Blood 1999). Here we characterized the dose intensity (DI) and tolerance of 6MP among patients with different TPMT genotypes in a more contemporary ALL regimen that included multiple agents in addition to 6MP and MTX. Our goal was to determine the tolerability of 6MP in patients with heterozygous vs wild-type TPMT in the context of multi-agent ALL therapy. Method: 388 children with ALL on the SJCRH Total XV protocol were evaluable for this study. TPMT genotype was analyzed at day 5 of remission induction using PCR and exome sequencing. Of the 388 patients, 347 (89%) were classified as wild-type and 41 (11%) carried a low activity allele, including *2, *3A, *3C and 677G〉A (Udaka, Genet Test 2005). No homozygous deficient patients were identified. It was generally recommended that patients with TPMT heterozygosity receive a starting 6MP dose of 50-60 mg/m2/d regardless of risk arm. Wild-type patients on the low-risk (LR) arm (n = 202) received daily 6MP at 75 mg/m2, while those on the standard/high-risk (SHR) arm (n = 186) started with 50 mg/m2/d in weeks 1-16, then increased to 75 mg/m2/d until week 120 (girls) or week 146 (boys). No 6MP was given during reinductions I (weeks 7-9) and II (weeks 17-19). The daily dosage, days of treatment, and DI of 6MP were analyzed for each phase of maintenance therapy. 6MP dosages were re-evaluated every 8-16 weeks to maintain a desired degree of leukopenia. Dosage was decreased if patients had myelosuppression (WBC 〈 1000/mm3, ANC 〈 300/mm3 and platelet 〈 5 × 104/mm3). Dosage increases were considered if patients missed less than 25% of therapy but had persistently high WBC (〉 4000/mm3) and ANC (〉 1000/mm3). In addition to the two reinductions, SHR patients received weekly asparaginase during weeks 1-19 and monthly cyclophosphamide/cytarabine until week 68. After week 20, patients received daily 6MP and weekly MTX with addition of monthly vincristine/dexamethasone (VCR/DEX) pulses until week 96, after which only 6MP and MTX were given. Result: The median cumulative DI was 83% (range 14-135%) in wild-type and 68% (range 5-102%) in heterozygotes, similar to that reported for SJCRH Total XII (Relling, Blood 1999). The DI of each phase differed by TPMT genotype (Fig 1). During week 10-16, wild-type patients on the SHR arm had lower median DI (77%, range 12-173%) than those on the LR arm (85%, range 0-110%; P = 6.4 × 10-5) despite the lower protocol dose in the SHR arm, supporting the practice of lowering the protocol 6MP dose during the early intensive weeks of therapy for the SHR arm, and suggesting the higher dose of 75 mg/m2/d is well tolerated even during early intensive therapy for the LR arm. The DI was similar in weeks 20-95 (6MP/MTX plus monthly VCR/DEX pulses) and in weeks 96-146 (6MP/MTX only) after stratifying by genotype and risk arm (Fig 1), suggesting that VCR/DEX pulses do not alter 6MP tolerance. TPMT heterozygotes received lower median daily 6MP doses (61 mg/m2) than wild-type (73 mg/m2; P = 3.2 × 10-7) during maintenance therapy. By using the lowered daily dose in heterozygotes, we prevented dose interruptions: the median percentage of days with no 6MP therapy was similar in heterozygotes and wild-type (12% vs 11% missed, P = 0.5). Conclusion: Using the approach of lowering the 6MP dose from 75 to 50 mg/m2/d during early intensive therapy for SHR patients results in reasonable DI in both groups. The lower tolerated daily dose of 6MP 60 mg/m2 in heterozygotes, compared to 75 mg/m2 in TPMT wild-type patients, supports the recommendation for a lower starting dose of 6MP for TPMT heterozygotes. 6MP DI was similar during phases that did vs did not include VCR/DEX, and similar in this contemporary regimen that includes VCR/DEX pulses compared to older studies without VCR/DEX pulses. Fig 1 Fig 1. *P-value of comparison between TPMT genotypes, and #P-value of comparison between weeks 20-95 and weeks 96-146. Disclosures Evans: St. Jude: In accordance with institutional policy (St. Jude), I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties. Relling:St. Jude: In accordance with institutional policy (St. Jude), I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties.