Peripheral Blood Blast Count Research Articles

Is Type of Monosomy (Total or Partial) Crucial for Prognostic Value of Monosomal Karyotype in AML Patients? – Preliminary Results of Retrospective Polish Adult Leukemia Group (PALG) Study,

Abstract 3537 Objectives:Cytogenetics is one of the most important prognostic factors in acute myeloid leukemia (AML). Breems et al., based on banding techniques (BT), have identified a monosomal karyotype (MK) to be associated with particularly poor survival. MK is defined by the presence of an autosomal monosomy in conjunction with at least one other autosomal monosomy or structural abnormality. However, classical BT may be not sufficient to confirm the “real” loss of a particular chromosome, especially in patients with complex karyotype (≥ 3 or ≥ 5 separate abnormalities) where parts of „missing” chromosomes could be involved in structural aberrations such as: additions, derivative chromosomes, rings, marker chromosomes and dicentrics. Molecular cytogenetic techniques, such as FISH, could be useful to verify if observed monosomy was “total” (loss of a complete chromosome) or “partial” (loss of only a part of chromosome including centromere).The aim of our study was to assess if the type of monosomy (total or partial), defined using FISH influences the prognosis of AML patients with MK.Materials and methods: Fifty newly diagnosed AML patients with MK, treated between January 2005 and January 2011 with PALG AML1/2004 and AML2/2004 protocols were included into the study. Cytogenetic analysis was performed on metaphases from bone marrow aspirates taken at diagnosis using standard BT. Karyotypes were centrally reviewed by two independent cytogeneticists and reported in accordance with the ISCN. In 41 cases FISH was performed with painting probes and in justified cases additionally with satellite probes and locus-specific probes to assess the type of monosomy. In 9 patients no confirmation by FISH was needed. Results:In 8/50 (16%) patients FISH verification proved the complex, but not monosomal (total or partial) karyotype. These patients were excluded from further study. The median age of 42 evaluable patients was 58 years (range 20–71 years). There were 26 males and 16 females. Twenty three (55%) patients received intensive induction chemotherapy according to PALG protocols (Holowiecki et al. Leukemia 2004), 8 patients - low dose cytarabine and 11 patients with high frailty index received treatment with hydroxyurea (n=9), and best supportive care (n=2).In 38/42 (90.5%) analyzed cases with MK abnormalities were complex. In all but one ≥5 separate aberrations were present. In 27/42 (64.3%) cases a total monosomy was confirmed by FISH (group A) and in 15/42 (35.7%) cases FISH revealed a monosomy to be the partial one (group B). Both groups A and B were comparable in terms of age, sex, immunophenotype and factors associated with tumor mass (leukemic bone marrow infiltration, WBC and peripheral blood blast count, as well as LDH activity). There was also no difference in the proportion of intensive and non-intensive treatment strategies between both groups. Six (26,1%) of intensively treated patients achieved complete remission (CR). The CR rate in patients with partial monosomy (40%) was higher than in total monosomy group (15,4%), however the difference was not significant (p=0.19; Fisher’s exact test). The median overall survival (OS) for all evaluable patients was 66 days (range 1–659 days) and the probability of OS at 1 yr was 14% (95% CI 4–23%). Total monosomy was the only factor associated with decreased probability of OS both in univariate (p=0.036) (Fig. 1) and multivariate (p=0.037) analyses in AML patients with MK. [Display omitted] Additionally, in patients with total monosomy the frequency of distinct monosomies was analyzed. The most frequent monosomies were monosomy 7 (n=13; 48%) and monosomy 18 (n=10; 37%). Other total monosomies included: monosomy 17 (n=3; 11%), monosomy 16 (n=2; 7%) and others (n=4). We did not observe any total monosomy 5. Conclusions:Our results indicate for the first time that partial monosomy is associated with significantly better OS than total monosomy in high risk AML with MK and provide further evidence for the heterogeneity of this defined cytogenetic group. These data also clearly demonstrate an important role of FISH method for precise evaluation of complex karyotype, which results in correct classification within MK group, as well as in adequate description of monosomy type. Disclosures:Wierzbowska:Genzyme: Membership on an entity's Board of Directors or advisory committees. Dmoszynska:Roche: Honoraria; Mundipharma:.

Survivin Is Highly Expressed in AML Stem Cells and Predicts Poor Clinical Outcome

Abstract 238Survivin, a member of the inhibitor of apoptosis (IAP) protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignant cells, including leukemic cells. We showed previously that targeting survivin expression in AML cells induces a cell proliferation defect and subsequent cell death involving the mitochondrial pathway. To assess its usefulness as a prognostic marker in AML, we profiled survivin expression in samples from 511 newly diagnosed AML patients using a validated reverse-phase protein array and correlated its protein expression levels with clinical outcomes. We found that survivin levels were higher in the bone marrow (BM) than in peripheral blood (PB) leukemic cells in 140-paired samples (p = 0.0001) consistent with its function in cell proliferation. Survivin levels did not correlate with cytogenetic groups, the mutational status of nucleophosmin or FLT-3, BM or PB blast counts, white blood count, or patient performance status. Interestingly, survivin levels were found to significantly predict shorter overall (P = 0.016) and event-free (P = 0.023) survival in multivariate Cox model analysis. Age, gender, white blast count, BM blasts, cytogenetics, and survivin levels were included in the final model. The groups expressing low and high survivin were defined by the median value. This was also true when data sets from BM and PB were analyzed separately. Further, we found that survivin levels were significantly higher in CD34+38− AML stem/progenitor cells than in bulk AML blasts and total CD34+ cells (2.06 and 1.91 fold, respectively, P < 0.001) when we analyzed survivin expression in 37 samples of isolated CD34+38− AML cells (see Figure). [Display omitted] These results suggest that survivin expression levels are prognostic in AML and that survivin is overexpressed in AML stem/progenitor cells, may play an important role in AML stem cells, and may thus be an important target in AML therapy. Disclosures:No relevant conflicts of interest to declare.

Phase I trial of a novel human monoclonal antibody mAb216 in patients with relapsed or refractory B-cell acute lymphoblastic leukemia

This phase I trial was conducted to determine the safety and pharmacokinetics of monoclonal antibody 216, a human monoclonal Immunoglobulin M antibody targeting a linear B-cell lactosamine antigen, administered alone and in combination with vincristine in patients with relapsed or refractory B-cell acute lymphoblastic leukemia, and to preliminarily assess tumor targeting and efficacy. Three cohorts of patients received escalating doses of monoclonal antibody 216 administered as an intravenous infusion. In the case of poor response to the first dose of monoclonal antibody 216 alone, defined as less than 75% reduction in peripheral blood blast count, a second dose of the antibody with vincristine was given between days 4 and 7. Responses were assessed weekly until day 35. Serum concentration of monoclonal antibody 216 was measured before and after infusion. Monoclonal antibody 216 targeting was determined with an anti-idiotypic antibody to monoclonal antibody 216 and preliminary efficacy was analyzed by changes in peripheral blood blasts. Thirteen patients were enrolled. One episode of grade 3 epistaxis was the only dose-limiting toxicity observed. All patients showed a poor response to the first monoclonal antibody 216 infusion with a decrease in peripheral blasts from 6-65% in 9 patients. In 8 patients, addition of vincristine to monoclonal antibody 216 resulted in an average reduction of the peripheral blasts of 81%. One patient without peripheral blasts achieved a hypoplastic marrow without evidence of leukemia after one infusion of monoclonal antibody 216 and monoclonal antibody 216/vincristine each. Monoclonal antibody 216 was detected on peripheral blasts in all patients. Treatment with monoclonal antibody 216 in combination with vincristine is feasible and well tolerated in patients with relapsed or refractory B-cell acute lymphoblastic leukemia. Binding of monoclonal antibody 216 to leukemic blasts was efficient, and favorable early responses were observed.

Serine protease (TPS): A diagnostic and prognostic marker in pediatric patients with acute non-lymphoblastic leukemia

The Serine protease, TPS (tryptase), is a specific marker for mast cells and mast cell-associated disorders. However, substantial amounts of TPS are also expressed in neoplastic myeloid, non-mast cell lineage. The aim of this study is determination and quantitation of TPS expression in patients with acute non-lymphoblastic leukemia (ANLL); to evaluate its prognostic value and its relevance as a genetic marker for detection of minimal residual blast cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect levels of TPS from 30 newly diagnosed ANLL children and 10 normal children served as controls. TPS levels for positive cases were reevaluated after induction chemotherapy; after onset of relapse or by the end of the study. Our results showed that the gene transcripts were detected in 56.7% of patients but were not expressed by normal controls. The highest frequency of TPS was recorded in patients with M4 showing significantly higher levels compared to other FAB (French–American–British Classification) subtypes. TPS levels were directly correlated to TLC, absolute blast counts in peripheral blood, levels of CD34 and CD117. After induction chemotherapy, levels of TPS decreased significantly in those who achieved complete remission while it increased significantly in relapsed patients, with a risk estimate of relapse six times higher in positive cases than TPS negative patients. 84.6% of the patients negative for TPS achieved complete remission with better disease free survival. In conclusion, TPS is expressed in a group of patients with ANLL thus serves as a disease related marker; it could be used as a prognostic indicator in evaluating remission status and early relapse as its elevated levels are associated with high risk of relapse; again, it is useful in predicting disease outcome.

Predictive Factors for All-Trans Retinoic Acid Related Differentiation Syndrome In Acute Promyelocytic Leukemia.

AbstractAbstract 1052 Background:All-trans retinoic acid (ATRA) used for the treatment of acute promyelocytic leukemia (APL) can lead to development of differentiation syndrome (DS), a possibly life threatening complication. DS is reported in 2–27% of APL patients treated with ATRA. Since ATRA is metabolized by cytochrome P450 (CYP) enzymes, we sought to identify drug interactions that might be associated with a higher risk for the development of DS in a series of APL patients treated with ATRA. We also evaluated overall incidence of DS, intensive care unit (ICU) admission rates, length of stay (LOS) in hospital as well as other predictive factors related to the incidence of DS. Patients:We identified 60 patients with confirmed APL treated at our institution between May 2004 and January 2010. All patients received ATRA 45mg/m2/day. Patients received concurrent chemotherapy with cytarabine and daunorubicin, idarubicin, arsenic or gemtuzumab as part of induction in 16 (27%), 32 (53%), 10 (17%), and 3 (5%) of cases respectively. Diagnosis of DS was made by the presence of the following symptoms based on the definition by Frankel et al. (Annals of Internal Medicine. 1992; 117: 292–296): dyspnea, unexplained fever, weight gain more than 5kg, unexplained hypotension, acute renal failure, hyperbilirubinemia and chest radiograph demonstrating interstitial infiltrates and pleural or pericardial effusion. Patients with ≥ 4 symptoms of DS were classified as severe DS, while patients with 2–3 symptoms were classified as having moderate DS. Patients were retrospectively evaluated for the development of DS for 5 weeks after initiation of ATRA. Medications were followed in patients until the development of DS. For patients who did not develop DS, medications were followed for 21 days. Results:Characteristics of the 60 APL patients included: Median age 44 years (18-79 years), 60% female, median white blood cell (WBC) count on admission 1.8 × 109/L (0.4-67.9 × 109/L). Twenty nine patients (48%, 90% confidence interval 37–60%) experienced DS (14 severe and 15 moderate). Dyspnea, unexplained fever and interstitial infiltrates were the most common features of DS. Twenty six of the 29 patients (90%) who met criteria for severe or moderate DS received treatment with dexamethasone. The mean time to development of DS was 9.4 days (range 1–31 days) with a median of 5 days. Seventeen patients developed DS ≤ 8 days after initiation of ATRA; the other 12 patients who developed DS did so between days 13 and 31 after initiation of ATRA. We did not find any difference in overall incidence of DS whether patients were on concurrent CYP 2C8, 2C9 or 3A4 inhibitors, inducers or substrates. Peripheral blood blast counts > 70% versus ≤ 70% on admission was associated with developing DS (p=0.05 using Fisher's exact test). In addition, patients with BMI > 40 kg/m2 versus ≤ 40 kg/m2 were more likely to experience DS (p=0.00068 using Fisher's exact test). There were no differences in rate of DS in regards to high WBC count, LDH, and albumin on admission. In addition, there were no differences in DS rate in patients receiving different chemotherapy regimens with ATRA. Thirteen (22%) patients were admitted to the ICU (31% in DS patients; 13% in no DS patients; NS). Out of the 29 patients with DS, there were 4 early deaths of which 2 were attributed to DS compared to no early deaths in the patients who did not develop DS (p=0.05). LOS was similar between patients with DS and without DS, 33 days (4-59 days) versus 34 days (19-53 days), respectively. Conclusion:DS may be underreported in the literature, given that this study is the first to find a high (48%) incidence of DS in APL patients treated with ATRA. Patients with peripheral blast counts >70% or BMI > 40 kg/m2 on admission had an increase risk of developing DS. Concurrent use of drugs metabolized in the same pathway as ATRA did not result in increase incidence of DS. Disclosures:No relevant conflicts of interest to declare.

Quantitative Effect of Age In Predicting Empirically-Defined Treatment-Related Mortality and Resistance In Newly Diagnosed AML: Case Against Age Alone as Primary Determinant of Treatment Assignment

AbstractAbstract 2191 Background:Treatment protocols for newly diagnosed AML typically use age (often 60 years) alone to restrict eligibility to either younger or older patients. Implied in this practice is the assumption that age is the principal predictor of therapeutic failure in AML due to either early treatment-related mortality (TRM) or resistance to therapy in patients who do not incur TRM. Yet, clinical observation and previous studies indicate that other prognostic factors modulate the effect of age on TRM and resistance, suggesting that age as sole or primary criterion for treatment allocation may be suboptimal. Methods:To test this hypothesis in newly-diagnosed non-APL AML, we quantified the relative effects of age and other covariates using 1,127 patients (median age: 57 years) treated on Southwest Oncology Group (SWOG) trials from 1986–2009 and 1,604 patients (median age: 61 years) treated on various protocols at M.D. Anderson Cancer Center (MDA) from 2000–2008. We calculated weekly hazard rates (the probability of death in week × given that the patient was alive at the beginning of the week) for both cohorts overall and in various age subgroups. We used the area under the receiver operator characteristic curve (AUC) to quantify the effects of covariates for prediction of TRM and resistance (no TRM but patient does not enter CR or relapses within 1 year of CR), where an AUC of 1 indicates that a covariate is perfect at prediction while an AUC of 0.5 indicates no prediction (i.e. it is no better than flipping a coin). Results:Despite the use of different treatment protocols, survival in the SWOG and MDA cohorts was virtually superimposable. In both cohorts, the maximum weekly hazard occurred at weeks 3 and 4 from start of treatment, after which it decreased. The maximum hazard was relatively independent of age and remained between weeks 3 and 5 in patients age <60 years, age 60–70 years, and age >70 years, respectively. The existence of such a discrete cut-point suggested that patients who die early are qualitatively distinct and prompted us to examine the relative effect of age and other covariates in patients who (a) died within the first 30 days of treatment (our empirically-based definition of TRM, 9% of MDA and 12 % of SWOG patients, respectively) and (b) survived at least 30 days but did not enter complete remission or relapsed within 1 year (“resistant”, 43% of MDA and 59% of SWOG patients, respectively). A model including age alone to predict early mortality had an AUC of 0.67, while a model including performance status (PS) alone had an AUC of 0.72. By comparison, a more refined model hat included PS, age, platelet count, cytogenetics, secondary AML, albumin, white blood cell count, peripheral blood blast count, and LDH yielded an AUC of 0.86. Elimination of age resulted in a model with an AUC that was only minimally lower (0.85). Prediction of resistance was more difficult with a model including age, secondary AML, cytogenetics, peripheral blood blasts, race, hemoglobin, and marrow neutrophils giving an AUC of only 0.70. Elimination of age had little effect (AUC 0.67) while age alone gave an AUC of 0.64. Conclusion:Age alone appears inadequate in predicting resistance and particularly TRM. The use of models based on several covariates improves predictive ability, but the ability to predict resistance is still limited, suggesting the value of randomized trials to assess treatment designed to reduce resistance. The observation that elimination of age has little effect on the predictive ability of such models, suggests that age is primarily a surrogate for other covariates. Disclosures:No relevant conflicts of interest to declare.

A Phase 1 Study of Combotox in Pediatric Patients With Refractory B-lineage Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Combotox is a 1:1 mixture of RFB4-dgA and HD37-dgA which are immunotoxins that target the CD22 and CD19 antigens, respectively. Combotox has different toxicities and targets than chemotherapy and is, thus, a new candidate for the treatment of patients with relapsed ALL. Preclinical data have demonstrated which Combotox is effective in killing pre-B-ALL cell lines and cells from patients with pre-B ALL. We designed and conducted a Phase 1 dose-escalation study using Combotox in children with refractory or relapsed B-lineage-ALL. Seventeen patients aged 1 to 16 years were enrolled in this multi-institution study. They were treated at 4-dose levels: 2 mg/m2, 4 mg/m2, 5 mg/m2, and 6 mg/m2. The maximum tolerated dose was 5 mg/m2 and graft versus host disease defined the maximum tolerated dose. Three patients experienced complete remission. Six additional patients experienced a decrease of >95% in their peripheral blood blast counts, and 1 patient experienced a decrease of 75%. Combotox can be safely administered to children with refractory leukemia. It has clinically important anticancer activity as a single agent. The recommended dose for future studies is 5 mg/m2/dose.

Non-Cycling Cells From Acute Myeloid Leukemia Patients Harbor the FLT3-ITD Mutation and Are Insensitive to TKI258, a Potent FLT3-Directed Inhibitor, in Vitro.

Abstract 479The identification of activating mutations in the FLT3 gene and their impact on prognosis has been crucial to the rationale behind the development of FLT3 inhibitors. While it has been shown that some leukemic cells with high tumorigenic potential exist mostly in a dormant state, it is unclear if this quiescent/non-cycling leukemia-initiating fraction carries the FLT3-ITD mutation and if it is successfully targeted by FLT3 inhibitors. As a paradigm, quiescent Ph+ stem cells in CML have been shown to exhibit resistance to bcr-abl targeted inhibitors. Additionally, results from clinical trials suggest that FLT3 inhibitors reduce the leukemic blast count in peripheral blood but are less successful in the bone marrow where factors regulating hematopoietic stem cell quiescence are active. In order to investigate the non-cycling and cycling human leukemic cell boundary, we devised a biological model that allowed us to distinguish non-cycling AML cells from cycling AML progenitors in human FLT3-ITD positive AML samples. CD34+ cells were isolated from AML samples using magnetic cell sorting, labeled with the cell membrane dye PKH26 to enable tracking of cell division, and cultured on murine stroma for 12 days. Non-cycling AML cells were then separated from cycling cells by FACS sorting and were found to retain a CD34+ primitive phenotype in contrast to expanding leukemic blasts. Fluorescence in situ hybridization analyses revealed that non-cycling cells carried leukemic gene rearrangements (trisomy 8, trisomy 13, t[3;21]and t[16;16] in our cases), and were therefore part of the original leukemic clone. PCR for the FLT3-ITD region showed that in four out of five cases, the FLT3-ITD mutation was present in the non-cycling fraction. To examine the distribution of FLT3-ITD to FLT3 wild type (WT) bearing cells, non-cycling AML cells were FACS sorted, DNA extracted and the PCR products subsequently cloned. Bacterial colonies were sequenced and colony-PCR used to determine the ratio of FLT3-ITD to WT bearing colonies for each patient. These data indicated that at least 25% of non-cycling cells (range 25%-100%) harbored the FLT3-ITD mutation. We then assessed the impact of a potent FLT3-directed inhibitor, TKI258 (Novartis), on leukemic cell expansion and the viability of non-cycling cells. TKI258 has been found to induce apoptosis of FLT3-ITD bearing cells of the human acute monocytic leukemia MV4;11 cell line. In our present study, CD34+ AML blasts from the same FLT3-ITD positive patient samples were grown in vitro in the presence of 0 μM, 0.3 μM (IC50 dose) and 1.25 μM TKI258. In stromal cultures, TKI258 significantly reduced leukemic cell expansion (range 2.13 to 20 fold for untreated cultures and 0.07 to 2.27 fold for 1.25 μM TKI258 treated cultures at day 12, p ≤ 0.05). In methylcellulose colony assays, TKI258 exposure resulted in dose-dependent suppression of colony formation of CD34+ FLT3-ITD positive leukemia cells (60% to 81% reduction in the mean plating efficiency of CD34+ AML cells at 0.3 μM TKI258). Despite this striking anti-proliferative effect, the majority of non-cycling cells from AML patients showed resistance to TKI258 (five out of six cases). In these samples, FLT3-ITD positive non-cycling cells could still be detected after treatment with the equivalent highest clinical dose (1.25 μM) of TKI258. Moreover, at a functional level, limiting-dilution experiments on non-cycling AML cells pre-treated with TKI258 showed no impairment in a modified leukemic cobblestone assay at four weeks compared to untreated non-cycling cells. These results suggest that the majority of non-cycling AML cells that harbor FLT3-ITD are unaffected by a FLT3 inhibitor and may constitute an as yet untargeted disease reservoir. Only one FLT3-ITD AML case showed exquisite sensitivity to TKI258 with elimination of the non-cycling fraction observed from 0.3 μM of TKI258 upwards. Possible explanations for this may include specific mutant receptor sensitivities or generic multi-drug resistance mechanisms operating in dormant cells. Interestingly, TKI258 selectively eradicated an ‘intermediate' dividing progenitor population in two of the insensitive cases, an indication that leukemic progenitors may be rendered sensitive to FLT3 inhibition on transiting the dormancy-cycling boundary. Further studies are needed to determine if these findings are representative of a generation of FLT3 inhibitors or specific for TKI258. Disclosures:No relevant conflicts of interest to declare.

Effective Management of Patients with Leukemic Transformation of Myelofibrosis.

Abstract 4967 Introduction Myelofibrosis (MF) is a clonal stem cell Philadelphia chromosome negative myeloproliferative neoplasm (MPN) characterized by progressive cytopenias, massive splenomegaly, early satiety, and a hypercatabolic state manifested by fevers, fatigue, and weight loss. Approximately 10-20% of MF patients will transform to acute myeloid leukemia (AML) over a 10-year period with a median overall survival at time of transformation of 2.6 months (Mesa 2005). In a multivariate analysis, peripheral blood blast count &gt; 3% and/or a platelet count &lt; 100 × 109/L at time of diagnosis of MF were independent risk factors for leukemic transformation (LT) (Huang 2008). Standard induction chemotherapy improved survival to 3.9-5 months (Mesa 2005, Tam 2008). Survival was decreased due to a high relapse rate and treatment-related mortality. Patients who received an allogeneic stem cell transplant early in the disease course had superior survival. We describe the outcome of ten consecutive patients with MF-LT at our institution. Patients and Methods A retrospective chart review was performed to evaluate patients with MF-LT, including clinical characteristics, therapies administered, and survival outcomes. Between the years of 2007 and 2009, ten MF-LT patients were identified in our institution. The median age was 66 (range 45-77) with 60 percent male and 40 percent female patients. Thirty percent (3/10) of patients were JAK2 V617F positive. Seven patients were cytogenetically abnormal and three had a normal karyotype. Chromosomal abnormalities included +1q (2 pts), +8 (2 pts), -7 (2 pts), inv 3 (1 pt), and t(6;9) (1 pt). Fifty percent of patients had a post-essential thrombocythemia related MF, while 30 percent had an MDS/MF overlap syndrome. Time to LT ranged from 3 months to 12 years with a median of 12 months. In three cases, there was discordance between bone marrow blasts by immunohistochemical staining and peripheral blood manual count. Six patients were treated with decitabine at 20 mg/m2 for 5 days (for two to ten cycles), and four received reduced intensity conditioning allogeneic stem cell transplantation (RIC-ASCT). Patients were eligible for RIC-ASCT if they were under 70 years of age, had no significant comorbidities, and had either an HLA compatible sibling or an unrelated donor with at most one mismatched allele. Results The median survival from diagnosis of LT with the use of decitabine has not yet been reached but exceeds 7.5 months in this patient population. Two out of six patients treated with decitabine died 6 and 8 months after LT. The remaining four patients are still alive at 6, 7, 9, and 36 months after LT and are actively receiving therapy. Four out of six patients who received decitabine had subjective improvement in fatigue as well as decrease in splenomegaly. Three out of six patients had a decrease in transfusion requirements. The four patients who underwent RIC-ASCT are all alive after a median follow-up of 16.5 months with no evidence of leukemia in the bone marrow. Conclusions These results demonstrate that single-agent decitabine for those who are not eligible for transplant or RIC-ASCT for eligible patients, leads to a significant improvement in overall survival. Further investigation using these therapeutic modalities is warranted given the aggressive nature of this disease. Furthermore, the noted discordance between bone marrow and peripheral blood blast percentage most likely reflects leukemic cell production originating at sites of extramedullary hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Danusertib Hydrochloride (PHA-739358), a Multi-Kinase Aurora Inhibitor, Elicits Clinical Benefit in Advanced Chronic Myeloid Leukemia and Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia .

Abstract 864 Background:Despite the availability of novel ABL tyrosine kinase inhibitors ( TKI ) in addition to imatinib mesylate, the acquisition of the T315I BCR-Abl mutation remains a major cause of resistance to registered therapeutic compounds. Some patients (Pts) may also fail therapy with ≥ 2 TKI and need additional treatment. PHA-739358 is a small ATP competitive molecule that specifically inhibits Aurora A, B and C kinases. PHA-739358 possesses high affinity binding capacity to both wild-type Abl and Abl/T315I in in vitro assays. Methods:An international, multicenter, open-label, single agent, non-comparative, phase I study is being conducted in adult Pts with advanced Chronic Myeloid Leukemia (Accelerated AP-CML/Blastic phase BP-CML) and Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) resistant or intolerant to imatinib and/or 2nd generation c-Abl therapy. Primary objective of the study is to determine the Maximum Tolerated Dose (MTD) and the Dose Limiting Toxicity (DLTs) during the first cycle. Two schedules were planned. Schedule A (PHA-739358 given as daily 3-hrs iv infusion for 7 consecutive days, every 2 wks) is open. Schedule B, not yet started, foresees a more aggressive approach and is currently being amended. Results:Twenty-three Pts with CML and Ph+ ALL have been treated so far (4 with AP-CML; 8 with BP-CML and 11 with Ph+ALL). Five dose levels have been tested: 90 (N=7); 120 (N=4); 150 (N=6); 180 (N=3) and 200 (N=3) mg/m2. Only one DLT occurred at 90 mg/m2 (NCI-CTC AE Gr3 fainting). Presently the MTD has not been defined. Fifteen out of 23 Pts have confirmation of BCR-Abl T315I mutation. A response occurred in 6/14 Pts, including 3 cytogenetic (1 complete, 1 partial, 1 minimal), 5 hematologic, and 1 clinical improvement (reduction in extramedullary disease mass). One severely pretreated (chemotherapy+Imatinib, SCT, Dasatinib and Donor Lymphocyte Infusion) pt with T315I mutated Ph+ALL reached Complete Hematological Response since Cycle (Cy) 4, Complete Cytogenetic Response since Cy 8 and Molecular Response (BCR/ABL undetectable transcripts) since Cy 9. Treatment continues after 10+ months. Last cycles were given every 4 wks due to mild/moderate transaminitis. This Pt at baseline showed: Peripheral Blood Blasts=0%; Bone Marrow (BM) blasts= 33%; % of Ph+ Metaphases = 80%. Two additional Pts (AP-CML and Ph+ ALL) achieved cytogenetic responses, one minimal and one partial reached at Cy2 and Cys 1-6, respectively. Another Pt (BP-CML) who received multiple transplants (3 times) and progressed after Imatinib, Dasatinib, Bosutinib, Nilotinib and Homoharringtonine, with 4 cycles of PHA-739358 had significant improvement of a large extramedullary lesion in the left neck impeding breathing and swallowing. Significant but transient reduction in the White Blood Cells (8/13 pts) and peripheral blood blast counts (3/13 Pts) were obtained with PHA-739358. An acceptable tolerability and safety profile characterized the study therapy: max non-hematological tox (regardless of causality) was as follows (most frequent Adverse events ≥ 30 %): diarrhoea 57% (CTC Gr 3 one Pt), pyrexia 50%; headache 43% (CTC Gr 3 one Pt); dyspnoea 36% (CTC Gr 3 one Pt ) and nausea 36%. Data analysis ongoing. Conclusions:The preliminary results obtained with our study show that PHA-739358 can elicit significant response with clinical benefit in severely pretreated populations of Pts affected with advanced leukemias resistant/intolerant to Imatinib and other 2nd generation TKI. Disclosures:Schafhausen:Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi-Aventis: Speakers Bureau; Novartis: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau. Latini:Nerviano Medical Sciences: Employment. Capolongo:Nerviano Medical Sciences: Employment. Laffranchi:Nerviano Medical Sciences: Employment. Comis:Nerviano Medical Sciences: Employment.

Reduced-Intensity Cord Blood Transplantation without Preceding Remission Induction Therapy Induces Durable Remission in Patients with Relapsed Acute Leukemia After the First Allogeneic Stem Cell Transplantation.

Abstract 4341Relapses in acute leukemia (AL) following allogeneic stem cell transplantation (allo-SCT), particularly those relapses within 1 year after transplantation, are often refractory to induction chemotherapy. Patients with this type of relapse rarely achieve long-term remission even after the second SCT. Cord blood transplantation, performed in a timely manner, may improve treatment outcomes in these patients. To assess the effectiveness of second cord blood transplantation (CBT) with a reduced intensity conditioning (RIC) regimen early after the diagnosis of leukemia relapse, we retrospectively analyzed outcomes from RIC-CBT without preceding remission induction therapy in 11 consecutive patients.Between 2007 and 2009, we performed CBTs with a RIC regimen for 11 patients with AL who relapsed after allo-SCT. Seven patients were diagnosed with acute myeloid leukemia and four with acute lymphoblastic leukemia. Median duration between the first and the second SCT was six months (range: 3-24 months). Median duration between the relapse and the second SCT was 39 days (range: 22-60 days). All but one patient didnot receive induction chemotherapy due to the low probability of achieving complete remission (CR),and another patient didnot receive induction chemotherapy due to low blast percentage in bone marrow. Mean blast counts in bone marrow and peripheral blood were 39.3% and 22.1%, respectively. All but one patient were conditioned with fludarabine (25 mg/m2/day) from Day -8 to Day -4, with L-PAM (40 mg/m2/day) from Day -3 to Day -2, and with TBI (4Gy) on Day -1. Only FK506 was administered to patients as a graft versus host disease (GVHD) prophylaxis. Mean concentrations of FK506 during the first, second, third, and fourth 10-day period after CBT were 8.9, 8.9, 10.2, and 9.8 ng/ml, respectively. Median follow-up period was 12 months after transplantation in patients who survived.Six patients were alive at the last follow-up with a 1-year and 2-year OS rate of 72.7% and 36.4%, respectively. Five of these 6 patients have been maintaining CR. Four of the 5 patients maintaining CR were considered to be at an extremely high risk for relapse because they relapsed within 8 months (median: 5.5 months, range: 5 to 8 months) after the first SCT. These 4 patients have maintained CR with a median follow-up of 12 months (range: 7 to 32 months). The 2-year probabilities of transplant-related mortality (TRM), relapse-free survival (RFS), and relapse were 27.3%, 40.9%, and 42.9%, respectively. RIC-CBT without preceding remission induction therapy using a relatively low concentration of FK506 as a GVHD prophylaxis may be a promising therapeutic option for patients with AL who relapse shortly after the first transplantation.Age/SexDiseaseDonor type of 1st SCTPreparative regimen of 1st SCTTime (months) from 1st SCT to relapseTime (days) from relapse to 2nd SCTBlast BMBlast PBStatus last follow upOverall Survival (months)53/FAMLMUDTBI-CY124231.8%3%death no relapse342/MAMLCBTBI-CY53749.4%6%alive CR3240/FAMLCBBU-CY-TBI53229.0%81%death relapse1538/MAMLMUDTBI-CY212864.8%80%death no relapse228/FAMLSIBFlu-Mel-TBI24606.4%1%alive CR2319/MALLSIBTBI-CY83550.0%49%death relapse1521/MALLMUDTBI-CY82273.8%1%alive CR750/MAMLMUDTBI-CY5399.0%2%alive CR945/FALLSIBTBI-CY64157.8%4%alive CR1555/FALL(Ph1)SIBTBI-CY44151.2%16%alive relapse621/FAMLRelatedTBI-CY6429.2%0%death no relapse3 Disclosures:No relevant conflicts of interest to declare.

Cytoplasmic localization of nucleophosmin in bone marrow blasts of acute myeloid leukemia patients is not completely concordant with NPM1 mutation and is not predictive of prognosis

Nucleophosmin (NPM1) gene mutations are reported to predict a favorable prognosis in acute myeloid leukemia (AML) patients. Aberrant cytoplasmic localization of nucleophosmin (NPM) protein is reported be a surrogate for NPM1 gene mutation. Using immunohistochemical (IHC) analysis, we assessed for NPM (clone 376) expression in formalin-fixed, formic acid-decalcified bone marrow biopsy specimens. DNA sequencing of exon 12 of NPM1 gene was performed in 104 patients. The study included 252 AML patients: 192 de novo AML, 33 AML preceded by either myelodysplastic syndrome or chronic myelomonocytic leukemia, and 27 therapy-related AML. The median age was 62 years and 115 patients were <or=60 years old. All patients received intensive chemotherapy. Cytoplasmic NPM was detected in 59 of 252 (23%) patients, including 48 of 192 (25%) de novo AML and 33 of 94 (35%) with a normal karyotype. DNA sequencing identified NPM1 mutations in 30 of 38 cases with cytoplasmic NPM and 10 of 66 cases with nuclear NPM. Cytoplasmic NPM was associated with young patient age (P=.024), FLT3/ITD (P=.005), CD34 negative blasts (P<.001), high peripheral blood blast count (P=.041), and high serum albumin level (P=.028). No statistical differences in overall or event-free survival were found on the basis of NPM localization. Similar results were obtained in patients<or=60 years old with normal karyotype and wild-type FLT3 (P=.768). IHC assessment for NPM localization did not predict prognosis in this patient cohort. The discordance between immunohistochemistry and DNA sequencing results indicates that DNA sequencing cannot be replaced by IHC assessment.

Decitabine and Gemtuzumab Ozogamicin in Acute Myelogenous Leukemia and High-Risk Myelodysplastic Syndrome

Background: Hypomethylating agents are approved for the treatment of myelodysplastic syndrome (MDS) and have activity in acute myelogenous leukemia (AML) (Issa, JP et al. Blood . 2004; 103:1635–40)(Silverman LR et al. Journal of Clinical Oncology 2006; 24: 3895–3903). Exposure of AML cells to hypomethylating agents increase their CD33 expression (Balain and Ball. Leukemia . 2006; 20:2093–2101). This establishes a rationale for the combination of hypomethylating agent with CD33 antibody for treatment of high-risk MDS and AML. A preliminary report of the combination of 5-azacitidine (Aza) and gemtuzumab ozogamicin (GO) in elderly patients with previously untreated AML and MDS showed promise (Nand, S. et al. Blood 2006; 108: Abstract 1981).Patients and Method: We have started enrolling patients in a phase 2 study of Decitabine (DAC) with GO in patients with AML and high-risk myelodysplastic syndrome (MDS). Treatment includes DAC 20 mg/m2/day intravenously (IV) × 5 days and GO 3 mg/m2 IV on day 5. GO is repeated on day 15±2 of first cycle if peripheral blood blast count is > 109/L. Six such cycles are planned every 4–8 weeks. Patients achieving clinically relevant response e.g. complete remission (CR), CR with incomplete platelet recovery (CRp), marrow clearance/reduction of blasts etc. can continue on DAC alone approximately every 4–8 weeks for up to 24 cycles of total therapy duration. Based on historical expectations patients are divided into four groups according to study design:relapsed AML 1st CR duration <= 1 year or beyond first relapse,relapsed AML 1st CR duration > 1 year,untreated AML (not eligible for standard induction)/MDS,previously treated MDS, secondary MDS or AML.Results: Thirty-seven patients have been treated till date; Group 1=19, group 2=1, Group 3=12 and group 4=5. Median age is 63.5 years (range, 26–82), performance status =1 (range, 0–3), prior therapy =1 (range, 0–7). Eighteen (49%) patients has adverse cytogenetics and 14 (38%) has prior malignancies. Two patients received day 15±2 administration of GO. Median number of treatment cycles is 2 (range, 1–5). Eight patients died while on study. Except for cytopenias, infections, infusion related reactions etc. no significant grade 3/4 toxicities have been encountered. Three patients have achieved CR/CRp and 5 patients achieved other clinically relevant responses with an overall response rate of 22%. Responses according to patient groups are; Group 1=2/19 (4 ongoing), group 2=1/1, group 3=2/12 (3 ongoing), group 4=3/5. Seven additional patients are continuing on study with stable disease. Accrual to the study is ongoing.Conclusion: The combination of DAC and GO appears promising particularly in less heavily pre-treated patients with high-risk MDS and AML.

Dasatinib in Children and Adolescents with Relapsed or Refractory Leukemia: Interim Results of the CA180-018 Phase I Study from the ITCC Consortium.

Relapsed/refractory leukemia in childhood carries a grave prognosis. Dasatinib (SPRYCEL®) is a potent oral kinase inhibitor of BCR-ABL, KIT, and SRC kinases. Dasatinib is approved for adults with Philadelphia chromosome-positive (Ph(+) CML and ALL, resistant or intolerant to prior imatinib therapy. An investigational phase I dose- finding study of dasatinib treatment of patients aged 1–21 years with various leukemia subtypes is currently being conducted in 12 European centers. The aim of the study is to establish a safe and effective dose for each of the leukemia sub-types. Patients were stratified into three treatment groups - Stratum 1: CML in chronic phase, resistant or intolerant to imatinib; Stratum 2/3: advanced-phase CML resistant or intolerant to imatinib, Ph(+) ALL, relapsed/ refractory after imatinib, or Ph(+) AML, ≥ 2nd relapse; Stratum 4: Ph(−) ALL, or Ph(−) AML in second/subsequent relapse. The starting dose was 60mg/m2 once daily for all strata. Intra-patient dose escalation was allowed for lack of initial response and dose reductions for toxicity. Current data reflect the first 41 patients (median age 11 years, range 1–21) treated from March 2006 through May 2008, including eight in Stratum 1, twelve in Stratum 2/3, and twenty-one in Stratum 4. The patients were heavily pretreated and prior therapy included chemotherapy (n=35), imatinib (n=20), and stem cell transplant (n=24). Dasatinib was well tolerated up to the current 120mg/m2 dose. Treatment-related toxicities were mostly mild to moderate in severity with nausea (34% grade 1/2; 2% grade 3/4) and diarrhea (15% grade 1/2; 0% grade 3/4) occurring most frequently. In Stratum 4, two dose-limiting toxicities were seen: anaphylaxis 5 hours after the first dose (60mg/m2) and upper-GI bleed on Day 6 of dasatinib dosing (120mg/ m2). Only one of the 41 patients experienced a malignant/hemorrhagic pleural effusion at 100mg/m2 dose. A maximum tolerated dose has not been established. PK studies were performed on samples from 32 patients after 60 mg/m2, 80 mg/m2 and 100 mg/m2 dosing. Absorption occurred rapidly (median Tmax 0.75 – 1.0 hour). The area under the curve (AUC) and maximum concentration (Cmax) proportionately increased with higher dose levels, but the difference was much greater between 60 and 80 mg/m2 than between 80 and 100 mg/m2. Complete Hematological Response (CHR) occurred in 75% of patients with CML-CP. Major Hematological Response (MaHR) was achieved in 25% of patients with advanced CML/Ph(+) and Ph(+) ALL/ AML. Major Cytogenetic Response occurred in 88% of CML-CP patients and 50% of patients with advanced Ph(+) CML and Ph(+) ALL/ AML. Stratum 4 observations included a temporary decrease in peripheral blood (PB) blast count in one Ph(−) ALL patient and in PB and bone marrow (BM) blast counts in two Ph(−) AML patients (one with AML7 and one with Down's syndrome). Additionally, three Ph(+) ALL patients had a CSF response. These interim data demonstrate a favorable safety profile for dasatinib. Clear efficacy was seen in patients with CML-CP, and other Ph(+) leukemias. Further exploration is needed in the Ph(−) leukemias in the pediatric population. Updated data will be presented.

Treating children with acute lymphoblastic leukemia and Down syndrome: Pharmacokinetics provides insight into vincristine therapy

Treating children with acute lymphoblastic leukemia and Down syndrome: Pharmacokinetics provides insight into vincristine therapy

Detection of Nucleophosmin Gene Mutations in Plasma from Patients with Acute Myeloid Leukemia: Clinical Significance and Implications.

Nucleotides insertion in the nucleophosphamin (NPM1) gene has been reported in about one third of patients with acute myeloid leukemia (AML). Multiple studies showed that the presence of NPM1 mutations associated with better outcome in patients with AML. Studies reported to date have analyzed leukemic cells obtained from bone marrow or peripheral blood. We tested for mutations in the NPM1 gene using peripheral blood plasma and compared results with clinical outcome from a single institution. Analyzing plasma from 98 newly diagnosed patient with AML showed NPM1 mutation in 24 (23%) of patient while only one (4%) of 28 previously untreated patients with myelodysplastic syndrome (MDS) showed NPM1 mutation. Compared with peripheral blood cells, 2 (8%) of the 24 positive patients were negative by cells; none were positive by cells and negative by plasma. Most of the mutations detected (45%) were in patients with FAB classification M2, M4 and M5. In addition to the reported 4 bp insertion, we also detected 4 bp deletion in one patient in cells and plasma. Patients with NPM1 mutation had a significantly higher white blood cell count (P = 0.0009) and a higher blast count in peripheral blood (P = 0.002) and in bone marrow (P = 0.002). Blasts in patients with NPM1 mutant expressed lower levels of HLA-DR (P = 0.005), CD13 (P = 0.02) and CD34 (P < 0.0001), but higher CD33 levels (P = 0.0004). Patients with NPM1 mutation appear to have better chance of responding to standard therapy (P = 0.06). Event free survival of patients with NPM1 mutation was longer (P = 0.056) than in patients with intermediate cytogenetic abnormalities. The most striking difference in survival was in patients who required >35 days to respond to therapy (Figure). Survival was significantly longer in patients with NPM1 mutation requiring >35 days to respond (P = 0.027). This data not only support that NPM1 plays a significant role in the biology and clinical behavior of AML, but also show that plasma DNA is enriched with leukemia-specific DNA and is a reliable source for testing. [Display omitted]

Wilm's Tumor 1 Gene Expression Is a Useful Marker for Minimal Residual Disease in Acute Myeloid Leukemia.

One of the difficulties in managing patients with acute myeloid leukemia (AML) is the detection of minimal residual disease. Only a fraction of AML patients have a distinct phenotype on flow cytometry or a genetic abnormality which can be followed as a sensitive marker for minimal residual disease (MRD). The Wilm's tumor - 1 gene (WT1) is overexpressed in leukemic blasts, but not in normal blood or marrow cells, and thus can be used as a marker for MRD in leukemia patients who otherwise do not have a characteristic immunophenotypic or molecular marker that can be monitored. We prospectively measured serial peripheral blood WT1 levels by RQ-PCR in 54 patients with AML. Of the 54 patients, 23 patients were newly diagnosed. The remaining 31 patients were in complete remission prior to a planned allogeneic transplant. Subsequent WT1 levels were obtained during routine evaluation both during and up to three years after completion of either chemotherapy and/or transplant. Using regression analysis, we found that a normalized WT1 level greater than 10−4 (as compared to a level of 1 in K562 cells) indicates a higher likelihood of the patient having active leukemia with a sensitivity of 87.5% and a specificity of 89.1%. The positive and negative predictive values are 71.19% and 95.89% respectively. We also compared WT1 levels with peripheral blood and bone marrow blasts to determine whether a correlation existed. We found a strong correlation between WT1 expression and bone marrow blasts with a correlation coefficient of 0.77 (p< 0.0001). A weak correlation was found with peripheral blood blast count (correlation coefficient = 0.35, p<0.0001). Our data implies WT1 can be a useful marker for minimal residual disease in patients with acute myeloid leukemia, especially in patients who do not have an unusual phenotype or molecular marker to monitor MRD.

KIT Mutations Define Characteristic Gene Expression Signatures in Core Binding Factor Leukemias.

Core binding factor (CBF) leukemias, characterized by either inv(16)/t(16;16) or t(8;21), constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, there exists substantial biological and clinical heterogeneity within these cytogenetic groups, which is not fully reflected by the current classification system. Recently, the identification of novel molecular markers, like the gain-of-function KIT mutations in exons 8 and 17 have been shown to confer higher relapse risk and adversely influence overall survival (Paschka et al, J Clin Onc 2006). To further investigate whether the presence of KIT mutations leads to a characteristic gene expression pattern in CBF AML we profiled gene expression in a large series (n=83) of AML patients with CBF leukemia (KIT WT n=59, KIT mutated n=24). By supervised hierarchical clustering we were able to define a KIT mutation associated gene signature consisting of 168 genes (p=0.005). Gene set enrichment analyses revealed several groups of pathways to be differentially regulated, thereby likely reflecting differences in pathogenic mechanisms which might lead to distinct prognoses of different subgroups of CBF leukemias. For example, in the KIT mutated group, cellular stress response pathways, as for example NFkappaB-signaling and TNF/stress related signaling, were downregulated, whereas the motility-associated Y branching of actin filaments was upregulated. In general, a great overlap of deregulated pathways was found between the overall set and the cytogenetic CBF subgroups inv(16) and t(8;21), respectively. The same was true for KIT mutations in exons 8 and 17 in comparison to all KIT mutations. However, as all these subgroups are characterized by distinct biology and clinics, each subgroup was also associated with a number of deregulated pathways that were not significantly affected in other subgroups. Apart from the expected upregulation of KIT in the KIT mutated group, we also found e.g. a down-regulation of TNF, NFkappaB and PRKCA. Overall, KIT mutations seem to entail a proliferative advantage (higher blast counts in KIT mutated cases; p=0.0015 and p=0.0157 for peripheral blood and bone marrow blast counts, respectively), an increased stem cell self renewal as suggested by the upregulation of LRP6 of the Wnt/LRP6 Signaling pathway and a deregulation of apoptosis. This latter finding is reflected by the NFkappaB-signaling pathway and TNF/stress related signaling, mentioned above, and also, e.g. by the upregulation of the Apoptotic Signaling in Response to DNA Damage pathway in KIT mutated inv(16) cases. Thus, gene expression profiling reveals novel insights into the potential pathomechanism of KIT mutations in CBF leukemias. However, while the leukemogenic relevance of these signatures remains to be validated, their existence nevertheless supports a prognostically relevant biological basis for the heterogeneity observed in CBF leukemia.

A Phase I Study of a Novel Human Monoclonal Antibody (mAb216) with Chemotherapy for the Treatment of Patients with Relapsed or Refractory B-Lineage Acute Lymphoblastic Leukemia.

BACKGROUND: Despite improvements in front-line therapy for adult ALL, most patients eventually relapse and do not tolerate or respond to reinduction therapy. Novel targeted therapies are needed that have both activity against adult ALL and a toxicity profile distinct from conventional chemotherapy. MAb216 is a naturally occurring human IgM monoclonal antibody derived from the VH4-34 (variable heavy chain) gene. It has shown promise as a novel therapy for B-ALL in preclinical studies. In vitro, mAb216 specifically binds and is cytotoxic to normal human B-lymphocytes and B-progenitor lymphoblasts from patients with ALL. Binding of mAb216 to its linear lactosamine ligand leads to formation of large membrane pores resulting in cell lysis. This non-classical apoptosis occurs in the absence of complement fixation but the cytotoxicity is enhanced by complement. The membrane pores may also facilitate entry of chemotherapeutic drugs into the leukemic blast providing an explanation for the enhanced cell killing that is seen when mAb216 is combined with vincristine.METHODS: The primary aims of the study are to determine the maximum-tolerated dose, and dose-limiting toxicities of mAb216 as a single agent and in combination with vincristine in patients with relapsed or refractory B-ALL. Secondary aims are to characterize the pharmacokinetic behavior of mAb216 and to preliminarily assess clinical efficacy. Binding of mAb216 to the patient's leukemic blasts is confirmed prior to enrollment. Two treatment courses of mAb216 are given with the same dose of antibody administered on days 0 and 7. In case of a suboptimal response (< 75% reduction in peripheral blood blasts) after the first dose of mAb216, the second dose is given in combination with vincristine. Five mAb216 dose levels are planned using a starting dose of 1.25 mg/kg and a standard 3+3 dose-escalation design. Clinical response is assessed by measuring the peripheral blood blast count weekly and by bone marrow biopsy if no blasts are present in the peripheral blood.RESULTS: Nine of 10 patients screened exhibited binding of mAb216 to their blasts and were enrolled in the study (median age 27; range 10–73). Four patients had relapsed after allogeneic bone marrow transplantation, four patients had relapsed after a median of four (range 2–5) prior therapies, and one patient had primary refractory disease. In these patients, doses up to 2.5 mg/kg of mAb216 have been well tolerated. At a dose of 1.25 mg/kg one patient experienced vomiting and grade 3 epistaxis three days after the infusion. At 2.5 mg/kg one patient developed hives during the infusion that resolved with diphenhydramine and hydrocortisone. MAb216 has not induced immune complex formation. The median half-life is 1.76 h (range 0.78–9.3 h) and strongly correlates with the binding affinity of mAb216 to the blasts. 7/9 patients experienced a reduction of their peripheral blast count between 8–61% after infusion of mAb216 alone. 7/9 patients received the second dose of mAb216 in combination with vincristine on day 7 or earlier. All seven patients, who had failed previous treatment with regimens containing vincristine, achieved a reduction of their peripheral blast count of 50–100%. One patient had a hypocellular bone marrow with no residual blasts on day 21.CONCLUSIONS: These results indicate that mAb216 alone and in combination with vincristine is a promising treatment for relapsed/refractory B-ALL. Patient recruitment and dose-escalation is ongoing.

Targeting CD22 in Childhood B-Precursor Acute Lymphoblastic Leukemia (Pre-B ALL): Pre-Clinical Studies and Phase I Trial of the Anti-CD22 Immunotoxin CAT-3888 (BL22).

Although most children with pre-B ALL are cured, treatment is associated with multiple toxicities and outcome after relapse is poor. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. We evaluated the B-lineage differentiation antigen CD22 and the anti-CD22 immunotoxin RFB4(dsFv)-PE38 (CAT-3888 or BL22) in childhood ALL.Methods: Blasts were obtained from children with pre-B ALL. Flow cytometry was used to determine CD22 expression and anti-CD22 antibody binding using the BD Quantibrite System. 72 hour in vitro protein synthesis cytotoxicity assays were conducted. Murine xenografts (human ALL cell line EU-1 into CB17 SCID mice) were treated with varying doses of CAT-3888. A pediatric Phase I trial of CAT-3888 was initiated.Results: All cases (n=69) were CD22 positive. Median CD22 site density (studied in 28 cases) was 4,099 sites/cell (range, 451 – 9,380). In vitro cytotoxicity to CAT-3888 was assessed in 43 cases and the median IC50 was 10 ng/ml. Murine xenografts treated with CAT-3888 had significant prolongation of leukemia free survival in a dose responsive fashion (p<0.05). 21 subjects (18 ALL, 3 lymphoma; 3–22 years of age) were treated on a Phase I trial of CAT-3888 at doses of 10–40 mcg/kg QOD for 3–6 doses with cycles repeated every 21–28 days. Treatment was well tolerated and adverse events attributed to CAT-3888 were reversible. Pharmacokinetic parameters appeared to be influenced by disease burden consistent with rapid drug binding by CD22 positive blasts. All subjects were heavily pre-treated and had progressive high burden disease at the time of treatment. Transient clinical activity was observed in 17 of 21 subjects as evidenced by decreased peripheral blood blast count (8), decreased marrow infiltration (6), decreased extramedullary infiltration (3), increased platelet count (2), increased neutrophil count (1), increased reticulocyte count (1), improved PET scan (1), decreased tumor-associated pain (2), and/or peripheral blast count stabilization (5). A dose response was apparent with 6 of 12 (50%) achieving disease stabilization at or above 30 mcg/kg (vs. 0/9 at lower doses). Three subjects developed high-titer neutralizing antibodies, 1 of which had pre-existing low titer antibodies from prior pseudomonas infection.Conclusions: CD22 represents a relevant target for childhood pre-B ALL. All cases tested were CD22+ and most blasts were highly sensitive to CAT-3888 in vitro at concentrations below clinically achievable levels. CAT-3888 showed a dose response effect and prolonged leukemia free survival in murine ALL xenografts. CAT-3888 is well tolerated in children and activity has been seen in most subjects treated on the Phase I trial. A new higher affinity anti-CD22 immunotoxin (CAT-8015 or HA22) is in development.