Relapsed ALL Research Articles

ARID5B Genetic Polymorphisms Contribute to Racial Disparities In Childhood Acute Lymphoblastic Leukemia: A Children's Oncology Group Study

AbstractAbstract 8Acute lymphoblastic leukemia (ALL) is the most common type of cancer in children. Substantial racial differences exist not only in the risk of developing childhood ALL, but also in the outcome of ALL therapy, e.g. children with Hispanic ethnic background exhibit the highest incidence of ALL and the lowest survival rate among major racial/ethnic groups in the US. Although both genetic and non-genetic factors are thought to be important, the exact cause(s) of such racial disparities remains largely unknown. Taking a genome-wide approach, we previously identified single nucleotide polymorphisms (SNPs) in the ARID5B gene that are strongly associated with ALL risk in whites (e.g. rs10821936, P=1.4×10-15, Nat. Genet. 2009, 41:1001), which was independently replicated in several studies (Nat. Genet. 2009, 41:1006; Blood 2010 115:1765). Interestingly, the ALL risk allele (allele C) at the ARID5B SNP is also associated with ALL risk in black children, but is much less prevalent in blacks than whites (allele C frequency, 18% in blacks and 34% in whites), providing a genetic basis for the lower risk of ALL in black children (Leukemia 2010 24:894). In this study, we evaluated the contribution of ARID5B genetic variations to the disparities in ALL incidence and outcome between Hispanic and white children. To identify genetic variations associated with ALL in each population, we first compared genotype frequencies at 51 ARID5B SNPs between ALL cases and ancestry-matched controls, i.e., between 330 Hispanic children with ALL and 134 Hispanic controls; and between 978 white children with ALL and 1,046 white controls. After adjusting genetic ancestry to control for population stratification, we observed that 10 of 51 ARID5B SNPs were significantly associated with ALL in both populations, and 6 and 10 SNPs were significant only in whites and Hispanics, respectively. In both race groups, the strongest association was observed at rs10821936 (P= 8.3×10-20 in whites and P=3.7×10-8 in Hispanics). Interestingly, the frequency of the ALL risk allele (allele C) at rs10821936 was higher in Hispanics (43%) than in whites (33%), consistent with the higher incidence of ALL in Hispanics. In fact, for all 10 SNPs significant in both populations, the risk alleles were consistently more common in Hispanics than whites and conferred greater risk of ALL (higher odds ratios in Hispanics than whites). Additionally, we performed forward selection-based multivariate analyses in each population in which ARID5B SNPs compete against each other on the basis of independent associations with ALL. In whites, rs10821936 was the first and only SNP that entered the multivariate model, suggesting a single causal variant at the ARID5B locus tagged by this SNP. In contrast, in Hispanics, 3 ARID5B SNPs were independently associated with ALL in the multivariate model even after adjusting for genotype at rs10821936. Moreover, these 3 ARID5B SNPs were located in linkage disequilibrium blocks distinct from that tagged by rs10821936, arguing for multiple possible causal variants in the ARID5B gene in Hispanics. Finally, we tested whether ARID5B polymorphisms are related to treatment outcome in 1,605 children enrolled on the COG P9904 and P9905 studies. Of 26 ARID5B SNPs associated with ALL risk, 5 were also associated ALL relapse (P<0.05), with the alleles related to disease risk always linked to poorer treatment outcomes. For instance, the T allele at rs6479778 was more frequent in ALL cases than in healthy controls (P=0.0029 in whites and P=0.0031 in Hispanics), and patients carrying the T allele also exhibited an increased risk of ALL relapse (P=1.3×10-4). Because the T allele is more common in Hispanics (24%) than whites (14%), this polymorphism is likely to contribute to the racial differences in both disease risk and treatment outcome of ALL. In conclusion, our results indicate that genetic polymorphisms in the ARID5B gene are important determinants of racial disparities in childhood ALL. Disclosures:Relling: St. Jude Children's Research Hospital: Employment, Patents & Royalties; Enzon Pharmaceuticals: Research Funding.

Liposomal Cytarabine (DepoCyte®) for the Prophylaxis and Treatment of Central Nervous System Involvement In Adult Acute Lymphoblastic Leukemia (ALL) - A Retrospective Analysis

AbstractAbstract 2122 Background:Central nervous system involvement in Acute Lymphoblastic Leukemia can be either primary, present at diagnosis or associated with relapse of the disease. Prevention and treatment of CNS relapse is an essential component of all ALL chemotherapy regimens. Liposomal cytarabine (DepoCyte) is a sustained release formulation of Ara-C with a homogeneous distribution throughout the neuroaxis and a prolonged half-life maintaining cytotoxic concentrations in the CSF for more than 14 days. DepoCyte obtained superior response rates, improved patient quality of life and improved the time to neurological progression compared to standard cytarabine in a randomized clinical trial (Glantz et al 1999). Materials and Methods:A retrospective analysis was carried out to evaluate feasibility, safety and efficacy of DepoCyte in the prophylaxis and treatment of ALL patients. 45 patients who received 50mg liposomal cytarabine either as a prophylaxis (N=24) or treatment (N=21) between March 2006 – December 2009 in 4 centers in Poland were included in the analysis.Baseline characteristics of patients who completed CNS prophylaxis with DepoCyte: median age 34 (range: 18–67 years), male/female ratio 16/8, B-cell ALL (n=17), T-cell ALL (n=4), Ph-positive ALL (n=3). In the treatment group median age was 35 (range 18–60), male/female ratio 12/9, B-cell ALL (n=15), T-cell ALL (n=3) and Ph-positive ALL (n=3). Results:In the prophylaxis group the average number of DepoCyte administrations was 2.6 (range 2–4). Oral or IV Dexamethasone for 5 days was given to all patients to prevent symptoms of arachnoiditis. With a median follow-up of 12 months (range: 3–27) only 1 pt developed combined systemic and CNS relapse. 25% of patients (6/24) experienced mild and transitory adverse events: headache (n=3), brain edema during methotrexate-containing consolidation (n=1) and post-puncture syndrome (n=2).In the treatment group 8 pts were in first isolated CNS relapse and 13 pts were in combined CNS and systemic relapse. All patients had neurological symptoms and blast cells identified in the cerebrospinal fluid with average cellularity 713/μL (range 20–2500/μL). In 2 patients CNS disease was confirmed by computed tomography. DepoCyte was administered intrathecally together with systemic chemotherapy in 18 patients. The treatment was planned to avoid concomitant administration of DepoCyte and other cytotoxic agents that cross the BBB. All patients received concurrent dexamethasone for prophylaxis of arachnoiditis. Neurological and cytological responses were obtained in all 21 pts (16 CRs and 5 PRs). No serious adverse events with DepoCyte were reported. Mild headache was the most commonly reported toxicity (10/21pts, 47.6%). 2 out of 21 (9.5%) heavily pretreated patients developed transient sacral radiculopathy. Conclusions:1) Implementation of liposomal cytarabine as IT prophylaxis in ALL patients reduces the total number of IT injections, is feasible and effective and has a favorable tolerance profile. 2) DepoCyte used for the treatment of leukaemic meningitis with concurrent systemic chemotherapy is a highly effective and feasible treatment in isolated and combined CNS relapse in ALL. 3) DepoCyte is generally well tolerated when concurrent dexamethasone is administered to alleviate symptoms of arachnoiditis and concomitant administration of agents that cross the BBB is avoided. Reference:Glantz M et al. Randomized trial of a slow release versus a standard formulation of cytarabine for the intrathecal treatment of lymphomatous meningitis. J Clin Oncol 1999; 17: 3110-17. Disclosures:No relevant conflicts of interest to declare.

Allogeneic Hematopoetic Stem Cell Transplantation (SCT) In Children with ALL: Outcome of Matched Sibling Donor SCT (MSD-SCT) Is Equivalent to Unrelated Well Matched Donor HSCT (MD-HSCT) A Report From the Prospective International Multicenter Trial ALL-SCT-BFM 2003

AbstractAbstract 530In 2003 the Berlin-Frankfurt-M ünster (BFM)-Study group initiated a prospective international multicenter trial (ALL-SCT-BFM 2003) for allogeneic hematopoetic stem cell transplantation (HSCT) in children with ALL. Main goals were 1. to answer the question, whether HSCT from a matched sibling donor (MSD) was equivalent to HSCT from a 9/10 or 10/10 HLA matched donor (MD) and 2. to standardize and harmonize the HSCT procedure. Another intention was to determine the efficacy of HSCT from HLA-mismatched donors (MMD) compared to HSCT from MD/MSD as well as the incidence of acute and chronic graft-versus-host disease (GvHD) and the antileukemic efficacy of the procedure.Between September 2003 and September 2009, 387 patients (188 in CR1, 199 ≥ CR2) were transplanted in 27 participating HSCT centers in Austria, Germany and Switzerland. Mean age of the patients at HSCT was 10 years (range 0.5 to 18). Indications for HSCT were poor response to induction treatment, either detected by morphology on day 33 or by minimal residual disease load measured after 12 weeks of treatment, cytogenetic aberrations [t(9;22), t(4;11)], early bone marrow relapse, or any subsequent ALL relapse. 97 patients received a MSD-HSCT, 251 patients a MD-HSCT, and 39 patients a MMD-HSCT. Median follow-up was 2.4 years. The compliance with HLA-typing procedures according to the trial guidelines (high resolution typing of HLA A, B, C, DRB1 and DQB1) improved over time to 100% since 2007. Unmanipulated bone marrow was used in 81% of MSD-HSCT and in 65% of MD-HSCT as determined by the protocol. The conditioning regimens consisted of TBI+VP16 for MSD, TBI+VP16+ATG for MD, and TBI+Flu+VP16+ATG for MMD; TBI was substituted by BU+Cy in children younger than 2 years). GvHD prophylaxis (CSA for MSD, and CSA plus MTX on day 1, 3 and 6 for MD) were given in the vast majority of patients according to the trial guidelines. Protocol deviations were mostly due to clinical reasons. Results:Acute GvHD (Grade III and IV) occurred in 10% of all patients, the 2-year cumulative incidence of extensive chronic GvHD was 15% after MSD and 12 % after MD HSCT. The 4-year probability of event-free survival (pEFS) after MSD-HSCT was equivalent to MD-HSCT (70% vs. 68%; p=0.37). The cumulative incidence of treatment related mortality (TRM) after 1 year was 5% for MSD and 8% for MD HSCT (n.s.). The 2-years cumulative incidence of relapse was 18% after MSD-HSCT and 20% after MD-HSCT (n.s.) For patients with very high risk of relapse the results for MD/MSD HSCT (n=187) and MMD HSCT (n=39) differed significantly (2-year pEFS 68% vs. 28%; p<0.001). The 2-year incidence of relapse was 23% after MSD/MD HSCT and 37% after MMD-HSCT (n.s.). The 1-year incidence of TRM was 8% after MSD/MD-HSCT and 22% after MMD-SCT (p=0.04). Overall, MMD HSCT showed a significant worse result with higher TRM and higher relapse rates. For patients beyond CR1 (n=25) transplanted from a MMD, the 2-year pEFS was 19% only.The results from this ALL-SCT-BFM 2003 trial, which is the largest prospective, international and multicenter HSCT trial ever performed in childhood ALL, demonstrate the feasibility of a harmonized HSCT approach across multiple international centers. We demonstrate that allogeneic HSCT from well HLA-matched unrelated donors or geno-identical sibling donors is an effective treatment option with acceptable toxicity in children with high risk ALL. Precise HLA typing and matching and ATG resulted in a low incidence of extensive chronic GvHD which is an important achievement for the quality of life in children and adolescents. These data will form a solid basis for subsequent ALL-HSCT trials, focussing on controlled modifications and interventions in patients at highest risk for relapse after HSCT. Disclosures:No relevant conflicts of interest to declare.

Anti-Leukemic Property of Sulphoraphane In Precursor B Cell Acute Lymphoblastic Leukemia

AbstractAbstract 3974Acute lymphoblastic leukemia (ALL) is the most common form of hematologic malignancy in children. In spite of significant advances achieved in the treatment of childhood ALL, one fifth of these patients still relapse after the standard treatment. Moreover, relapse ALL is the second most common cause of cancer-related deaths in children. The development of novel therapies is prevented by a limited understanding of the exact pathobiology. There are emerging evidences that the transcription factor KLF4 has a tumor suppressor property in ALL. Recently, a gene expression classifier study in pediatric precursor B-cell ALL (pre-B ALL) showed that KLF4 expression was significantly reduced in high risk ALL patients with positive MRD after induction. This finding suggests a possible role of this cell cycle inhibitor on the development, expansion and drug-resistant of leukemic cells. Several studies demonstrate that overexpression of KLF4 in normal B cells and BCR transformed B cells show increased apoptosis and reduced proliferation. Furthermore, we recently described that KLF4 inhibits proliferation of naïve lymphocytes by activating p21 (Yamada, et al, 2009). Sulphoraphane (SF; 4-methylsulfonylbutyl isothiocyanate) is a dietary compound derived from Cruciferae vegetables with anti-carcinogenic activity in colon cancer by upregulating KLF4 and p21 among other genes. Thus, we hypothesized that SF could also exhibit anti-leukemic activity in human-derived acute lymphoblastic leukemia cells via the activation of KLF4. The pre-B ALL cell lines (Nalm6, REH, RS-4, SUP-B15) and an EBV transformed B cell line were treated with different concentrations of SF (0-40 μM) for 24–48 hours. Then, cell number was estimated using an ATP-based viability method. Flow cytometric analysis of ANNEXIN-V/7-AAD binding as well as CFSE dilution was used to measure apoptosis and proliferation respectively. We found that SF induced cytotoxicity in Nalm-6, REH and RS-4 cell lines in a dose and time dependent manner. This cytotoxic effect was less pronounced in EBV-transformed B cell line. SF treatment led to increased ANNEXIN-V and 7-AAD positive cells (82% apoptotic cells in SF-treated versus 9% in DMSO control). Further, SF-treated cells displayed significantly less proliferation in comparison to DMSO controls thus suggesting that SF inhibits cellular proliferation. Preliminary data also suggest that SF-mediated apoptosis is caused by upregulation of KLF4. In conclusion, Sulphoraphane exhibits an anti-leukemic property by inducing apoptosis and abrogating proliferation in pre-B ALL cell lines. Thus, sulphoraphane could potentially be used as an adjunctive therapy in a subgroup of pre-B ALL patients who have decreased expression of KLF4. Disclosures:No relevant conflicts of interest to declare.

Dose Escalation of Clofarabine In Combination with Cytarabine and Total Body Irradiation (TBI) Followed by Allogeneic Stem Cell Transplantation (AlloSCT) In Children, Adolescents and Young Adults (CAYA) with Poor-Risk Acute Leukemia

AbstractAbstract 36 Introduction:Despite dramatic improvements in cure rates for pediatric ALL and AML, the prognosis remains poor for pediatric patients with ALL or AML in third complete remission (CR3) or with relapsed or refractory disease (10-20% EFS). Single-agent clofarabine has demonstrated activity in CAYA with relapsed or refractory ALL and AML (Jeha et al., Blood, 2004, JCO, 2006 and JCO, 2009). Clofarabine is a potent inhibitor of DNA polymerase and ribonucleotide reductase; biochemical and clinical studies suggest synergy with cytarabine (Faderl et al., Blood 2005). Clofarabine in combination with cytarabine has been studied in adults with AML and MDS and results in greater rates of CR than clofarabine alone (Faderl et al., Blood, 2006 and Blood, 2008). The combination of clofarabine and cytarabine in CAYA with relapsed acute leukemia appears to be safe and well-tolerated (Cooper et al., ASH, 2009). Objectives:To determine the maximum tolerated dose (MTD) of clofarabine in combination with cytarabine and TBI followed by AlloSCT and to assess the safety, progression-free survival (PFS) and overall survival (OS) associated with this regimen in CAYA with poor-risk acute leukemia. Methods:We instituted a multi-center phase I/II study of a novel conditioning regimen consisting of clofarabine, cytarabine and TBI prior to AlloSCT in CAYA with ALL or AML in CR3, relapse, or induction failure (IF). Herein we report results from the phase I portion of the study. Eligible patients received 5 days (day-9 to day-5) of clofarabine (dose escalation with 40 mg/m2 [n=3], 46 mg/m2 [n=3], 52 mg/m2 [n=6]) and sequential cytarabine 1000 mg/m2 (day-10 to day-5) and TBI (1200cGy) followed by AlloSCT from matched related or unrelated donors. Unrelated donor AlloSCT recipients also received R-ATG 2 mg/kg (day-4 to -2). GVHD prophylaxis consisted of tacrolimus and MMF as we have recently described (Bhatia/Cairo et al., BBMT, 2009). Probabilities of engraftment, acute and chronic GVHD, PFS and OS were computed using the Kaplan-Meier method. Results:Twelve patients enrolled, median age 11 years (range 5–18), 10/2 M/F, 9 ALL (6 CR3, 2 refractory relapse, 1 CR2 with history of IF), 3 AML (2 primary IF, 1 treatment-related AML in CR3), 6 with history of IF, 6 related donors (5 bone marrow [BM], 1 PBSCs), 6 unrelated donors (3 BM, 2 umbilical cord blood [UCB], 1 PBSCs). HLA matching was 5/6 (n=4) or 6/6 (n=4) at HLA-A, B and DRB1 for related donors and unrelated UCB donors and 9/10 (n=1) or 10/10 (n=3) at HLA-A, B, C, DRB1 and DQB1 for unrelated BM or PBSC donors. Median total nucleated cell dose (TNC) was 4.76×108/kg (2.30-14.56) and CD34 dose 5.04×106/kg (2.41-10.10) for patients receiving BM or PBSCs and 4.78×107/kg (4.00-5.55) and 4.04×105/kg (1.41-6.66), respectively, for patients receiving UCB grafts. An MTD of clofarabine was not exceeded at 52mg/m2/dose. No serious adverse events related to clofarabine were observed. One patient developed grade III hearing loss in the setting of atypical PRES, unlikely related to clofarabine, with normal cochlear function and abnormal auditory brainstem response.All engrafted neutrophils at median day 14 (12-29) and 90% of evaluable patients engrafted platelets at median day 36 (16-97). All achieved 100% whole blood donor chimerism by day +30. Day 100 transplant related mortality is 0%. The probability of grade II-IV acute GVHD was 43% (CI95: 13–71%); no patients developed chronic GVHD. Two patients died days +137 and +182 from multi-organ failure in the setting of infection and acute GVHD. Two experienced progressive disease on days +87 and +126 and received further therapy off study; one remains alive and stable. Eight are alive in continuous CR at a median of 209 days (40-770). Probabilities of PFS and OS are 53.3% (CI95: 18–80%) and 61.0% (CI95: 20–86%), respectively. Conclusions:Myeloablative conditioning with clofarabine, cytarabine and TBI followed by AlloSCT from related or unrelated donors is well tolerated in CAYA with poor-risk ALL and AML. Clofarabine 52 mg/m2/day × 5 days in combination with cytarabine and TBI can safely be used in the phase II study. Preliminary results from the phase I cohort are encouraging with respect to the risk of early relapse, despite poor-risk underlying disease. Efficacy data and measurements of change in minimal residual disease in the phase II study will provide a broader experience at the MTD to assess the role of this conditioning regimen in CAYA with poor-risk acute leukemia. Disclosures:Talano:Genzyme: Membership on an entity's Board of Directors or advisory committees. Cairo:Genzyme: Research Funding, Speakers Bureau.

Pattern of Relapse in Childhood ALL: Challenges and Lessons From a Uniform Treatment Protocol

This retrospective analysis of 254 children less than 15 years of age treated with MCP-841 protocol from June 1992 to June 2002 was undertaken to identify the pattern of relapse and determine management lacunae. Two hundred twenty-three (87.8%) children achieved a complete remission of whom 40 (17.9%) relapsed. The mean age of relapsed patients was 6.5 years. The male/female ratio was 9:1. There were 23 (57.5%) isolated bone marrow (BM), 7 (17.5%) isolated central nervous system (CNS), 2 (5%) isolated testicular, 5 (12.5%) BM+testes and 1 each of BM+CNS, CNS+testes, and isolated bone relapses. Twenty-seven children (67.5%) relapsed on-therapy whereas 13 (32.5%) relapsed posttherapy. All 9 CNS relapses occurred on-therapy whereas 5/8 (62.5%) of testicular relapses occurred posttherapy. Lymphadenopathy was the only significant predictor for relapse. High-risk features such as age less than 1 year and greater than 10 years (P=0.047) and white cell count greater than 50.0 x 10(9)/L (P=0.044) were significantly more frequent in patients with early on-therapy relapse than in patients with off-therapy relapse. The overall survival in the entire study cohort was 67+/-3.5%. Modest survival outcome, relapse while on chemotherapy and the higher incidence of CNS and testicular relapse indicate the need for reappraisal of our treatment protocol. There is a need of identifying risk factors and high-risk groups in our set of patients and risk-stratified intensification of chemotherapy in them.

Autologous purified peripheral blood SCT in childhood low-risk relapsed ALL

The treatment of childhood B-cell-precursor ALL after isolated-extramedullary or late relapse is controversial. Most approaches are based on chemotherapy or allogeneic transplantation. The aim of this report is to assess the long-term outcome of children with 'low-risk' relapsed ALL treated according to a prospective purified auto-transplantation protocol. From January 1997 to March 2004, at a single pediatric Center, 30 ALL consecutive children, lacking an HLA-identical sibling, were treated according to the autologous purified peripheral blood stem cell protocol after isolated-extramedullary (7) or late medullary (24) relapse. After the 'DIAVE' mobilizing regimen a median of 11.6 × 10(6)CD34+/Kg (range 3.9-27.4) were collected. Leukaphereses were depleted by 99% of CD19+cells (range 98-100) by means of a double step immunological purification. The conditioning regimen included TBI. No early severe complications nor transplant-related deaths occurred; late effects, as expected, mostly consisted in endocrinological issues and were assessed at a median follow-up of 8.5 years. Five-year-EFS and survival were 68.5% (s.e. 7.9) and 85.7% (s.e. 5.9), respectively, for the 35 eligible patients and 70.0% (s.e. 8.4) and 86.7% (s.e. 6.2) for the 30 patients actually transplanted as per protocol. The outcome of this series favorably compares with historical data regarding both autologous transplantation and standard salvage chemotherapy.

Alterations of TP53 are associated with poor outcome in relapsed childhood acute lymphoblastic leukemia

Prognosis of children with relapsed ALL has improved by treatment protocols using clinical markers. Further improvement will rely on the identification of molecular markers. Inactivation of the TP53 gene in cancer is associated with poor outcome. However, its role in relapsed ALL is still controversial. To clarify the significance of TP53 inactivation, we analysed 159 patients with first relapse enrolled in the ALL-REZ BFM 2002 trial for copy number and sequence alteration of the TP53 gene. TP53 alterations were observed in 17.46% (22/126) of relapsed B-cell precursor (BCP) and 18.18% (6/33) of relapsed T-cell ALL. Within BCP ALL bi- and monoallelic alterations of TP53 by loss and/or mutation were associated with poor response to chemotherapy and low event free survival (p&lt;0.01). Forward tracking of TP53 sequence alteration in a nonresponder with monoallelic loss of TP53, but no mutation at relapse diagnoses, revealed the formation of an additional mutation during treatment. In summary, our data shows that TP53 alteration identifies relapse patients at high risk of treatment failure and therefore represents a potential molecular marker for future risk stratification of ALL relapse.

Prevalence and prognostic significance of chromosome 21 amplifications in children with relapsed acute lymphoblastic leukemia: the ALL-REZ BFM study group

Survival after relapse of childhood ALL is currently predicted from clinical factors; however, the underlying molecular determinants are poorly understood. To identify chromosomal alterations associated with prognosis, we screened 223 patients with first relapse of B-cell precursor ALL by multiplex ligation-dependent probe amplification (MLPA). Gains of chromosome 21 (chr21) were the most frequent alterations (41%) that were further classified into 3 groups: gain of chr21 (+21, 15%), gain of chr21 and a pattern of additional gains consistent with high hyperdiploidy (+21/HHD, 21%) as well as intrachromosomal amplification (iAMP21, 5%). Concerning time of relapse, all 3 groups had less early relapses than patients without an amplification of chr21. iAMP21 patients showed entirely late relapses. All 3 groups had a significant higher rate of treatment related death. The cumulative incidence of relapses was higher in patients with +21 or iAMP21 than in +21/HHD patients indicating that HHD positively influences outcome. Collectively, our results show that chr21 amplifications are the most frequent chromosomal alterations in relapsed ALL and are associated with prognosis and outcome.

Mismatch repair and the downstream target genes, PAX5 and Ikaros, in childhood acute lymphoblastic leukemia

The mismatch repair (MMR) pathway is a post-replicative DNA repair process and MMR deficiency is a common feature of ALL cell lines. In this study we have investigated MMR deficiency in a large cohort of primary relapsed ALL ( n = 40) and investigated coding microsatellites (MS) of the lymphoid transcription factors, PAX5 and IKZF1 as downstream target genes. Only one patient showed MMR deficiency, as evidenced by microsatellite instability, which was acquired at relapse and was associated with reduced expression of both MLH1 and MSH2. Coding MS in candidate target genes including PAX5, IKZF1, BAX and TGFBRII were all wild type in this patient but the MMR-deficient cell line REH, was confirmed to have a coding MS in both PAX5 and TGFBRII. Whilst MMR deficiency is not highly prevalent in primary ALL, optimisation of the drug regimen to omit/replace thioguanines should be considered for children with MMR deficiency and/or reduced expression of key pathway components.

Early disease predictor for ALL relapse

Early disease predictor for ALL relapse

MECp, a Salvage Regimen with Drugs Not Employed as Frontline Therapy, Allowed to Obtain a High CR Rate and to Bring to Unrelated Allogeneic Transplantation a High Proportion of Adult Patients with Relapsed ALL.

Abstract 4101 Background and aimThe only curative option for adult patients with refractory or relapsed ALL is allogeneic haematopoietic stem cell transplant (allo-HSCT), which can offer a 28-34% long term survival in transplanted patients. However the actual feasibility of allo-HSCT is only 20-30% in unselected patients because of the low rate (30-50%) of complete remission (CR) achieved with salvage regimens (Tavernier, 2007- Thomas, 1999), the high rate of early relapse (Martino, 1999) and the difficulties in finding a suitable donor before progression (Davies, 1996). Hence, relapsed ALL can be actually cured in less than 10% of unselected adult patients. Using a second line treatment capable of obtaining a higher proportion of CR of longer duration may improve the dismal overall prognosis of patients. We report on the efficacy and toxicity profile of the combination of 6-metilprednisolone, mitoxantrone, etoposide and high-dose cytarabine (MECp), a salvage regimen containing cytostatic drugs to which patients had not been exposed during first line therapy, except for cytarabine at lower doses. Patients and MethodsBetween October 2000 and May 2009, 18 refractory/relapsed ALL patients were treated at our Institution with MECp regimen, consisting of a single course of etoposide 80mg/mq/die iv, cytarabine 1000mg/mq/die iv for 6 hours and mitoxantrone 6mg/mq/die iv 9 hours after cytarabine infusion for 6 days associated to metilprednisolone 50 mg/mq/die for 21 days, subsequently tapered to zero over one week. Three patients received an experimental sequential pulsed chemotherapy program in a multiinstitutional setting. At diagnosis, all patients had been treated according to the NILG-ALL 00/09 program (Bassan, Blood 2009). Four had been refractory to induction therapy and 14 had relapsed after a median of 12 months (range 3-43), 11 while on consolidation/maintenance, one after allo-HSCT, two after 3 and 12 months from the end of maintenance. There were 10 males and 8 females with a median age of 28 (range 17-64). ALL lineage was B in 9 cases (4 pro-B, 4 common, 1 pre B), T in 8 (5 pro-T, 3 cortical-TIII) and biphenotypic in 1. Molecular studies showed MLL/AF4 rearrangement in 3 and bcr/abl rearrangement in 3 cases, all before tyrosine-kinase inhibitors were available. Karyotypic abnormalities were present in 10 of 16 evaluable cases. Patients were treated in single/double bed rooms with reverse isolation. In 3 cases treatment duration was reduced to 4 days. ResultsCR was obtained in 13 of 18 patient (72,2%), independently of immunophenotype and time to relapse. CR rate was 100% in all ten patients with karyotypic abnormalities. Three patients (16%) died in aplasia during treatment, 2 of septic shock and 1 of unexplained shock. Two patients (T-ALL) were resistant. Recovery of neutrophils (>0,5×109/L) and platelets (>20×109/L) required a median of 22 days (range 17-37) and 28 days (range 21-45) from the start of therapy, respectively. Infections were documented in 9 of 18 (50%), being fatal in 2 (11%). Non-haematologic toxicity, mainly mucositis, was negligible. The median duration of CR was 5 months (range 2-5) which allowed 9 of 13 CR patients (69%) to undergo allo-HSCT (7 MUD, 1 HLA-identical sibling and 1 cord blood) after a median of 4 months (range 2-7) from CR. Reason for not being transplanted was failure of donor search in 4 patients who relapsed a median of 4 months. Causes of death included progressive disease in 7 patients, in 3 cases after HSCT, and transplant-related toxicity in 3 patients. The median survival of patients achieving CR was 13 months (range 7-33) and overall survival of the entire cohort was 8 months (range 1-33m). ConclusionsWith MECp, a combination of drugs not used during previous first-line therapy, a higher CR rate (72%) than commonly reported could be obtained, with acceptable toxicity. CR duration was long enough to allow 44,4% of patients to receive a non-family donor allo-HSCT, which is presently the best, yet still unsatisfactory, treatment option for adult patients with refractory/relapsed ALL. Disclosures:No relevant conflicts of interest to declare.

A Phase 1 Study of Dose-Dense 5-Aza-2′-Deoxycitidine (decitabine) in Relapse Refractory Acute Lymphocytic Leukemia (ALL).

Abstract 2030Poster Board II-7Aberrant DNA methylation of multiple promoter associated CpG islands is frequent in primary ALL and predicts for poor prognosis in adult and pediatric disease. Treatment of ALL cell lines with the hypomethylating agent decitabine results in induction of global and gene specific hypomethylation, reactivation of epigenetically silenced genes and induction of apoptosis at low concentrations and prolonged exposures (Leuk Res 2005;29:739-48). Prior studies of decitabine in myeloid leukemias indicated that induction of global and gene specific hypomethylation using a 5-day schedule of decitabine is transient peaking 7 to 10 days after initiation of therapy. In view of the proliferative nature of ALL and in vitro modeling results, we designed a phase 1 study of decitabine administered daily x 5 every other week in patients with relapsed or refractory ALL. The main objective of the study is to determine the safety, activity, pharmacodynamic effects and optimal dose (based on clinical activity, toxicity profile and hypomethylating properties) of decitabine in relapsed refractory ALL. The study design follows a standard “3+3” rule with an expansion cohort of N=10 patients at the optimal dose. Patients of any age with relapsed refractory ALL are elegible regardless of performance status or organ function. Initial dose level of decitabine was 10 mg/m2 IV infused over 1 hour daily x 5 days every other week with courses of therapy repeated every 28 days. Use of steroids was allowed during the first course of therapy at the discretion of treating physician. 23 patients have been treated in 7 dose levels (10, 20, 40, 60, 80, 100 and 120 mg/m2 IV QD x 5 every other week). Cumulative doses per course ranged from 100 to 1200 mg/m2. Patient characteristics are: median age 36 years (range 8-67), median WBC 5.3 (range 0.2 to 97), median % peripheral blasts 23% (0-97), cytogenetics diploid in 4 (17%), Ph + 2 (8%), complex 17 (73%), phenotype preB/B in 15 (65%). Median number of prior therapies was 3 (range 1 to 7). No severe drug related grade 3 or 4 toxicity was observed at any dose level. Frequent toxicities included diarrhea, fatigue and liver function abnormalities that were limited and probably related to disease. Overall response rate was 23% (6 pts) including 1 CRp (complete remission with incomplete platelet recovery) and 5 complete marrow responses (blasts less than 5%). All responses lasted at least 4 weeks. Responses were observed at multiple dose leves (#1, 2, 4, 5, 7). Global and gene specific methylation was analyzed on days 0, 2,5, 14,16,19 and 28 of cycle 1. Samples were collected from 18 consenting patients. Global methylation was analyzed using the LINE bisulfite pyrosequencing assay. Median day 0 methylation was 63%, declined to 55% (p=0.01) on day 14 and increased to 61% on day 28. The most effective dose in inducing global hypomethylation was 60 mg/m2 : 61% baseline to 21% on day 28. The following genes were analyzed for gene specific methylation: p73, p15, p15, HES5, Notch3 and Jag1. Induction of hypomethylation was observed in informative patients, a process associated with gene expression reactivation. The analysis is not powered to detect association between response and hypomethylating effect. Finally, depletion of DNMT1 was measured using a Western blot assay. Depletion was only observed in 1 patient treated at 60 mg/m2 that had achieved a response. In summary, single agent decitabine is safe at higher doses than used in myeloid leukemias with clinical activity in patients with advanced refractory relapsed ALL. Of importance, hypomethylating effect is observed at cumulative doses of up to 1200 mg/m2 with a maximal effect at 600 mg/m2, doses that are considered cytotoxic in myeloid leukemias. The study continues at the expansion cohort of 60 mg/m2 IV QD x 5 every other week, the dose considered to be optimal based on toxicity, response and hypomethylating effects. Two patients have been treated but are early for assessment. A parallel study of decitabine combined with hyperCVAD is ongoing in patients that do not respond or progress after single agent decitabine. The activity of decitabine should be tested in patients in first relapse ALL. Disclosures:Off Label Use: Decitabine is not approved for treatment of ALL.

AAML0523: A Report From the Children's Oncology Group On the Safety of Clofarabine in Combination with Cytarabine in Pediatric Patients with Relapsed Acute Leukemia.

Abstract 3076Poster Board III-13 BackgroundDespite improvements in survival rates for pediatric ALL and AML, many children still relapse with dismal outcomes. It is critical to develop non-anthracycline containing salvage regimens that induce remission and allow for hematopoietic stem cell transplantation (HSCT). Clofarabine is a nucleoside analog that potently inhibits ribonucleotide reductase and DNA polymerase αa. The biochemical modulation of cytarabine by clofarabine via inhibition of ribonucleotide reductase is well established, and this combination has been studied in adults with relapsed AML. Study DesignThe Children's Oncology Group instituted a Phase I/II study testing the combination of clofarabine and cytarabine in pediatric patients with AML in first relapse and ALL in second or subsequent relapse. We report on the toxicity profile in the Phase I portion of the study. Cytarabine was administered on days 1-5 at a dose of 1 gm/m2 and clofarabine was administered on days 2-6 in varying doses. Cytarabine was given 4 hours after the start of clofarabine to optimize the biochemical modulation of ara-CTP. Patients could receive up to 2 cycles on study. The Phase I portion of the study consisted of a single dose escalation/de-escalation of clofarabine with a fixed dose of cytarabine. For each dose level, 10 patients were enrolled. The first cohort of patients received clofarabine at 40 mg/m2/day (the adult single-agent MTD). Based on safety data on the first cohort, the dose of clofarabine would either be escalated to 52 mg/m2/day (single-agent pediatric MTD) or deescalated to a dose of 30 mg/m2/day. The MTD determined in this dose finding phase would determine the dose for the Phase II efficacy portion of the study. ResultsOf the 10 patients enrolled on the first cohort, 8 patients had ALL (6 in second relapse, 2 refractory to reinduction) and 2 had AML. Ten patients completed one cycle and 5 patients completed two cycles. In both cycles, the most common toxicities ≥ Grade 3 were fever/neutropenia (7/15 occurrences), infection (6/15), transaminitis (5/15), abdominal pain (3/15), and hypokalemia (4/15). Two ALL patients with prior HSCT had dose-limiting toxicities (DLT's): one with grade 4 fungal infection and pneumonitis and another with grade 4 fungal infection. Neither patient was on antifungal prophylaxis. The MTD was not met, and 10 patients were enrolled on the second cohort (52 mg/m2): 3 patients with ALL in second relapse and 7 with AML. Ten patients completed cycle 1 and 7 completed two cycles. In both cycles, the most common toxicities ≥Grade 3 were diarrhea (3/17), nausea (3/17), febrile neutropenia (5/17), infection (10/17), hyperglycemia (3/17), and hypokalemia (3/17). In this cohort, one AML patient experienced DLT's of prolonged bone marrow aplasia, Grade 4 hypokalemia, Grade 3 nausea, and Grade 3 dehydration. The table below summarizes count recovery data at both dose levels. The median cycle length for patients without ANC and platelet recovery at the 40 mg/m2 level was 22 and 30 days respectively after one course and 19 and 17.5 days respectively after 2 courses. At the 52 mg/m2 dose level, the median cycle length for patients without ANC and platelet recovery was 20 days for each after one course and 23 and 25 days respectively after 2 courses. These numbers suggest that patients without marrow recovery had progressive disease, received alternate chemotherapy, or proceeded to HSCT prior to count recovery, rather than drug-induced prolonged bone marrow aplasia. ConclusionsThe combination of clofarabine and cytarabine is safe and tolerable in pediatric patients with relapsed acute leukemia. The MTD of clofarabine in combination with cytarabine in pediatric patients with relapsed acute leukemia is 52 mg/m2/day.Dose: 40 mg/m2Course 1Course 2# of patients with recoverymedianrange# of patients with recoverymedianrangeALL patients (n=8)Time to ANC >750/μl (days)52318-3632116-22Time to platelets >75,000/μl (days)32513-28221.521-22AML patients (n=2)Time to ANC >1,000/μl (days)23836-4013333Time to platelets >100,000/μl (days)1363613333Dose: 52 mg/m2Course 1Course 2# of patients with recoverymedianrange# of patients with recoverymedianrangeALL patients (n=3)Time to ANC >750/μl (days)0--0--Time to platelets >75,000/μl (days)0--0--AML patients (n=7)Time to ANC >1,000/μl (days)23324-4215757Time to platelet s >100,000/μl (days)22421-270-- DisclosuresWhitlock:Genzyme: Consultancy.

The Serum Fas and Fas Ligand levels in Childhood Acute Leukemias.

Abstract 4724 AimAbnormalities and alterations in apoptosis mechanism may lead to cancer development. Cystean proteases enzymes, called caspases, appear to be involved in both the initial signaling events. There are many proteins that trigger intrinsic and extrinsic pathway and induce apoptosis signals. Fas and its specific ligand that known as Fas Ligand are the best defined dead receptors and have functions in apoptosis regulation with many tumor types. Fas binds the ligand on the cytotoxic T cells and start apoptosis. Objectives of this study were to determine serum levels of Fas and Fas Ligand at the time of diagnosis in childhood acute leukemias that may be play important role in apoptosis mechanism. Patients and MethodsIn this study, we investigated serum Fas and Fas Ligand levels by using ELISA method in childhood acute leukemias. Twenty-nine cases with acute lymphoblastic leukemia and twenty-three cases with acute myeloblastic leukemia at the ages of 1-18 years are included this study. The age distrubition of the control group varied 1-15 years consisted of twenty-seven children. We investigated serum Fas and Fas Ligand levels at the time of diagnosis from peripheral blood samples. ResultsThe comparison of the mean values of Fas and Fas Ligand levels in acute leukemia patients groups and control group have shown important difference as statistically (p<0,05). The mean values of Fas and Fas Ligand levels were higher in ALL and AML patients. The comparison of the mean values of Fas and Fas ligand levels in ALL and AML patients have shown no difference (p>0,05). The comparison of the Fas levels in ALL patients according to immunophenotypes; CALLA(+) B-ALL have higher mean level than T-ALL and shown important difference as statistically (p<0,05). The comparison of the mean values of Fas level at the diagnosis in ALL patients who had relapsed and patients who had remission have shown important difference (p<0,05). The mean values of Fas level were found higher in relapsed ALL patients. In these results showed that Fas and Fas ligand may play important role in apoptosis mechanism. Disclosures:No relevant conflicts of interest to declare.

Chemokines in Leukemic Infiltration of the Central Nervous System in Childhood Acute Lymphoblastic Leukemia.

Abstract 1627Poster Board I-653 BackgroundLeukemic blasts from B-cell acute lymphoblastic leukemia (B-ALL) and T-ALL circulate through the blood stream and may infiltrate different organs. Extramedullary organs may act as sanctuaries for lymphoblasts, preventing the exposure to adequate levels of chemotherapeutic drugs. Typical extramedullary relapses are seen in testes and in central nervous system (CNS). We aimed at determining whether chemokines may play a role in the infiltration of the CNS by leukemic blasts in childhood ALL. We studied the expression of chemokine receptors in ALL blasts in marrow, as well as the levels of chemokine ligands in the cerebrospinal fluid (CSF) of children with B- or T-ALL. If chemokines had a role in CNS leukemic infiltration, the following should be confirmed: leukemic blasts should express high levels of the chemokine receptor/s for which high levels of its corresponding ligand/s were detected at the CNS. MethodsThis prospective study was approved by the local ethical committees for clinical research. Samples from 80 children in 10 Spanish pediatric oncology units were obtained. We detected the presence of leukemic blasts in CFS by flow cytometry. We defined leukemic infiltration of CFS samples as the presence of cells with the same immunophenotype as the leukemia in the marrow aspirates. We detected the expression levels of 9 CCR and 6 CXCR molecules in ALL blasts by flow cytometry in marrow aspirates. Levels of chemokine ligands were quantitated by Cytometric Bead Array or by commercial ELISA kits in CSF samples. ResultsWe found that chemokine receptors expression and levels of chemokine ligands varied depending upon the lineage (T versus B), the maturation state (pre-T versus T; pro-B versus pre-B versus common-B) and the risk-status (high versus non-high) of the leukemia. T-ALL patients with high levels of CNS leukemic infiltration expressed significantly higher levels of CXCL10 compared to the same parameters of T-ALL patients with low/absent levels of CNS disease (p=0,049). Common B-ALL patients with high levels of CNS leukemic infiltration expressed higher levels of CCL22 compared to that of common B-ALL patients with low/absent levels of CNS disease (p=0,059). Among the 4 patients with a CNS relapse, we detected higher levels of CXCR3 (p=0,0038) and of its ligand, CXCL10 (p=0,0169), compared to patients who did not relapse. ConclusionsOur study suggests that the CXCR3/CXCL10 axis may be involved in the CNS relapse of high-risk ALL in children: high expression of CXCR3 on marrow blasts plus high levels of CXCL10 in the CNS was associated with leukemic CNS relapse. DisclosuresNo relevant conflicts of interest to declare.

MicroRNA Patterns Associated with Clinical Prognostic Parameters and CNS Relapse Prediction in Pediatric Acute Leukemia

BackgroundRecent reports have indicated that microRNAs (miRNAs) play a critical role in malignancies, and regulations in the progress of adult leukemia. The role of miRNAs in pediatric leukemia still needs to be established. The purpose of this study was to investigate the aberrantly expressed miRNAs in pediatric acute leukemia and demonstrate miRNA patterns that are pediatric-specific and prognostic parameter-associated.Methodology/Principal FindingsA total of 111 pediatric bone marrow samples, including 99 patients and 12 normal donors, were enrolled in this study. Of those samples, 36 patients and 7 normal samples were used as a test cohort for the evaluation of miRNA profiling; 63 pediatric patients and 5 normal donors were used as a validation cohort to confirm the miRNA differential expression. Pediatric ALL- and AML-specific microRNA expression patterns were identified in this study. The most highly expressed miRNAs in pediatric ALL were miR-34a, miR-128a, miR-128b, and miR-146a, while the highly expressed miRNAs in pediatric AML were miR-100, miR-125b, miR-335, miR-146a, and miR-99a, which are significantly different from those reported for adult CLL and AML. miR-125b and miR-126 may serve as favorable prognosticators for M3 and M2 patients, respectively. Importantly, we identified a “miRNA cascade” associated with central nervous system (CNS) relapse in ALL. Additionally, miRNA patterns associated with prednisone response, specific risk group, and relapse of ALL were also identified.Conclusions/SignificanceThere are existing pediatric-associated and prognostic parameter-associated miRNAs that are independent of cell lineage and could provide therapeutic direction for individual risk-adapted therapy for pediatric leukemia patients.

Achievement of complete donor-type chimerism and remission with dasatinib in Philadelphia chromosome-positive ALL relapsing after allogeneic transplantation

Achievement of complete donor-type chimerism and remission with dasatinib in Philadelphia chromosome-positive ALL relapsing after allogeneic transplantation

Lactic acidosis and hypoglycemia with ALL relapse following engrafted bone marrow transplant

Lactic acidosis together with hypoglycemia in the face of hematologic malignancy is a grave development. A 7-year-old male with pre-B-cell ALL following hematopoietic cell transplant was admitted to our hospital in his second relapse. On hospital days 4 and 5, he developed refractory hypoglycemia, lactic acidosis, central respiratory failure, and acute renal failure. Bicarbonate infusion, B vitamins, and hemodialysis were not effective. Care was withdrawn on hospital day 9. Further understanding of the mechanisms that cause the combined onset of lactic acidosis and hypoglycemia will help clinicians in implementing timely therapies that may reduce mortality.

A Prognostic Scoring System for Patients ≤ 60 Years of Age with Acute Lymphocytic Leukemia in First Relapse.

The prognosis of adult pts with ALL in first relapse is poor. We analyzed pt characteristics at the time of relapse to define a prognostic score for pts ≤ 60 yrs of age.Methods: Pts 18–60 years of age with ALL (WHO criteria) in first relapse treated at the Cleveland Clinic between the years 1990–2007 were evaluated. Pts were identified through the Health Data Service's Hospital Abstract and BMT databases using the ICD-9 code for ALL. Pts with residual leukemia after their initial induction or those receiving allogeneic bone marrow transplant (alloBMT) in first CR were not included. Relapsed disease was defined as the reappearance of leukemic blasts in the blood or bone marrow (BM) (> 5%) or in any extramedullary site. Cox proportional hazards analysis was used to identify univariate and multivariate factors significantly affecting overall survival (OS) in all pts including those proceeding to alloBMT. Factors evaluated were: age, cytogenetic (CG) risk group, disease status, time from diagnosis to relapse, lactate dehydrogenase (LDH) at the time of relapse, gender, and HLA match (for related or unrelated donors). An HLA match was defined as a 6/6 allele match based on 2 A, B, and DRB1 markers. Factors significant in univariate analysis were used in the multivariate model.Results: Seventy-seven pts were analyzed. The median age at relapse was 32 yrs [range 18–60] and median time from diagnosis to first relapse 14.8 months [0.8–41.6]. Twenty-six percent of pts had poor risk cytogenetics (CG), 47% normal, 14% miscellaneous abnormalities, and 13% unknown CG as defined by CALGB criteria. Seventy-eight percent of pts had ALL derived from B-cell, 18% T-cell, 1% mixed, and 3% unknown lineage. Ninety-one percent of pts relapsed with blood/BM disease and 21% with central nervous system (CNS) involvement. Nine percent relapsed with leukemia isolated to an extramedullary site. Thirty-seven percent of pts achieved a CR following salvage therapy. Forty-six pts (60%) received an alloBMT, with 24 pts undergoing transplant from a matched-related donor. Forty-eight percent of pts were in CR at the time of alloBMT. The estimated OS for all pts was 41%, 23%, and 6% at 1, 2, and 5 yrs post-relapse. However, the OS was significantly better for those receiving BMT (58%, 33%, and 9% at 1, 2, and 5 yrs) compared to those not receiving an alloBMT (9%, 5%, and 0%) [p< 0.001]. Factors adversely affecting OS in pts proceeding to BMT on both univariate and multivariate analysis were: older age, HLA mismatch, leukemia present at the time of BMT, and higher LDH at the time of relapse [per 200 unit increase (HR 1.77, CI 1.30–2.38, p< 0.001)]. Higher LDH at the time of relapse and older age at the time of diagnosis were predictors of worse OS for all pts, in addition to being predictive for those proceeding to BMT. For pts < age 45 with an LDH at the time of relapse < 1.4 times the upper limits of normal (ULN), 79% proceeded to BMT, and the CR rate to salvage therapy was 67%. Based on this data, we were able to develop a prognostic model. For all pts, a scoring system of 0–3 was used with 1 point for each of the following: age at diagnosis 345, LDH 3 1.4 times ULN, not proceeding to BMT (Table 1). Among pts who proceeded to BMT, a similar scoring system was used with 1 point for each of the following: age at diagnosis > 45 years, LDH at the time of relapse > 1.4 times ULN, leukemia present at the time of BMT (Table 1). For pts proceeding to BMT with a score of 2 (based on LDH and age), the median OS was only 4.9 months. The only long-term survivor was a pt who had achieved a CR to salvage therapy.Table 1 Prognostic Score for Overall SurvivalPrognostic ScoreAll PtsPts Proceeding to BMT3 vs. < 3HR=6.77, CI 1.81–25.4, p=0.004HR 14.2, CI 2.37–85.8, p=0.004≥ 2 vs. < 2HR=3.04, CI 1.58–5.83, p=0.001HR 2.16, CI 0.89–5.23, p=0.09Conclusions: LDH > 1.4 ULN at the time of relapse predicts poor OS and may be incorporated, with age, into a prognostic score that also predicts a pt's response to salvage therapy and likelihood of proceeding to BMT. Pts with a score of 2 have a poor prognosis, even with BMT, and should be considered for innovative approaches such as Phase 1 clinical trials. This prognostic scoring system will require prospective validation with an independent data set.