ALBERT

Description: Introduction. Chronic myeloid leukemia(CML) is a very rare disease in children and adolescents. The international registry of CML in children and adolescents gives us the opportunity to assess the prognostic relevance of variant t(9 ;22) and additional cytogenetic abnormalities at diagnosis on prognosis. Patients and Methods. We used clinical and biological characteristics and outcome of 239 children and adolescents less than 18 years to assess the impact of additional cytogenetic abnormalities and variant t(9 ;22) on the rate and the time to achieve a complete cytogenetic response (CCyR). Cytogenetic analysis was performed 24 or 48 hour culture on G-banded metaphases. Results. Overall, 17/239 children (7.1%) presented with additional cytogenetic finding at diagnosis: 5/239 children (2.1%) had a variant of the t(9;22), 10 children (4.2%) had other additional cytogenetic (numerical/structural) abnormalities (ACA), and 2/239 (0.8%) had both. Characteristics of the patients and phase of the disease are reported in Table 1. In the 7 patients with variant t(9;22), one (chromosome 1 in 3 patients, chromosome 8 in 2 patients and chromosome 14 in one patient) or two further chromosomes (chromosomes 8 and 17) were involved in 6 and one patient(s), respectively. In the 12 patients with ACA, 9 patients have one type of ACA, 2 patients have 2 type of ACA and one patient have 3 type of ACA. Regarding the patients in chronic phase at diagnosis (n=219), the cumulative incidence of complete cytogenetic response at 18 months was 88% (95% CI: 82 % - 93%) and 70% (95% CI: 42% - 93%) for patients without variant t(9;22) or ACA and for those with variant t(9;22) and/or ACA (p=0.151), respectively. Three deaths were recorded (among them one patient had ACA at initial diagnosis). With a median follow up of 43 months (range 1 – 161) the probability of 3 years overall survival was 99% (95% CI: 94 % - 100%) and 88% (95% CI: 39 % - 98%) for patients in chronic phase without variant t(9;22) or ACA and for those with variant t(9;22) and/or ACA (p=0.042), respectively. Conclusion. Additional chromosomal abnormalities at diagnosis in children with CML in chronic phase may have an impact on the outcome. A larger cohort of patient and a longer follow up is needed to confirm such findings. Table 1. Cytogenetic No variant t(9;22) or ACA Variant t(9;22) ACA Variant t(9;22) and ACA Number of patients 222 5 10 2 Sex (% males) 56% 60% 50% 100% Median age, yrs (range) 12 (1-18) 14 (9-17) 12 (5-16) 6 and 17 Phase of the disease (n) chronic accelerated blastic 208 12 2 3 1 1 7 1 2 1 0 1 Acknowledgment: The I-CML Ped study is supported by an unrestricted grant from Novartis Pharmaceutical Company Disclosures No relevant conflicts of interest to declare.

Description: Background: Patients with acute lymphoblastic leukemia (ALL) are treated with different chemotherapeutic drugs, which can be transported by ABC-transporters, including six multidrug resistance-associated proteins (MRPs). New insights suggest that a drug resistance profile, in which multiple transporters act in concert, is more likely to predict prognosis than one single transporter. The aim of this study was to investigate the expression profile of MRP1-6 in ALL in both children and adults and determine its impact on prognosis. Methods: mRNA expression of MRP1-6 was analyzed by real-time PCR in human de novo ALL samples from 56 children and 49 adults. Of these patients, 32 showed a T-lineage and 73 a B-lineage immunophenotype. Results: Adults showed a significantly higher expression of MRP1 (median value 1.08 vs. 0.69), MRP2 (0.96 vs. 0.53) and MRP3 (0.31 vs. 0.07) than children. However, no difference in expression levels was observed between children and adults who developed a relapse. High expression of one MRP in individual patients was correlated with increased expression of multiple MRPs. The ability to discriminate between patients who will remain in continuous complete remission and patients who will relapse was determined by receiver operating characteristic (ROC) curves. When children and adults were analyzed together, the area under the ROC curves showed that MRP1 (AUC 0.72, p=0.002), MRP2 (AUC 0.71, p=0.002), MRP3 (AUC 0.72, p=0.001), MRP5 (AUC=0.70, p=0.004) and MRP6 (AUC=0.68, p=0.009) predicted relapse. Using median values as cutoff for high versus low expression, high expression of all six MRP genes, except MRP4, was significantly associated with reduced event-free survival in children and adults (Kaplan-Meier curves with Log-rank tests). Conclusion: Our results suggest that a profile with high mRNA expression of MRP1, MRP2, MRP3, MRP5 and MRP6 is involved in drug resistance in ALL. This unfavorable profile is more often observed in adults, and is associated with reduced event-free survival.

Description: Background Treatment outcome in acute lymphoblastic leukemia (ALL) has improved over the past 30 years, with overall survival rates of ∼45% in adults and ∼85% in children. Gross cytogenetic abnormalities, including numerical changes and chromosomal translocations, are of considerable prognostic value in both pediatric and adult ALL. In addition, we and others have recently identified novel molecular markers associated with a poor outcome in ALL, including deletions of the lymphoid transcription factor IKZF1. In order to identify downstream signaling events associated with these genetic alterations, we performed an integrated analysis of genomic abnormalities, including copy number alterations, sequence mutations and chromosomal translocations, with alterations in protein expression and modification. Methods A cohort of 91 precursor B-ALL cases treated at M.D. Anderson Cancer Center in Houston, USA, including 82 newly diagnosed cases and 5 diagnosis-relapse pairs was used for this study. The cohort consisted of 6 children (age 1-6), 30 young adults (age 15-39) and 45 adults (age〉39), and 20 patients carried a BCR-ABL1 chromosomal translocation. Copy number alterations in eight genes frequently deleted in ALL (IKZF1, PAX5, EBF1, RB1, CRLF2, CDKN2A/2B, BTG1, and ETV6) were determined by multiplex ligation-dependent probe amplification analysis. IKZF1 deletions were associated with relapse (Pearson's chi-square test, p=0.009), and the presence of BCR-ABL1 translocation (p=0.032). Protein expression and modification levels were determined by probing Reverse Phase Protein Arrays (RPPA) containing protein lysates of all above samples with 128 rigorously validated antibodies including 34 phospho-specific antibodies. Hierarchical clustering analysis was used to determine which (phospho)proteins are differently expressed in genetic subsets of ALL. The significance of correlations was determined using two-sample t-test, with correction for multiple testing (Beta-Uniform Mixture model). Results We identified clustering of cases with a BCR-ABL1 chromosomal translocation (p=0.01; false discovery rate (FDR)=0.1), IKZF1-deletions (p=0.01, FDR=0.072), RB1-deletions (p=0.03, FDR=0.43) and EBF1 deletions (p=0.05, FDR=0.63). As expected RB1 deletion positive cases were characterized by decreased levels of (phospho)-RB1 and increased levels of cyclin E, illustrating the validity of our approach. EBF1-deleted cases showed relatively high levels of SHIP1, SSBP2 and phospho-STAT5, and lower levels of FAK and LYN. The protein signatures of BCR-ABL1-positive cases and IKZF1-deletion positive cases largely overlapped, and were characterized by elevated levels of (phospho)PKCα, SMAD1, phospho-STAT3, and phospho-STAT5 and lower levels of LYN and cyclinD3 (Figure 1). In total 70% of the BCR-ABL1-positive cases carried an IKZF1 deletion and several BCR-ABL1-negative cases with similar RPPA signature could be identified, all of which were IKZF1-deletion positive. These cases may represent the “BCR-ABL1-like” cases that were previously identified using gene expression signatures (Mullighan et al. 2009, NEJM 360:470-480; Den Boer et al. 2009, Lancet Oncol. 10:125-134), and could reflect activation of cAbl or other cellular tyrosine kinases. Together, we conclude that integrated analysis of genetic and proteomic aberrations identified protein signatures downstream of recurrent mutational events in ALL, a strategy that promises to facilitate the discovery of novel therapeutic targets in ALL and may aid in the identification of (high risk) patients that would benefit from tyrosine kinase inhibition. Disclosures: No relevant conflicts of interest to declare.

Description: Aims: An international registry (I-CML-Ped Study)was established to assess epidemiology, management and outcome of CML in the pediatric population. Methods: All national pediatric study groups were invited to include newly diagnosed children and adolescents less than 18 years with CML diagnosed later than January 2000. Results: Since January 2011, 351 patients from 12 countries have been registered. Clinical and biological data at initial diagnosis are available in 278 patients.There was a male preponderance (57%). The median age at diagnosis was 12.4 years (range, 9 months -17.5 years); 6% of the patients were younger than 4 years. At time of diagnosis 92% of the children were in chronic phase, 8% in accelerated phase and 1% in blastic phase according to the European Leukemia Net criteria. The Sokal risk group distribution was: 18% low, 31% intermediate and 51% high risk. The majority of the patients showed a Lansky score of 100 (59%) or 90 (21%). Splenomegaly was present in 76% of patients. The median of the spleen size below the costal margin was 11 cm (range: 1 to 25 cm). The median of the leukocyte count was 235x109/L (range: 5 to 1038). Additional chromosomal abnormalities in Ph-positive cells were observed in 6 % of the patients. The BCR-ABL transcript type was available in 227 patients: b3a2 54%, b2a2 38%, b3a2-b2a2 6% and b2a3 2%. The median follow-up time is 39 months (range, 0.5-161). Eight deaths were recorded. The estimated overall survival rate at 60 months is 95% (95%CI 89-97). Irrespective of treatment and follow-up, 124 (73%) of 169 assessable patients for cytogenetic response achieved complete cytogenetic response (CCyR). Exploratory analyses were performed in newly diagnosed patients regarding clinical and biological factors influencing the achievement of CCyR 12 months after starting imatinib. In univariate analyses, the Eutos score, the spleen size, the hematocrit level, the lymphocyte count and immature cells in peripheral blood, the percentage of granulocytes and monocytes in the marrow were identified as potential prognostic factors. However, only the percentage of the granulocytes in the marrow was identified as independent factor of achievement of CCyR at 12 months in multivariate analysis. Data collection and quality control regarding molecular assessment are ongoing. Conclusion: The data indicates that children and adolescents with CML presented with clinical and biological differences compared to adult patients with CML. Identification of prognostic factors is needed to optimize the strategy in the pediatric population. Acknowledgment: The I-CML Ped study is supported by an unrestricted grant from Novartis Pharmaceutical Company Disclosures No relevant conflicts of interest to declare.

Description: Prognosis of refractory and relapsed ALL is poor and improvement requires the introduction of agents with a new mechanism of action. Bortezomib (BTZ) as a proteasome inhibitor is such an agent, and was safe as single agent in phase I studies in children (Blaney 2004; Horton 2007). Messinger et al. (2012) reported a single-arm study that BTZ can be combined safely with conventional drugs; the combination was remarkably effective. BTZ results in sensitization of malignant cells to anticancer agents, both in vitro for leukemias as well as for multiple myeloma patients. In patients with ALL, this effect regarding glucocorticoids has not been addressed yet. It also has not been studied whether BTZ reaches the cerebrospinal fluid (CSF), which is relevant in pediatric leukemias in view of the frequent leptomeningeal involvement. In the setting of the lack of clinical experience with BTZ in children in Europe, we developed a European multicentre feasibility/phase II study in refractory or relapsed ALL, in which all patients get BTZ (NTR 1881, EUDRaCT 2009-014037-25, ITCC 021). A randomisation is done for BTZ to start “early”, on day 1 of treatment, or “late”, on day 8 of treatment. Bortezomib is given as iv push for 4 doses at 1.3 mg/m2/dose, thus in group “early” on days 1, 4, 8 and 11 and in group “late” on days 8, 11, 15 and 18. In addition, all patients receive dexamethasone (10 mg/m2/day in 3 doses for 2 weeks, orally or iv) and vincristine (1.5 mg/m2/dose with a maximum of 2 mg as 1-hour infusion on days 8 and 15), and one intrathecal administration of methotrexate (dose age-adjusted) on day 1. Eligible patients have 2nd or greater relapsed ALL, 1st relapsed ALL after allogeneic stem cell transplantation (allo-SCT) in 1st complete remission (CR1), or refractory 1st relapsed ALL, bone marrow involvement and at least 100 leukemic cells per ul blood. Exclusion criteria includes symptomatic CNS leukemia, among other factors. It is planned to have 24 fully evaluable patients. This interim analysis is limited to a description of pharmacokinetic (PK) data, especially concerning the CSF. As per June 1, 2013 a total of 14 patients has been enrolled, 9 boys and 5 girls, median 8.7 years of age (range, 1.6-16.2). Most had 2nd relapsed ALL (n=9), others 1st relapsed ALL following allo-SCT in CR1 (n=3) or refractory 1st relapsed ALL (n=2). Regarding PK in the peripheral blood, there was remarkable intra- and inter-individual variability in peak plasma concentrations, between patients ranging from 4.7 to 2920 ng/ml 15 minutes after the first administration of BTZ, median 12.4 ng/ml (18.7 ng/ml in the group with BTZ “early”, 12.1 ng/ml in the group with BTZ “late”). Peak levels after the fourth administration were higher, median 41.1 ng/ml (29.5 ng/ml in the group with BTZ “early” and 158.9 ng/ml in the group with BTZ “late”). There was a 10-fold interindividual variation in the area-under the concentration versus time curve until 72 h (AUC[0-72h]) after administration. Median ratio of AUC[0-72h] fourth dose / AUC[0-72h] first dose was 2.7 (range 0.9 – 9.3), which is indicative of accumulation. In all patients, PK of BTZ was studied in the CSF 15 minutes after administration of the first and third dose (group “early” only) of BTZ, as well as 4 days (group “late”) or one week (group “early”) after the last administration. In general, no BTZ was detected with a lower detection limit of 0.1 ng/ml. In 4 patients some BTZ was detected in CSF, at 0.2 – 0.4 – 1.7 - 5.2 ng/ml. Of potential interest, the latter patient also had the highest peak plasma level of BTZ (2920 ng/ml) and the highest AUC. Future analyses in the complete cohort of 24 randomised patients will focus on more extensive population PK and pharmacodynamic analyses, as well as on efficacy of BTZ. The higher peak plasma levels after the fourth dose suggest decreased clearance (especially in the group which received bortezomib “late”), which indeed has been reported in adults and which requires careful monitoring of toxicity over time. Mean peak plasma concentrations in adults were reported to be 173 ng/ml, and thus seem higher. Meanwhile, BTZ does not or hardly penetrate the cerebrospinal fluid and is unlikely to be a drug that significantly adds to the treatment of leptomeningeal involvement in leukemia. Financially supported by the Dutch Foundation Children Cancer-free. Disclosures: Off Label Use: bortezomib in pediatric relapsed acute lymphoblastic leukemia.

Description: Abstract 3471 Poster Board III-359 One important cytogenetic subgroup of pediatric acute myeloid leukemia (AML) is characterized by translocations of chromosome 11q23, which accounts for 15 to 20% of all cases with an evaluable chromosome analysis. In most of these cases, the mixed lineage leukemia (MLL) gene is involved. More than 50 fusion translocation partners of the MLL gene have been identified and outcome differs by translocation partner, suggesting differences in the biological background. So far these biological differences have not been unravelled. Therefore, we investigated the gene expression profiles of MLL-rearranged subgroups in pediatric AML in order to discover and identify the role of differentially expressed genes. Affymetrix Human Genome U133 plus 2.0 microarrays were used to generate gene expression profiles of 257 pediatric AML cases, which included 21 pediatric AML cases with t(9;11)(p22;q23) and 33 with other MLL-rearrangements. With these profiles, we were able to identify a specific gene expression signature for t(9;11)(p22;q23) using an empirical Bayes linear regression model (Bioconductor package: Limma). This signature was mainly determined by overexpression of the BRE (brain and reproductive organ-expressed) gene. The mean average VSN normalized expression for BRE in the t(9;11)(p22;q23) subgroup was 3.7-fold higher compared with that in other MLL-rearranged cases (p

Description: Pediatric acute myeloid leukemia (AML) is a heterogeneous disease, which is classified according to the WHO classification, based on morphology, immunophenotyping and non-random genetic aberrations. AML is hypothesized to arise from two different types of genetic aberrations, i.e. type-I (proliferation enhancing) mutations and type-II (differentiation impairing) mutations. To detect genetic aberrations multiple techniques such as conventional karyotyping, FISH and RT-PCR are being used. In addition to conventional karyotyping, the latter two techniques revealed a higher frequency of aberrations. Still, failures or false negative results should be taken into account. Recent studies have focused on the potential of gene expression profiling (GEP) to classify acute leukemias. To study the clinical value of classification by GEP, we first used a double-loop cross validation (CV) to avoid over-fitting of GEP data and, subsequently, addressed whether the identified GEP was suitable to classify pediatric AML cases in a second independent group of cases. Affymetrix Human Genome U133 plus 2.0 microarrays were used to generate gene expression profiles of 257 children with AML, with high blast counts, if necessary, after enrichment (~80% or more) and good quality RNA. Probe set intensities were normalized using the variance-stabilizing normalization (VSN) implemented in R (version 2.2.0). The patient group was divided into a test cohort (n=170) and an independent validation cohort (n=87). The test cohort was used to construct the classifier using two levels of CV: the minimum number of predictive genes was estimated using a 10-fold CV on random subsets of about 113 (~2/3 of total) patients; the accuracy of the obtained classifier is estimated on the remaining 57 (~1/3) patients. Candidate genes to represent the GEP in the classifier were those genes that discriminated AML subtypes according to an empirical Bayes linear regression model (Bioconductor package: Limma). To construct a reliable classifier it was sufficient to use 75 probe sets, representing the top 15 discriminating probe sets for MLL-gene rearranged AML, t(8;21), inv(16), t(15;17) and t(7;12). These subtypes represented ~50% of the included patients. The remaining patients either had normal cytogenetics, random aberrations or no data available (cytogenetic failure). Due to the heterogeneity of these remaining groups discriminative probe sets were not found. This classifier could reliably predict the 5 subtypes with a median accuracy of 93%. Validation of the classifier on the independent cohort confirmed that the sensitivity and accuracy was more than 99%. No gene expression signatures could be found for the molecular aberrations NPM1, CEBPa, MLL-PTD, FLT3, C-KIT, RAS or PTPN1, possibly due to the small number of cases. However, specific gene expression signatures were found for FLT3-ITD within the subset of cases with t(15;17) or normal cytogenetics. Importantly, a high expression of HOXB-cluster related genes was found in cases with FLT3-ITD and normal cytogenetics. In conclusion, GEP can correctly predict several important cytogenetic subtypes of pediatric AML, including cases that are currently classified using different cytogenetic techniques and cases with failed cytogenetic analysis. Prospective studies are needed to validate the use of GEP in the classification of pediatric AML, especially to provide information on its utility in clinical practice. Increasing numbers in rare subtypes may result in the discovery of genes discriminative for them, and may foster GEP as a new diagnostic tool.

Description: Objectives: A decade after being licensed for treatment of CML in minors, the TKI imatinib (IMA) is well known for it's inhibitory off-target effects on activity and proliferative capacity of osteoclasts and osteoblasts resulting in impaired bone remodeling (Vandyke K et al 2010 Blood 115:766; Tauer JT et al Blood 2011:118). This causes longitudinal growth retardation in not outgrown individuals (Millot F et al 2009 Blood 114:863; Shima H et al 2011 Pediatrics 159:676; Bansal D et al 2012 Ped Blood Cancer 59:481) which can be aggravated by a disrupted growth hormone:IGF-I axis as a possible additional off-target effect exerted by TKI treatment (Ulmer A et al 2013 Klin Padiatr 225:120; Bansal D et al 2012 Ped Blood Cancer 59:481). Starting a pediatric trial in the year 2006 which recruits approx. 15 pediatric patients (pts) with CML annually, we investigated to what extend growth is impaired depending on sex, age, and pubertal stage at start of IMA treatment in a pediatric cohort. Methods: 102 pts (54 male / 48 female; median age 12 years, range: 1-18 years) at diagnosis of CML receiving IMA as upfront treatment were enrolled retrospectively in this analysis from centers in Germany and participating countries during 02/2006 to 06/2014. Height standard deviation scores (SDS) were derived from WHO-AnthroPlus, version 1.04 software, a global growth-monitoring tool providing normal range values for the age cohorts from birth till 19 years. 81 out of 102 pts fulfilled the criteria for continuous assessment of growth scheduled at three months intervals during IMA exposure. 21 pts were analyzed at intervals ≠ 3 month. Pts excluded comprised individuals shifted to a 2nd generation TKI, or cumulative interruptions of drug intake exceeding 4 weeks, or pts undergoing stem cell transplantation. Results: The mean and median duration of IMA exposure was 12 months and 9 months, respectively (range: 0–98 month). 27/102 pts (13 male, 14 female) were prepubertal (age: 14 years; 18 male, 11 female). In comparison to mean SDS at diagnosis a mean decrease in height of 0.48 SDS per year was observed in the total cohort during the first three years of treatment, being more pronounced in prepubertal pts. In pts diagnosed shortly before or at puberty a mean reduction of 0.75 SDS per year during the first three years were observed. Older teenagers revealed no change in body height z-score during TKI treatment compared to height z-score at diagnosis. Discussion: Growth retardation is a significant adverse effect of IMA in children with CML affecting predominantly prepubertal children. Possible medical interventions still need to be investigated. Acknowledgment: Supported by grant DFG SU122-3/1 to MS. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1624 Poster Board I-650 Activating type I mutations provide cells with proliferative and survival advantages. Together with type II abnormalities, which cause a differentiation arrest and an increase in self-renewal properties, they cooperate to cause acute myeloid leukemia (AML). Currently, many novel drugs have been developed to specifically target these mutations and the mutations are also used as marker of malignant cells in MRD detection. It is thus of importance to accurately determine the mutation status of AML samples. Our previous study showed frequent shifts in FLT3/ITD mutations between initial and relapse samples. Losing an FLT3/ITD mutation at relapse was associated with prolonged time to relapse, whereas acquiring an FLT3/ITD at relapse was associated with a shorter time to relapse (Cloos et al. Leukemia, 2006). We now extended the mutation analysis of paired initial and relapsed pediatric samples to study the stability of type I and II mutations including RAS oncogenes (e.g. N-RAS, K-RAS), receptor tyrosine kinases (FLT3 and KIT), WT1, NPM1, PTPN11 and CEBPá genes. Samples were analyzed of 70 pediatric AML patients who were treated with protocols of the BFM-AML Study Group or Dutch Childhood Oncology Group between 1992 and 2004. Using capillary gel electrophoresis based fragment analysis, we analyzed the patient samples for insertions/deletions in exons 14, 15 and 20 of FLT3, exon 11 of KIT, exon 12 of NPM1 and 3 hotspots in the CEBPá gene. FRET based melting curve analysis or high resolution melting curve analysis were used to detect point mutations in exons 8, 9 and 17 of KIT, exons 3 and 13 of PTPN11, codon 12/13 and 61 mutations of N-RAS and codon 12/13 mutations of K-RAS. Exons 7,8,9 and 10 of WT1 were screened using multiplex ligation probe amplification. Instabilities were found in 25 out of 70 (36%) patients (Table 1). In none of the cases a mutation at diagnosis predicted time to relapse. However, when patients were stratified according to the presence of a type I/II mutation at relapse, independent of its presence at diagnosis, patients who had a FLT3/ITD or RAS mutation at relapse, experienced a significantly shorter mean time to relapse than patients without a mutation at relapse (Table 2). One possible explanation for the observed shifts in type I/II aberrations is the expansion at relapse of minor leukemic sub-clones, present, but not detected at diagnosis. Of a patient of whom we had enough cryopreserved cells from diagnosis samples available to FACSsort small amounts of cells (