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Correction: Cancer Res 2008;68:2557-60 from Role of the Aggresome Pathway in Cancer: Targeting Histone Deacetylase 6–Dependent Protein Degradation

Misfolded or aggregated proteins have two fates: they are either refolded with the help of chaperones or degraded by the proteasome. Cells also have an alternative pathway that involves intracellular “storage bins” for misfolded intracellular proteins known as aggresomes. Aggresomes recruit motor proteins that transport misfolded or aggregated proteins to chaperones and proteasomes for subsequent destruction. There is emerging evidence that inhibiting the aggresome pathway leads to accumulation of misfolded proteins and apoptosis in tumor cells through autophagy. We discuss the role of aggresomes in cancer and the potential to target this pathway for therapy. [Cancer Res 2008;68(8):2557–60]

Background: Panobinostat demonstrates activity against pediatric cancers in vitro. A phase I trial in children with refractory hematologic malignancies was conducted. Study design: The trial evalua...

Genome-wide association studies of childhood acute lymphoblastic leukemia (ALL) have identified regions of association at PIP4K2A and upstream of BMI1 at chromosome 10p12.31-12.2. The contribution of both loci to ALL risk and underlying functional variants remain to be elucidated. We carried out single nucleotide polymorphism (SNP) imputation across chromosome 10p12.31-12.2 in Latino and non-Latino white ALL cases and controls from two independent California childhood leukemia studies, and additional Genetic Epidemiology Research on Aging study controls. Ethnicity-stratified association analyses were performed using logistic regression, with meta-analysis including 3,133 cases (1,949 Latino, 1,184 non-Latino white) and 12,135 controls (8,584 Latino, 3,551 non-Latino white). SNP associations were identified at both BMI1 and PIP4K2A. After adjusting for the lead PIP4K2A SNP, genome-wide significant associations remained at BMI1, and vice-versa (pmeta < 10-10 ), supporting independent effects. Lead SNPs differed by ethnicity at both peaks. We sought functional variants in tight linkage disequilibrium with both the lead Latino SNP among Admixed Americans and lead non-Latino white SNP among Europeans. This pinpointed rs11591377 (pmeta = 2.1 x 10-10 ) upstream of BMI1, residing within a hematopoietic stem cell enhancer of BMI1, and which showed significant preferential binding of the risk allele to MYBL2 (p = 1.73 x 10-5 ) and p300 (p = 1.55 x 10-3 ) transcription factors using binomial tests on ChIP-Seq data from a SNP heterozygote. At PIP4K2A, we identified rs4748812 (pmeta = 1.3 x 10-15 ), which alters a RUNX1 binding motif and demonstrated chromosomal looping to the PIP4K2A promoter. Fine-mapping chromosome 10p12 in a multi-ethnic ALL GWAS confirmed independent associations and identified putative functional variants upstream of BMI1 and at PIP4K2A.

Chronic myeloid leukemia (CML) accounts for 2-9% of leukemias in children and adolescents, and occurs with much greater frequency in adults. Compared to adults, children with CML tend to present with higher white blood cell counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML are due to unique molecular characteristics that differ from adult CML. To test this hypothesis, we compared the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy age-matched CD34+ cells. CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric healthy (n=10), and adult healthy (n=10) bone marrow samples. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes. Four comparisons were performed: (1) pediatric CML vs pediatric healthy, (2) adult CML vs adult healthy, (3) pediatric CML vs adult CML, and (4) pediatric healthy vs adult healthy. A False Discovery Rate of ≤ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes (DEGs) in each comparison. To identify potentially unique pathways based on DEG, pathway over-representation was calculated with either goana from the limma package or clueGO. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients. Median WBC counts were 273,000 and 143,000 in pediatric and adult patients respectively. A total of 1352 genes were differentially expressed in either adult or pediatric CML CD34+ cells compared to healthy CD34+ cells, 174 of which were expressed similarly in pediatric and adult CML CD34+ cells (54 up- and 120 down-regulated). There were 746 differentially expressed genes (325 up- and 421 down-regulated) in adult CML CD34+ cells compared to adult healthy CD34+ cells, and 432 differentially expressed genes (156 up- and 276 down-regulated) in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. In direct comparison of pediatric and adult CML CD34+ cells, 446 genes (270 up and 176 down) were dysregulated in pediatric CML CD34+ cells. Pathway analysis showed that Rho signaling pathway was downregulated in pediatric CML CD34+ cells and several genes in Rho pathway were uniquely dysregulated. ARHGAP27 and VAV2 were significantly upregulated in adult CML CD34+ cells by 3.7-fold (p=0.0453) and 11-fold (p=0.0072), respectively, compared to pediatric CML CD34+ cells. In addition, several genes involved in the NADPH oxidase pathway, one of the best-characterized Rho GTPase-regulated systems, were differently expressed in CML. NCF1, CYBB, and S100A8 were significantly upregulated in adult CML CD34+ cells by 4-fold (p=0.0045), 3.26-fold (p These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the molecular biology of CML across the ages will provide new insights into the pathogenesis of pediatric CML and potentially inform future treatment decisions. Disclosures Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria. Hijiya: Novartis: Consultancy; Stemline Therapeutics: Consultancy.

Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10-15% of pediatric myeloid leukemias and 2-9% of all pediatric leukemias. There are several unique characteristics of CML diagnosed in children, adolescents, and young adults, compared to adults. They present with higher white blood counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that differ from adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells. Methods CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric normal (n=10) and adult normal (n=10) bone marrow samples. Total RNA was isolated from cells, and cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values (TMM) from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes (LogFC). We performed three comparisons: (1) Pediatric CML vs Normal, (2) Adult CML vs Normal, and (3) Pediatric CML vs Adult CML. A False Discovery Rate (FDR) of £ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes in each comparison. Over-representation analysis was used to identify potentially unique pathways based on differentially expressed genes. Clinical and demographic features at diagnosis were extracted for pediatric and adult CML patients and compared using Fisher's exact test (categorical variables) or Wilcoxon rank sum test (continuous variables). Results Pediatric patients were diagnosed with CML at a median of 11 years (interquartile range (IQR): 10-14) compared to 54 years (IQR: 33-62) for adult patients. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients, whereas the white blood cell count and phase at diagnosis did not differ. We found 606 genes (210 up- and 396 down-regulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric normal controls. Interestingly, transcriptional regulators involved in blood cell differentiation including GATA1, TAL1, and KLF1 were differentially enriched in pediatric CML. In comparing adult CML patients to normal adult CD34+ cells, we found 920 genes (379 up- and 541 down-regulated) differentially expressed. Among all dysregulated genes we identified (1352 genes), 174 genes (54 up- and 120-down-regulated) overlapped when comparing pediatric and adult CML patients. Significantly enriched pathways in both adult and pediatric CML cells included PI3K/AKT signaling, MAPK signaling, and Notch/Wnt signaling, which have been previously reported. We found 437 unique genes that were dysregulated only in pediatric CML (270 up- and 167 down-regulated). Notch/Wnt signaling and Rho signaling pathways were significantly enriched. DLC1, a tumor suppressor gene that encodes a RhoGTPase-activating protein, has been known to be downregulated in solid tumors and hematologic malignancies. Interestingly, our data showed that DLC1 is significantly upregulated by 3-fold (p=0.0238) in pediatric CML, but not adult CML CD34+ cells. In addition, we observed that ABR, an inducer of C/EBPa that encodes an activator of RhoGEF and GTPase, was significantly downregulated by 2-fold (p=0.0119) in pediatric but not in adult CML CD34+ cells. Conclusion These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the particular biology of pediatric CML might impact the treatment of those patients in the future. Disclosures Gotlib: Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: co-chair of the Study Steering Committee and Research Funding; Blueprint Medicines Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair of the Response Adjudication Committee and Research Funding, Research Funding.

Previous studies on maternal nutrition and childhood leukaemia risk have focused on the role of specific nutrients such as folate and have not considered broader measures of diet quality, which may better capture intake of diverse nutrients known to impact fetal development. We examined the relationship between maternal diet quality before pregnancy, as summarised by a diet quality index, and risk of childhood acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML) in a case–control study in California. Dietary intake in the year before pregnancy was assessed using FFQ in 681 ALL cases, 103 AML cases and 1076 matched controls. Conditional logistic regression was used to estimate OR and 95 % CI for diet quality continuous score and quartiles (Q1–Q4). Higher maternal diet quality score was associated with reduced risk of ALL (OR 0·66; 95 % CI 0·47, 0·93 for Q4v.Q1) and possibly AML (OR 0·42; 95 % CI 0·15, 1·15 for Q4v.Q1). No single index component appeared to account for the association. The association of maternal diet quality with risk of ALL was stronger in children diagnosed under the age of 5 years and in children of women who did not report using vitamin supplements before pregnancy. These findings suggest that the joint effects of many dietary components may be important in influencing childhood leukaemia risk.

Obesity is associated with poorer event-free survival (EFS) in pediatric acute lymphoblastic leukemia (ALL). Persistent minimal residual disease (MRD) in the bone marrow as measured by multidimensional flow cytometry (MDF) is a key early prognostic indicator and is strongly associated with EFS. We therefore hypothesized that obesity during induction would be associated with positive end-of-induction MRD (≥0.01%). We analyzed MDF of end-induction bone marrow samples from a historical cohort of 198 children newly diagnosed with B-precursor ALL (BP-ALL) and treated with Children's Oncology Group induction regimens. We assessed the influence of body mass index on risk for positive end-induction MRD in the bone marrow. In our cohort of BP-ALL, 30 children (15.2%) were overweight and 41 (20.7%) were obese at diagnosis. Independent of established predictors of treatment response, obesity during induction was associated with significantly greater risk for persistent MRD (odds ratio, 2.57; 95% confidence interval, 1.19 to 5.54; P = .016). Obesity and overweight were associated with poorer EFS irrespective of end-induction MRD (P = .012). Obese children with newly diagnosed BP-ALL are at increased risk for positive end-induction MRD and poorer EFS.

Purpose The Children’s Oncology Group AAML0631 trial for newly diagnosed pediatric acute promyelocytic leukemia (APL) was a phase III historically controlled trial to determine the survival of patients receiving arsenic trioxide (ATO) consolidation and reduced doses of anthracyclines. Patients and Methods Patients age 2 to 21 years with de novo APL confirmed by PML-RARα polymerase chain reaction were stratified as standard risk (SR) or high risk (HR) on the basis of diagnostic WBC count. All patients received all-trans retinoic acid (ATRA) during induction, each consolidation course, and maintenance. All patients received two cycles of ATO therapy during consolidation 1, an additional two (SR) or three (HR) consolidation courses that included high-dose cytarabine and anthracycline, and maintenance therapy comprising ATRA, oral methotrexate, and mercaptopurine. Results One hundred one patients (66 SR and 35 HR) were evaluable for outcome. The 3-year overall survival was 94%, and event-free survival (EFS) was 91%. For SR and HR patients with APL, the overall survival was 98% versus 86% ( P = .003), and EFS was 95% versus 83% ( P = .03), respectively. The EFS for SR patients in AAML0631 was noninferior to that of patients in the AIDA 0493 historical control, which used a significantly higher anthracycline dose and did not include ATO consolidation. Relapse risk for patients in AAML0631 from end consolidation 1 (after ATO treatment) was only 4% at 3 years and did not differ significantly between SR and HR patients. Conclusion ATO consolidation cycles were well tolerated in pediatric patients with APL and allowed significant reduction in cumulative anthracycline doses while maintaining excellent survival and a low relapse risk for both SR and HR patients with APL.

Background: Tobacco smoke contains carcinogens known to damage somatic and germ cells. We investigated the effect of tobacco smoke on the risk of childhood acute lymphoblastic leukemia (ALL) and myeloid leukemia (AML), especially subtypes of prenatal origin such as ALL with translocation t(12;21) or high-hyperdiploidy (51–67 chromosomes). Methods: We collected information on exposures to tobacco smoking before conception, during pregnancy, and after birth in 767 ALL cases, 135 AML cases, and 1,139 controls (1996–2008). Among cases, chromosome translocations, deletions, or aneuploidy were identified by conventional karyotype and fluorescence in situ hybridization. Results: Multivariable regression analyses for ALL and AML overall showed no definite evidence of associations with self-reported (yes/no) parental prenatal active smoking and child's passive smoking. However, children with history of paternal prenatal smoking combined with postnatal passive smoking had a 1.5-fold increased risk of ALL [95% confidence interval (CI), 1.01–2.23], compared to those without smoking history (ORs for pre- or postnatal smoking only were close to one). This joint effect was seen for B-cell precursor ALL with t(12;21) (OR = 2.08; 95% CI, 1.04–4.16), but not high hyperdiploid B-cell ALL. Similarly, child's passive smoking was associated with an elevated risk of AML with chromosome structural changes (OR = 2.76; 95% CI, 1.01–7.58), but not aneuploidy. Conclusions: Our data suggest that exposure to tobacco smoking was associated with increased risks of childhood ALL and AML; and risks varied by timing of exposure (before and/or after birth) and cytogenetic subtype, based on imprecise estimates. Impact: Parents should limit exposures to tobacco smoke before and after the child's birth. Cancer Epidemiol Biomarkers Prev; 22(9); 1600–11. ©2013 AACR.

Background Children with relapsed acute lymphoblastic leukemia (ALL) typically receive vincristine-prednisone-L-asparaginase-doxorubicin reinduction chemotherapy similar to contemporary induction regimens. However, up to 20% of patients are unable to receive vincristine-prednisone-L-asparaginase-doxorubicin secondary to asparaginase intolerance. We report our experience with a promising reinduction regimen for children with relapsed ALL who are unable to receive asparaginase. Patients and methods This is a single institution, retrospective review of the safety and activity of bortezomib, dexamethasone, mitoxantrone, and vinorelbine (BDMV) in patients with relapsed ALL. Complete remission and adverse events after reinduction were study endpoints. Patients treated with BDMV between 2012 and 2015 were identified. Response and adverse events (AEs) were assessed by review of medical records. Standard response criteria were used and AEs were graded based on NCI CTCAEv4.0. Results Seven of 10 patients achieved complete remission after 1 cycle of BDMV, with 4 achieving minimal residual disease negativity. The most common ≥grade 3 nonhematological toxicities were infection (91%), gastrointestinal (45%), metabolic (45%), and cardiovascular (9%). Conclusions BDMV is an active reinduction regimen for children with relapsed ALL who cannot receive asparaginase. The toxicity profile is as expected for this patient population. Further prospective clinical trials are warranted to evaluate the safety and efficacy of BDMV.

Background: Although the accumulation of white adipose tissue (WAT) is a risk factor for disease, brown adipose tissue (BAT) has been suggested to have a protective role against obesity. Objective: We studied whether changes in BAT were related to changes in the amounts of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in children treated for malignancy. Design: We examined the effect of BAT activity on weight, SAT, and VAT in 32 pediatric patients with cancer whose positron emission tomography–computed tomography (PET-CT) scans at diagnosis showed no BATactivity. Changes in weight, SAT, and VAT from diagnosis to remission for children with metabolically active BATat disease-free follow-up (BAT+) were compared with those in children without visualized BAT when free of disease (BAT2). Results: Follow-up PET-CT studies (4.7 6 2.4 mo later) after successful treatment of the cancer showed BAT+ in 19 patients but no active BAT (BAT2) in 13 patients. BAT+ patients, in comparison with BAT2 patients, gained significantly less weight (3.3 6 6.6% compared with 11.0 6 11.6%; P = 0.02) and had significantly less SAT (18.2 6 26.5% compared with 67.4 6 71.7%; P = 0.01) and VAT (22.6 6 33.5% compared with 131.6 6 171.8%; P = 0.01) during treatment. Multiple regression analysis indicated that the inverse relations between BAT activation and measures of weight, SAT, and VAT persisted even after age, glucocorticoid treatment, and the season when the PET-CT scans were obtained were accounted for. Conclusion: The activation of BAT in pediatric patients undergoing treatment of malignancy is associated with significantly less adipose accumulation. This trial was registered at clinicaltrials.gov as NCT01517581. Am J Clin Nutr 2012;95:1144–9.

Purpose To determine the efficacy of imatinib in children with newly diagnosed chronic phase (CP) chronic myelogenous leukemia (CML). Methods This was an open label, multi-center phase II clinical trial. Courses were defined as consecutive 28-day intervals. Oral imatinib was administered daily at 340 mg/m2 without interruption in the absence of toxicity. Results Fifty-one children received 978 28-day courses of imatinib. The most common toxicities encountered were hematologic. Forty-one patients (80%) achieved a complete hematologic response by the end of course 2. Nineteen children (38%) obtained a complete cytogenetic response (CCyR) at the end of course 3. Overall, 72% achieved CCyR at a median time of 5.6 months. The rate of complete molecular response (>3 log reduction) was 27%. Progression-free and overall survival at 3 years were 72% ± 6.4% and 92% ± 3.9%, respectively. Conclusions Daily oral imatinib at a dose of 340 mg/m2 is well tolerated in children. In addition, imatinib therapy is effective in inducing a high percent of hematologic, cytogenetic and molecular responses, comparable to adults with CML. (This study was registered at ClinicalTrials.gov under identifier NCT00030394.). Pediatr Blood Cancer 2011;57:56–62. © 2011 Wiley-Liss, Inc.

Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10 to 15% of children with myeloid leukemia and 2 to 9% of all pediatric leukemias. Prior to the discovery of tyrosine kinase inhibitors (TKI) such as imatinib, stem cell transplantation was the only curative treatment for both adults and children with CML. However, due to the small numbers of patients, standardized treatment approaches for pediatric CML have not been established. There are several unique characteristics of CML diagnosed in children and adolescents, and young adults (AYA; 16-29 years), compared to adults. Children and AYA with CML present with a higher white blood count and have larger spleens, higher peripheral blast counts, and lower hemoglobin levels, suggesting that the biology of pediatric CML is different than adult CML. In addition, potential side effects of TKIs unique to pediatric CML patients include impaired bone growth, fertility and immune function, however none have been extensively studied. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that are absent in adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells. Methods CD34+ cells were isolated from pediatric CML (n=7), adult CML (n=8), pediatric normal (n=2) and adult normal (n=3) bone marrow samples. Total RNA was isolated from cells, and then cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. We aligned reads using the HISAT2 alignment software, and mapped to genes with HT-Seq. We removed genes that had zero reads across all the samples, resulting in a set of 4,696 genes that were detected in one or more samples. In case of technical replicates, we used mean of replicates. We performed three differential expression comparisons with edgeR: (1) Pediatric CML vs Adult CML, (2) Adult CML vs Adult Normal, and (3) Pediatric CML vs Pediatric Normal. We used a False Discovery Rate (FDR) of £ 20% and absolute log2 fold-change ³ 1 for selecting differentially expressed genes in each comparison. We used Fisher's exact test to identify significant KEGG pathways for the differentially expressed genes in each comparison. Results Pediatric CML vs Adult CML We found 24 differentially expressed genes (15 over- and 9 under-expressed). Though no pathway was found to be significant at the false discovery rate (FDR) £ 20%, we identified a number of sub-pathways that are relevant. For example, the Chemokine Signaling pathway shows at the top of the list (ordered by raw p-value) because of two genes, XCR1 and HCK, associated with VEGF and MAPK pathways involved in cell proliferation, angiogenesis, DNA repair, and cancer pathogenesis. Adult CML vs Adult Normal We found 60 genes (30 over- and 30 under-expressed) differentially expressed when comparing adult CML patients to normal adults. Ten genes overlapped with 24 genes we identified when comparing pediatric and adult CML patients. We found 11 pathways as significant at FDR £ 10%. Multiple pathways, including Cell adhesion, allograft rejection, Graft versus Host Disease, and Type I diabetes pathways, showed downregulation of MHC, with subsequent downstream reduction in expression of apoptosis-related genes. The IL-17 pathway makes sense, as MAPK, well-known to be associated with various cancers, is down-regulated. Lastly, in the NK pathway the gene DAP12 is up-regulated. This gene is known as a tyrosine kinase binding protein, and although tyrosine kinase inhibitors are the standard treatment for CML, the role of DAP12 in relation to leukemia has not yet been described. Pediatric CML vs Pediatric Normal We found 509 genes (350 over- and 159 under-expressed) differentially expressed in pediatric CML patients compared to normal. Interestingly, transcriptional regulators are differentially enriched in the hematopoietic stem cell differentiation function group including GATA1, GATA2, KLF1 and KLF2. RFC is down-regulated. RFC is a mismatch repair gene known to be involved in colorectal cancer. Many of the significant pathways are involved in glucose and fatty acid metabolism. Our pilot study identified novel molecular features of pediatric CML bone marrow stem cells, providing new insights into the novel biomarkers and pathogenesis of pediatric CML. Disclosures Gotlib: Blueprint Medicines: Consultancy, Honoraria, Research Funding; Promedior: Research Funding; Deciphera: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Kartos: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Duplication 22q11.2 syndrome is the result of a microduplication of the same chromosomal region that is deleted in DiGeorge and Velocardiofacial syndromes. We describe a patient with dysmorphic features who was diagnosed with pre-B acute lymphoblastic leukemia, and developed cystinuria and pancreatitis during treatment. Duplication 22q11.2 has not been previously described in association with hematologic abnormalities. Chromosomal microarray technology was used to diagnose duplication 22q11.2 syndrome. In this era of advanced genomics, this technology has become an important method for helping to determine the molecular basis of diseases, best treatments and ultimately patient outcomes.

Misfolded or unfolded proteins are often refolded with the help of chaperones or degraded by the 26S proteasome. An alternative fate of these proteins is the aggresome pathway. The microtubule-organizing center (MTOC) transports unfolded proteins to lysosomes and are degraded through autophagy. Histone deacetylase 6 (HDAC6) deacetylates alpha-tubulin, which is thought to be a component of the MTOC. Recently, two small molecule inhibitors of the aggresome pathway and HDAC6 have been described. One inhibitor, tubacin, prevents deacetylation of alpha-tubulin and produces accumulation of polyubiquitinated proteins and apoptosis. Tubacin acts synergistically with the proteasome inhibitor, bortezomib, to induce cytotoxicity in one type of hematologic malignancy, multiple myeloma. The other, LBH589, is a pan HDAC inhibitor and hydroxamic acid derivative that induces apoptosis of multiple myeloma cells resistant to conventional therapies. In this review, we summarize recent reports on targeting the aggresome pathway and HDAC6 in hematologic malignancies.

Misfolded or aggregated proteins have two fates: they are either refolded with the help of chaperones or degraded by the proteasome. Cells also have an alternative pathway that involves intracellular “storage bins” for misfolded intracellular proteins known as aggresomes. Aggresomes recruit motor proteins that transport misfolded or aggregated proteins to chaperones and proteasomes for subsequent destruction. There is emerging evidence that inhibiting the aggresome pathway leads to accumulation of misfolded proteins and apoptosis in tumor cells through autophagy. We discuss the role of aggresomes in cancer and the potential to target this pathway for therapy. [Cancer Res 2008;68(8):2557–60]

Background To determine the response rate and toxicity of docetaxel when administered as a 60 mg/m2 dose by 1 hr intravenous (IV) infusion weekly × 3 weeks in children with relapsed acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). Procedure Patients who were under the age of 22-year-old at the time of the original ALL or AML diagnosis and in a second relapse were accrued from August 2002 to May 2005 for this Children's Oncology Group (COG) phase 2 study (ADVL0023). Ten patients with ALL and two patients with AML were enrolled. Results There were no complete or partial responses observed. The most common grade 3 or 4 toxicities were hematologic followed by febrile neutropenia. One patient developed a dose limiting elevation in serum bilirubin, but no other significant hepatotoxicity was observed. Conclusions Docetaxel was not effective therapy for children with relapsed ALL at the dose and schedule tested. Pediatr Blood Cancer 2008;50:533–536. © 2007 Wiley-Liss, Inc.

Genome-wide association studies (GWAS) have identified SNPs in six genes that are associated with childhood acute lymphoblastic leukemia (ALL). A lead SNP was found to occur on chromosome 9p21.3, a region that is deleted in 30% of childhood ALLs, suggesting the presence of causal polymorphisms linked to ALL risk. We used SNP genotyping and imputation-based fine-mapping of a multiethnic ALL case–control population (Ncases = 1,464, Ncontrols = 3,279) to identify variants of large effect within 9p21.3. We identified a CDKN2A missense variant (rs3731249) with 2% allele frequency in controls that confers three-fold increased risk of ALL in children of European ancestry (OR, 2.99; P = 1.51 × 10−9) and Hispanic children (OR, 2.77; P = 3.78 × 10−4). Moreover, of 17 patients whose tumors displayed allelic imbalance at CDKN2A, 14 preferentially retained the risk allele and lost the protective allele (PBinomial = 0.006), suggesting that the risk allele provides a selective advantage during tumor growth. Notably, the CDKN2A variant was not significantly associated with melanoma, glioblastoma, or pancreatic cancer risk, implying that this polymorphism specifically confers ALL risk but not general cancer risk. Taken together, our findings demonstrate that coding polymorphisms of large effect can underlie GWAS “hits” and that inherited polymorphisms may undergo directional selection during clonal expansion of tumors. Cancer Res; 75(22); 4884–94. ©2015 AACR.

Background Intensifying treatment for pediatric acute myeloid leukemia (AML) has improved survival, with infections now being a leading cause of morbidity. Because quinolone prophylaxis is effective in adults with AML and in transplant populations, ciprofloxacin prophylaxis (CPx) was introduced as the standard for pediatric AML. We report here the impact of CPx in this population. Methods Prevalence of fever and neutropenia, frequency and pathogen spectrum of infections, antibiotic use, supportive care and mortality before and after implementation of CPx were retrospectively compared in children with AML. Results The cohort included 35 patients with de novo and 10 with relapsed AML, who together underwent 153 chemotherapy courses. Fever and neutropenia resulting in the use of empiric antibiotics occurred in 90% of chemotherapy courses (137/153); this was associated with proven bacteremia in 26%. The use of CPx did not change the incidence of febrile or infectious episodes, number of days of fever or antibiotic treatment or mortality. CPx was associated with a significant decrease in infections caused by Gram-negative rods (13.4% vs 4.7%) but a concomitant significant increase in bacteremia caused by viridans streptococci (12% vs 28%), resulting in no significant overall difference in the incidence of bacteremia between the 2 groups (35.9% vs 31.5%). Conclusions CPx neither alter the incidence of overall bacteremia nor change the pattern of fever or use of supportive care. Our experience supports further investigation into the use of extended-spectrum quinolone prophylaxis during therapy for pediatric AML.

BackgroundGenome-wide association studies focusing on European-ancestry populations have identified ALL risk loci on IKZF1, ARID5B, and CEBPE. To capture the impacts of these genes on ALL risk in the California Hispanic population, we comprehensively assessed the variation within the genes and further assessed the joint effects between the genetic variation and surrogates for early-life infections (the presence of older siblings, daycare attendance, and ear infections).MethodsGenotypic data for 323 Hispanic ALL cases and 454 controls from the California Childhood Leukemia Study were generated using Illumina OmniExpress v1 platform. Logistic regression assuming a log-additive model estimated odds ratios (OR) associated with each SNP, adjusted for age, sex, and the first five principal components. In addition, we examined potential interactions between six ALL risk alleles and surrogates for early-life infections using logistic regression models that included an interaction term.ResultsSignificant associations between genotypes at IKZF1, ARID5B, and CEBPE and ALL risk were identified: rs7780012, OR 0.50, 95% confidence interval (CI) 0.35-0.71 (p = 0.004); rs7089424, OR 2.12, 95% CI 1.70-2.65 (p = 1.16 × 10(-9)); rs4982731, OR 1.69, 95% CI 1.37-2.08 (p = 2.35 × 10(-6)), respectively. Evidence for multiplicative interactions between genetic variants and surrogates for early-life infections with ALL risk was not observed.ConclusionsConsistent with findings in non-Hispanic White population, our study showed that variants within IKZF1, ARID5B, and CEBPE were associated with increased ALL risk, and the effects for ARID5B and CEBPE were most prominent in the high-hyperdiploid ALL subtype in the California Hispanic population. Results implicate the ARID5B, CEBPE, and IKZF1 genes in the pathogenesis of childhood ALL.

Background Survival for acute promyelocytic leukemia (APL) has improved dramatically in recent decades through use of targeted therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Early deaths, principally due to complications of APL coagulopathy, remain a significant barrier to cure. White blood cell (WBC) count at diagnosis is a well validated risk marker for early death and relapse. While APL is characterized by t(15;17), mutations of the FLT3 gene are prevalent. Prior reports in both adult and pediatric APL have demonstrated variable impact of FLT3 mutation status on outcomes. In a pediatric cohort from the Intergroup Study CALGB 9710, our group previously found an association of FLT3 mutations with early death. The more recent Children's Oncology Group (COG) APL trial AAML0631 included addition of two courses of arsenic trioxide (ATO) during consolidation. Relapse rate on this study was low, but survival, particularly for the high risk patients, was impacted by early deaths. In a prior analysis we reported that a significant proportion of patients on AAML0631 experienced clinically significant bleeding and clotting events. Here we report our analysis of the prevalence of FLT3 mutations and their association with outcomes for pediatric APL patients treated on the AAML0631 study. Methods Diagnostic bone marrow samples were collected from consenting patients with newly diagnosed APL enrolling onto COG AAML0631. FLT3 mutation testing of these bone marrow samples included evaluation for internal tandem duplications (FLT3/ITD) of the juxta-membrane domain and point mutations of the activating loop (FLT3/ALM) of the tyrosine kinase domain. FLT3 mutation status was correlated with clinically significant bleeding/clotting, early death during induction and relapse. Significance of correlation was determined using the chi-square test and relapse risk for patients who achieved CR by end of Induction 1 was calculated using cumulative incidence. Eligible patients were age ≥ 2 years and < 22 years and required confirmation of PML-RARA by PCR testing. Risk group was defined by WBC at diagnosis: standard risk (SR) WBC < 10,000 and high risk (HR) WBC ≥ 10,000. Treatment regimen has been previously reported [Kutny et al, ASH Abstract, 2015]. Results There were 101 evaluable patients on AAML0631 including 35 HR APL and 66 SR APL. A total of 94 patients had specimens available for FLT3 mutation testing. Patients with unknown FLT3 status (3 HR and 4 SR) were excluded from the following analyses. FLT3 mutations were present in 43% (N=40) including 35% (N=33) with FLT3/ITD, 4% (N=4) with FLT3/ALM, and 3% (N=3) with both FLT3/ITD and FLT3/ALM. The median WBC was 24,450 for FLT3 mutant APL compared to 7,130 for FLT3 wild type, and the rate of FLT3 mutation was significantly higher in HR APL compared to SR APL (66% vs. 31%, P=0.001). Four patients died during induction (all HR APL with WBC of 16200-173800), but early death did not correlate with FLT3 mutation (2/4 patients had FLT3 mutations). We analyzed association of FLT3 mutations with prevalence of clinically significant (Grade III-IV) bleeding or clotting events during induction. There was no difference in prevalence of coagulopathy events (N= 19) between those with and without FLT3 mutations (18% vs. 22%, P=0.57). While OS and EFS were not significantly different, the relapse rate from end of Consolidation 1 was significantly higher at 6% (N=3) in FLT3 mutant patients versus 0% in FLT3 wild-type (P=0.039) [Figure 1]. Conclusions FLT3 mutations are highly prevalent (43%) in pediatric APL. The COG AAML0631 study represents the second largest group of pediatric APL patients with outcome data treated on a cooperative group trial and to our knowledge the current report is the largest group of pediatric APL patients evaluated for outcome based on FLT3 mutation status. This study failed to show an association of FLT3 mutations with early death or bleeding/clotting events in induction. With ATO consolidation, the relapse rate was low on this trial. However, it is of great interest that the relapse rate following ATO consolidation was significantly higher in FLT3 mutant patients. Evaluation of a larger (international) cohort of pediatric APL patients is warranted to further define of role of FLT3 in relapse risk, but the current results give further support to consideration of integrating FLT3 inhibitors into relapsed/refractory APL treatment. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Results from cooperative group trials demonstrating the effectiveness of arsenic trioxide in the treatment of newly diagnosed APL have thus far been restricted to trials consisting of primarily adult patients. The Children's Oncology Group trial AAML0631 incorporated two cycles of arsenic trioxide (ATO) during consolidation with approximately 40% reduction in anthracycline dosing compared to the Italian AIDA0493 trial which has the largest cohort and best phase III results published for pediatric APL (Testi et al Blood 2005). Eligibility criteria included age ≥ 2 to < 22 years, de novo APL confirmed by PML-RARA PCR and no prior therapy. Diagnostic white blood cell count (WBC) determined risk group as standard risk (SR) for WBC < 10,000 and high risk (HR) for WBC ≥ 10,000. ATRA was given at the pediatric dose of 12.5 mg/m2/dose PO BID on days 1-30 of induction and days 1-14 of each consolidation course and each maintenance cycle. Other therapy was as follows: Induction- idarubicin 12 mg/m2/dose for 3 doses; Consolidation 1- 2 cycles of ATO 0.15 mg/kg/day IV 5 days each week for 5 weeks; Consolidation 2- cytarabine 1,000 mg/m2/dose IV q12 hours days 1-3, mitoxantrone 10 mg/m2/dose IV days 3, 4; Consolidation 3- idarubicin 5 mg/m2/dose IV days 1, 3, 5; Consolidation 4 (HR APL only)- cytarabine 1,000 mg/m2/dose IV q12 hours days 1-3, idarubicin 10 mg/m2/dose IV day 4; Maintenance (12 weeks/cycle for 9 cycles)- 6-mercaptopurine 50 mg/m2/day PO, methotrexate 25 mg/m2 PO once weekly. Intrathecal cytarabine was given once during each consolidation 2-4 and cycle 1 of maintenance. Between 3/2009 and 11/2012, 108 patients enrolled and 101 (66 SR and 35 HR) were evaluable for outcome (4 PML-RARA PCR negative, 3 local consent issues). Hematologic CR after induction (without central review) was 81%. The molecular (RQ-PCR) remission rate for those tested at end of Consolidation was 100%. For all patients, overall survival (OS) was 94% and event free survival (EFS) was 92% at 2 years. Comparing SR to HR, the OS was 98% vs. 86% (p=0.004) and EFS was 97% vs. 83% (p=0.014) (Figure 1). The predetermined statistical plan was to compare the EFS results to AIDA0493 which had 2 year EFS of 91% for SR and 79% for HR APL. Relapse risk on AAML0631 from end consolidation I (following ATO treatment) was 2% at 2 years and did not differ significantly between patients with SR vs. HR APL. Events on this trial included 3 relapses, 1 second malignant neoplasm (squamous cell carcinoma in situ) and 7 deaths (6 HR APL, 1 SR APL) including one death post-relapse. Four deaths during induction (all HR APL) included multiple etiologies, but coagulopathy was a factor in each. There were 2 post-induction on-therapy deaths: SR APL patient with accidental (non-chemotherapy) drug overdose during Consolidation 3 and HR APL patient with gram negative sepsis during Consolidation 2. Three relapses included: combined molecular bone marrow (detected by PCR) and CNS relapse in a SR APL patient during maintenance cycle 7 (24 months post diagnosis), combined hematologic bone marrow and CNS relapse in a HR APL patient during maintenance cycle 6 (23 months post diagnosis), and molecular bone marrow relapse in a HR APL patient after therapy completion (38 months post diagnosis) who later died of stem cell transplant complications. Both patients with molecular relapse had low level PML-RARA transcript detection (under 0.001 NCN) during maintenance beginning 1 and 16 months prior to relapse. No other patients had similar "borderline" results. While 28 patients had a diagnostic LP performed in pre-therapy evaluation, 7 patients met the protocol definition of CNS disease (CNS2 N=4, or CNS3 N=3). The 2 patients with relapse involving the CNS did not have CNS disease at diagnosis. Historically, AIDA0493 represents the largest (N=107) and best published outcomes for a major pediatric phase III trial in APL. The current trial of 101 pediatric patients achieved higher EFS rates in both SR and HR APL through incorporation of ATO while significantly reducing anthracycline doses. The reported CR rate was lower than historical comparisons, but this was likely due to difficulty in assessing differentiating and recovering bone marrow cells after APL induction. The excellent molecular remission rate at end of consolidation and the low relapse rate confirm the effectiveness of this therapy. These results strongly support use of ATO in pediatric patients with newly diagnosed APL. Disclosures Off Label Use: Arsenic Trioxide in pediatric patients with newly diagnosed APL. Gemtuzumab Ozogamicin in Pediatric AML..

Duplication 22q11.2 syndrome is the result of a microduplication of the same chromosomal region that is deleted in DiGeorge and Velocardiofacial syndromes. We describe a patient with dysmorphic features who was diagnosed with pre-B acute lymphoblastic leukemia, and developed cystinuria and pancreatitis during treatment. Duplication 22q11.2 has not been previously described in association with hematologic abnormalities. Chromosomal microarray technology was used to diagnose duplication 22q11.2 syndrome. In this era of advanced genomics, this technology has become an important method for helping to determine the molecular basis of diseases, best treatments and ultimately patient outcomes.

INTRODUCTION Infant acute lymphoblastic leukemia (ALL) is considered a high-risk entity. Patients diagnosed in the first 3 months of life have especially high mortality. By morphology, infant ALL is classified as a lymphoid lineage leukemia; however, its physiologic behavior has brought many to consider it a pathologic hybrid between lymphoid leukemia and myeloid leukemias. As such, standard of care currently employs the use of chemotherapeutic agents used commonly in ALL protocols and agents typically reserved for the treatment of myelogenous lineage leukemias. The role of hematopoietic stem cell transplantation and graft-versus-leukemia effect in these patients has not been well studied. CASE PRESENTATION An earlier healthy 9-week-old Hispanic male diagnosed with precursor B-cell lymphoblastic leukemia was treated with protocol P9407 and matched sibling hematopoietic stem cell transplantation. Relapse was noted on posttransplant day +114 with blasts on peripheral blood smear. The sole antigraft-versus-host disease (GVHD) agent, cyclosporine, was discontinued. Blast clearance from the peripheral blood was obtained by posttransplant day +128 with the appearance of skin and liver GVHD at posttransplant day +181. Bone marrow examination on posttransplant day +205 revealed normal marrow with no evidence of leukemic cells. He remains disease free more than 2 years posttransplant. CONCLUSION Traditionally, graft-versus-leukemia effect was thought to contribute therapeutically little to the treatment of ALL by hematopoietic stem cell transplantation (HSCT). The effects of graft-versus-leukemia immunologic phenomenon in our patient with infant acute lymphoblastic leukemia underscore the potential that infant ALL may not be entirely the same biologic entity as standard pediatric ALL and may be more responsive than understood earlier. Therapeutic response and appearance of GVHD after the withdrawal of immunosuppression in this patient provides evidence that graft-versus-leukemia effect may play a role in disease control in infant ALL after HSCT. Patients who relapse after the HSCT may be salvaged with the withdrawal of immunosuppression. This suggests that other immunotherapeutic interventions in the context of relapse may offer potential clinical benefit in this disease.

Purpose To evaluate the effect of obesity (defined as a body mass index > 95th percentile for age and sex at diagnosis) on outcome of pediatric acute lymphoblastic leukemia (ALL). Patients and Methods We retrospectively analyzed data from 4,260 patients with newly diagnosed ALL enrolled from 1988 to 1995 onto five concurrent Children's Cancer Group studies. Results were verified in a second cohort of 1,733 patients enrolled onto a sixth study from 1996 to 2002. Results The 1988 to 1995 cohort included 343 obese and 3,971 nonobese patients. The 5-year event-free survival rate and risk of relapse in obese versus nonobese patients were 72% ± 2.4% v 77% ± 0.6% (P = .02) and 26 ± 2.4 v 20 ± 0.6 (P = .02), respectively. After adjusting for other prognostic variables, obesity's hazard ratios (HRs) of events and relapses were 1.36 (95% CI, 1.04 to 1.77; P = .021) and 1.29 (95% CI, 1.02 to 1.56; P = .04), respectively. The effect of obesity was prominent in the 1,003 patients ≥ 10 years old at diagnosis; in this subset, obesity's adjusted HRs of events and relapses were 1.5 (95% CI, 1.1 to 2.1; P = .009) and 1.5 (95% CI, 1.2 to 2.1; P = .013), respectively. In a second cohort of 1,160 patients ≥ 10 years old, obesity's adjusted HRs of events and relapses were 1.42 (95% CI, 1.03 to 1.96; P = .032) and 1.65 (95% CI, 1.13 to 2.41; P = .009), respectively. The effect of obesity on outcome was unrelated to changes in chemotherapy doses, length of intervals between chemotherapy cycles, or incidence and severity of therapy-related toxicity. Conclusion Obesity at diagnosis independently predicts likelihood of relapse and cure in preteenagers and adolescents with ALL.

Purpose Re-induction outcomes vary for children with acute lymphoblastic leukemia (ALL) and marrow relapse. We explored possible relationships among asparaginase (ASNase) activity levels, asparagine (ASN) depletion, anti-ASNase antibody titers, and response to re-induction therapy in children and adolescents with ALL and an ‘early’ first marrow relapse. Patients and methods After appropriate informed consent, we enrolled children and adolescents 1–21 years old with ALL and first marrow relapse within 12 months of completion of primary therapy. Induction therapy included intramuscular pegylated ASNase on Days 2 and 16. We assessed ASNase activity, anti-ASNase antibody titers against native and pegylated (E. coli) ASNase, and amino acid levels of asparagine (ASN) and glutamine (GLN) on Days 0, 14, and 35 of re-induction. Results Ninety-three patients were at least partially assessable. Among 21 patients with M1 marrow status at Day 35, the median Day 14 ASN level was

Background Anthracyclines have historically been the backbone of chemotherapy regimens used to treat newly diagnosed APL in both children and adults, but large cumulative doses of these drugs are associated with long term cardiotoxicity especially in children. The Children’s Oncology Group designed trial AAML0631 to reduce the cumulative anthracycline dosing in children with APL while incorporating two courses of arsenic trioxide (ATO) during consolidation. The majority of the experience with ATO in clinical trials in newly diagnosed APL has been in adult patients. Here we present data on the tolerability of ATO consolidation in pediatric APL patients along with the results of induction therapy and targeted toxicities during induction and consolidation. Because some patients are still on maintenance therapy, survival outcome is not presented. Methods Eligibility criteria for this non-randomized study included age > 2 years and < 22 years, de novo APL with confirmation of PML-RARA translocation by PCR and no prior APL directed therapy or secondary APL. Patients were stratified by white blood cell count (WBC) at diagnosis into standard risk (SR)- WBC < 10,000 and high risk (HR)- WBC > 10,000. Induction consisted of idarubicin 12 mg/m2/dose for 3 doses and all-trans retinoic acid (ATRA) 12.5 mg/m2/dose (no max dose) PO BID on days 1-30. Consolidation 1 included 2 cycles of ATO 0.15 mg/kg/day (no max dose) IV over 2 hours on Monday through Friday for 5 weeks and ATRA 12.5 mg/m2/dose PO BID on days 1-14. Consolidation 2 included cytarabine 1,000 mg/m2/dose IV every 12 hours for 6 doses on days 1-3, mitoxantrone 10 mg/m2/dose IV on days 3 and 4 and ATRA 12.5 mg/m2/dose PO BID on days 1-14. Consolidation 3 included Idarubicin 5 mg/m2/dose IV on days 1, 3 and 5 and ATRA 12.5 mg/m2/dose PO BID on days 1-14. Consolidation 4 was given to HR patients and included cytarabine 1,000 mg/m2/dose IV every 12 hours for 6 doses on days 1-3, idarubicin 10 mg/m2/dose IV on day 4 and ATRA 12.5 mg/m2/dose PO BID on days 1-14. All patients received 9 cycles of maintenance therapy consisting of 12 week cycles of 6-mercaptopurine 50 mg/m2/day PO, methotrexate 25 mg/m2 once weekly PO and ATRA 12.5 mg/m2/dose PO BID on days 1-14. Intrathecal cytarabine was given once during each consolidation 2-4 and during the first cycle of maintenance. Results Between 3/2009 and 11/2012, 108 patients were enrolled on AAML0631. A total of six patients were found to be ineligible due to being RQ-PCR negative for PML-RARA (n=3) or local issues with consent (n=3). Of the 102 eligible patients, 67 had standard risk APL and 35 had high risk APL. Regarding toxicity during induction, 4 deaths occurred with 3 occurring on day 2 of induction and 1 occurring on day 10 of induction. Patients with HR APL had a significantly increased risk of induction death (4/35 = 11.4%) compared to patients with SR APL (0/67; P=0.012). Deaths during consolidation included one HR patient death due to sepsis during consolidation 2 and one SR patient death due to suicide during consolidation 4. Adverse events (AE) reporting included non-hematologic toxicity grade ≥3 and all grades of cardiac toxicity. Cardiac AEs during the 2 ATO cycles included 18% (n=17/92) of patients experiencing at least 1 grade 1/2 event with the majority (94%, 16/17) being prolongation of the QTc interval. Only 1 patient experienced a grade 3/4 cardiac toxicity during these cycles and there were no cardiac related deaths. Hepatotoxicity during ATO cycles included few patients experiencing at least 1 grade 3/4 event including elevations of ALT (4%, n=4/92), AST (3%, n=3/92) and bilirubin (1%, n=1/92). For grade ≥3 AEs reported the percentage of patients experiencing at least one toxicity was 27% during the two cycles of consolidation 1 which was significantly less than the 81% in induction (P Conclusions We were particularly interested in the toxicity and tolerability of two cycles of ATO in a pediatric population. While cardiac toxicity is a concern with this medication, we found minimal significant cardiac complications. Hepatotoxicity has also been a concern with ATO but we did not find this to be a significant problem in our group of patients. In conclusion, ATO which has been reported to be a highly effective agent was found to be well tolerated in our large pediatric cohort of newly diagnosed APL patients. Disclosures Off Label Use: Idarubicin and Mitoxantrone- labeled for use in AML/APL for adults (not pediatrics) Arsenic Trioxide- labeled for use in relapsed/refractory APL (not de novo APL) Mercaptopurine and Methotrexate- labeled for use in pediatric ALL (not APL).

Background: Approximately 3540 children are diagnosed with leukemia in the United States yearly (Bhatia and Robison, Oncology of Infancy and Childhood, 2008). Cooperative group trials have increased survival, particularly for acute lymphoblastic leukemia (ALL), but successful treatment of recurrent leukemia remains an unmet medical need. Resistance pathways and epigenetic alterations suggest a role for histone deacetylase (HDAC) inhibitors in children with leukemia (Burke and Bhatla, Frontiers in Pediatrics, 2014). Panobinostat is an orally administered pan-deacetylase inhibitor with activity against HDACs at concentrations in the nanomolar range (Atadja, Cancer Letters, 2009), and for which there is pre-clinical evidence of activity in pediatric leukemia (Stubbs, et al., ASH 2010). Panobinostat shows promise in a variety of adult hematologic malignancies (Khot et al., Expert Opinion on Investigational Drugs, 2013). We undertook a phase I trial of panobinostat in children with recurrent hematologic malignancies, and herein report the safety and pharmacokinetics (PK) from enrolled children with leukemia. Methods: T2009-012 is a first-in-child study coordinated by Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL). Children with relapsed or refractory leukemia between the ages of 1 and 21 years were enrolled to a multi-center, single agent trial of panobinostat dosed once per day three days per week for four successive weeks. Dose escalation was a standard 3+3 design with three dose levels planned. Subjects underwent lumbar puncture with prophylactic chemotherapy at treatment start and after a 28 day course. Blood was sampled pre-dose, at 0.5, 1, 6, 24 and 28-48 hours following the first dose. PK was obtained from blood on patients concurrently with optional specimens obtained from cerebrospinal fluid (CSF) on Day 29. Subjects who received fewer than 11 of the 12 planned doses and did not experience a dose limiting toxicity (DLT) were considered not evaluable for DLT, but were included in the summary of toxicities. Serial ECGs were monitored. Results: Seventeen subjects were enrolled with a diagnosis of acute leukemia, 10 with ALL and 7 with acute myelogenous leukemia (AML). Five were enrolled at dose level 1, 24 mg/m2/dose, 6 at dose level 2, 30 mg/m2/dose, and 6 at dose level 3, 34 mg/m2/dose. There have been no DLTs. Nine subjects are evaluable for DLT and 4 subjects were taken off study early due to increasing blast count. No subjects required removal from protocol therapy for QTc prolongation. One subject with infant ALL was removed early for progressive Aspergillus infection, 1 subject only received 10 doses owing to electrolyte abnormalities, and 2 subjects had nausea and vomiting after administration of 4 doses and did not continue. Grade 3/4 adverse events occurring in more than 20% of subjects included anemia in 82%, diarrhea in 24%, febrile neutropenia in 65%, hypokalemia in 41%, and hypophosphatemia in 24%. Concentration-time profiles were obtained from 9 subjects ages 16 months to 14 years in the 3 dose levels. Mean ± SE of PK for all subjects were Cmax 28.8 ± 6.1 ng/mL, Tmax 2.0 ± 0.8 hours, and T1/2 12.8 ± 3.0 hours. Two toddlers had the highest dose-normalized AUC0-inf and lowest oral clearance. Apparent oral clearance proportionally increased with increase in BSA. To date, 4 CSF specimens have been evaluated and found to have panobinostat below the lower limit of the quantification of 0.1 ng/mL, despite appreciable levels in the plasma. Two subjects on dose level two began a second cycle of therapy; one completed a second cycle for MLL rearranged leukemia and one discontinued study participation in the second cycle to undergo hematopoietic stem cell transplant for secondary AML after achieving a CRp in the first cycle. Conclusions: Panobinostat was tolerated these heavily pre-treated patients without unanticipated toxicities. PK in larger children and adults appears similar but PK in smaller children needs to be further explored. Penetration of panobinostat into the CSF was negligible. Two of 17 patients were able to receive a second cycle of therapy, but 4 had to be withdrawn early because of rapid increase in blast counts. Future trials will explore combination therapy in children with refractory hematologic malignancies, particularly those known to be driven by epigenetic mechanisms, in order to better control risk of rapid progression and improve efficacy through synergy. Disclosures Off Label Use: panobinostat for leukemia. Manley:Seattle Genetics, Inc.: Employment, Equity Ownership. Thomson:Epizyme, Inc: Employment.

The authors of the above-referenced article did not correctly use quotation marks to indicate that they were directly quoting a number of statements made originally in the article below: Hassles with taking out the garbage: Aggravating aggresomes. R. Garcia-Mata, Y.-S. Gao, E. Sztul, Traffic 3(6) (2002) 388–396. Furthermore, the authors did not correctly cite or reference as quotations statements that came directly from an earlier article published by the same lab

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Despite effective chemotherapy, 25 to 30% of children will relapse. In adults, less than 30% of patients with ALL are cured. Therefore, it is critical that we identify novel therapies to treat ALL. We are studying the effects of a small molecule compound known as tubacin (tubulin acetylation inducer) that selectively inhibits histone deacetylase 6 (HDAC6) resulting in increased acetylation of alpha-tubulin by inhibiting one of the two catalytic domains of HDAC6. We found that treatment of both pre-B and T-ALL cell lines with tubacin inhibits growth at very low micromolar concentrations (Jurkat IC50=1μM, Loucy IC50=3μM, REH IC50=2μM, Nalm6 IC50=5μM). We also determined that there is a therapeutic window, since tubacin inhibits the growth of normal bone marrow progenitor cells in methylcellulose colony assays at 20μM and normal human lymphocytes cultured in IL-2 at an IC50 of 16μM. We next tested the effects of tubacin in vivo. SCID mice injected with pre-B ALL Nalm-6 cells were treated with tubacin intraperitoneally at 50 mg/kg/day. Preliminary data using bioluminescence imaging in SCID mouse models showed that tubacin inhibited leukemic progression in vivo. To understand the mechanism of tubacin in ALL cells, we examined both apoptosis and cell cycle regulation by PARP cleavage, activation of caspases, and propidium iodide staining with FACs analysis. Tubacin induced apoptosis of pre-B and T-ALL cells within 12 hours of treatment. There was no effect on cell cycle progression, Retinoblastoma protein phosphorylation, or p21 upregulation, which have been observed with other HDAC inhibitors. Unlike in myeloma cells, tubacin did not increase JunK/SAPK activation or accumulation of acetylated HSP90 in ALL cells. Tubacin treatment resulted in accumulation of acetylated alpha-tubulin after 1 hour and an increase in polyubiquitinated proteins after 7 hours. To address potential mechanisms of tubacin in ALL, we tested whether Na+/K+ ATPase could be contributing to apoptosis. Previous work has shown that treatment with L-glutamate dissociates the Na+/K+ ATPase complex from acetylated tubulin and restores ATPase enzymatic activity. We hypothesized that the accumulation of acetylated tubulin could potentially inhibit the activity of the cytosolic Na/K ATPase pump, which could be reversed by treatment with 1mM sodium glutamate. Preliminary data demonstrate that we can partially rescue the effects of tubacin on PARP cleavage with sodium glutamate. These results suggest that tubacin induces apoptosis through a novel pathway in ALL cells and provide rationale for targeting the aggresome pathway to treat ALL in the future.

FLT3 is a receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with acute myelogenous leukemia (AML) and confer a poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations in the FLT3 kinase activation loop, e.g. D835V. We are studying the signaling pathways associated with a small molecule multi-targeted receptor tyrosine kinase inhibitor (RTKI), ABT-869. To determine the effects of ABT-869 in vitro and in vivo, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation was used as an isolated FLT-3 mutant model system. In vitro, ABT-869 is effective in inhibiting the proliferation of Ba/F3 Flt-3 ITD mutant cells (IC50 value of 1 nM) when compared to Ba/F3 Flt-3 D835V mutant (IC50 value between 1 and 10 μM) and Ba/F3 Flt-3 wildtype (WT) cells (IC50 value of 10 μM). Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis occurred in Ba/F3 Flt-3 ITD mutant cells treated with 1μM ABT-869 for 24 hours (42.8%) when compared to untreated (4.7%) or vehicle control (4.0%) cells. Ba/F3 Flt-3 D835V mutant cell lines demonstrated a 12.5% rate of apoptosis at 1μM, compared to untreated (1.99%) and vehicle control (2.1%) cell lines. Propidium iodide staining of treated Ba/F3 Flt-3 WT cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cells or DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells, following 6 hours of treatment with 1 to 100 nM ABT-869, whereas no cleavage was observed in Ba/F3 WT cells treated with ABT-869. To study the effects of ABT-869 in vivo, we treated SCID mice injected with Ba/F3 Flt-3 ITD, Ba/F3 Flt-3 D835V, or Ba/F3 Flt-3 WT cells and monitored disease progression using bioluminescence imaging. The mice injected with the Ba/F3 FLT-3 ITD mutant cells and treated with vehicle control developed metastases and had a median survival time of 2 weeks. In contrast, the ABT-869 treated group had slower disease progression with median survival of 6.2 weeks (P

Children with acute myeloid leukemia (AML) have 50% overall survival despite aggressive chemotherapy and bone marrow transplantation. Similarly, only one third of adults diagnosed with AML will be cured. AML blast cells from approximately 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains internal tandem duplications in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and members of the VEGF and PDGF receptor families. We previously demonstrated that ABT-869 in vitro induces apoptosis of AML cell lines harboring the FLT3-ITD and primary AML cells, and in vivo in tumors from MV-411 xenograft models. Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and propidium iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. Toxic effects were not observed on normal hematopoietic progenitor cells in methylcellulose-based colony assays at concentrations that were effective in AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-411 with oral preparations of ABT-869. Complete regression of MV4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Tumors from mice treated with ABT-869 showed decreased proliferation by Ki67 and increased apoptosis by TUNEL staining. We also observed that the mice treated with ABT-869 the day after injection of AML cells remained tumor-free for over 3 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. ABT-869 also suppresses the growth of Molm-13 (human AML cell line that expresses both FLT3-ITD and wt FLT3) at an IC50 between 1 and 10nM. To examine the effects of ABT-869 in vivo, we employed a murine bone marrow transplantation model. After chemical ablation of the bone marrow, SCID mice were injected with Molm-13 cells through the tail vein to allow engraftment. We observed that mice treated with an oral preparation of ABT-869 at 40 mg/kg/day prevented the engraftment of Molm-13 cells. The SCID mice that were not administered ABT-869 demonstrated clinical engraftment with hind leg paralysis and chloroma formation. Chloroma formation was confirmed by immunohistochemical staining with CD33 and CD45. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. We will also determine if there is any difference in efficacy in relation to the FLT3 status of each primary AML sample. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic at the doses studied, and provide rationale for the treatment and prevention of relapse in AML patients.

FLT3 is an receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of the differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with Acute myelogenous leukemia (AML) and confer poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations on the FLT3 kinase activation loop (e.g. D835V). Our laboratory is studying the signaling pathways associated with a newly identified multi-targeted tyrosine kinase receptor small molecule inhibitor (RTKI), ABT-869. Recently published work in our laboratory showed that using ABT-869 to treat MV4-11, a human AML FLT-3 ITD mutant cell line, resulted in the inhibition of phosphorylation of FLT-3 with a downstream inhibitory effect on the activation of STAT5, ERK, and Pim-1. Cell viability assays determined that MV-411 cells responded to ABT-869 in a concentration dependent manner (IC50 = 10nM). Apoptosis studies also showed an induction of apoptosis in ABT-869 treated cells. In vivo studies involving xenograft injections of MV-411 cells into SCID mice and subsequent treatment with ABT-869 demonstrated regression of tumor formation. In this study, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation is used as an isolated Flt-3 mutant model system. In vitro, ABT-869 is effective in inhibiting the proliferation of Ba/F3 Flt-3 ITD mutant cells when compared to Ba/F3 Flt-3 D835V mutant and Ba/F3 Flt-3 WT cells. Trypan Blue Exclusion and Alamar Blue assays were used to demonstrate that there is 50% inhibition of growth and proliferation (IC50) of Ba/F3 FLT3 ITD mutant cells at a concentration of 1nM after 48 hours of treatment. Ba/F3 FLT3 D835V mutant cells show an IC50 between 1μM and 10μM after 48 hours of treatment. In contrast, Ba/F3 FLT3 WT cells demonstrate an IC50 of 10μM only after 72 hours of treatment. Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis (41.2%) occurred in Ba/F3 Flt-3 ITD mutant cells treated with 10nM ABT-869 after 24 hours when compared to untreated (6.5%) or vehicle control (6.1%) cells. Staining of Ba/F3 Flt-3 WT treated cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cell only and DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells following treatment with ABT-869 whereas no cleavage was observed with Ba/F3 WT cells treated with ABT-869. In vivo, the activity of ABT-869 treatment of SCID mice injected with Baf3 Flt-3 ITD, Baf3 Flt-3 D835V, or Baf3 Flt-3 WT cells is also being evaluated. Using bioluminescence imaging, it was determined that Ba/F3 FLT-3 ITD mutant and Ba/F3 Flt-3 D835Vmutant cell lines result in metastases and subsequent death in SCID mice after 2 weeks for ITD and 5 weeks for D835V, whereas mice injected with Ba/F3 WT survive longer than 5 weeks. Preliminary data demonstrated that ABT-869 prolonged survival in mice injected with the Ba/F3 FLT3-ITD cells compared to controls. Our preclinical data demonstrate that ABT-869 is effective specifically with FLT-3 ITD mutant cell lines in an isolated system. These studies provide rationale for the treatment of AML patients and the prevention of relapse.

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Despite effective chemotherapy, 20% of patients will relapse. Therefore, it is critical that we identify novel therapies to treat ALL. We are studying a new small molecule compound known as tubacin (tubulin acetylation inducer) that selectively inhibits histone deacetylase 6 (HDAC6). HDAC6 binds to polyubiquitinated misfolded proteins and to dynein motor proteins, including alpha-tubulin, thereby recruiting misfolded or unwanted proteins to aggresomes and subsequent degradation by the lysosome. Tubacin was discovered through a chemical genetic screen of a 7,392 small molecule library and was found to induce acetylation of alpha-tubulin by inhibiting one of the two catalytic domains of HDAC6. This inhibition disrupts the interaction of HDAC6 with dynein resulting in marked accumulation of ubiquitinated proteins. Previous work demonstrated that treatment of multiple myeloma cells with tubacin inhibited growth at low micromolar concentrations. Tubacin does not appear to affect global histone acetylation, gene expression, or cell cycle regulation. To determine the effects of tubacin in human ALL cells, we treated both B- and T-cell ALL cell lines with varying concentrations of the drug and performed MTT assays. In T-ALL cells (Jurkat, Loucy), the IC50 of tubacin was found to be 1 to 3 uM, while in B-cell ALL cells (REH, Nalm-6), the IC50 was 2 to 5uM. Tubacin induced apoptosis of Jurkat and Loucy cells stained with Annexin V and propidium iodide. Within 12 hours, we observed increased protein polyubiquitination and PARP cleavage, but no difference in Rb phosphorylation in cells treated with tubacin. Furthermore, tubacin treatment increased acetylation of alpha-tubulin in Loucy and Jurkat cells within 3 hours. To study whether tubacin was toxic to normal hematopoietic cells, we treated human bone marrow cells cultured in methylcellulose containing IL-3, IL-6, and Stem Cell Factor with varying concentrations of tubacin. The IC50 of 20uM was determined from numbers of colonies plated in triplicate. Similarly, treatment of normal human lymphocytes cultured in IL-2, demonstrated an IC50 of 16uM. Finally, we examined the effects of tubacin on growth of primary ALL cells. Bone marrow cells from three patients with B-cell ALL at diagnosis were cultured in tubacin at varying concentrations and MTT assays were performed. In two of the three ALL samples, the IC50 was less than 5uM. Experiments to study the effects of tubacin in mouse models of leukemia are in progress. Our results suggest that inhibition of HDAC6 and the aggresome pathway provides a novel approach to treat ALL.

13010 Background: Using the linear PK first order (FO) compartmental approach, the methodical and computational uniformity in modeling various linear systems (ERW) is the dominant characteristic of the population PK analyses. However, saturation of the enzymatic reaction results in nonlinear kinetics based on the MM equation, i.e., the deamination of ASN by ERW, which complicates the PK-PD model. The PK with FO & the MM PK-PD model is theoretically better when single dose of ERW is given. To simulate the PK-PD data and to project patient (pt) data using this model, we used simultaneous integration of the FO+MM equations. Methods: In the 1990’s, ERW was used in pediatric ALL pt (n=23, 1st cohort evaluated for PK-PD). The current study was started to assess if the parallel MM+FO (MM+FO) PK-PD model fit the limited ERW (25K) data better than the MM model, and to validate the MM+FO model and its population parameter estimates. Results: The population PK-PD model best-fitted serum ERW & ASN-time pairs obtained in 23 and from 5 pediatric pt with ALL, using nonlinear mixed-effects modeling (NONMEM). The validity of the MM+FO population PK-PD model and the estimated parameters were tested using the naive prediction method. Patients were administered ERW 6K or 25 K IU/m2 when allergy to E. coli formulations appeared. High correlation between ERW peak serum levels calculated from limited individual pt’ KM (900 μM) & Vmax values in 5 pediatric pt (2nd cohort) and the observed ERW trough levels & its substrate (ASN) were found. The T1/2 averaged 16 hr (25K ERW), and the trough level of 0.1–0.2 IU/ml was correlated with ASN st cohort. When simulations on population parameters were conducted, the MM+FO predicted the multiple dose steady-state serum ERW & ASN levels nicely. Therefore, the MM+FO model was clearly superior to either the FO or the MM PD models. Moreover, simulations compared favorably ERW 25K Q2 days x3 doses & x2 weeks vs. Pegaspargase 2,5K Q2 weeks. Conclusions: The NONMEM PK-PD model for ERW fitted the simultaneous analyses of data from different doses and regimens better than either standard Two-stage or MM could. No significant financial relationships to disclose.