non-DS Acute Lymphoblastic Leukemia Research Articles

Epigenome-Wide Association Study of Acute Lymphoblastic Leukemia in Children with Down Syndrome

Background: Down syndrome (DS) is associated with an up to 30-fold increased risk of B-cell acute lymphoblastic leukemia (ALL), and DS-ALL patients have worse overall survival and increased long-term treatment-related health conditions compared with non-DS ALL patients. In a recent genome-wide association study of DS-ALL, established ALL genetic risk loci were associated with DS-ALL, with several single nucleotide polymorphisms (SNPs) conferring a larger effect on ALL risk in the context of DS than in euploidy. We performed an epigenome-wide association study (EWAS) to elucidate whether epigenetic differences at birth are associated with risk of subsequent DS-ALL.Methods: The DS-ALL Discovery Study included 147 DS-ALL cases and 198 DS controls from the International Study of Down Syndrome Acute Leukemia, with newborn dried bloodspots (DBS) obtained from California (n=326) and Washington state (n=19) biobanks. The DS-ALL Replication Study included 24 DS-ALL cases and 24 DS controls with newborn DBS from the Michigan Neonatal Biobank. DNA was isolated from DBS, bisulfite converted, and assayed using Illumina Infinium MethylationEPIC Beadchip genome-wide DNA methylation arrays. Raw data were processed using “minfi” and “noob” packages in R. Reference-based deconvolution of blood cell proportions was performed using the Identifying Optimal DNA methylation Libraries (IDOL) algorithm, using DNA methylation data from cord blood reference samples, to estimate proportions of B cells, T cells (CD4+ and CD8+), monocytes, granulocytes, natural killer cells, and nucleated red blood cells. We compared each cell type proportion between DS-ALL cases and DS controls using linear regression adjusting for sex, plate, and principal components (PCs) to account for genetic ancestry. To identify single CpG probes associated with DS-ALL risk, we performed a multiethnic EWAS of DS-ALL in each study using linear regression adjusting for sex, plate, and PCs related to: 1) cell-type proportions and 2) genetic ancestry. Differentially methylated regions (DMRs) were identified using DMRcate and comb-p methods. In the Discovery Study, genome-wide SNP array data were available for 131 cases and 130 controls, and data from targeted sequencing of somatic mutations in exons 2/3 of GATA1 were available for 184/198 DS controls.Results: Deconvolution of blood cell proportions in the DS-ALL Discovery Study showed significantly higher B cell proportions in newborns with DS who later developed ALL (mean=0.0128, sd=0.0151) compared with DS controls (mean=0.00826, sd=0.0115) (P=6.4x10 -4, coefficient=0.0052). A significantly higher B cell proportion at birth was also found in DS-ALL cases in the independent Replication Study (cases mean=0.048, sd=0.024; controls mean=0.039, sd=0.028; P=0.03, coefficient=0.015). In the Discovery Study, the B cell difference remained significant (P=5.8x10 -3) with a similar effect size (coefficient=0.0045) after removal of GATA1 mutation-positive DS controls (n=30). We also investigated whether DS-ALL risk SNPs at ARID5B, IKZF1, GATA3, and CDKN2A may confound the association, but the increased B cell proportions in DS-ALL remained significant and effect estimates slightly increased in SNP genotype-adjusted models (coefficient range:0.0055-0.0059). In the EWAS of DS-ALL, 9 CpGs reached epigenome-wide significance (P<7.67x10 -8), including 2 CpGs overlapping the promoter of the tumor suppressor gene TRIM13, frequently deleted in B-CLL, although none of these showed evidence of association (P<0.05) in the Replication Study. We identified 125 DMRs associated with DS-ALL in the Discovery Study. For 3 DMRs, overlapping genes HOPX, SMIM24, and PPP1R10, all implicated in normal and leukemic stem cell function, there were multiple significant CpGs in the Replication Study (P<0.05) all with effects in the same direction as the Discovery Study DMRs.Conclusions: Increased B cell proportions in newborns with DS may be a risk factor for development of DS-ALL in childhood. This finding, based on DNA methylation data, requires confirmation using conventional cell count measures, and should be explored as a novel biomarker for ALL risk in the non-DS population. Single CpGs and DMRs associated with DS-ALL risk in our Discovery Study require further investigation, including in additional ALL case-control studies in DS and non-DS populations. DisclosuresMa: Celgene/Bristol Myers Squibb: Consultancy, Research Funding.

Integrated Genetic/Epigenetic Analysis Revealed High Heterogeneity of Acute Lymphoblastic Leukemia in Down Syndrome

BackgroundChildren with Down syndrome (DS), which caused by an extra copy of chromosome 21, have a 20-fold increased risk for acute lymphoblastic leukemia (ALL) compared with non-DS children. Recent genome-wide studies have revealed that DS-ALL have uncommon genetic alterations such as mutations in JAK2 , NRAS , and KRAS , mutations or overexpression of CRLF2 , suggesting the unique biological features of DS-ALL distinct from non-DS ALL. However, most of previous studies regarding molecular analysis of DS-ALL have been limited to genomic and transcriptomic characterizations, and no study showed comprehensive genetic/epigenetic overview in DS-ALL.Materials and MethodsOur cohort included 179 pediatric B-cell precursor ALL samples (61 DS-ALL and 118 non-DS-ALL). Biological subtypes are detailed in Table 1. We performed targeted deep sequencing for seven ALL-related genes in 61 DS-ALL, SNP array analysis in 43 DS-ALL, and whole transcriptome sequencing (WTS) in 25 DS-ALL and 118 non-DS-ALL samples. WTS data from 153 ALL in TARGET database were also used in this study as a validation cohort. Additionally, methylation array analysis was performed in 36 DS-ALL, 17 non-DS-ALL, and 20 DS patients without ALL. To validate gene mutations detected by WTS, Sanger sequencing was also conducted. To cluster gene expression profiling, we applied the hierarchical clustering method for expression profiling and consensus clustering for methylation profiling.ResultsTargeted deep sequencing revealed frequent gene mutations of JAK2 (23%) and RAS (31%) pathway. SNP array analysis elucidated several recurrent chromosomal deletions affecting actionable gene loci; IKZF1 (33%), PAX5 (30%), EBF1 (15%), CDKN2A (34%), and RB1 (7%). Gains of whole chromosome X (18%) and 8 (10%) were also recurrently identified. Expression profiling stratified 143 samples into 6 clusters (E1-E6 clusters), which were well correlated with biological subtypes of ALL (Figure 1). Intriguingly, DS-ALL cases were clustered into four clusters, suggesting the heterogeneity of DS-ALL. Cluster E3 included one DS-ALL, which was clustered to the branch of PAX5 -rearranged cases. SNP array analysis revealed this case had focal amplification of PAX5 . All DS-ALL cases with ETV6 - RUNX1 fusion were classified into cluster E4, which also included all ETV6 - RUNX1 fusion cases of non-DS-ALL. This cluster included one DS-ALL case without ETV6 - RUNX1 fusion. This particular case had a homozygous deletion of ETV6, implicating ETV6 - RUNX1 -like signature. DS-ALL with high hyperdiploidy (HeH) were classified into cluster E5, which also included most of HeH samples of non-DS-ALL. Among other 5 DS-ALL cases in this cluster, 2 cases showed IGH - CEBPA fusion and IGH - CEBPD fusion, respectively. Cluster E6 was characterized by BCR - ABL1 fusion and Ph-like expression profiles. In our cohort, we identified 7 DS-ALL cases as Ph-like signature, which included all JAK2 mutated samples. In this cluster, PAX5 mutation and IKZF1 mutation were detected in 2 DS-ALL cases, respectively. Notably, by using 250 genes in our hierarchical clustering, TARGET database samples were divided to similar 6 clusters (Figure 2). On the other hand, Methylation array analysis revealed unique hypermethylation of promoter regions of RUNX1 in DS-ALL. Methylation of this regions were confirmed in B-cell of DS without ALL, but not in non-DS-ALL. Furthermore, methylation array analysis categorized DS-ALL into 3 clusters (Figure 3). Cluster M1 included IGH - CEBPA fusion and IGH - CEBPD fusion cases. This cluster had worse prognosis than other 2 clusters.ConclusionOur integrated analysis enlightened the genetic and epigenetic heterogeneity of DS-ALL. Importantly, DS-ALL had genetic features corresponding to each biological subtypes same as non-DS-ALL. Our analyses also elucidated that DS-ALL had unique subtypes such as IGH - CEBPA fusion, IGH - CEBPD fusion, or PAX5 alterations. It may be a reason for worse prognoses of DS-ALL. Our result indicated risk classification or therapy of DS-ALL should be tailored by each biological subtype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Minimal Residual Disease, Long-Term Outcome, and &lt;i&gt;IKZF1&lt;/i&gt; Deletions in Down Syndrome Acute Lymphoblastic Leukemia in a Matched Cohort Study

Background: Down syndrome (DS) patients with acute lymphoblastic leukemia (ALL) are at a higher risk of treatment-related mortality and of relapse, which is influenced by a higher incidence of unfavorable genetic aberrations, such as IKZF1 deletion. We aimed to investigate the potential underlying effect of DS versus the impact of adverse cancer genetics on clinical outcome. Method: The association between DS and minimal residual disease (MRD) and clinical outcome was evaluated in a cohort of 136 DS ALL and 407 non-DS ALL pediatric patients matched for clinical risk factors and genetics, including IKZF1 deletion. Findings: The percentage of patients in the higher MRD category (≥1E-4) at the end of induction treatment did not significantly differ between DS ALL (38%) and matched non-DS ALL (39%; p=0·88). DS ALL was associated with a higher relapse risk compared with matched non-DS ALL in the IKZF1 -deleted group (cause-specific hazard ratio [HRcs], 4·3; 95% confidence interval [CI], 1·6 to 11, p=0·003), but not in the IKZF1 -wildtype group (HRcs, 1·0; 95% CI, 0·48 to 2·1; p=0·99). Our matched cohort confirmed that in addition to more induction deaths (6% versus 0·8%), DS ALL was associated with a higher risk of post-induction treatment-related mortality (HRcs, 5·0; 95% CI, 2·3 to 11; p<0·001). Interpretation: Our matched study shows that the increased relapse risk in DS ALL is not exclusively due to adverse cancer genetics, but DS itself provides an additional risk in IKZF1 -deleted cases, suggesting an interplay between the germline environment and this specific poor-risk somatic aberration. Funding Information: Stichting Kinder Oncologisch Centrum Rotterdam (SKOCR), NHMRC Australia, The Cancer Council NSW, Tour de Cure, Blood Cancer UK, UK MRC, Children with Cancer, and Swedish Society for Pediatric Cancer. Declaration of Interests: K.S: speaker and/or Advisory Board Honoraria from Jazz Pharmaceuticals and Servier; speaker fee from Amgen and Medscape; Educational grant from Servier. All other authors declare no conflict of interest. Ethics Approval Statement: The ethical review boards of each participating country or center approved treatment protocols according to local law and regulations. Written informed consent was obtained from patients, parents or guardians.

Outcomes of Patients with Down Syndrome and CRLF2-Overexpressing Acute Lymphoblastic Leukemia (ALL): A Report from the Children's Oncology Group (COG)

Background: Patients with Down syndrome (DS) have an approximately 10-fold increased risk of developing ALL, and the spectrum of genetic alterations differs from that of non-DS ALL. Rearrangement and/or overexpression of cytokine receptor-like factor 2 (CRLF2+) occurs in 50% of DS ALL, compared to only 5-10% CRLF2+ cases in non-DS children and adolescents. JAK2 or JAK1 mutations co-occur in about half of CRLF2+ cases in both DS and non-DS ALL. The prognostic significance of CRLF2+ ALL also appears to differ in the limited data reported to date, with less adverse impact in patients with DS compared to non-DS ALL. Here, we report the clinical characteristics and prognostic significance of B-ALL with CRLF2 overexpression and JAK alterations in children and adolescents/young adults (AYA) with DS who were treated on Children's Oncology Group (COG) clinical trials from 2003-2016. Methods: We analyzed clinical, laboratory, and outcome data for 317 patients with DS B-ALL treated on standard risk (SR) trials AALL0331 and AALL0932 and high risk (HR) trials AALL0232 and AALL1131, for whom CRLF2 status and rearrangement partners (IGH or P2RY8) were ascertained by flow cytometric assessment of surface expression; fluorescence in situ hybridization; and/or polymerase chain reaction (PCR) testing. JAK mutations were ascertained in a subset by PCR and sequencing. Minimal residual disease (MRD) was assessed by flow cytometry at the end of induction (EOI) and at end of consolidation (EOC) for a subset of EOI MRD+ patients. Results: We identified 168/317 (53.0%) CRLF2+ cases, and among those assessed for CRLF2 partner, 17/73 (23.3%) were IGH-CRLF2 and 56/73 (76.7%) were P2RY8-CRLF2. In the subset of 165 cases tested for JAK mutations (85 CRLF2- and 80 CRLF2+), 42/165 (25.4%) had JAK mutations, all of which co-occurred in CRLF2+ cases. CRLF2 positivity was significantly associated with younger age at diagnosis: 140/168 (83.3%) of CRLF2+ cases were under 10 years old, versus 106/149 (71.1%) of CRLF2- cases, p&amp;lt;0.01. P2RY8-CRLF2 cases were significantly more likely than IGH-CRLF2 cases to be associated with age under 10 years at diagnosis (92.9% vs 29.4%, p&amp;lt;0.0001), initial white blood count (WBC) &amp;lt;50x103 (80.4% vs 52.9%, p&amp;lt;0.024), National Cancer Institute (NCI) standard risk status (73.2% vs 23.5%, p&amp;lt;0.0002), and day 29 MRD &amp;lt;0.01% (67.9% vs 37.5%, p&amp;lt;0.028). There were no significant associations between JAK mutation status and any clinical features assessed (sex, initial WBC, central nervous system or testicular involvement, NCI risk group, or EOI/EOC MRD). Among patients with neutral cytogenetics (defined as neither favorable [ETV6-RUNX1, double trisomies of chromosomes 4 and 10] nor unfavorable [BCR-ABL1, KMT2A-rearranged, hypodiploid, iAMP21]), survival did not significantly differ between CRLF2+ and CRLF2- patients (Figure 1A,B), either for 5-year event-free survival (EFS) (79.1 +3.6% vs 77.6 +4.7%, p=0.856) or 5-year overall survival (OS) (89.5 ±2.8% vs 84.9 ±4.1%, p=0.674). There were also no significant differences in EFS or OS based on JAK mutation status or CRLF2 partner, although there were trends toward poorer outcomes in the CRLF2+/JAK+ and IGH-CRLF2 subgroups (Figure 1C). Finally, there was no significant difference in 5-year cumulative incidence of relapse (CIR) according to CRLF2 status (CRLF2+ 16.0 +3.0%, CRLF2- 10.6 +2.6%, p=0.179), although there were non-significant trends toward more relapses in CRLF2+ cases overall and in the NCI HR subgroup analysis. Discussion: Whereas CRLF2 and JAK alterations are associated with higher MRD, poorer survival, and increased CIR in patients with high-risk ALL without DS, these alterations do not demonstrate strong adverse prognostic impact in children and AYAs with DS-ALL treated on recent frontline COG trials, although larger sample sizes are needed to adequately assess for possible poorer prognoses associated with the CRLF2+/JAK+ and IGH-CRLF2 subgroups. Regardless, given the frequency of these targetable lesions and the increased risk of relapse and chemotherapy-associated toxicities in patients with DS-ALL, targeted therapies currently under investigation for these genetic lesions may be beneficial to replace some intensive blocks of therapy in DS-ALL with CRLF2 and/or JAK alterations, both to enhance anti-leukemic efficacy and decrease intensive chemotherapy-associated toxicities. Figure 1 Disclosures Tasian: Gilead Sciences: Research Funding; Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Mullighan:Illumina: Consultancy, Honoraria, Speakers Bureau; AbbVie, Inc.: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau. Raetz:Celgene: Other: DSMB member; Pfizer: Other: Institutional research funding. Loh:Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees. Hunger:Novartis: Consultancy; Amgen: Current equity holder in publicly-traded company, Honoraria.

Trisomy 21 Driven Pro-Inflammatory Signalling in Fetal Bone Marrow May Play a Role in Perturbed B-Lymphopoiesis and Acute Lymphoblastic Leukemia of Down Syndrome

Introduction: Children with Down syndrome (DS) have a markedly increased risk of acute lymphoblastic leukemia (ALL), suggesting that trisomy 21 (T21) has specific effects on hematopoietic stem and progenitor cell (HSPC) biology in early life. Data from human fetal liver (FL) indicates that T21 alters fetal hematopoiesis, causing multiple defects in lympho-myelopoiesis. The impact of T21 on fetal B lymphopoiesis and how this may underpin the increase in ALL is not well known. We have recently found that fetal bone marrow (FBM) rather than FL is the main site of B lymphopoiesis; with a marked enrichment of fetal-specific progenitors (early lymphoid progenitors, ELP and PreProB progenitors) that lie upstream of adult type ProB progenitors (O'Byrne et al, Blood, in press). Previous preliminary data suggested that B progenitors were also reduced in T21 FBM (Roy et al, Blood. 124, 4331). Aim: To dissect putative molecular mechanisms responsible for the defects in T21 FBM B-lymphopoiesis and its association with childhood DS ALL. Methods: Second trimester human FBM and paediatric ALL samples were obtained from the Human Developmental Biology Resource and UK Childhood Leukaemia Cell Bank respectively. Multiparameter flow cytometry/sorting, transcriptome analysis by RNA-sequencing and microarray, and stromal co-culture assays were used to characterize HSPC and mesenchymal stromal cells (MSC) from normal (NM) disomic (n=21-35) and T21 (n=7-12) human FBM; RNASeq was performed on cytogenetically matched non-DS (n=13) and DS ALL (n=7). Results: In contrast to NM FBM, fetal specific progenitors were virtually absent (CD34+CD10-CD19-CD127+ ELP 2.8±0.4% vs. 0.8±0.4% of CD34+ cells) or very severely reduced (CD34+CD10-CD19+ PreProB 12.8±1 vs 2.6±0.7%) in T21 FBM. This was despite a &gt;4-fold increase in the frequency of immunophenotypic HSC (4.2±1.2% vs 0.9±0.2% of CD34+ cells) and similar frequencies of MPP and LMPP in T21 FBM. As in adult BM, the vast majority of B progenitors in T21 FBM were CD34+CD10+CD19+ ProB progenitors with a frequency (28.8±8.3%) similar to NM FBM (30.3±2.3% of CD34+ cells). Thus, T21 causes a severe block in B-progenitor commitment at the LMPP stage, in tandem with a compensatory expansion of ProB progenitors. Consistent with this, T21 FBM HSC, MPP and LMPP had reduced B cell potential in vitro compared to NM FBM in MS5 co-cultures. RNAseq of NM (n=3) and T21 (n=3) FBM HSPC demonstrated global transcriptomic disruption by T21, with increased gene expression in HSC, MPP, LMPP and ProB progenitors. Cell cycle genes were enriched in T21 ProB progenitors. Despite these functional and global gene expression differences, expression of key B-lineage commitment genes was maintained suggesting the defect in B-lymphopoiesis may be secondary to lineage skewing of multipotent progenitors towards a non-B lymphoid fate and/or mediated by extrinsic factors. GSEA pointed to a role for multiple inflammatory pathways in T21 hematopoiesis with dysregulation of IFNα, IL6 and TGFβ signalling pathways in T21 HSC/LMPP. To investigate the role of the T21 microenvironment, we co-cultured NM HSC, MPP and LMPP with T21 or NM primary FBM MSC. T21 FBM MSC (n=3) had reduced capacity to support B cell differentiation in vitro consistent with perturbation of MSC function by T21. Similar to T21 FBM HSPC, transcriptomic analysis of T21 FBM MSC by microarray showed enrichment for IFNα signalling compared to NM; and T21 HSPC and MSC both showed increased gene expression for IFNα receptors IFNAR1 and IFNAR2, which are encoded on chromosome 21. Since IFNα was undetectable by ELISA of conditioned media from NM and T21 MSC, differences in secreted IFNα from MSC are unlikely to fully explain the increased IFN signalling in T21 HSPC and MSC. This suggests that T21 may drive autocrine rather than paracrine IFN signalling in FBM cells. Finally, RNASeq showed perturbed inflammatory signalling in DS ALL compared to non-DS ALL, suggesting a role for T21-driven inflammatory pathways in the biology of DS ALL. Conclusions: These data show that T21 severely impairs B lymphopoiesis in FBM and is associated with expression of proinflammatory gene expression programs in T21 FBM HSPC and MSC and DS ALL. The compensatory expansion of T21 FBM ProB progenitors, through self-renewal or via an alternative differentiation pathway; with concomitant T21-driven proinflammatory signalling may underpin the increased risk of B progenitor ALL in childhood. Disclosures No relevant conflicts of interest to declare.

A Novel Role for HMGN1 in Down Syndrome Acute Lymphoblastic Leukemia

Background Down Syndrome (DS) Acute Lymphoblastic Leukemia (ALL) patients have extremely poor outcomes with mortality rates four times greater than non-DS ALL patients within their first two years of diagnosis. They are more suspectible to treatment related toxicities and experience higher relapse rates compared to other ALL patients. Approximately 60% of DS-ALL patients harbor rearrangement of cytokine receptor like factor 2 (CRLF2r), specifically P2RY8-CRLF2, and/or the CRLF2 F232C activating mutation. These lesions are considered poor risk and currently no targeted therapy exist. How increased chromosome 21 gene dosage affect disease phenotype is not yet fully elucidated. However, the high mobility group nucleosome-binding domain-containing protein 1 (HMGN1) on chromosome 21, which competes with histone H1 to bind the nucleosome and results in gene activation may be a candidate for targeted therapy in DS-ALL. Methods We aimed to determine the role of HMGN1 in CRLF2r DS-ALL. To model CRLF2r DS-ALL, the trisomy 21 cell line, SET-2, was transduced with a retroviral vector encoding the CRLF2 F232C activating mutation. Gene knockdown of HMGN1 using CRISPR/Cas9 was performed in the SET-2 CRLF2r line and the non-trisomy-21, non-CRLF2 expressing Jurkat line. Individual knockdowns of another two genes on chromosome 21, DYRK1A and ERG were also performed. Knockdown of JAK2 was used as a control as it is critical for CRLF2 signaling. CellTiter-Glo was used to investigate proliferation of knockdown lines to test the hypothesis that HMGN1 is essential for CRLF2r DS-ALL cell proliferation. Lentiviral vectors encoding the P2RY8-CRLF2 fusion gene, CRLF2 F232C activating mutation or an overexpression construct of HMGN1 were transduced into BaF3 cells individually or in combination to test the hypothesis that overexpressing HMGN1 is associated with activation of CRLF2. Quantitative PCR (qRTPCR) for CRLF2 and flow cytometry for the CRLF2/IL7Rα receptor (TSLPR) were used to determine the effect of increased HMGN1 on CRLF2 expression. AnnexinV/7-AAD cell death assays were performed to determine if the effects of HMGN1 could be reduced by the demethylase inhibitor GSK-J4. Results Knockdown of HMGN1 resulted in an 80-90% decrease in HMGN1 protein expression in SET-2 CRLF2 and Jurkat lines compared to the Cas9 controls. While knockdowns of DYRK1A and ERG did not impair the proliferation of SET-2 CRLF2 cells, HMGN1 and JAK2 knockdowns led to a complete proliferation arrest over a period of 120hrs (p=&amp;lt;0.001, n=3), demonstrating their effect on cell division. However, no change in proliferation was observed in the Jurkat knockdown lines. Overexpression of either HMGN1 or P2RY8-CRLF2 alone in BaF3 cells did not result in cytokine independent transformation. However, cytokine independence was triggered in BaF3 cells when HMGN1 and P2RY8-CRLF2 were co-expressed (p=&amp;lt;0.001, n=3); demonstrating a role for HMGN1 in leukemic transformation. Importantly, the overexpression of HMGN1 in the BaF3 P2RY8-CRLF2 line increased the mRNA expression of CRLF2 by 5.8-fold compared to the BaF3 P2RY8-CRLF2 line without HMGN1 (p=0.034, n=3) and increased the mean fluorescence intensity of TSLPR by flow cytometry from 42 to 308 (p=0.008, n=3) (figure 1.a-c) indicating a novel role for HMGN1 in P2RY8-CRLF2 activation. While there are no pharmacological inhibitors for HMGN1, Lane et al. (2014) have shown that the restoration of H3K27 methylation using the demethylase inhibitor GSK-J4 was able to prevent DS-ALL cells from repassaging. Therefore, we have employed the inhibitor GSK-J4 to determine if it can reduce cell survival in HMGN1 overexpressed BaF3 cells. Specific inhibition of BaF3 P2RY8-CRLF2 HMGN1 cells was evident by decreased cell viability at a concentration of 3.8µM compared to BaF3 P2RY8-CRLF2 or BaF3 HMGN1 lines (p=&amp;lt;0.001, n=3) (figure 4.d). Thus, demonstrating a role for HMGN1 in the modification of the P2RY8-CRLF2 methylome and suggesting HMGN1 as a potential therapeutic target. Conclusion These data support the hypotheses that HMGN1 has a significant role in DS-ALL cell proliferation and that overexpression of HMGN1 results in activation of P2RY8-CRLF2. This is the first report of a novel role for HMGN1 in P2RY8-CRLF2 activation and leukemic transformation in CRLF2r DS-ALL. Additionally, we show that HMGN1 is a potential candidate for the development of a pharmacological inhibitor for CRLF2r DS-ALL. Disclosures Yeung: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau.

Inherited genetic susceptibility to acute lymphoblastic leukemia in Down syndrome

AbstractChildren with Down syndrome (DS) have a 20-fold increased risk of acute lymphoblastic leukemia (ALL) and distinct somatic features, including CRLF2 rearrangement in ∼50% of cases; however, the role of inherited genetic variation in DS-ALL susceptibility is unknown. We report the first genome-wide association study of DS-ALL, comprising a meta-analysis of 4 independent studies, with 542 DS-ALL cases and 1192 DS controls. We identified 4 susceptibility loci at genome-wide significance: rs58923657 near IKZF1 (odds ratio [OR], 2.02; Pmeta = 5.32 × 10−15), rs3731249 in CDKN2A (OR, 3.63; Pmeta = 3.91 × 10−10), rs7090445 in ARID5B (OR, 1.60; Pmeta = 8.44 × 10−9), and rs3781093 in GATA3 (OR, 1.73; Pmeta = 2.89 × 10−8). We performed DS-ALL vs non-DS ALL case-case analyses, comparing risk allele frequencies at these and other established susceptibility loci (BMI1, PIP4K2A, and CEBPE) and found significant association with DS status for CDKN2A (OR, 1.58; Pmeta = 4.1 × 10−4). This association was maintained in separate regression models, both adjusting for and stratifying on CRLF2 overexpression and other molecular subgroups, indicating an increased penetrance of CDKN2A risk alleles in children with DS. Finally, we investigated functional significance of the IKZF1 risk locus, and demonstrated mapping to a B-cell super-enhancer, and risk allele association with decreased enhancer activity and differential protein binding. IKZF1 knockdown resulted in significantly higher proliferation in DS than non-DS lymphoblastoid cell lines. Our findings demonstrate a higher penetrance of the CDKN2A risk locus in DS and serve as a basis for further biological insights into DS-ALL etiology.

Excellent Outcome of Children with Down Syndrome (DS) and Acute Lymphoblastic Leukemia (ALL) Treated on Dana-Farber Cancer Institute (DFCI) ALL Consortium Protocols 00-001 and 05-001

▪BackgroundChildren with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) are shown to have increased therapy-related morbidity and mortality. Hence, therapy modifications and/or dose-reductions are common treatment strategies for this patient (pt) population. Dana-Farber Cancer Institute (DFCI) ALL Consortium protocols have used same risk-stratified treatment for children with and without DS and ALL.Aim:To define the toxicity profile and outcome of children with DS and de novo ALL treated on DFCI ALL Consortium therapy protocols 00-001 and 05-001 using therapy identical to non-DS patients.Methods:Demographic, clinical and outcome data of DS and non-DS patients enrolled on the DFCI ALL protocols 00-001 (2000-2004) and 05-001 (2005-2011) were analyzed. Risk categorization and protocol therapy have previously been described (J Clin Oncol 2013; 31:1202-10; Lancet Oncol 2015;16:1677-90). On both protocols, DS ALL pts were treated identically to non-DS pts without any dose reduction or modification, except for the option for DS ALL pts to receive 3 doses of leucovorin after IT methotrexate. Fisher’s exact test was used to compare toxicities in the DS and non-DS pts and Gray test was used to compare the cumulative incidence of fracture and osteonecrosis. Overall survival (OS) was defined as time from registration to death. Event-free survival (EFS) was defined as time from registration to first event (defined as induction failure, relapse, second malignant neoplasm (SMN) or death due to any cause). Induction failure and induction death were included as events at time zero. Disease-free survival (DFS) was defined as time from complete remission (CR) to relapse, SMN or death. Pts without an event were censored at the last known follow-up. The Kaplan-Meier method was used for survival estimation and Greenwood’s formula for calculation of 95% confidence interval (CI) of survival estimates. Outcome of DS patients was also examined using Ponte di Legno (PdL) risk group [Low risk (LR) was defined as age at diagnosis ≤ 6 yr. and white cell count < 10X109/L and, remainder as high risk (HR)].(Blood 2014;123:70-7). Two-sided p values <0.05 were considered significant.Results:Of 1286 eligible pts aged 1-18 yrs. with de novo ALL enrolled on protocols 00-001 and 05-001, 38 (3%) had DS. There was no difference in demographic or presenting clinical features between DS and non-DS ALL pts except immunophenotype (absence of T-ALL in DS vs 11.7% in non-DS, p=0.017) and presence of high hyperdiploidy (51-65 chromosomes) (8.8% in DS vs 25.1% in non-DS, p=0.027) (Table 1). Two DS-ALL pts withdrew from the study after achieving CR. There was no difference in the CR rates (DS: 100% vs non-DS: 95.2%, p=0.47) or proportion of pts with low end of induction minimal residual disease (MRD) between DS and non-DS groups (p=0.73). Toxicities were comparable except DS pts had significantly higher rates of ≥Grade 3 mucositis (data available for protocol 05-001 only) (DS: 52.0% vs. non-DS: 12.0%, p<0.001), non-CNS thrombosis/bleed (18.4% vs. 8.2%; p=0.036), and seizure (15.8% vs. 4.7%, p=0.010). DS pts also had marginally higher rate of bacterial and fungal infections (55.3% vs. 41.3%, p=0.096) (Table 2). All 38 DS pts achieved a CR and there were 4 relapses with 1 death due to disease. There were no treatment-related deaths in DS-ALL pts. With a median follow-up of 6.2 yrs. the 5-yr EFS, DFS, and OS of DS pts were similar to non-DS pts (90.7% [81.1-100.0] vs. 83.7% [81.7-85.9]; 90.7% [81.1-100.0] vs. 87.4% [85.5-89.3]; 97.1% [91.8-100.0] vs. 91.4% [89.8-93.0]), with the 95% CI overlapping for each comparison (Figures 1a and 1b). There was no difference in outcomes of DS-ALL PdL LR pts (n=13) compared to PdL HR pts (n=25) (5-yr EFS 90.0% [73.2-100.0]. vs. 91.0% [79.9-100.0]; 5-yr OS 100.0% [100.0-100.0] vs. 95.8% [88.2-100.0]).Conclusion:DS pts treated on DFCI ALL Consortium protocols without dose reduction or modifications achieved similar outcomes to non-DS pts. DS pts had a higher frequency of mucositis, infection, and seizures, but did not experience any treatment-related deaths. Other than a higher risk of thrombotic complications, they did not develop excessive toxicity to asparaginase. The low rates of relapse and toxicity-related mortality support the approach of unified therapy protocol for DS and non-DS ALL pts with emphasis on supportive care interventions to prevent toxicities. [Display omitted] [Display omitted] [Display omitted] DisclosuresAsselin:Jazz Pharmaceuticals: Consultancy, Speakers Bureau; Sigma Tau Pharamceuticals: Consultancy.

Integrated Genomic Analysis of Down Syndrome Acute Lymphoblastic Leukemia Reveals Recurrent Cancer Gene Alterations and Evidence of Frequent Subclonal Driver Events

Introduction: Children with Down syndrome (DS) have a 10-20 fold increased risk of acute lymphoblastic leukemia (ALL) and suffer significantly poorer outcomes due to increased relapse and treatment-related mortality. DS-ALL exhibits a distinct spectrum of cytogenetic alterations, with a lower incidence of common recurrent ALL alterations, a higher incidence of alterations involving CRLF2 and JAK2, and a higher proportion of cases lacking a known oncogenic driver compared to non-DS ALL. We hypothesized that identifying genetic drivers in DS-ALL will facilitate improved risk-based treatment stratification and lead to novel therapeutic targets that may be harnessed to improve outcomes in this vulnerable population.Methods: We interrogated diagnostic tumor (bone marrow or peripheral blood) and matched remission ("normal") blood samples from a cohort of 63 DS-ALL cases (65% male; median age 5 years, range 1-21 years) obtained from Texas Children’s Cancer Center and the Children’s Oncology Group. Samples were selected to include ~1/3 CRLF2-rearranged (CRLF2-R) and ~2/3 CRLF2-wild-type (CRLF2-WT) cases. Whole exome sequencing (WES) and copy number analysis using the OmniExpress array were performed on both tumor and normal DNA and transcriptome sequencing (RNAseq) was conducted on tumor RNA, all using the Illumina platform and existing informatic pipelines at the Baylor College of Medicine Human Genome Sequencing Center. A median of >97% of targeted bases with at least 20x coverage (range 93.5-98.1%) was observed for WES, and an average of 55.4 million paired reads were generated per RNAseq.Results: Analysis of RNAseq data (n=60) using deFuse and SOAPfuse algorithms revealed known ALL-associated gene rearrangements in 37/60 (62%) of cases, including CRLF2-R (21/60, 35%) involving P2RY8 (n=17) or IGH (n=4); ETV6-RUNX1 fusions (14/60, 23%) and TCF3 fusions (PBX1 x 2 and a novel in-frame fusion with FLI1 x 1). WES (n=62) identified a median of 19 nonsilent somatic mutations per case (range 6-228). We did not find evidence of somatic or germline mismatch repair gene mutations in the 3 cases with >50 somatic mutations. WES findings included recurrent activating hotspot mutations in JAK2 (predominantly p.R683G/S) in 14 cases (23%) and CRLF2 (p.F232C) in 2 cases, all of which also had CRLF2-R. Somatic RAS pathway mutations (13 KRAS, 7 NRAS, and 2 PTPN11) were found in 20/62 cases (32%), 4 CRLF2-R and 16 CRLF2-WT, including 2 cases also harboring JAK2 activating mutations. Recurrent mutations in epigenetic or chromatin remodeling genes (e.g. KMT2C, SETD2, KDM6A, WHSC1, DOT1L) were observed in ~25% of patients, as were mutations in other known ALL genes (e.g. FLT3, IL7R, IKZF1, PAX5). Interestingly, inactivating mutations in the deubiquitinase USP9X, also recently reported in acute promyelocytic leukemia, were identified in 5/62 (8%) cases. Evaluation for internal tandem duplications (ITDs) demonstrated typical in-frame events in FLT3 (n=2) and an out-of-frame ITD in SETD2. Copy number analysis revealed frequent losses in cell cycle genes (CDKN2A/B, BTG1, RB1) and other known B-cell development genes (IKZF1, PAX5). Other putative candidate alterations detected by RNAseq, WES, and array are currently being validated by orthogonal platforms. We observed multiple subclonal driver events in >10% of patients, including one case with CRLF2-R and mutations in CRLF2, JAK2, KRAS, and NRAS; and a second case with CRLF2 and ETV6-RUNX1 rearrangements and a PTPN11 hotspot mutation. Six cases exhibited 2 different JAK2, KRAS, or NRAS mutations: in all four cases where evaluation was feasible, these mutations were present on different sequencing reads, suggesting the existence of distinct subclones. Clinical features (gender, age, initial white blood count) did not appear to co-cluster with any of the recurrent alterations.Conclusions: These findings detail the landscape of genomic and transcriptomic alterations in the largest cohort of DS-ALL comprehensively characterized to date. Our data confirm the frequency and occasional polyclonal nature of JAK2 and RAS pathway mutations, and demonstrate that mutations in these pathways are not mutually exclusive. We also identified several novel, recurrent mutations and fusions. Future analyses of additional cohorts will be needed to assess recurrence frequency and prognostic impact of these alterations. [Display omitted] DisclosuresSchore:Baxalta: Honoraria; Millennium Pharmaceuticals, Inc: Research Funding; Onyx/Amgen: Research Funding; Merck: Research Funding.

CRLF2 Overexpression Demonstrates Enhanced Leukemogenicity in Down Syndrome Hematopoietic Cells

Introduction: Children with Down syndrome (DS) are 10-20 times more likely than children without DS to develop acute lymphoblastic leukemia (ALL), and they demonstrate a distinctive spectrum of genetic alterations. Approximately 50% of DS-ALL cases demonstrate CRLF2 rearrangements (CRLF2-R), an approximately 10-fold higher frequency than in non-DS ALL. We sought to identify the functional basis for the increased incidence of ALL, and specifically CRLF2-R ALL, in children with DS.Methods: We created retroviral vectors which induce overexpression of CRLF2 and green fluorescent protein (GFP) for transduction into bone marrow (BM) cells isolated from the Dp16(1)Yey (Dp16) mouse model of DS, which is trisomic for the approximately 115 human chromosome 21 gene orthologs present on mouse chromosome 16. Transduced BM cells from Dp16 and wild-type (WT) control mice were co-cultured with OP9 stromal cells for one week to promote B-lymphoid lineage development, and then characterized by flow cytometric Hardy fraction analysis, or grown in B-lymphoid-promoting methylcellulose medium for colony growth assays.Results: We achieved efficient transduction (80-95%) of Dp16 and WT BM enriched for hematopoietic stem cells (HSCs) with CRLF2-GFP+ and control GFP+ viruses. Following OP9 co-culture, transduced HSCs were characterized by Hardy fraction analysis. CRLF2-GFP+ Dp16 lymphoid cells demonstrated significantly higher percentages of immature Fraction A (pre-pro-B) cells compared with GFP+ Dp16 cells (39.9% vs 15.7%, p=0.004, Fig. 1A). This CRLF2-GFP-induced immature immunophenotype was more pronounced in Dp16 versus WT HSCs, with a significantly higher percentage of Fraction A cells (39.9% in Dp16 vs 24.0% in WT, p=0.0002) and a significantly lower percentage of more mature Fraction B (pro-B) cells (24.3% in Dp16 vs 49.1% in WT, p=0.02, Fig. 1A,B). In methylcellulose colony assays, CRLF2-GFP+ Dp16 cells yielded a 36-fold increase in B cell colonies compared to GFP+ Dp16 cells (Fig 1C). Again, the effect of CRLF2 transduction was magnified in the Dp16 versus WT background. CRLF2-GFP+ WT cells demonstrated only a 2.9-fold increase in B cell colonies (Fig 1C).Conclusions: Here we demonstrate that CRLF2 overexpression results in a more immature B-lineage immunophenotype and increased lymphoid colony growth in vitro, and that these effects are significantly greater in a murine DS versus WT genetic background. Experiments to investigate the pathways involved and to evaluate these effects in vivo are ongoing. This work provides functional evidence of the enhanced leukemogenicity of CRLF2 overexpression in DS-ALL, and creates a tractable model system for additional future genetic studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Outcome of transplantation for acute lymphoblastic leukemia in children with down syndrome

We report on 27 patients with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) who received allogeneic hematopoietic cell transplantation (HCT) between 2000 and 2009. Seventy-eight percent of patients received myeloablative conditioning and 52% underwent transplantation in second remission. Disease-free survival (DFS) was 24% at a median of 3 years. Post-transplant leukemic relapse was more frequent than expected for children with DS-ALL (54%) than for non-DS ALL. These data suggest leukemic relapse rather than transplant toxicity is the most important cause of treatment failure. Advancements in leukemia control are especially needed for improvement in HCT outcomes for DS-ALL.

Physicians Compliance During Maintenance Therapy in Children with Down Syndrome and Acute Lymphoblastic Leukemia.

AbstractAbstract 2577Children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) have an inferior prognosis compared to non-DS ALL patients. We reviewed Methotrexate (MTX)/Mercaptopurine (6MP) maintenance therapy data for children with DS treated according to the NOPHO ALL92 or the NOPHO ALL2000 protocols between 1992 and 2007. The five year event-free survival (pEFS5y) for the DS patients was inferior to the non-DS patients (0.50 ± 0.07 vs 0.77 ± 0.01, p<0.001). The 48 DS patients in 1st remission at the beginning of maintenance therapy had pEFS10y below the 522 non-DS control patients (pEFS10y: 0.58, 95%-CI 0.43–0.77 vs. 0.83, 95%-CI 0.80–0.86, p<0.0001). The DS patients received lower median doses of MTX (Median: 11.8 vs 15.4, p<0.0001) and 6MP (median: 45.6 vs. 59.4, p<0.0001). In Cox regression analysis male gender, presence of DS, and high median maintenance therapy white blood cell levels (mWBC) were associated with increased risk for relapse. The mWBC hazard ratio for DS-ALL patients was 2.0, p<0.0005). This supports that DS children with ALL should be treated as vigorously as non-DS ALL patients. Obtaining WBC levels within target range could potentially increase their cure rates. [Display omitted] Non-DS513464443433418DS472519126 Disclosures:No relevant conflicts of interest to declare.

Physicians compliance during maintenance therapy in children with Down syndrome and acute lymphoblastic leukemia

Children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) have an inferior prognosis compared with non-DS ALL patients. We reviewed methotrexate (MTX)/mercaptopurine (6MP) maintenance therapy data for children with DS treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL92 or the NOPHO ALL2000 protocols between 1992 and 2007. The 5-year event-free survival probability (pEFS(5 yr)) for the 66 DS patients was inferior to the 2602 non-DS patients (0.50 ± 0.07 vs 0.77 ± 0.01 (P<0.001)). The 48 DS patients in first remission at the beginning of maintenance therapy had pEFS(10 yr) below that of the 522 non-DS control patients (pEFS(10 yr): 0.58 (95% confidence interval (CI) 0.43-0.77) vs 0.83 (95% CI 0.80-0.86), respectively (P<0.0001)). The DS patients received lower median doses of MTX (median: 11.8 vs 15.4 (P<0.0001)) and 6MP (median: 43.6 vs 59.4 (P<0.0001)). In Cox regression analysis, male gender, presence of DS and high median maintenance therapy white blood cell levels (mWBC) were associated with increased risk for relapse. DS-ALL patients with mWBC above or below 3.5 × 10(9)/l (protocol target) had pEFS(10 yr) of 0.31 and 0.72 (P=0.02), and the mWBC hazard ratio for DS-ALL patients was 2.0 (P<0.0005). We conclude that insufficient treatment intensity during maintenance therapy of DS-ALL patients may contribute to their poor prognosis.

Myelosuppression and infectious complications in children with down syndrome and acute lymphoblastic leukemia

Children with Down syndrome (DS) bear an increased risk of acute lymphoblastic leukemia (ALL) and treatment complications. We compared blood counts and toxicities in 22 DS and 44 non-DS ALL patients. Patients with DS had deeper, longer neutrophil and monocyte count nadirs; more toxicities (HR 2.0, P = 0.0005); longer hospitalizations (HR 1.4, P < 0.0001); and more frequent microbiologically documented infections (HR 5.7, P = 0.0019), mucositis (HR 29.0, P = 0.0006), and cellulitis (HR 3.0, P = 0.033). Severe neutropenia, monocytopenia, and increased cellulitis in DS-ALL suggest the importance of skin hygiene, vigilance and aggressive treatment of cutaneous infections.

Genomic profiling in Down syndrome acute lymphoblastic leukemia identifies histone gene deletions associated with altered methylation profiles

Patients with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) have distinct clinical and biological features. Whereas most DS-ALL cases lack the sentinel cytogenetic lesions that guide risk assignment in childhood ALL, JAK2 mutations and CRLF2 overexpression are highly enriched. To further characterize the unique biology of DS-ALL, we performed genome-wide profiling of 58 DS-ALL and 68 non-DS (NDS) ALL cases by DNA copy number, loss of heterozygosity, gene expression and methylation analyses. We report a novel deletion within the 6p22 histone gene cluster as significantly more frequent in DS-ALL, occurring in 11 DS (22%) and only 2 NDS cases (3.1%) (Fisher's exact P=0.002). Homozygous deletions yielded significantly lower histone expression levels, and were associated with higher methylation levels, distinct spatial localization of methylated promoters and enrichment of highly methylated genes for specific pathways and transcription factor-binding motifs. Gene expression profiling demonstrated heterogeneity of DS-ALL cases overall, with supervised analysis defining a 45-transcript signature associated with CRLF2 overexpression. Further characterization of pathways associated with histone deletions may identify opportunities for novel targeted interventions.

Deletions in B-Cell Development Genes in Down Syndrome ALL: IKAROS Causes Poor Outcome

Abstract 3963Poster Board III-899Children with Down Syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL). DS ALL patients differ in presenting characteristics from ALL patients without DS (non-DS ALL). Recent evidence suggests that a unique genetic event may characterize DS ALL, i.e. an activating mutation localized at R683 in the Janus Kinase 2 (JAK2) gene, which occurs in 18% of DS ALL cases, and differs from the JAK2 mutations that are typically found in Myeloproliferatieve diseases (Bercovich et al, The Lancet 2008). However, Mullighan et al. recently reported also JAK R683 mutations in non-DS high risk ALL (PNAS, 2009). Furthermore, we and others described deletions in B-cell development genes in high-risk ALL (Den Boer et al, Lancet Oncology 2009, and Mullighan, NEJM 2009). One of these genes, IKZF1, which encodes the lymphoid transcription factor IKAROS, was found to be an indicator of poor prognosis in this high-risk group. In the present study, we studied deletions in B-cell development genes in DS ALL, utilizing array-Comparative Genomic Hybridisation (array-CGH, 105K Agilent). Moreover, genomic DNA was PCR-amplified with specific primers to detect the isoform 6 of IKAROS, which consists of a deletion of exon 3-6 resulting in expression of a dominant-negative form of IKAROS, with intact homodimerization but reduced DNA-binding capacity. We used direct sequencing for mutation screening of the pseudo-kinase and kinase domains of JAK2. Of 34 DS ALL patients treated according to the DCOG treatment protocols samples were available. All 34 patients had B-cell precursor ALL. Median follow up time was 5.7 years (range 1.2 – 15.4 years).In total, 19/34 (56%) DS ALL patients had one or more deletions in B-cell development genes (Table 1). Affected genes included the transcription factors IKZF1 (41%, n=14), PAX5 (12%, n=4) and VPREB1 (18%, n=6). These aberrations were not mutually exclusive. No deletions were found in EBF1 and TCF3 (E2A). Deletions in the PAX5 gene were part of larger deletions (≥0.5 million bases), whereas the other genes were mainly affected by focal deletions (<0.5 million bases). In 10/14 cases with IKZF1 abnormalities, the Isoform 6 was detected, whereas in 4 patients the entire IKZF1 gene was deleted due to a larger deletion on chromosome 7p. There were 2 cases with hyperdiploidy and 2 with a TEL/AML rearrangement; 3 of them had a deletion in one of the named transcription factors. One DS patient with a Philadelphia chromosome had a focal deletion in IKZF1 resulting in isoform 6, as well as a deletion in PAX5 and VPREB1. JAK2 mutations were detected in 5/34 patients (15%). Only one of the JAK2 R683 mutated DS ALL patients had a deletion of IKZF. Patients with an IKZF1 deletion had a significantly worse outcome when compared to patients without IKZF1 deletion (pEFS 57% vs. 95%; p=0.005), pDFS (62% vs. 95%; p=0.01) and pOS (71% vs. 95%; p=0.04). For PAX5 deleted cases, the pOS was 50% vs. 90% in non-PAX5 deleted cases (p=0.03), but the differences for pEFS (50% vs. 83%; p=0.14) and pDFS (50.0% vs. 86%; p=0.06) were not significant. None of the JAK2 mutated patients had an event. Multivariate Cox regression analysis including age, WBC, JAK2, TEL/AML, IKZF1, PAX5 and VPREB1 showed that deletion of IKZF1 was the only independent prognostic factor for event free survival (RR 23.5; p=0.03). In fact, 6 of the 7 patients (86%) with an event had a deletion of IKZF1, of whom 2 had a deletion of the entire IKZF1 gene, and 4 patients had a focal deletion resulting in isoform 6. In conclusion, we found deletions in B-cell development genes in a comparable frequency as in high-risk types of ALL without DS. Especially a high incidence of IKAROS deletions was found, which identified an independent poor prognostic group within the DS ALL patients. The high frequency of IKZF1 deletions may in part be responsible for the worse prognosis of DS ALL compared to non-DS ALL as reported by some groups.Chromosomal locationNumber*Cytogenetics- Hyperdiploidy-2/29 (6.9%)- BCR/ABLt(9;22)1/26 (3.8%)- TEL/AMLt(12;21)2/34 (5.9%)JAK2 R683 mutation9p245/34 (14.7%)B-cell development genes- IKZF17p1214/34 (41.2%)- VPREB122q11.226/34 (17.6%)- PAX59p13.24/34 (11.7%)- EBF15q33.30- TCF319p13.30*Cytogenetic information was not always available for all patients Disclosures:No relevant conflicts of interest to declare.

Gene Expression Profiling in Down Syndrome Acute Lymphoblastic Leukemia Identifies Distinct Profiles Associated with CRLF2 Expression Status.

Abstract 2389Poster Board II-366Children with Down syndrome (DS) have a 10 to 20-fold increased risk of developing acute lymphoblastic leukemia (ALL), and they have experienced poorer outcomes on recent major protocols worldwide. The cytogenetic abnormalities which are generally common in childhood ALL and contribute to risk-based treatment assignment are markedly less frequent in children with DS-ALL. Recently, activating mutations in Janus kinase 2 (JAK2) have been identified in approximately 20% of DS-ALL, and interstitial deletions involving cytokine receptor-like factor 2 (CRLF2) in approximately 50% of DS-ALL. Global gene expression profiling may provide insights into the biologic consequences of these molecular lesions. We performed microarray analysis of RNA from diagnostic bone marrow samples in 23 DS-ALL and 26 non-DS ALL cases using the Affymetrix Human Genome U133 Plus 2.0 array. CRLF2 expression was high in 10 of the 23 DS-ALL cases, 3 of which also bore JAK2 mutations, and in a single non-DS ALL case. Unsupervised hierarchical clustering analysis demonstrated clustering of non-DS ALL cases belonging to known cytogenetic subgroups such as E2A-PBX1, MLL rearrangement, and high hyperdiploidy. In contrast, neither DS-ALL cases overall nor the JAK2-mutated or high CRLF2 expressing cases formed a cohesive cluster. Supervised analysis identified 43 genes that were differentially expressed between CRLF2 high versus low cases with a false discovery rate <10%. Several of the most highly differentially expressed genes were validated by quantitative real-time PCR. These included three genes with high expression in CRLF2-high cases: chemokine (C-C motif) ligand 17 (CCL17) (p=0.01), V-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES1) (p=0.007), and Iroquois homeobox 2 (IRX2) (p=0.008); and one gene with expression inversely correlated with CRLF2 expression: dual specificity phosphatase 6 (DUSP6) (p=0.0015). Our findings suggest that DS-ALL does not form a single distinct biologic subgroup, but nearly half of DS-ALL cases are defined by high CRLF2 expression, a substantial enrichment for this lesion compared to its prevalence in non-DS ALL. Identification of downstream pathways may identify opportunities for targeted intervention, including interactions with other cytokines and activation of the JAK-STAT pathway. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Gene Expression Profiling Differentiates Childhood Acute Lymphoblastic Leukemia in Down Syndrome Versus Non-Down Syndrome Patients.

Children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) form a unique biological subset. These patients have generally inferior outcomes in many studies, and an increased incidence of treatment-related toxicities. Cases of DS ALL have a much lower frequency of recurrent prognostically significant chromosomal abnormalities than cases of ALL in the general pediatric population. Global gene expression profiling provides an opportunity to gain insights into pathogenesis and potential therapeutic targets in DS ALL. We performed microarray analysis of RNA from bone marrow samples obtained at diagnosis in 30 DS ALL and 24 non-DS ALL cases using the Affymetrix Human Genome U133 Plus 2.0 array. Unsupervised hierarchical clustering separated cases into two main groups, one of which included 21 of 30 DS samples (Fisher's exact test, p = 0.013), suggesting inherent biologic similarities. Non-DS samples clustered according to known cytogenetic features. Consistent with recently published data, a subset (13%) of our DS ALL cases were found to have JAK2 mutations. Cases of DS ALL bearing JAK2 mutations did not form a distinct subcluster, suggesting that the JAK2 pathway may be dysregulated via other events in cases of DS ALL with wild-type JAK2. Two-sample comparison of DS versus non-DS ALL cases demonstrated differential expression of 513 genes with p values <0.001 (Figure 1). Oxidative phosphorylation pathway genes were most over-represented among differentially expressed genes, with 35 of 115 genes in this pathway demonstrating down-regulation in DS compared to non-DS ALL (Bonferroni corrected p value < 1 ×10−9), including several cytochrome c oxidase and ubiquinone subunits. Our data indicate that DS ALL blasts may utilize oxidative phosphorylation to a lesser extent than non-DS ALL, a feature which could be exploited therapeutically.The top 513 genes differentially expressed in DS versus non-DS ALL (Benjamini-Hochberg corrected p values < 0.001) are displayed in a heatmap where genes relatively overexpressed in DS ALL are depicted in yellow, and relatively underexpressed in DS ALL in red. DS ALL cases are indicated by red circles and non-DS ALL cases by white circles. Four DS ALL cases bearing a JAK2 mutation at arginine 683 are indicated by black stars. [Display omitted]

Differential in vitro cytotoxicity does not explain increased host toxicities from chemotherapy in Down syndrome acute lymphoblastic leukemia

Treatment-related toxicities such as mucositis and infections are both more frequent and more severe in children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) compared to non-DS ALL. Altered methotrexate pharmacodynamics play a role, but severe toxicities also occur in treatment courses that lack methotrexate. We hypothesized that this might be attributable to heightened cytotoxic effects of other ALL chemotherapeutic agents on DS versus non-DS host tissues. Panels of DS and non-DS lymphoblastoid cell lines (LCLs) and primary fibroblast cell lines were treated with asparaginase, dexamethasone, doxorubicin, mafosfamide and vincristine. LCL survival was assessed using the MTT assay, and fibroblast proliferation using the clonogenic survival assay. No significant differences were observed between DS and non-DS cell lines using either assay. Both DS and non-DS cell lines were resistant to dexamethasone at the maximal concentrations tested, and did not differ significantly in sensitivity to the other drugs studied. Thus, heightened in vitro cytotoxicity does not appear to account for the increased treatment-related toxicities observed in patients with DS ALL.

Comparison of the biology of Down syndrome (DS) acute lymphoblastic leukemia (ALL) and non-DS ALL: Children’s Oncology Group (COG) study P9900

10003 Background: DS ALL is a heterogeneous disorder with inferior survival compared to non-DS. Controversy exists regarding the relative frequencies of sentinel cytogenetic lesions in children with DS and ALL. Methods: COG P9900 was a laboratory classification study for ALL therapeutic trials from 12/99 to 2/05. Flow cytometry for DNA index; molecular testing for TEL-AML1, E2A-PBX1, BCR-ABL and FISH testing for trisomies 4 and 10 and for MLL rearrangements were done centrally for risk group stratification in a prospective manner to assign continuing treatment. Results: Eighty of 2811 (3%) consecutively enrolled eligible B-precursor ALL patients (pts) had DS. Age > or < than 10 years of age, gender, presenting white blood cell count (WBC) and NCI risk group were similar between ALL patients with/without DS. However, the genetic lesions present in DS-ALL patients and the event-free survival (EFS) and overall survival (OS) differed from those of non-DS patients. No BCR/ABL or MLL translocations were found in the DS pts. TEL-AML1 fusion was present in 2.5% (2/80) of DS-ALL pts vs. 24% (651/2710) (p<0.0001) of non-DS-ALL pts. Trisomies of chromosomes 4 and 10 were present in 7.7% (6/78) of DS-ALL pts vs. 24% (643/2689) (p=0.0004) of non-DS-ALL pts. Five-year EFS and OS were inferior in pts with DS-ALL; 70% vs. 76% (p=0.078), and 86% vs. 90% (p=0.0333). However, when pts with MLL, BCR/ABL, TEL-AML 1 and trisomies 4 and 10 were excluded, the EFS and OS for both groups were equivalent with EFS for DS 68% vs. 70% for non-DS (p=0.8174), and OS for DS 87% vs. 85% for non-DS (p=0.8519). Conclusions: The inferior outcome of patients with ALL and DS is related to a much lower incidence of favorable genetic features (TEL- AML 1 and trisomies of chromosomes 4 and 10). Further investigation into the distinct pathogenesis of DS ALL is warranted to improve treatment response. No significant financial relationships to disclose.