Down Syndrome Acute Lymphoblastic Leukemia Research Articles

Asparaginase-Associated Toxicities and Hypersensitivity Reactions in Pediatric and Adolescent Down Syndrome Patients with Acute Lymphoblastic Leukemia or Lymphoma

Children with Down syndrome acute lymphoblastic leukemia (DS-ALL) have worse prognosis compared to those with non-DS-ALL, largely due to higher treatment related mortality. Pegaspargase (PEG) is integral in protocols for the treatment of pediatric ALL and lymphoblastic lymphoma (LBL), and full exposure to all doses of PEG can be limited by toxicities, including hypersensitivity reactions (HSR) due to the production of anti-asparaginase antibodies rendering PEG therapy ineffective. To date, minimal data has been reported on the use of PEG in children with DS-ALL/LBL. We conducted a prospective, multicenter study to evaluate the efficacy of premedicating pediatric and adolescent ALL/LBL patients receiving PEG with antihistamines-H1 and -H2 blockers with subsequent therapeutic drug monitoring (TDM) to better identify patients with true HSR versus those with non-antibody mediated infusion reactions. Here, we report on the incidence of HSRs and PEG-associated toxicities in the cohort of children with DS-ALL/LBL. Eligible patients from six centers received PEG, as per a Children's Oncology Group protocol, beginning in induction therapy. An H1 blocker (diphenhydramine) and H2 blocker (ranitidine, famotidine or cimetidine) were administered prior to PEG administration in consolidation and subsequent cycles. TDM was performed at 7 and 14 days post each PEG. PEG was administered at 2500 International units/m 2/dose that was capped for obesity or age > 22 years. Patients were monitored for HSR and other PEG-related toxicities using CTCAE version 5 criteria. Therapeutic asparaginase activity (AA) was defined as ≥ 0.1 IU/mL (≥100 IU/L) at 7 days post infusion and greater than the lower level of quantification at 14 days. We observed for silent inactivation (subtherapeutic levels without symptoms) and accelerated clearance (levels below quantification at 14 days without symptoms). Descriptive statistics summarized patient characteristics and mixed effects modeling procedure assessed mean AA levels at 7 and 14 days with specification of repeat measures and compound symmetry covariance structure. Out of 148 patients, 9 DS (8 B-ALL, 1 B-LBL) were enrolled between 10/2019 - 3/2022. The median age at diagnosis was 5.0 years [IQR: 4.3, 14.9], 4 were National Cancer Institute (NCI) High Risk (HR) at diagnosis, 4 were Hispanic and none were obese. ALL 9 DS patients received a total of 26 doses of PEG at the standard dose without dose capping required. The mean unadjusted AA levels at 7 and 14 days post PEG were 0.76 IU/ml (SE=0.11) and 0.54 IU/ml (SE=0.11), respectively, p<.001. None of the DS patients experienced HSR or silent inactivation. Accelerated clearance was observed in 1 patient. Nineteen episodes of grade >3 PEG-associated toxicity were observed in 5 DS-ALL patients (5/9; 55.6%). PEG-associated toxicity was observed more frequently in older patients whose mean age was 13 years (SD=6.2) versus 4.3 years in those without toxicity (SDE=0.9), p=.028. Four of the DS-ALL who experienced toxicities were NCI HR at diagnosis and > 10 years of age. For the 19 episodes of toxicities: 6 occurred during induction, 9 during consolidation, and 4 during delayed intensification. The most common toxicity observed was hepatoxicity (increased alanine transaminase) which was experienced by 3 DS-ALL patients. There were no grade > 3 hyperbilirubinemia. All 4 DS-ALL patients who experienced PEG toxicity during induction also had toxicity with subsequent cycles. One DS-ALL patient had a dose limiting toxicity (grade 3 pancreatic pseudocyst), requiring discontinuation of subsequent PEG therapy. There were no grade 4 or 5 toxicities observed. All 4 DS patients who did not experience toxicities were NCI Standard Risk patients. All DS patients had undetectable minimal residual disease (MRD) that was < 0.01 at the end of induction therapy. We noted that in this small DS-ALL/LBL cohort, none of the patients experienced HSR, and all achieved therapeutic AA at both time points of 7 and 10 days post PEG. Five (55.6%) of the DS-ALL patients experienced at least one grade > 3 asparaginase toxicity, and they were older than those without toxicity. Most toxicity occurred during the consolidation phase and no grade 4 or 5 toxicities were observed. Given the PEG-associated toxicity observed in DS ALL/LBL, we are conducting pharmacokinetic analysis to propose a lower dose of PEG which may minimize toxicity while maintaining therapeutic efficacy.

Outcome of ALL in Adult Patient with Down Syndrome, Single Center Experience

Background: Individuals with down syndrome (DS) have an approximately 20-fold increased risk of developing acute lymphoblastic leukemia (ALL) and significantly worse outcomes due to a higher relapse rate and treatment related-toxicities including severe mucositis and prolonged hospitalization. Previous studies showed that, among the pediatric age group, the transplant-related mortality (TRM) of allogenic HSCT in DS-ALL is 39%, though subsequent studies attributed treatment failure to leukemia relapse. Chimeric antigen receptor therapy is a newly emergent therapy, used on a limited number of children. Currently, there is insufficient data regarding the outcome and optimal treatment strategy for adult patients with DS-ALL at time of diagnosis or relapse. Method: We retrospectively reviewed the outcome of adult patients who were initially treated or continued their treatments after being transferred from pediatric institutes to Princess Margaret Cancer Centre over the last 20 years. Adult patients with DS and de-novo ALL (diagnosed after the age of 18 years) and those with late relapsed DS-ALL who had not previously received intensive asparaginase therapy were treated according to a modified Dana Farber Cancer Institute (DFCI) ALL protocol. This protocol includes an at least four-fold higher total dose of E. coli-derived asparaginase compared to other adult regimens. Reduced doses of methotrexate were used due to the increased risk of mucositis reported in children. After 2018, relapsed patients received either CAR-T or Blinatumomab. Results: Thirteen adult patients with DS-ALL were treated at our center. Nine of them were diagnosed with de novo adult DS-ALL and four with late relapsed disease as adults after previous treatment for childhood DS-ALL. There was no CNS involvement in all patients at diagnosis or relapse. The median age of de-novo adult DS-ALL was 30 years (range 21-46 years). Seven patients (77.7%) achieved complete remission (CR) after initial induction with DFCI; two patients (22.3%) died within 60 days of induction due to sepsis. Meanwhile, the median age of patients with late relapses was 12.5 years (range 7-15 years) at initial diagnosis, and 23.5 years (15-36 years) at relapse. Four of the seven (57%) de-novo adult DS-ALL patients relapsed after CR1 of 11, 35, 36, 48 months. Two of them received palliative therapy, one received re-induction with HyperCVAD, and one with Blinatumomab, achieving CR2 of 3 and 8.5 months, respectively. Unfortunately, all relapsed de-novo DS-ALL patients have died. The remaining three of the seven (43%) patients are alive and in continuous CR1 at 129, 36 and 33 months. Two of the four patients with late relapse DS-ALL received re-induction with DFCI, achieving CR2 of 8 and 15 months, followed by a second refractory relapse. The third patient died during re-induction by HyperCVAD due to septic shock. The last patient received CART-Cell therapy and achieved CR2 for 27 months. He is currently in remission after one cycle of Blinatumomab. The overall and relapse-free survival of adult patients with DS-ALL at 3 years was 77% and 69.2% respectively, and thus shows inferior results when compared to a similarly treated population of adults (aged 18-35 years) without DS (3-year OS 83%, 3-year RFS 77%) at our center. De-novo DS-ALL overall and relapse-free survival was 66.6% and 55.5%, respectively. Conclusion:To our knowledge, we are describing the largest single-centre retrospective study involving DS in adult ALL patients. We found that the observed barriers to successful treatment were similar to those in pediatric cases. Pediatric chemotherapeutic regimens can be used for adult DS-ALL but are associated with more toxicities and higher relapse rates when compared to adult without DS. CAR-T therapy should be considered a first-line treatment at the time of relapse. Blinatumomab and Inotuzumab can be used as third-line treatments for second relapse. Ultimately, patients who are fit for allogenic bone marrow transplant, should consider it as a curative option during second remission. Future studies involving patients with DS-ALL should explore the use of Blinatumomab and Inotuzumab in combination with first-line chemotherapies.

HMGN1 expression Predisposes Down Syndrome Patients to Develop P2RY8-CRLF2 acute Lymphoblastic Leukemia

IntroductionChildren with Down Syndrome (DS) frequently develop hematological malignancies including acute lymphoblastic leukemia (ALL), however, the genomic basis for the predisposition to DS-ALL remains unknown. DS-ALL is associated with increased rates of relapse, chemotherapy related toxicity and poor overall survival compared to non-DS patients. The P2RY8-CRLF2 (CRLF2r) gene fusion, the result of deletion of the pseudoautosomal region 1 (PAR1) on the X/Y chromosome (chr), has been identified in 60% of DS-ALL (+21) patients and 30% of iAMP21 (intrachromosomal amplification of chr21) ALL patients compared to only 5-16% of other B-ALL patients. The high mobility group nucleosome binding protein 1, encoded by HMGN1 on chr21 may play a role in DS-ALL leukemogenesis, due to its demethylase activity associated with transcriptional activation.MethodsmRNA seq was performed on the blast cells of 76 ALL patients using the Universal Plus mRNA-seq with NuQuant kit. FusionCatcher, SOAPfuse and JAFFA were used to identify fusions and featureCounts used for mRNA expression of STAR (v2.7.3a) aligned BAM files. CRISPR/Cas9 gRNAs targeting P2RY8 intron 1 or CRLF2 5' UTR created the 320 KB (PAR1) deletion. Cas9/gRNAs were transduced into Jurkat cells ± HMGN1 overexpression, activated with doxycycline and stained with TSLPR (CRLF2/IL-7Rα dimer) for single cell sort and clonal expansion. Phosphorylated proteins were measured by intracellular flow cytometry and SYBR Green RQ-PCR was used for mRNA expression.ResultsSignificantly higher HMGN1 expression was identified in CRLF2r ALL patients, compared to the control (BCR-ABL1+ patients. Fig. 1A; p<0.0001). There was no significant difference in HMGN2 (p=0.7881) or JAK2 (p=0.1171) control genes between groups. Of the CRLF2r ALL patients, 26% harbored +21, and 5% iAMP21, strongly suggesting a relationship between chr21, HMGN1 and the CRLF2r fusion.To further understand the role of HMGN1 in CRLF2r development, HMGN1 was overexpressed (1.5-fold; HMGN1H) in Jurkat Cas9 cells to represent a trisomy level of expression. gRNAs targeting P2RY8 and CRLF2 were activated in cells ± HMGN1H with undirected repair. TSLPR surface and CRLF2 mRNA expression were higher in CRLF2r + HMGN1H cells compared to CRLF2r only cells (Fig. 1B,C; p=0.034 and <0.001 respectively). This suggests higher HMGN1 expression significantly predisposed to PAR1 deletion and formation of CRLF2r fusion after a double stranded DNA break (DSB) versus repair where HMGN1 expression is normal. This is consistent with higher HMGN1 expression observed in patients with CRLF2r. There was also increased gene editing activity in CRLF2r + HMGN1H cells, identified through T7 gene editing assay, compared to CRLF2r only cells.CRLF2r + HMGN1H cells also demonstrated significantly increased pSTAT5 (MFI: 2359 ± 1; Fig. 1D), pAKT (MFI: 2339 ± 6; Fig. 1E) and pERK (MFI: 2478 ± 48; Fig. 1F) compared to CRLF2r only cells (MFI pSTAT5: 1910 ± 10; pAKT: 1727 ± 14; pERK: 1946 ± 6; all p<0.001), consistent with the data observed in CRLF2r patients, and confirming cooperation between CRLF2 and HMGN1. CRLF2r + HMGN1H cells demonstrated reduced H3K27me3 (MFI: 1 ± 0.07) compared to CRLF2r only cells (MFI: 1.6 ± 0.03; p=0.003) and higher expression levels of STAT5 target genes (BCL2, CDKN1,MCL1and MYC).ConclusionThe P2RY8-CRLF2 (CRLF2r) gene fusionis prevalent in DS, +21 and iAMP21 ALL patients. Here, we demonstrate that CRLF2r is associated with high expression of HMGN1 (chr21)in ALL patient cells . Using CRISPR/Cas9 in an in vitro model, we demonstrate that enforced high expression of HMGN1 alters DSB repair mechanism, favoring PAR1 deletion and the subsequent formation of the P2RY8-CRLF2 gene fusion, with attendant higher expression of STAT5 target genes. Understanding the role of HMGN1 in the disproportionate number of DS-ALL patients who are diagnosed with CRLF2r has the potential to lead to novel therapeutic interventions, in this high-risk group of patients where efficacious therapeutic options are currently poor. [Display omitted] DisclosuresYeung: BMS: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria. White: Novartis: Research Funding; BMS: Honoraria, Research Funding.

Tisagenlecleucel Therapy Is Safe and Effective for Children with Down Syndrome with ALL in First Relapse

Introduction: Children with Down Syndrome (DS) have a 20-fold increased incidence of acute lymphoblastic leukemia (ALL) and a 70% higher risk of relapse than children with ALL and no DS. Treatment for DS children with relapsed ALL is challenging due to the high treatment-related mortality associated with both intensive chemotherapy and hematopoietic stem cell transplantation (HSCT) as well as high rates of second relapse. Outcomes for relapsed ALL in DS are dismal, with recent studies citing an event-free survival (EFS) as low as 17%. Alternative treatment approaches are needed for this patient population. Treatment with anti-CD19 chimeric antigen receptor T-cells (CARTs) have shown high remission rates in patients with multiple relapsed/refractory ALL with an acceptable safety profile. CARTs are generally not indicated for first relapse of ALL; DS-specific indications for CARTs do not exist. In Ontario, due to the specific susceptibility of children with DS-ALL, CARTs were made available to this population upon first relapse. We describe the population-based Canadian experience using CARTs for first relapse of DS-ALL.Methods: Efficacy and safety data on all children referred for CARTs at the Hospital for Sick Children, in Toronto, Canada were retrospectively collected in a central database and validated by treating clinicians. Here we report all patients with DS suffering a first relapse of ALL at age <18 years referred for commercially available CARTs (tisagenlecleucel) between 2019 and 2021.Results: Five patients with DS received tisagenlecleucel for first relapse of ALL during the study period. Median age at original diagnosis was 6.3 years (range 3.4-11.5). Four patients presenting with National Cancer Institute (NCI) standard risk and one with NCI high-risk disease were initially treated on Children's Oncology Group based protocols. Four patients presented with relapse >36 months from diagnosis and one patient with early relapse. Mononuclear cells were successfully collected for all patients on the first attempt. Patients were bridged from collection to infusion with low intensity chemotherapy regimens. Tisagenlecleucel was successfully manufactured for all patients and infused at a median of 69 days (range 56-101) from collection of T cells. Four patients developed CRS: two patients grade 1, one patient grade 2, and one patient grade 4 requiring ICU care and treatment with tocilizumab and steroid. All patients achieved minimal residual disease (MRD) negative remission one month after infusion, with B-cell aplasia. Two patients showed early (< 6months post infusion) B-cell recovery consistent with CAR T-cell loss; both received off-label reinfusions of tisagenlecleucel and again achieved B-cell aplasia. One patient had a seizure with the first Fludarabine dose of lymphodepleting (LD) chemotherapy for re-infusion and was found to have elevated intracranial pressure. After treatment with Topiramate and anti-seizure prophylaxis the patient tolerated LD chemotherapy without further neurological events. No patient received further ALL therapy; none went on to HSCT. One patient suffered a CD19-negative relapse 7 months post infusion and was transitioned to palliative-intent treatment. Two patients developed prolonged neutropenia with one patient showing recovery (ANC>1x10E9/l) at 5 months post infusion and the patient who had relapsed disease remained neutropenic until relapse. Infectious complications in the first 6 months post infusion were rare. One patient, who was not neutropenic, required two hospital admissions for line related sepsis. With a median follow-up time of 519 days (range 197-785), the 1-year estimated EFS and overall survival were both 80% (95 th confidence interval 52-100%; Figure 1).Conclusions: Tisagenlecleucel was safe and effective in a small cohort of children with DS and first relapse of CD19+ ALL, with achievement of long-term remissions without further therapy, and an EFS superior to that seen in historical cohorts. Rate of acute complications were not increased compared with reported outcomes in patients without DS. Further evaluation of tisagenlecleucel in this setting is warranted. [Display omitted] DisclosuresKrueger: KITE/Gilead: Consultancy; Novartis: Consultancy; SOBI: Consultancy. Whitlock: Sobi Pharmaceuticals: Consultancy; Novartis: Research Funding; Amgen; Jazz Pharmaceuticals: Honoraria. Gupta: Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure:tisagenlecleucel for early B-cell recovery

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.

STAT3 Is Activated By DYRK1A and Is a Potential Therapeutic Target in B-ALL

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that mediates signal transduction from the extracellular surface to the nucleus. Canonically, STAT3 is phosphorylated at Tyrosine 705 (Y705) by JAK family kinases, which promotes its dimerization and subsequent localization to the nucleus. However, the role of Serine 727 (S727) phosphorylation in regulating STAT3 activity varies across cell types and remains unclear in hematopoietic tissues particularly. Several studies indicate that phosphorylation at S727 is critical for optimal STAT3 function. For example, astrogliogenesis is regulated by enhancing STAT3 activity by phosphorylation of S727 by DYRK1A. Of note, DYRK1A is overexpressed in Down syndrome-acute lymphoblastic leukemia (DS-ALL), and has previously been found to phosphorylate substrates in order to prime them for downstream phosphorylation events. Given these findings, we hypothesized that the DYRK1A phosphorylation of STAT3 at S727 is critical for promoting DS-ALL. Furthermore, certain subtypes of ALL have high rates of JAK2 activation, namely DS-ALL and Philadelphia-like ALL (Ph-like ALL); we propose that STAT3 can effectively be targeted specifically in these subtypes.In order to elucidate the role of DYRK1A phosphorylation of STAT3, we treated cytokine-deprived murine pre-B cells with EHT1610, a selective DYRK1 inhibitor, or vehicle and then pulsed the cells with JAK-STAT activating cytokines. EHT1610-treated cells had diminished S727 phosphorylation compared to vehicle, regardless of cytokine pulse; however, only vehicle-treated cells regained Y705 phosphorylation after cytokine pulse. This suggests that S727 phosphorylation is cytokine-independent and is critical for maintenance of Y705 phosphorylation. We then generated flag-tagged STAT3 S727 phospho-mimetic (S727D/E) and phospho-deficient (S727A) alleles and transduced them into pre-B cells. We observed that the degree of Y705 phosphorylation is dependent on S727, as cells expressing S727A have reduced Y705 phosphorylation compared to wild-type STAT3. Additionally, overexpression of the phospho-deficient allele conferred a significant proliferative impairment compared to the phospho-mimetic alleles.As DS-ALL and Ph-like ALL often have JAK2-activating mutations, we next aimed to determine if loss of S727 phosphorylation would decrease ALL cell growth. Indeed, two human Ph-like ALL cell lines, MHH-CALL4 and MUTZ5, displayed decreased proliferation when overexpressing the S727A mutant. These cell lines were also sensitive to treatment with C188-9, a small molecule STAT3 inhibitor that is in clinical trials for various solid tumors. Additionally, we treated primary patient ALL samples with amplification of HSA21 segments ex vivo and found that DS-ALL samples were preferentially sensitive to STAT3 inhibition compared to HD-ALL or iAMP-ALL, suggesting that STAT3 is specifically a target in JAK2-activated ALL.Our study provides new and significant insights into the regulation of STAT3 by DYRK1A, and presents a new therapeutic target for ALL cells with JAK2 activating mutations. DisclosuresBourquin:Amgen: Other: Travel Support. Crispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

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.

IKZF1 and 22q11.22 Deletions and PDGFRA Gains Are Associated with Poor Outcome in Down Syndrome Acute Lymphoblastic Leukemia

AbstractAbstract 289▪▪This icon denotes a clinically relevant abstractChildren with Down syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL), and consistently demonstrate poorer outcomes due to higher rates of both relapse and treatment-related mortality compared to other children with ALL. The biology of ALL in DS is unique, with lower frequency of the classic cytogenetic lesions generally observed in childhood ALL, and increased frequency of JAK2 mutations and CRLF2 rearrangements, which are not clearly associated with adverse prognosis in DS-ALL. In order to improve risk stratification and identify potential novel therapeutic targets in this vulnerable population, we analyzed 90 DS-ALL cases for prognostically significant copy number abnormalities (CNAs) in a collaborative cohort from the Children’s Oncology Group (n=30), St. Jude Children’s Research Hospital (n=22), AIEOP (n=16), Texas Children’s Cancer Center (n=10), UKALL 2003 (n=6) and an archival UK sample (n=1), and Utah’s Primary Children’s Medical Center (n=5). Copy number profiling was performed using 500K, 6.0, CytoScan HD, and OncoScan FFPE Express arrays (Affymetrix), and Human CNV370-Duo arrays (Illumina). Gene expression profiling was performed using U133 Plus2.0 arrays (Affymetrix). Copy number was analyzed with Nexus Copy Number (BioDiscovery, Inc.) and gene expression was analyzed with Partek Genomics Suite (Partek, Inc.) and Gene Set Enrichment Analysis (Broad Institute). Deletions of a focal region on 22q11.22 (present in 28.9% of cases, similar to the incidence previously reported in a non-DS cohort [Mangum et al, ASH 2011:741]), and deletions of IKZF1 (present in 20.0% of cases), were significantly associated with poor event-free survival (EFS) (5-year EFS was 88.6 ± 6.3% in wild-type cases [n=31], 68.1 ± 13.3% in cases with deletion of 22q11.22 only [n=15], and 60.0 ± 21.9% in those with deletion of IKZF1 only [n=5]; combined deletion [n=6] was associated with an even poorer EFS (33.3 ± 19.3%, p<0.0001, Figure 1A). Patients were observed to have focal deletions on 22q11.22 spanning up to 235 kb; the most common recurring shared region is just under 10 kb in length, and does not contain known coding genes. Increased number of copies of a 14 kb region in the platelet-derived growth factor receptor alpha gene (PDGFRA), located at 4q12, occurred in 35.5% of cases performed on microarrays containing evaluable probes in this region, and were significantly associated with poor outcome (4-year EFS 87.5 ± 8.3% in wild-type cases [n=20] and 33.9 ± 17.6% in PDGFRA gain cases [n=11], p = 0.004, Figure 1B). Five cases contained 3 copies of PDGFRA, and six cases had at least 4 copies, with no correlation between outcome and number of copies gained. PDGFRA rearrangements and point mutations have been reported in other malignancies, but copy number gain is a novel mechanism of alteration. Cases with a focal 22q11.22 deletion were associated with an increased frequency of CRLF2 rearrangement (75% versus 47%, Fisher’s exact p=0.028). No other differences in age, initial WBC, or CRLF2 rearrangement in cases with versus without the focal 22q11.22 deletion, IKZF1 deletion, or PDGFRA gain were present, nor were there significant differences in incidence of focal PDGFRA gains in cases with versus without IKZF1 or 22q11.22 deletions. Gene expression profiling in 27 evaluable cases demonstrated upregulation of genes with kinase activity in 12 cases with either focal 22q11.22 or IKZF1 deletions. This study represents one of the largest collaborative cohorts of DS-ALL for genomic profiling, confirms the poor prognosis associated with IKZF1 deletion (Buitenkamp et al., Leukemia 2012), and identifies two additional genomic loci strongly associated with poor prognosis. If validated in additional DS-ALL cohorts, these findings suggest key lesions that contribute to the poor outcomes observed in this population. These specific genomic changes may improve risk stratification of treatment in children with DS and ALL, and may lead to enhanced sensitivity to tyrosine kinase inhibitors in the estimated 60% of cases bearing one or more of these three lesions. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Outcome of Treatment for Relapsed Acute Lymphoblastic Leukemia in Children with Down Syndrome

AbstractAbstract ▪669▪This icon denotes a clinically relevant abstract Background.Children with Down syndrome (DS) have a higher risk for developing not only acute lymphoblastic leukemia (ALL) but also significant adverse effects of chemotherapy compared to the overall pediatric population. Currently, it is unknown how children with DS, who develop a relapse of ALL, respond to treatment protocols that were optimized in the pediatric population without DS. We hypothesized that a concomitant diagnosis of DS is an independent prognostic factor of survival after treatment for relapsed ALL and that decreased tolerance of therapy impairs the success of relapse treatment in children with DS. Patients and Methods.The probability of event-free (EFS) and overall survival (OS) and the causes of treatment failure were determined for 51 children with DS and a matched cohort of 102 children without DS among 2736 children and young adults (up to 22 years of age) who were treated for relapsed ALL on a series of clinical trials conducted by the ALL-REZ-BFM Study Group between 1983 and 2012. Results.Among children with DS, who were enrolled on clinical trials of the ALL-REZ-BFM Study Group, ALL relapse more frequently exhibited favorable prognostic characteristics compared to the unmatched population of 2579 patients without DS. High risk forms of relapse were less frequent among children with DS (risk group S4, 10 vs. 28%, p<0.001) including relapse at a very early and early time point (35 vs. 56%, p<0.001). A higher proportion of relapse in DS involved the bone marrow (94 vs. 83% p<0.001). Transcripts of ETV6-RUNX1 or BCR-ABL1 were detected in none of the cases of relapsed ALL in DS compared to 20% (231/895, p<0.001) and 6% (83/1379, p=0.02) of cases in the population without DS. Treatment for relapsed ALL in children with DS less frequently included irradiation (of the central nervous system or total body) and hematopoietic stem cell transplantation compared to the matched non-DS group (28 vs. 70%, p<0.001, and 15 vs. 40%, p=0.002), respectively. Despite the apparent favorable risk profile, EFS and OS were lower in children with DS than the matched control group without DS (EFS 16 ± 08% vs. 39 ± 06%, p=0.005; OS 17 ± 08% vs. 48 ± 06%, p<0.001). Fatal adverse events of treatment developed more frequently in children with DS than the control group (36 ± 07% vs. 9 ± 03%, p<0.001). In contrast, the cumulative incidence of a subsequent relapse was similar in both groups (32 ± 07%, DS vs. 36 ± 05%, non-DS, p=0.373). The proportion of children with DS registered on clinical trials for relapsed ALL after frontline treatment on ALL-BFM trials showed an increase over time (from 1 registered vs. 1 non registered patient in ALL-BFM 81 to 18 registered vs. 8 non registered patients in ALL-BFM 2000). Furthermore, the proportion of patients with high risk relapse features (S4 group) and DS increased from 5 to 15%. During the more recent study period (2000–2012) EFS and OS were no longer significantly different in children with and without DS (EFS 30 ± 09% vs. 35 ± 09%, p=0.403; OS 30 ± 11% vs. 51 ± 08%, p=0.158).DS was an independent prognostic factor of outcome after relapse of ALL in multivariate analysis. Conclusion.A higher rate of induction deaths and treatment-related mortality was the main barrier to successful outcomes of relapse therapy in children with DS whereas relapse rates were not different from patients without DS. An increased representation of children with DS including those with high risk features in recent time suggests that access to clinical trials for relapsed ALL has been successfully broadened for children with DS. Specific optimization of treatment modifications and supportive care have improved survival in children with DS and are suggested to further decrease the number of fatal treatment-related events during relapse therapy in this group. Disclosures:No relevant conflicts of interest to declare.

Outcome in children with Down's syndrome and acute lymphoblastic leukemia: role of IKZF1 deletions and CRLF2 aberrations

Children with Down's syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL) and have a low frequency of established genetic aberrations. We aimed to determine which genetic abnormalities are involved in DS ALL. We studied the frequency and prognostic value of deletions in B-cell development genes and aberrations of janus kinase 2 (JAK2) and cytokine receptor-like factor 2 (CRLF2) using array-comparative genomic hybridization, and multiplex ligation-dependent probe amplification in a population-based cohort of 34 Dutch Childhood Oncology Group DS ALL samples. A population-based cohort of 88 DS samples from the UK trials was used to validate survival estimates for IKZF1 and CRLF2 abnormalities. In total, 50% of DS ALL patients had ≥1 deletion in the B-cell development genes: PAX5 (12%), VPREB1 (18%) and IKZF1 (35%). JAK2 was mutated in 15% of patients, genomic CRLF2 rearrangements in 62%. Outcome was significantly worse in patients with IKZF1 deletions (6-year event-free survival (EFS) 45 ± 16% vs 95 ± 4%; P=0.002), which was confirmed in the validation cohort (6-year EFS 21 ± 12% vs 58 ± 11%; P=0.002). This IKZF1 deletion was a strong independent predictor for outcome (hazard ratio EFS 3.05; P=0.001). Neither CRLF2 nor JAK2 were predictors for worse prognosis. If confirmed in prospective series, IKZF1 deletions may be used for risk-group stratification in DS ALL.

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 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]

Prevalence and Clinical Correlates of JAK2 Mutations in Pediatric Down Syndrome Acute Lymphoblastic Leukemia.

Approximately three-quarters of pediatric acute lymphoblastic leukemia (ALL) cases harbor recurrent, prognostically significant chromosomal abnormalities which affect assignment of risk group and therapeutic regimen, but these abnormalities occur far less often in children with Down syndrome (DS) and ALL. Recently, novel somatic activating mutations in Janus kinase 2 (JAK2) were reported to occur uniquely in DS ALL, affecting 18% of patients (Bercovich et al, in press). Here we report the identification and clinical correlates of JAK2 mutations in an independent cohort of cases. We sequenced JAK2 exon 16 and identified mutations in 10 of 53 DS ALL cases (18.9%). All were heterozygous point mutations affecting arginine 683 (Table 1). No JAK2 mutations were identified in the 25 available paired germline DS samples, including five germline samples associated with JAK2 mutated ALL cases, indicating that mutations were somatic in these cases. Eight of the JAK2 mutations (80%) occurred in males (p<0.03), whereas the full cohort contained 24 males (45%). No significant correlation was observed between JAK2 mutation status and age, initial white blood count (WBC), or 5-year event-free survival or overall survival. Our results confirm the 18% prevalence of JAK2 mutations at arginine 683 in DS ALL, but differ from the prior report in finding a male predominance of JAK2 mutations, and no association with age or initial WBC, differences which may be attributable to sample size. These results confirm that activation of the JAK-STAT pathway via a specific JAK2 mutation occurs in approximately one-fifth of cases of DS ALL.SequenceAmino acidCases (n)AGA (wild-type)Arginine43GGAGlycine6AGTSerine2AGCSerine1ACAThreonine1Table 1.JAK2mutation status in pediatric DS ALL

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.

Acute lymphoblastic leukemia and Down syndrome

The presenting features and treatment outcome of 120 patients with Down syndrome (DS) and childhood acute lymphoblastic leukemia (ALL) were compared with 6237 non-DS patients treated in the same years. We reviewed the database of 6 consecutive Italian Association of Pediatric Hematology and Oncology (AIEOP)-ALL trials conducted between 1982 and 2004. Features of DS patients were compared with those of non-DS patients. The 120 DS patients (1.9%) were more often girls (P = .027), aged > or = 10 years (P = .014), and high risk according to National Cancer Institute (NCI) criteria (P = .045). The distribution of white blood cell count did not differ (P = .32). DS patients belonged less frequently to the current high-risk group (P = .017). In all but 1 case they demonstrated B-cell precursor (BCP) immunophenotype (P < or = .001). TEL/AML1 molecular fusion transcript was found in only 1 of 44 (2.2%) tested patients. Induction death occurred more often in DS patients (4.2%, P = .009), but not failure to achieve remission. Leukemia relapse occurred in 31.6% of DS patients (vs 23.5%; P = .003), usually in the marrow. Remission death was more frequent in DS patients (4.2%, P = .03). Ten-year event-free survival and survival were significantly worse compared with non-DS patients (P < 0.001). DS patients diagnosed since 1995 had a better outcome (P = .06) than those diagnosed in previous years, but still had worse outcomes than non-DS patients (P = .04). Event-free survival of DS patients at NCI standard risk was lower than that of non-DS patients (P = .006). Presenting features of childhood ALL in DS differ from those in non-DS patients. They are almost invariably characterized by BCP phenotype, and are often TEL/AML1 negative. Treatment results, although not as good as for non-DS patients, improved progressively, with modern therapy and support allowing 75% to survive.

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.

Down syndrome, drug metabolism and chromosome 21.

It has been recognized that chromosomal abnormalities in childhood leukemia, are linked to both leukemogenesis and segregate patients into prognostic treatment groups. This is best exemplified in cases of children with Down syndrome (DS), who have significantly higher risks of developing leukemia compared to non-DS children and distinctive treatment outcomes, particularly in cases of acute myeloid leukemia (AML). The high event-free survival (EFS) rates of DS AML patients and in particular, patients with megakaryocytic leukemia (AMkL), at least in part reflects an increased sensitivity to cytosine arabinoside (ara-C) secondary to increased expression of the chromosome 21-localized gene, cystathionine-beta-synthase, and potentially global mechanisms which increase the susceptibility of cells to undergo apoptosis. Somatic mutations of the X-linked transcription factor gene, GATA1, have been detected uniformly and exclusively in DS AMkL cases, which may lead to altered expression of GATA1 target genes and alter the metabolism of drugs including ara-C. Hyperdiploid acute lymphoblastic leukemia (ALL) cells with extra copies of chromosome 21, generate higher levels of the active methotrexate (MTX) metabolite, MTX polyglutamates. This is on account of increased intracellular transport of MTX via the reduced folate carrier (RFC) whose gene is localized to chromosome 21 and may also account for the increased MTX-associated toxicity of DS ALL patients. Microarray technology should lead to the identification of additional gene targets linked to the treatment response of specific cytogenetic leukemia subgroups.

Different drug sensitivity profiles of acute myeloid and lymphoblastic leukemia and normal peripheral blood mononuclear cells in children with and without Down syndrome

Children with Down syndrome (DS) have an increased risk for leukemia. The prognosis for DS acute myeloid leukemia (AML) is better than for non-DS AML, but the clinical outcome of DS acute lymphoblastic leukemia (ALL) is equal to that of non-DS ALL. Differences in prognosis may reflect differences in cellular drug resistance. In vitro drug resistance profiles were successfully investigated on leukemic cells from 13 patients with DS AML and 9 patients with DS ALL and were compared with reference data from 151 non-DS AML and 430 non-DS B-cell precursor (BCP) ALL. DS AML cells were significantly more sensitive to cytarabine (median, 12-fold), the anthracyclines (2-7-fold), mitoxantrone (9-fold), amsacrine (16-fold), etoposide (20-fold), 6-thioguanine (3-fold), busulfan (5-fold), vincristine (23-fold), and prednisolone (more than 1.1-fold), than non-DS AML cells. Compared with DS ALL, DS AML cells were significantly more sensitive to cytarabine only (21-fold). After short-term exposure to methotrexate, DS AML cells were 21-fold more resistant than non-DS AML cells, but no difference was observed after continuous exposure. DS ALL cells and non-DS BCP-ALL cells were equally sensitive to all drugs, including methotrexate. Normal peripheral blood mononuclear cells from DS and non-DS children without leukemia showed highly resistant drug profiles. It was concluded that the better prognosis of DS AML might, at least partially, be explained by a specific, relatively sensitive drug-resistance profile, reflecting the unique biology of this disease. (Blood. 2002;99:245-251)