Syndrome AML Research Articles

Integrative Genome and Transcriptome Sequencing Analysis Indicates Genetic and Epigenetic Dysregulation in DS-AML

Down Syndrome related AML is driven by GATA1 alterations in patients with trisomy 21. Using exome or targeted sequencing, prior studies identified GATA1 alterations in ~80-90% of patients. We performed whole genome sequencing coupled with whole transcriptome sequencing on 207 cases to study the genomic basis of Down Syndrome AML. We detected GATA1 alterations in 96.1% of cases, with additional structural rearrangements in GATA1 in 15% of cases that may have been missed in prior studies. For GATA1, although substitutions and small indels occur nearly uniformly across exon 2, we observed that internal tandem duplications (ITDs) are significantly enriched in the second half of exon 2, suggesting a specific mutational mechanism for these cases. We also detected exon 3 (instead of exon 2) deletion in one case, where the resultant truncated GATA1 protein is different from the commonly recognized sGATA1. Our study further revealed that mutations in genes mediating JAK-STAT signaling (JAK1/JAK2/JAK3), RAS pathway (KRAS/NRAS/NF1), Cohesin complex (STAG2/CTCF/RAD21/SMC3), as well as MBNL1, KANSL1, IRX1, and NFIA. By integrating the genome with transcriptome sequencing data, we discovered that GATA1 exon 2 skipping to be a significant event in DS-AML. Although exon 2 is nearly completely skipped in cases with exon 2 deletion or splice site alterations, significant exon 2 skipping is also observed in cases where the alteration is small and is in the middle of exon 2. Further, exon 2 skipping is also observed in cases wither the alteration is a single base substitution that results in stop codon in the middle of exon 2 (therefore splicing is unlikely affected by these alterations in such cases). Based on this observation, we propose a hybrid genetic-epigenetic model on the development of Down Syndrome AML. In DS-AML, GATA1 exon 2 skipping during transcription/splicing might be a developmentally (epigenetically) regulated event in early fetal development that is destined to be silenced in the postnatal period that results in activation of the canonical long GATA1. This epigenetic silencing of sGATA1 is overridden by secondary genetic events, thus maintaining sGATA1 as the predominant GATA1 protein. Notably, the sGATA1 have variable isoforms as a result of exon 2 or exon 3 loss. In summary, our integrated whole genome and transcriptome approach has resulted in discovery of novel and high-prevalence structural alterations in GATA1 and potentially novel genes involved in pathogenesis of DS-AML. Our integrated analysis provided evidence on the epigenetic/developmental regulation of GATA1 exon 2 skipping via alterative splicing in the context of trisomy 21 that warrants further investigation.

Cancer Predisposition Genetic Testing Among Children with Hematologic Malignancies

Introduction: There is growing recognition of germline cancer predisposition syndromes (CPS) that increase risk of hematologic malignancies including leukemias, lymphomas, bone marrow failure syndromes and myeloproliferative diseases. Somatic (tumor) testing can identify potential germline variants based on multiple factors including variant allele fraction, sequence characteristics, and other findings; however, subsequent germline testing is required to characterize the lesion. Paired tumor and germline testing (T/N testing) is more accurate to diagnose CPS and may mitigate loss to follow-up, reduce time to CPS diagnosis, and possibly alleviate unnecessary anxiety due to suspected but ultimately disproved germline conditions. Previously, our center performed tumor-only diagnostic testing for patients with newly diagnosed hematologic malignancies. In 2020 we transitioned to paired T/N testing for all new hematologic malignancy diagnoses with patient/caregiver consent. Here we report the frequency of CPS diagnoses suggested by tumor-only testing and subsequently confirmed with germline testing, as well as CPS diagnoses identified from the outset by T/N testing. Methods: Data was automatically abstracted from the Children's Hospital of Philadelphia (CHOP) Diagnostic Genomics Database (FileMaker and Nexus) for 1057 pediatric, adolescent, and young adult patients who underwent testing with the CHOP Comprehensive Hematologic Malignancy panel between 2016 and 2023. This panel interrogates 117 genes for single nucleotide variants, insertions, deletions, and copy number variants. Of those tested, 932 patients underwent tumor-only testing, and 125 patients underwent paired T/N testing. Characteristics of patients with possible pre-established genetic syndromes (e.g. Trisomy 21) were cross-checked via manual chart review. Results: Schematic representation of patient testing is summarized in Figure 1. Among 932 patients who underwent tumor-only testing, 98 were identified to have potential or previously established germline pathogenic variants. Of these, 9 had known genetic syndromes and 8 had non-cancer related genetic diagnoses (e.g. Usher syndrome). Of the 81 patients with a suspected CPS, 26 were referred from an outside hospital with unavailable follow-up data. Among suspected germline patients from our center (n=55), 34 (62%) did not undergo confirmatory testing. Of the 21 (38%) who underwent confirmatory testing, 2 (10%) were diagnosed with a CPS. For patients with T/N testing, 16 of 125 (7%) were identified to have germline conditions; 8 were pre-established diagnoses and 4 were non-cancer related genetic diagnoses. Thus, 4 (3%) of patients with T/N testing were newly diagnosed with a CPS. New CPS diagnoses included: ETV6 thrombocytopenia and leukemia predisposition syndrome, CBL syndrome, GATA2 MDS/AML predisposition syndrome, Noonan syndrome, and CEBPA AML predisposition syndrome. Conclusion: At our center, 62% of patients with suspected germline conditions on tumor-only testing had no follow up testing documented in the available medical record. These data suggest that paired T/N testing at the time of hematologic malignancy diagnosis assists in mitigating loss-to-follow up for patients with possible CPS. Additionally, T/N testing may streamline result interpretation, reduce time to diagnosis, aid in treatment decisions, and guide cascade testing for potentially affected family members. When possible, genetic testing decisions and results should be discussed with a trained genetic counselor. Over time, increasing utilization of paired T/N testing may also enhance understanding of CPS-associated genes that increase risk of hematologic malignancies.

Prognostic Relevance of Minimal Residual Disease in Therapy Related and Secondary Acute Myeloid Leukemia Receiving CPX-351 or Fludarabine-Based Induction

Background: The high prognostic value of minimal residual disease (MRD) assessment in Acute Myeloid Leukemia patients (AML) undergoing intensive induction therapy has been confirmed by several studies. The most widely adopted MRD techniques include multicolor flow cytometry (MFC), RT-PCR for recurrent genetic lesion and, for patients lacking specific markers, RT-PCR for the pan-leukemic marker WT1. However, mostly younger patients receiving a conventional 3+7 induction are included in clinical studies evaluating MRD in AML. As a consequence, secondary AML, evolving from a previous myelodisplastic syndrome (s-AML) or therapy-related AML (t-AML), which usually affect elderly AML patients are usually under-represented in those trials. Recently, CPX-351 was approved for the treatment of s-AML and t-AML, following the positive results of the Phase III trial by Lancet et al. However, as MRD was not assessed in this trial, only a limited ammount of data are available on the kinetics and the prognostic value of MRD in elderly s- AML and t-AML patients receiving CPX-351 induction. Aims: In order to assess the prognostic value and to define the best time points for MRD assessment, MFC-based and WT1 based MRD was evaluated in a cohort of elderly s-AML or t-AML patients receiving CPX-351 induction. Timepoints for assessment were after induction (TP1), after first consolidation (TP2) and at the end of treatment (TP3). As a comparison, we also evaluated MRD in a cohort of elderly s-AML and t-AML patients who received an age-adjusted fludarabine-containing regimen (FLAI3). MRD analysis in this cohort was performed with the same methodics and at the same timepoints. Methods: A total of 151 elderly (>60 year, median age 68, range 60-77) patients were analyzed in this study. Patients were treated between January 2005 and January 2020 in our Center, either with CPX-351 (n=50) or fludarabine- high dose cytarabine-idarubicin (FLAI), with (n=72) or without (n =29) gemtuzumab-ozogamicin (GO). MRD was analyzed in all patients achieving hematological complete remission (CR) with both MFC and WT1 expression levels. All patients were affected by s-AML or t-AML, defined according to the WHO 2016 criteria. Results: After first induction cycle, 95 patients (59%) achieved CR. Patients treated with CPX-351 had a significantly higher CR rate, if compared to patients receiving FLAI (40/50, 80% and 55/101, 54.5%, respectively, p<0.05). Adding GO to FLAI did not increase CR rate. Overall, in the two cohorts, an MRD negative CR was achieved in 51 (53.7%) and 53 responding patients (55.8%) assessed with MFC or WT1-based methods, respectively. The probability of achieving MFC MRD negativity was significantly higher among patients treated with CPX-351, when compared to patients receiving FLAI with or without GO (MFC MRD negativity rate of 26/40, 65% and 25/55, 45%, respectively, p<0.05). In both cohort, the most informative timepoint in terms of survival prediciton was after first cycle (TP1). WT1-based MRD assessment led to superimposable results. In multivariate analysis, MRD was significantly correlated with an higher probability of longer Overall Survival (OS) in all treatment group (2-year OS of 34% and 77% in patients with or without residual MFC MRD after induction, respectively, p<0.05). Allogeneic stem cell transplantation consolidation (HSCT) was also significantly correlated with longer OS in both treatment groups (p<0.05). Notably, 12/40 (30%) CR patients treated with CPX 351 underwent HSCT, whereas only 8 CR patients proceeded to HSCT in the FLAI group (15%) Conclusions: MRD assessment has a strong prognostic value also in the context of elderly s-AML and t-AML patients. WT1-based and MFC-based MRD assessment lead to similar conclusions thus allowing to obtain MRD data from virtually all AML patients treated in the selected time period. Overall, CPX-351 resulted in higher CR rate with deeper MRD responses, if compared to FLAI3. This increased efficacy translated in a significantly higher number of elderly AML patients proceeding to HSCT in CR, which is the only curative approach in this particular AML setting.

PB1849: TRIAL IN PROGRESS: PHASE IB/II STUDY OF SIREMADLIN IN COMBINATION WITH VENETOCLAX + AZACITIDINE IN PATIENTS WITH ACUTE MYELOID LEUKEMIA (AML) WHO ARE INELIGIBLE FOR INTENSIVE CHEMOTHERAPY

Background: Treatment options for patients with AML who are not candidates for intensive chemotherapy (IC) are limited. The addition of venetoclax to hypomethylating agents demonstrated improved remission rates and survival; however, relapse rates are high, and most patients remain measurable residual disease (MRD) positive (DiNardo CD, N Engl J Med, 2020). Murine double minute-2 (MDM2) is a key negative regulator of tumor protein 53 (p53). Inhibition of the p53-MDM2 interaction results in up-regulation of the tumor-suppressive effect of p53. Siremadlin is a selective inhibitor of the p53-MDM2 interaction. Siremadlin + venetoclax has shown durable antitumor responses in p53-wild-type AML patient-derived xenograft models (Wang Y, Cancer Res, 2019). Findings from a first-in-human Phase I study with siremadlin demonstrated a tolerable safety profile and preliminary antitumor activity in patients with AML (Stein EM, Clin Cancer Res, 2021; NCT02143635). Preliminary results from the ongoing Phase Ib study exploring siremadlin in combination with venetoclax showed promising antileukemic activity in patients with relapsed/refractory AML (Wei AH, ASH 2021; NCT03940352). Aims: Here we describe the study design of a Phase Ib/II study to assess the safety and efficacy of the novel combination of siremadlin + venetoclax + azacitidine in patients with AML who are unfit for IC (NCT05155709). Methods: The study will enroll approximately 55 adult patients with AML who are unfit for IC due to older age (≥75 y) or presence of comorbidities (cardiac or pulmonary, Eastern Cooperative Oncology Group performance status of 2 or 3). Patients with prior myelodysplastic syndrome, myelofibrosis, essential thrombocythemia, polycythemia, or therapy-related AML may also be eligible. Patients with TP53 mutation or del17p are excluded. Two subpopulations will be evaluated in 2 arms: patients with prior suboptimal response (no complete remission [CR], CR with partial/incomplete hematologic recovery [CRh/CRi], or morphologic leukemia-free state) after 2-4 cycles of first-line venetoclax + azacitidine (Arm 1) and patients with newly diagnosed AML with high-risk clinical features that confer a low likelihood of response to venetoclax + hypomethylating agents (adverse genetic risk stratification per European Leukemia Network, 2017; Arm 2). The study will consist of a safety run-in (Part 1) and expansion phase (Part 2; Figure). The safety run-in will evaluate safety across 2 siremadlin dose levels (20 or 30 mg) from Day (D)1 to D5 in each 28-d treatment cycle in combination with venetoclax and azacitidine. In Arm 1, pts will continue receiving their venetoclax and azacitidine doses that were evaluated as tolerable prior to study enrollment. In Arm 2, venetoclax will be ramped up over 3 days to a target daily dose of 400 mg and azacitidine (75 mg/m2) will be administered from D1–D7 or from D1–D5, and D8 and D9). Upon confirmation of the recommended dose for expansion (RDE, determined for each arm separately), enrollment for the expansion phase (Part 2) will open; patients will be treated with siremadlin at the RDE in combination with venetoclax + azacitidine. The primary endpoints are incidence of dose-limiting toxicities (Arm 1 and Arm 2 separately) and CR rate (Arm 1 only). Secondary endpoints include safety and tolerability; CR rate (Arm 2 only); CR/CRh and CR/CRi rates; duration of CR, CRi, and CRh; overall survival; early mortality; MRD; and pharmacokinetics. Results: None, trial in progress. Image:Summary/Conclusion: None, trial in progress.

Treatment patterns and outcomes of 2310 patients with secondary acute myeloid leukemia: a PETHEMA registry study

AbstractSecondary acute myeloid leukemia (sAML) comprises a heterogeneous group of patients and is associated with poor overall survival (OS). We analyze the characteristics, treatment patterns, and outcomes of adult patients with sAML in the Programa Español de Tratamientos en Hematología (PETHEMA) registry. Overall, 6211 (72.9%) were de novo and 2310 (27.1%) had sAML, divided into myelodysplastic syndrome AML (MDS-AML, 44%), MDS/myeloproliferative AML (MDS/MPN-AML, 10%), MPN-AML (11%), therapy-related AML (t-AML, 25%), and antecedent neoplasia without prior chemotherapy/radiotherapy (neo-AML, 9%). Compared with de novo, patients with sAML were older (median age, 69 years), had more Eastern Cooperative Oncology Group ≥2 (35%) or high-risk cytogenetics (40%), less FMS-like tyrosine kinase 3 internal tandem duplication (11%), and nucleophosmin 1 (NPM1) mutations (21%) and received less intensive chemotherapy regimens (38%) (all P < .001). Median OS was higher for de novo than sAML (10.9 vs 5.6 months; P < .001) and shorter in sAML after hematologic disorder (MDS, MDS/MPN, or MPN) compared with t-AML and neo-AML (5.3 vs 6.1 vs 5.7 months, respectively; P = .04). After intensive chemotherapy, median OS was better among patients with de novo and neo-AML (17.2 and 14.6 months, respectively). No OS differences were observed after hypomethylating agents according to type of AML. sAML was an independent adverse prognostic factor for OS. We confirmed high prevalence and adverse features of sAML and established its independent adverse prognostic value. This trial was registered at www.clinicaltrials.gov as #NCT02607059.

Bcl2 Inhibitor and DNA Hypomethylating Agent Prolong Survival in Patient-Derived Xenograft Model of Down Syndrome Myeloid Leukemia By Synergistic Downregulation of Cytokine Signaling

Background:Down syndrome is known for its leukemia predisposition effects. Children with Down syndrome myeloid leukemia who fail chemotherapy, have a poor outcome. No targeted therapies are available for this population. We have generated and extensively characterized several patient-derived xenograft (PDX) models of Down syndrome AML (Barwe et al., 134:2683, Blood, 2019). We evaluated the efficacy of a combination of DNA hypomethylating agent, azacitidine and Bcl2 inhibitor, venetoclax in PDX model of Down syndrome AML. Although this drug combination is used in the clinic for the treatment of adult AML, their combinatorial mechanism of action is not well known.Methods:NTPL-386 (3 x 10 6 cells) were injected intravenously in NSG-SGM3 mice. Disease progression was monitored by determination of the percentage of human chimerism in peripheral blood by flow cytometry. Mice were randomly assigned to treatment groups (n=5) on day 13 when human CD45+ cells were detectable in blood. Azacitidine (2.5 mg/Kg) was administered intraperitoneally on days 13-19 and venetoclax (100 mg/Kg) was delivered orally on days 13-40 (Fig. 1A). Mice were monitored daily for pre-determined experimental endpoints and were euthanized when any of the endpoints were attained. Kaplan-Meier survival plots were generated. AML cells were harvested from treated mice and lysed, RNA was isolated and sequenced. Pathway analysis was performed to identify the mechanism of action of these two drugs in combination.Results:Azacitidine treatment for one week increased median survival by 8 days compared to untreated mice (Fig. 1B). Venetoclax treatment for 4 weeks was more effective than azacitidine and extended survival by 27 days (P<0.005, compared to untreated). We observed that the combination of venetoclax and azacitidine was much more efficient and prolonged survival by 39 days in this PDX model of refractory Down syndrome AML (P<0.005 and P<0.05 compared to single agent azacitidine or venetoclax respectively). This combination performed better than chemotherapy consisting of cytarabine and daunorubicin, which showed a 11-day improvement in median survival compared to untreated in our previous study (Barwe et al., 134:2683, Blood, 2019).To understand the mechanism of synergism between these two drugs, we conducted transcriptome analysis on cells harvested from mice (n=3 per group) receiving venetoclax and azacitidine, alone or in combination. The log fold changes for each treatment with respect to untreated were calculated and filtered based on FDR>0.05. The genes that were differentially regulated in the combination treatment (Fig. 1C, blue circle in the Venn diagram) were subjected to ToppGene analysis. A heatmap of the differentially regulated genes shows clustering of the replicates from individual mice (Fig. 1D). ‘Hematopoietic cell lineage’ and ‘cytokine signaling in immune system’ were the top modulated pathways in the combination treatment. A heatmap of the logFC of the differentially regulated genes from this pathway showed the modulation of these genes in the combination in comparison with individual drug treatment (Fig. 1E). The differentially regulated genes in the combination treatment that showed high variance from the range defined by individual drug treatment belonged to one of three categories - cell surface proteins, cytokines, and stem cell markers. These data indicate that the synergistic downregulation of cytokine signaling is likely responsible for the combinatorial effect of venetoclax and azacitidine.Conclusion:The combination of venetoclax and azacitidine was effective in prolonging survival in a highly refractory Down syndrome AML PDX model (NTPL-386) generated in the laboratory. The top genes differentially regulated by the combination treatment which varied the most in magnitude from the individual drug treatment identified genes that are likely to contribute to the synergism between the two drugs. [Display omitted] DisclosuresBarwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

Prognostic Impact of Minimal Residual Disease Assessment in Elderly Patients with Secondary Acute Myeloid Leukemia. a Comparison between CPX-351 and Intensified Fludarabine-Based Regimens

Background: Minimal residual disease (MRD) assessment retains high prognostic value in Acute Myeloid Leukemia patients (AML) undergoing intensive induction therapy. Widely available MRD techniques include multicolour flow cytometry (MFC), RT-PCR for recurrent genetic lesion and, for patients lacking specific markers, RT-PCR for the pan- leukemic marker WT1. However, most of the data on the prognostic value of MRD come from trials including younger patients treated with conventional 3+7 regimen. AML arising from a previous myelodisplastic syndrome (s-AML) and therapy-related AML (t-AML) are usually under-represented in trial involving younger patients and are unlikely to respond to conventional induction. Few data are therefore available on the kinetics and the prognostic value of MRD in elderly s- AML and t-AML patients, particularly in the context of alternative induction regimens.Aims: We evaluated MRD in a cohort of elderly s-AML or t-AML patients receiving induction therapy either with a fludarabine-containing regimen or CPX-351, in order to compare the probability of achieving MRD negativity, to disclose the prognostic value of MRD in this setting and to define the best time-points for MRD assessments.Methods: A total of 136 elderly (>60 year, median age 67, range 60-75) patients were analyzed in this study. Patients were treated between January 2005 and January 2020 in our Center, either with CPX-351 (n=35) or fludarabine- high dose cytarabine-idarubicin (FLAI), with (n=72) or without (n =29) gemtuzumab-ozogamicin (GO). MRD was retrospectively analyzed in all patients achieving hematological complete remission (CR) with both MFC and WT1 expression levels.All patients were affected by s-AML or t-AML, defined according to the WHO 2016 criteria.Results: After induction, CR was achieved in 83 patients (61%). CR rate was 28/35 in patients treated with CPX-351 (80%), significantly higher when compared to patients receiving FLAI (55/101, 54.5%, p<0.05). The addition of GO to FLAI did not increase CR rate. Among CR patients, a total of 41 (49.4%) and 44 patients (53%) achieved MRD negativity, with MFC or WT1-based methods, respectively. MFC MRD negativity probability was higher among patients receiving CPX-351 as induction therapy (MFC MRD negativity rate of 16/28, 57% and 25/55, 45% in CR patients who received CPX-351 or FLAI, respectively, p<0.05). Adding GO to FLAI did not improve MRD negativity probability.Moreover, MRD showed significant prognostic value in terms of Overall Survival in all treatment group (2-year OS of 74 and 36% in patients with or without residual MFC MRD after induction, respectively, p<0.05). WT1-based MRD lead to similar results.Conclusion: In conclusion MRD assessment retains a strong prognostic value and may help to identify patients with suboptimal response to treatment. The higher rate of MRD negativity observed with CPX-351 may be related with a more efficient anti-leukemic activity of the drug in this particular setting. The evaluation of MRD with both MFC and WT1-based assessment lead to superimposable conclusions and allowed us to obtain MRD data from virtually all AML patients treated in the selected time period. DisclosuresNo relevant conflicts of interest to declare.

Epidemiology, Cytogenetic and Molecular Abnormalities and Outcome of Childhood Acute Myeloid Leukemia- an Israeli Retrospective Multicenter Study

IntroductionAcute myeloid leukemia (AML) contributes to 18% of childhood leukemia cases and about 50% of all deaths of pediatric leukemia in the United States .In high-income countries, intensive therapy has increased survival rates to 60-70% . Contributing factors to the improved survival include refinement in the diagnosis of AML and advancement in therapeutic approaches.In light of the ethnic diversity in the Israeli population, we reviewed patient's data and investigate whether the clinical, biologic and molecular features differ between Israeli Arab and Jewish AML patients.Patients and MethodsWe performed a retrospective epidemiological and clinical study including patients younger than 18 years of age, treated at all six Israeli pediatric oncology centers between 2000-2015. Down syndrome and secondary AML were excluded . The clinical characteristics such as gender, age, subtype of FAB, leukocyte count at diagnosis, cytogenetics and molecular markers were analyzed in correlation with their prognosis between different characteristics groups and different ethnicity. Survival analyses were calculated by Kaplan-Meier survival curves and the log rank test. Multivariate analyses on categorized data were performed using Cox proportional hazards model.ResultsWe analyzed 175 patients, 46% females and 54%, males, 15 % younger than 2 years old, 35% from 2 to 10 years and 50% older than 10 years (median age 9.3±5.6 years ). According to FAB subtype, the majority subtypes were M2 and M4 for 26% and 27% respectively , 40% of patients presented WBC count below 10 X 109/L at diagnosis while 13% of patients higher than 100 X 109/L. The most common cytogenetic abnormalities in these children, including t(8;21)(q22;q22), inv(16)(p13.1q22) and 11q23/MLL-rearranged abnormalities were detected, with the occurrence of 18% , 8% and 13% respectively.Acute promyelocytic leukemia (APL) was diagnosed in 14% of the children. Molecular data such as FLT3 were not evaluated due to small numbers.No statistical difference was observed regarding ethnicity as to age, white blood cell (WBC) and central nervous system (CNS) involvement. However, the Arab patients presented with a significantly higher incidence of M2 (42% vs 23% p=0.035), while the Jewish population demonstrated significantly higher incidence of M4 (33% vs. 10% p=0.011). The high incidence of M3 was common to both ethnic groups. In addition, cytogenetic subtype distribution was similar in both ethnic groups.Out of 175 patients, 114 patients were treated in accordance with the same protocol, namely BFM98 . The demographic characteristics of these 114 pts, as well as their WBC, FAB and cytogenetics were distributed similarly to the whole cohort.Among the 114 AML patients, 105 pts achieved complete remission (CR) (92%) after one course of chemotherapy. Of those who achieved CR after 2 induction cycles, 62 pts are alive and free of disease, 38 pts relapsed, 4 pts died in CR (3 due to bacterial infection and 1 due to post transplant lymphoproliferative disease) , one patient developed myelodysplastic syndrome after treatment for APL . Six patients did not go into remission after two induction phases. Three patients had early death (within six weeks of diagnosis), all due to bleeding- 2 were APL and one with MLL split and hyperleukocytosis.The median follow-up was 6.3 years (0.7y-16y), with a 5-year event free survival (EFS) and overall survival (OS) of 54% and 67% respectively.There was no prognostic significance in EFS and OS related to gender, age and ethnicity.As to risk stratification, there was a statistical difference in EFS and OS in the standard risk (SR) and the high risk (HR) group: 5 year EFS SR group 83% and 39% for HR group (p<0.0001) 5 year OS for SR group 98% and 52% for HR group (p<0.0001).ConclusionsIn this retrospective study of pediatric AML patients in Israel,the clinical, laboratory and cytogenetic characteristics were not significantly different between Jewish and Arab patients, with a similar outcome. We report a 14% incidence of APL - an incidence that is higher than that reported for the European and American populations. It may be of interest to further investigate this increased rate of APL in the Israeli population and possibly identify underlying genetic predisposing factors.The pts treated according to AML-BFM-98 protocol without pCRT have a 5y EFS and OS of 54% and 67%, respectively. These outcomes are similar to those reported for BFM98. DisclosuresNo relevant conflicts of interest to declare.

The Efficacy and Safety of Super High-Dose Cytarabine Based Strategies in Childhood Acute Myeloid Leukemia Treatment

Objective The aim of this study was to evaluate the effect of super high-dose cytarabine (SHDAC), a regimen first created by the author, on the five-year overall survival (OS) and event-free survival (EFS) of children with AML. Methods From July 2001 to April 2016, a total of 25 pediatric patients with newly diagnosed AML (exclueding secondary AML、APL and Down Syndrome AML) were enrolled. Patients received consolidate chemotherapy with the same regimen of high-dose cytarabine (3g/m2 every 12 hours by continuous infusion for 6 days) after remission induction chemotherapy. Then phase II consolidation therapy (also known as early intensive treatment) was performed according to the regimen of high-dose cytarabine (3g/m2 every 12 hours on days 1, 3, and 5 for four cycles, a total of 108g/m2) in 16 cases, while the other 9 cases received the treatment with another regimen (H3A7, M3A7 and VP16 combined with cytarabine alternately at intervals ranging from 2 to 6 months). The total treatment course spanned about three years, with regular post-treatment follow-up tracking for more than five years. Results Originally 26 cases were enrolled, but 1 case was not relieved after two courses of treatment and quitted therapy, so only 25 cases were enrolled finally. The total remission rate was 96%. There were 12 cases (48%) achieving complete remission within one course, 12 cases (48%) within two consecutive remission course and one case (48%) ,only one case(4%) achieving complete remission within with in five courses. The five-year OS and EFS of these 25 AML patients in this study were 77±0.98% and 77±1.22% respectively. Four patients (16%) died (1 treatment-related (4%), 3 relapse (12%) including 3 marrow relapse and 1 combined with testicular relapse). The average relapse time was 29.6 months and the median time was 14.8 months. Conclusion The application of super high-dose cytarabine (a total of 108g/m2 cytarabine, with a regimen of 3g/m2 every 12 hours by continuous infusion for 6 days as consolidation chemotherapy and 3g/m2 every 12 hours on days 1, 3, and 5 for four cycles as the early intensive treatment) is clinically safe and effective, with minimal hospitalization expense index (HEI). For patients with financial difficulties, especially those in developing countries, super high-dose cytarabine shows landslide advantages and practicality. Therefore, there should be more collaborative clinical applications as well as research upon related mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Down Syndrome AML Is Unique in Phenotype Both at Diagnosis and in Post Chemotherapy Regeneration

Abstract Background: Down syndrome (DS) acute myeloid leukemia (DS-AML) is predominantly found in young children (under the age of 4) and responds well to chemotherapy. The difference in clinical features suggests that despite the predominance of megakaryoblastic disease, DS-AML blasts are different from non-DS AML blasts with megakaryoblastic morphology (AMKL). The immunophenotypes of DS-AML blasts at diagnosis were compared to those of non-DS AML patients using unsupervised hierarchical clustering analysis (HCA) and assessed for their immunophenotypic overlap with known morphologic subgroups. In addition, residual disease monitoring of non-DS AML patients using "Difference from Normal" flow cytometry has been applied to DS-AML with unexpected results. Methods: A standardized antibody panel was used to define quantitative gene product (antigen) expression in bone marrow (BM) specimens from patients with AML at diagnosis and following chemotherapy. The quantitative phenotypic expression of multiple cell surface markers and physical characteristics defined a position in 15 dimensional data space for each patient. HCA was performed to mathematically cluster DS-AML patients enrolled on Children's Oncology Group study AAML1531 with non-DS patients from study AAML0531 based on 15 dimensional profiles. Five patients with DS who were older than 4 years of age and therefore treated on AAML0531 were also included. Data regarding morphology were appended to the data set. The same antibody panel was used to assess measurable residual disease (MRD) in 147 follow up BM aspirates from 50 DS patients ages 1 to 21 undergoing treatment for either AML or B-ALL (n=46, n=4 respectively) who were not included in the clinical studies. When possible, cell sorting was performed for FISH or SNP/CGH microarray studies. Results: Diagnostic Clustering: HCA of 17 DS-AML diagnostic phenotypes with 769 non-DS AML patients revealed a tight immunophenotypic correlation between 12 (71%) DS-AML patients (Figure 1). Further analysis of this diagnostic subset showed a consistent mean antigen expression profile that was distinct from non-DS AML diagnostic phenotypes. Similarly, the DS-AML patients clustered separately from the 5 older DS patients (&gt;4 years of age) with AML, who were treated on AAML0531 and exhibited consistently different identifying diagnostic markers despite the shared constitutional trisomy 21. Additionally, the phenotype associated with the DS-AML cluster does not match that of patients with AMKL morphology. The largest immunophenotypic cluster of non-DS patients with AMKL morphology were classified with the RAM phenotype[1], which segregate into a mathematically unique branch of the dendogram distinct from the DS-cluster. Observation of a consistent DS-specific non-leukemic myeloid regenerative phenotype: Of the 147 post chemotherapy specimens submitted for MRD analysis off clinical study, 141 (96%) had a clearly identifiable CD34+/CD56+ population present between 0.06-6.0% total non-erythroid composition. A second abnormal population expressing CD34+ but not HLA-DR was also observed in these patients but at a lower level (0.02-2.66%) compared to the CD34+/CD56+ cell population. Two patients had a reproducible absence of 56+, DR- progenitors across all follow up specimens (6/147) and may represent unique clinical cases with disease features beyond our knowledge. In four DS patients undergoing treatment for B-ALL, the same immunophenotypic features (CD34+/CD56+ and negative HLA-DR) were observed. Genetic data: In 4 cases, cell sorting of CD34+/CD56+ post treatment progenitors revealed a lack of cytogenetic markers present in the clone of leukemic blasts at diagnosis. Conclusions: The blasts of patient with typical DS-AML (i.e under the age of 4 years at diagnosis) are immunophenotypically distinct from those of non-DS-AML patients and exhibit consistent immunophenotypic features that differ from DS patients with AML who are older than 4 years of age. The normal myeloid progenitors in DS patients who have undergone chemotherapy for AML or ALL express an immunophenotype that is different from normal BM of non-DS patients recovering from chemotherapy. [1] Brodersen, Lisa Eidenschink, et al. "A Recurrent Immunophenotype at Diagnosis Independently Identifies High Risk Pediatric AML: A Report from the Children's Oncology Group Trial AAML0531." Disclosures Wilson: Hematologics: Employment. Brodersen:Hematologics: Employment. Zehentner:Hematologics: Employment, Equity Ownership. Pardo:Hematologics, Inc: Employment. Wells:Hematologics: Employment, Equity Ownership. Loken:Hematologics: Employment, Equity Ownership.

Survival Advantage and Comparable Toxicity in Reduced-Toxicity Treosulfan-Based versus Reduced-Intensity Busulfan-Based Conditioning Regimen in Myelodysplastic Syndrome and Acute Myeloid Leukemia Patients after Allogeneic Hematopoietic Cell Transplantation

Treosulfan has been incorporated in conditioning regimens for sustained remission without substantial toxicity and treatment-related mortality (TRM). We aimed to analyze the safety and efficacy of a fludarabine 150 mg/m2 and treosulfan 42 g/m2 (FluTreo) conditioning regimen in medically infirm patients. Outcomes were compared with those of a similar historical group treated with fludarabine 150 mg/m2 to 180 mg/m2, busulfan 6.4 mg/kg, and antithymocyte globulin (ATG) 5 mg/kg to 7.5 mg/kg (FluBuATG). Thirty-one consecutive patients with acute myeloid leukemia (AML; n = 21), myelodysplastic syndrome (MDS; n = 6), or treatment-related AML (n = 4) received FluTreo conditioning. The historical group consisted of 26 consecutive patients treated with FluBuATG. In the FluTreo group, engraftment was prompt in all patients and 74% achieved >99% donor chimerism by day +30. No grades III or IV organ toxicities were noted. One-year cumulative incidences (CI) of acute and chronic graft-versus-host disease (GVHD) were 19.4% and 58.4%. The groups were similar for age, disease risk, lines of treatment, hematopoietic cell transplantation–specific comorbidity index, and acute or chronic GVHD incidence, except that there were more matched unrelated donor recipients in the FluTreo group (P < .001). With 20 (range, 2 to 36) months follow-up for FluTreo and 14 (range, 2 to 136) for FluBuATG, the 1-year cumulative overall survival (OS) probability was 76% versus 57%, respectively (P = .026); 1-year disease-free survival (DFS) was 79% versus 38% (P < .001). In multivariate analysis, the only significantly favorable factor for OS and DFS was FluTreo (P = .010 and P = .012). The CI of relapse mortality was markedly decreased in FluTreo versus FluBuATG (7.4% versus 42.3%, P < .001). In conclusion, the treosulfan-based regimen resulted in favorable OS and DFS with acceptable toxicity and low relapse rates compared with busulfan-based conditioning.

Frequency and Prognostic Relevance of Gene Mutations in Pediatric AML Patients At First Relapse.

AbstractAbstract 2480Relapsed pediatric AML patients respond poorly to conventional re-induction therapy and as a consequence long term survival rates of these patients are low (up to 36%). Accurate characterization and risk group stratification at first relapse may facilitate personalized, targeted re-induction therapies for these patients and ultimately improve outcome. Gene mutations provide putative targets in personalized treatment (e.g. FLT3/ITD, KIT or RAS mutations) and their incidence and prognostic relevance in pediatric AML at first relapse remains to be elucidated. Changes in mutational status occur during therapy; hence, assessment of mutations at first relapse is warranted to establish the accurate mutation status of the leukemic cells before the start of salvage treatment.Mutational status at first relapse as well as clinical and karyotypic data were retrospectively analyzed in a large set (n = 240) of relapsed non-FAB M3, non-Down syndrome AML patients younger than 19 years. The majority of patients (88%) were uniformly treated at first relapse according to the ‘Relapsed AML 2001/01' protocol of the International Berlin-Frankfurt-Münster (BFM) study group that involved two re-induction courses of chemotherapy with FLAG as standard treatment with or without liposomal daunorubicin (randomization), followed by allogeneic stem cell transplantation in most cases. Other patients received FLAG based (5%) or other high dose cytarabin based therapy (6%).We screened the relapse samples for hotspot mutations in a selected panel of genes (FLT3, WT1, KIT, N-RAS, K-RAS, NPM1) relevant for AML and found one or more mutations in 139 out of 240 patients (57.9%). Gene mutations were mutually exclusive in 73 out of 139 patients carrying mutations (52.5%), while in 66 out of 139 patients (47.5%) two or more gene mutations were observed. FLT3/ITD mutations coincided with 50% of NPM1 and 53% of WT1 exon 7 mutated cases (p=.003 and p<.0001 respectively). The frequencies of mutations at first relapse are summarized in the Table below including their impact on event free and overall survival, according to uni-variate analysis. In multivariate analyses, we included mutations and other variables (e.g. FAB type, WBC) with a uni-variate P value below 0.2 to exclude confounding factors. From these analyses, three independent factors significantly increased the risk of a second relapse (RFS after first relapse diagnosis); WT1 mutations (HR=11.2, P<.0001), WT1 single nucleotide variants (HR 3.4, P=.003) and FAB type M7 (HR= 2.2, P=.027). Different independent factors were associated with dismal overall survival after first relapse including FLT3/ITD mutations (HR=2.2, P=.028) and high WBC (HR=3.2, P=.004) at first relapse.In conclusion, mutations in the studied panel of genes in this large cohort of pediatric relapsed AML patients were frequent. Overall, we detected receptor tyrosine kinase mutations in more than 25% of the patients, indicating the large proportion of relapsed patients that is eligible for targeted receptor tyrosine kinase inhibitor treatment. The gene mutations studied here provide strong prognostic factors for dismal outcome after first relapse. The relevance of these markers in predicting outcome should be validated in a prospective setting. Accurate risk group stratification of relapsed AML patients should be based on clinical and molecular characterization performed at first relapse. In addition, mutations in drugable genes can identify patients that are eligible for personalized, targeted re-induction strategies.TableUni-variate analysis of the association of relevant gene mutations in pediatric AML at first relapse with outcome after 1st relapse, ranked according to frequency.FrequencyRFS after 1st relapseOS after 1st relapseHazard ratioCIPHazard ratioCIPFLT3/ITD17.8%1.20.6–2.30.5861.40.9–2.40.153WT1 mutations10.2%5.02.0–12.40.0012.00.9–4.10.077WT1 SNV10.2%2.01.1–3.80.0251.00.6–1.70.989KIT9.0%0.50.2–1.30.1370.60.3–1.10.114KRAS7.5%1.60.8–3.30.1760.80.4–1.50.422NRAS c12/136.4%0.60.2–1.90.3761.20.6–2.20.658NPM15.7%0.20.1–1.70.1560.70.3–1.80.519NRAS c611.9%0.10.0–3970.5112.50.9–6.80.072ITD = internal tandem duplication, SNV = single nucleotide variant, c = codon, CI = 95% confidence interval Disclosures:No relevant conflicts of interest to declare.

Combined Targeting of the MET and FGF Receptor Tyrosine Kinases Induces Sustained AML Cell Death by Preventing Compensatory Upregulation of HGF in Response to MET Kinase Inhibition

Abstract 1405▪▪This icon denotes a clinically relevant abstractDespite improvements in the treatment of AML, high-risk disease including 8p11 myeloproliferative syndrome/stem cell leukemia remains largely refractory to current therapy, and is mostly fatal. Identification of effective therapeutic targets by using candidate gene approaches has been limited by the number and variety of genetic defects associated with AML. Based on the results of a genome-wide functional screen in AML using a retroviral library of short hairpin RNAs (shRNAs), we discovered that hepatocyte growth factor (HGF) is aberrantly expressed in about 40% of patients with AML, leading to activation of its receptor MET. Autocrine activation of MET is required for survival of AML cells, as genetic and pharmacologic inhibition of HGF or MET induces apoptosis and inhibits cell growth. However, AML cells treated for more than one week with the MET kinase inhibitor crizotinib develop resistance as a result of 13-fold upregulation of HGF expression, leading to restoration of MET pathway activity, and emphasizing autocrine HGF production as the basis for MET signaling addiction. Using simple thermodynamic models, we find that ligand-mediated receptor activation due to increased ligand expression effectively antagonizes the effects of targeted kinase inhibitors, causing complete restoration of aberrant signaling activity. Here we show that this mechanism of resistance can be overcome by combining MET kinase inhibition with depletion of HGF or targeting of pathways required for compensatory HGF upregulation. In the case of KG-1 cells derived from a patient with 8p11 myeloproliferative syndrome AML with chromosomal translocation mediated activation of FGFR1, we find that signaling downstream of the FGFR is required for the upregulation of HGF that occurs in response to MET kinase inhibition. Sustained MET inhibition due to the blockade of compensatory HGF upregulation leads to durable logarithmic cell kill as a result of combined treatment with the MET kinase inhibitor crizotinib and FGFR kinase inhibitor PD173074. In addition, we find that aberrant expression of HGF is highly correlated with expression of FGF2 (Pearson’s correlation = 0.79, p < 0.001), with 10% of all patients exhibiting co-expression of HGF and FGF2. This co-expression leads to autocrine co-activation of MET and FGFR1, which can be effectively targeted using combined treatment with MET and FGFR kinase inhibitors. In all, these findings suggest a therapeutic strategy for patients with co-activation of MET and FGFR, including patients with 8p11 myeloproliferative syndrome AML, establish a paradigm of effective therapeutic targeting of aberrant HGF/MET signaling in AML, and emphasize the importance of targeting compensatory ligand-dependent receptor tyrosine kinase activation, as required for the development of rational combination therapy. Disclosures:Christensen:Pfizer: Employment. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding.

Access to allogeneic hematopoietic SCT for patients with MDS or relapsed AML treated according to protocols of the Dutch Childhood Oncology Group

To investigate whether all patients in need of an allogeneic hematopoietic SCT (HSCT) are offered one, we retrospectively investigated the policy for all children diagnosed with myelodysplastic syndrome (n=90) or relapsed AML (n=75) between 1998 and 2008. These children are registered at diagnosis and treated according to protocols of the Dutch Childhood Oncology Group, which provides accurate disease incidence data and protocol-indicated appropriateness for HSCT. For 48 (30%) patients, a family donor was identified; for 90 (57%) patients, an unrelated donor (UD) search was performed; and for 21 (13%) patients, no UD search was initiated. Reasons for not initiating an UD search include: progressive disease (n=10), conserve quality of life (n=1), stable disease (n=3), immunosuppressive therapy (n=2), patient death (n=3), patient lives abroad (n=1) and second relapse (n=1). On the basis of the time interval between date of diagnosis and date of death/last follow-up, for eight (5%) patients, it may be questioned why an UD search was not performed. The fact that 95% of all children are given the option of an allogeneic HSCT is encouraging and reasons not to transplant seem fair in most cases.

Primary Treatment of Waldenstrom's Macroglobulinemia with Dexamethasone, Rituximab and Cyclophosphamide (DRC): Long Term Follow-up Analysis of a Phase II Study.

Abstract 2887Poster Board II-863Between November 2002 and April 2006, 72 patients with Waldenstrom's Macroglobulinemia (WM) were enrolled into this multicenter trial of primary treatment with DRC which consisted of dexamethasone 20 mg IV followed by rituximab 375mg/m2 IV on day 1 and oral cyclophosphamide 100 mg/m2 bid on days 1 to 5 (total dose 1000 mg/m2). DRC courses were repeated every 21 days for six courses and then patients without progressive disease were observed without treatment. Patient characteristics, toxicity and response data have been reported previously (Dimopoulos et al, J Clin Oncol 2007; 25:3344): 83% of patients achieved a response including 7% complete, 67% partial and 9% minor responses. In June 2009 we updated this study (minimum follow-up >3 years) in order to assess time to progression, time to next treatment, type of second-line treatment and response to this, overall survival (OS) and cause-specific survival (CSS) in which deaths unrelated to WM or complications of treatment were censored. Second line treatment was administered to patients who experienced progressive disease and also met criteria for treatment requirement based on consensus recommendations (Kyle et al, Sem Oncol 2003; 30:116). As of June 2009, 42 patients fulfilled the criteria for progressive disease (Kimby et al, Clin Lymphoma Myeloma 2006; 6:380) but 14 patients have not yet required second line treatment. The median time to progression was 35 months (95% Confidence Interval: 22-48 months) and the median time to next treatment requirement was 51 months. Among several factors who were analyzed for their possible correlation with shorter time to progression, only lymphadenopathy was significant (p<0.01), while splenomegaly was of borderline significance (p=0.06). Among 28 patients who received second line treatment, 19 patients were retreated with either rituximab alone (n=7) with DRC (n=8) or with rituximab combined with other agents (n=4) and 16 patients (84%) achieved a minor response or better. The remaining 9 patients were treated with alkylating agents (n=3), with nucleoside analogues (n=3) with bortezomib (n=2) or with high dose therapy (n=1) and 6 patients achieved a minor response or better. So far 27 patients (38%) have died including 11 patients from unrelated causes (3 lung cancer, 1 bladder cancer, 1 melanoma, 1 gastric cancer, 1 pancreatic cancer, 2 congestive heart failure, 1 cerebral stroke and 1 pancreatitis). No patient developed myelodysplastic syndrome or secondary AML and one patient developed diffuse large B-cell lymphoma. The probability for 5-year OS and CSS is 59% and 74%, respectively. The International Prognostic Staging System (IPSS) is predictive for both OS and CSS. The probability for 5-year OS is 100%, 67% and 43% for patients with low-, intermediate- and high-risk WM according to IPSS (p<0.01). Furthermore, the probability for 5-year CSS is 100%, 81% and 62% for patients with IPSS low-, intermediate- and high-risk WM (p=0.05). We conclude that this long-term follow-up analysis of the original phase II study showed that the DRC regimen is associated with a significant median time to progression and that most patients who develop disease progression respond again to rituximab-based regimens. So far, this regimen has not been associated with development of secondary myelodysplasia. The DRC regimen represents an active and safe treatment choice for patients with symptomatic WM. Disclosures:No relevant conflicts of interest to declare.

The Predictive Value of TP53 FISH Analysis for Treatment Response and Survival in Cytogenetic Subgroups of AML Patients.

Abstract 2617▪▪This icon denotes an abstract that is clinically relevant.Poster Board II-593In recent years, a number of studies emphasized AML with complex aberrant karyotype as a distinct biological entity which is characterized by a unique gene expression pattern, with very frequent alteration in TP53 and a median overall survival (OS) of less than 6 months. However, the definition of a complex karyotype (CK) or whether monosomal karyotype (MK) provides a better prognostic prediction than CK is not well established. In the present study, we examined whether a prompt and simple Fluorescence in situ hybridization (FISH) test for TP53 deletion (presence or absence of 17p13) at diagnosis, has a predictive value for response to therapy and overall survival in AML patients (pts) with CK or chromosomal deletions (either complete or partial of any other chromosome). Between 2000 and 2009 we analyzed 38 patients with newly diagnosed AML, with age ranged from 18 to 82 years (median 52 years). On cytogenetic analysis from bone marrow at diagnosis, 16 (42%) pts had a CK (≥3 chromosome abnormalities), 16 pts (42%) had a normal karyotype (NK) and 6 (16%) pts had other monosomy or deletion of any chromosomal region. Seven patients had a post myelodysplastic syndrome AML and 1 post therapy for a prior malignancy. For induction treatment patients received idarubicine, 12 mg/m2/d (age 18– 55y) or mitoxantron 10 mg/m2/d intravenous (IV) on days 1-3 (age 55–68y) and cytarabine, 100mg/m2/d by continuous IV infusion on days 1 through 7. Pts entering complete remission (CR) received three courses of consolidation with high dose cytarabine. Patients with a histocompatibale donor, which did not received CR or relapsed, were allografted. Elderly patients, 69–82 years old, were treated with one induction chemotherapy which included Mylotarg (3mg/m2/2h on day 1) plus cytarabine (100 mg/m2/24h on days 2-8). Patients at this group of age did not received consolidation therapy.Of the16 pts with CK AML, 14 were treated with chemotherapy but only 3 pts (21%) achieved CR, 4 pts died during induction therapy. By comparison, of the 16 pts with NK AML, 15 pts were treated with chemotherapy, 11 pts achieved CR (73%), 3 pts died during induction therapy. Of the 6 AML pts with monosomy or deletion in cytogenetic analysis, 3 pts (50%) achieved CR (p =0.0067). In all NK AML patients examined the TP53 FISH was normal (mean 4% of cells examined), where as in patient with CK 75% of pts had TP53 deletion (mean 51%), and in the group with chromosomal deletion 50% of pts showed a loss of one TP53 (mean 18%) p=0.0016. Of note, in 13 pts (34%) with TP53 deletion by FISH analysis, cytogenetic analysis found no anomaly of 17p chromosome. In a stepwise logistic regression model FISH groups significantly entered the model in first step, and no other variable did (p=0.0056, C=0.833). In pts in which two copies of TP53 were found in FISH analysis ('10%) the median survival time is 440 days, whereas in pts with TP53 deletion was detected the median survival time was 189 days. The OS in pts with deletion of TP53 was significantly shorter as compared to pts that TP53 deletion was not found (p=0.025). However, a multivariate analysis including age, karyotype, deletion of TP53 and allogeneic transplantation showed that only CK and allogeneic transplantation are independent prognostic factors in this analysis.In conclusion, TP53 status by FISH at diagnosis is important due to the following findings:The test is sensitive, rapid and simple.2. TP53 deletions are frequent in AML with CK and tend to segregate with additional chromosomal deletions.TP53 status at diagnosis has a predictive value with respect to chemotherapy response and OS in AML pts with CK and chromosomal deletions. Disclosures:No relevant conflicts of interest to declare.

Congenital Amniotic Band Syndrome and MLL Rearranged Neonatal Leukemia in an Infant with Multiple Pre-Conception and In Utero Exposures.

Introduction: Patients with birth defects have a higher incidence of cancer; however, an association of amniotic band syndrome (vascular disruption syndrome) with congenital leukemia has never been reported. We characterized a case of MLL-rearranged leukemia in a neonate affected with this birth defect.Methods: The MLL translocation was characterized in peripheral AML blasts by karyotype analysis, multicolor FISH, Southern blot analysis and genomic and cDNA panhandle PCR. NQO1 genotype was determined by real-time PCR.Clinical history and results: The infant was born at 38 weeks gestation by C-section for suboccipital encephalocele to a 21 year-old gravida 3 para 2 mother with a history of cigarette, marijuana, cocaine and opiate use, and antidepressant, antipsychotic, barbiturate, caffeine and proton pump inhibitor treatment during pregnancy. Drug screen at delivery was positive for opioids and barbiturates. In addition to the encephalocele, a circular constriction of the right arm, consistent with amniotic band syndrome, and blueberry muffin lesions were noted at delivery. The CBC showed mild thrombocytopenia that resolved the next day. Congenital infection was excluded. The encephalocele was repaired. At 19–20 days of age the infant became septic and hepatosplenomegaly and hyperleukocytosis were observed. The peripheral smear and flow cytomery revealed acute myeloid leukemia with monocytic differentiation (CD45+ population positive for CD4, CD14, CD33, CD38, HLA-DR). Karyotype analysis showed a complex structural abnormality disrupting chromosomes 4, 11 and 19 involving MLL. M-FISH showed insertion of chromosome 11 material into a chromosome 19 and translocation between chromosomes 11q and 4q. The infant received cytosine arabinoside and daunomycin but succumbed to AML, sepsis and multi-organ failure within 4 days. Autopsy showed marrow, viscera, brain and skin infiltration with AML and Chiari type III brain malformation. Southern blot analysis detected two MLL bcr rearrangements. Panhandle PCR demonstrated fusion of MLL intron 6 and intron 1 of ELL from band 19p13.1. Short sequence homologies at the breakpoint junction suggested DNA damage resolution by NHEJ repair. The corresponding transcript joined MLL exon 6 to ELL exon 2. The infant was wild-type at NQO1 C609T.Conclusions: This is the first association of amniotic band syndrome and congenital AML, both of which are rare conditions. Although the cause(s) are unknown, both conditions originate in utero and maternal exposures during pregnancy may be relevant. There was a history of maternal fetal loss, which is a risk factor for leukemia in infants. The NQO1 substrate p-benzoquinone in cigarette smoke is a topoisomerase II poison, but the infant did not harbor the NQO1 variant that predisposes to leukemia. Cocaine is an in utero exposure implicated in amniotic band syndrome. The occurrence of amniotic band syndrome and congenital AML in this infant raises questions about potential host predisposition or gene-environment interactions common to both conditions. Alternatively, both rare conditions may have occurred by chance alone in the setting of the many in utero exposures.

Developmental Stage-Selective Effect of Somatically Mutated GATA-1 in Down Syndrome AML M7--a Potential Basis for Transient Myeloproliferative Disorder.

Transient myeloproliferative disorder (TMD) is a self-limited disorder of newborns with Down Syndrome (DS). Recently, acquired mutations in GATA-1 have been identified in megakaryoblasts from nearly all DS neonates with TMD, and in virtually all DS patients with the related acute megakaryoblastic leukemia (DS-AMKL, or DS-AML M7). These mutations lead to production of an N-terminus truncated GATA-1 protein (GATA-1s). Here we address the biological properties of GATA-1s. Two alternate hypotheses may account for the temporal nature of TMD and restriction of DS-AMKL to infants. First, GATA-1 somatic mutation under the DS background might occur only during a narrow developmental window and additional mutations are needed to sustain a premalignant clone; alternatively, GATA-1 mutation might occur at any time, but only fetal progenitors are sensitive to effects of GATA-1s. Our findings provide support for the latter possibility. In our study, we have generated ES cells and mice harboring a GATA-1 knockin allele with a truncation at this domain (GATA-1ΔN). ES cells bearing GATA-1ΔN exhibit slightly perturbed erythropoiesis, but generate a unique type of large thrombopoietin-dependent megakaryocytes-containing colonies, when differentiated in vitro. Remarkably, adult mutant mice have normal platelet and red blood cell counts, and their corresponding progenitors. During embryonic development, however, mutant embryos are anemic in E12.5 fetal livers, but return to normal shortly thereafter. Mutant fetal livers contain elevated numbers of CD41+ cells throughout embryonic development, especially during mid-gestation. Despite these abnormalities in proliferation, differentiation/maturation of both erythrocytes and megakaryocytes are relatively normal. Strikingly, in in vitro hematopoietic colony assays, cells from yolk sacs and early-mid stage fetal livers give rise to a large number of macroscopic hyperproliferative megakaryocytic colonies (CFU-MKs). This unique type of macroscopic CFU-MK is greatly reduced in number after mid-fetal liver stage and not detectable after birth. Interestingly, wild type mid-gestation fetal liver cells ectopically expressing GATA-1s through retroviral transduction also give rise to similar macroscopic CFU-MK colonies, as do wild type ES cells expressing GATA-1s from a GATA-1 regulatory element, when differentiated in vitro. Overall, our data suggest that the GATA-1 N-terminal domain controls proliferation of megakaryocytes, but has a more limited role in their differentiation. The N-terminal truncation of GATA-1, therefore, uncouples proliferation from differentiation, and does so dominantly. The biological effects of GATA-1s appear restricted to a unique, previously unrecognized yolk sac/fetal liver progenitor, a finding that we propose accounts for the transient nature of TMD and the infantile restriction of DS-AMKL. This observation may also pertain to other infant leukemia syndromes where the target cell may prove to be an embryonic/fetal progenitor rather than an adult myeloid progenitor. Our results thus support a model for the pathogenesis of TMD/DS-AMKL in which trisomy 21 in DS patients provides a “sensitized” background that favors exaggerated expansion of this fetal-type progenitor harboring GATA-1s. This is predicted to lead to TMD, which then evolves into full-blown DS-AMKL following additional, as yet unknown, somatic mutation(s).