Mutations within the Activation Loop Domain of FLT3 in Two Pediatric Patients with Refractory Infant Acute Myeloid Leukemia

Approximately 24% of all pediatric acute myeloid leukemia (AML) cases have mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor gene. FLT3-TKD point mutations are rare in pediatrics and often occur in younger patients and in combination with 11q23 abnormalities. There is a paucity of data related to their prognostic implications in children. We describe 2 pediatric patients with FLT3-activating mutations as a feature of their AML. Both were diagnosed in infancy. The first experienced induction failure and had refractory disease without expression of FLT3-TKD mutation on subsequent bone marrow evaluations. His disease also harbored a KMT2A-PICALM gene rearrangement. He died of invasive fungal disease nine months after diagnosis. The second had a post-induction remission but developed swelling of the left calcaneus shown on biopsy to be a myeloid sarcoma positive for a new BRAF V600E mutation in addition to his known KMT2A rearrangement but without FLT3-TKD mutation. Despite multiple courses of therapy including BRAF/MEK-inhibition, he died of progressive disease nine months after diagnosis. FLT3 inhibition was not utilized in either patient as studies have largely focused on its role in internal tandem duplication (ITD) mutations and because the mutation was no longer detectable in either patient on subsequent evaluation. However, these cases add to the suggestion that these mutations confer a worse prognosis in pediatric AML patients.

Clinical and biological features of acute myeloid leukemia with MLL gene rearrangements in children and results of therapy according to protocols AML-MM-2000/2006 in the Republic of Belarus

Objective of the study . Analysis of the treatment outcomes of patients with MLL rearrangements in the Republic of Belarus within protocols AML-MM-2000 and AML-MM-2006.Materials and methods . The study included 151 patients with newly diagnosed acute myeloid leukemia (AML) who were treated according to protocol AML-MM-2000 and AML-MM-2006. 11q23 abnormalities were detected in 40 (26.5 %) out of 151 patients.Results . The performed analysis of the survival outcomes of patients with 11q23 depending on the protocol showed that the probability of 5-year event-free survival (EFS) was significantly better (p = 0.0110) in children receiving treatment under protocol AML-MM-2006 (86 ± 13 %) compared with that of the patients included in protocol AML-MM-2000 (23 ± 12 %). Using protocol AML-MM-2006 allowed reducing the cumulative incidence of relapse (CIR) in this cohort from 46.2 ± 15.1 to 14.3 ± 14.3 % (p = 0.1609). EFS probability in recipients of allogeneic hematopoietic stem cell transplantation (alloHSCT) was 100 %, whereas in the group without alloHSCT – 31 ± 12 %, p = 0.0359. The treatment outcomes of patients with t(1;11) are comparable to those with CBF leukemia. The risk of relapse in patients with t(10;11) is higher than in the rest of the 11q23 cohort (62.5 ± 19.2 % versus 21.9 ± 7.5 %; p = 0.0136). CIR in patients with t(9;11) decreased from 42.8 % in protocol AML-MM-2000 to 15.4 % in protocol AML-MM-2006 (p = 0.1411).Conclusion . For the described cohort of patients alloHSCT is the best option for post-remission therapy. The worst prognosis is determined in patients with t(10;11), whereas the presence of t(1;11) is a favorable prognostic factor. Using the arm with cladribine showed to be effective in patients with t(9;11). To obtain reliable outcomes, we consider it reasonable to continue the study with the use of cladribine in patients with t(9;11).

Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations

Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities.

Recurrent Genetic Abnormalities Are Reflected in Phenotype at Diagnosis in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group Protocol AAML0531

Background: Acute myeloid leukemia (AML) is a heterogeneous disease with high genomic and phenotypic complexity. Although a relationship between phenotype and t(15;17) is recognized, the effect of other genotypic abnormalities on phenotype remains to be elucidated. Objective: To explore the relationships between genotypic and phenotypic abnormalities in diagnostic specimens in pediatric AML. Methods: Of 1022 newly diagnosed pediatric patients with de novo AML enrolled on protocol AAML0531, 769 satisfied three criteria for this study: (1) submission of a blood or bone marrow sample for multidimensional flow cytometry (MDF) at diagnosis, (2) consent to blood bank specimen testing and (3) leukemia comprising >10% of non-erythroid cells by MDF. The diagnostic AML tumor population was identified by gating on CD45 vs log-SSC. A fifteen-dimensional immunophenotypic expression profile (IEP) was defined by computing mean fluorescent intensities, antigen intensity coefficient of variations, and light scatter characteristics of each leukemia without analyst imposed cut-offs. Unsupervised hierarchical clustering was performed to mathematically cluster patients with similar IEPs. The underlying karyotypic and mutational profile of each patient was compared to the IEPs to identify clusters of patients with relationships between phenotype and identifiable genetic abnormalities. Supervised bagged tree-based models were used to quantify the importance of each cell-surface antigen in identifying these clusters. Levene's test was used to assess the homogeneity of mean cell-surface antigen expression for important markers. Results: Analysis of hierarchical clustering results revealed 7 immunophenotypic clusters (A-G) of patients with relationships between IEP and 4 common genetic abnormalities (Figure 1). Cluster A and Cluster B were both strongly associated with the t(8;21) karyotype (Figure 1). 54/62 patients in Cluster A (87%) and 24/26 patients in Cluster B (92%) were positive for t(8;21). The most important cell-surface antigens in identifying patients with the Cluster A associated phenotype were dim CD33, expression of CD56, expression of CD34 and expression of HLA-DR. The mean antigen expression of these markers was significantly more homogenous for patients in Cluster A compared to all other patients for CD33 (p = 0.02), CD34 (p < 0.01) and HLA-DR (p<0.01). Mean expression of CD56 was notably more heterogeneous (p = 0.40) but positive in 91% of patients in this cluster. The most important cell-surface antigens in identifying the Cluster B associated phenotype were dim CD13, expression of CD34, expression of HLA-DR and dim CD33. The mean expression of these markers was significantly more homogeneous for patients in Cluster B compared to all other patients (all p values <0.01). In contrast to Cluster A, CD56 MFIs were negative for patients in Cluster B. Collectively, 72% of all patients with t(8;21) segregated into Cluster A (50%) or Cluster B (22%). Cluster C was strongly associated with chromosome 16 abnormalities (Figure 1). Of patients in this cluster, 50/55 (91%) were positive for inv(16)/t(16;16). The mean high-expression of CD13 was the most important antigen in identifying patients with this phenotype, and patients in Cluster C had more consistent mean CD13 expression compared to all other patients (p<0.01). 53% of all patients with chromosome 16 abnormalities comprised this cluster. Cluster D was strongly associated with 11q23 abnormalities (Figure 1). Of patients in this cluster, 105/135 (78%) were positive for 11q23 translocations. The most discriminating markers in identifying these patients were dim-to-negative CD34 and dim-to-negative CD117. 69% of all patients with 11q23 abnormalities comprised this cluster. Clusters E, F and G were associated with FLT3 ITD mutations (Figure 1). Of patients in these clusters, 44%, 49% and 53% were positive for FLT3 ITD mutations. Cumulatively, 63% of all patients with FLT3 ITD mutations were grouped into one of these three clusters. Conclusion: Hierarchical clustering algorithms identify IEPs associated with common genetic abnormalities in pediatric AML, providing insight into the consistent dysregulation of cell-surface gene product expression in patients with common genetic abnormalities. Further analysis may reveal that IEPs can identify patients otherwise classified as good or standard risk that will have a poor outcome. Disclosures Voigt: Hematologics Inc.: Employment. Brodersen:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Loken:Hematologics Inc.: Equity Ownership.

Value of Cytogenetic Abnormalities in Adult Patients with Philadelphia Chromosome (Ph)-Negative Acute Lymphoblastic Leukemia (ALL) Treated in the Pediatric-Inspired Trials from the Group for Research on Adult ALL (GRAALL)

Background: Numerous recurrent chromosomal abnormalities have been described in adult Ph-negative ALL, often observed in small patient cohorts. In the largest MRC/ECOG study (Moorman, Blood 2007), t(4;11)(q21;q23), 14q32 involvement, complex karyotype (≥5 abnormalities), and low hypodiploidy/near triploidy (Ho-Tr) were associated with shorter event-free survival (EFS), while patients with high hyperdiploidy or del(9p) had a better outcome. We aimed to confirm these observations in 955 adult patients (15-60y; median, 35y) with Ph-negative ALL treated in the pediatric-inspired GRAALL-2003/2005 trials. Patients and Methods: Overall, a karyotype was performed for 946 (611 BCP-ALL, and 335 T-ALL), successful for 811 (523 BCP-ALL and 288 T-ALL) and abnormal in 590 patients (387 BCP-ALL and 203 T-ALL). FISH and/or PCR screening for relevant abnormalities and DNA index were also performed, finally allowing for the identification of cytogenetic abnormalities in 677/955 patients (71%). All were centrally reviewed. Ultimately, 857/955 patients (90%; 542 BCP-ALL and 315 T-ALL) could be classified in 18 exclusive primary cytogenetic subsets as detailed below. Endpoints were cumulative incidence of failure (CIF, including primary refractoriness and relapse) and EFS. With a median follow-up of 4 years, 5-year CIF and EFS were estimated in these patients at 31% and 51%, respectively. As some abnormalities, including MLL rearrangements, Ho/Tr, t(1;19)(q23;p13)/TCF3 and complex karyotypes were used to stratify allogeneic stem cell transplantation (SCT) in GRAALL trials, some comparisons were repeated after censoring patients transplanted in first CR at SCT time. Results: The 542 informative BCP-ALL patients were classified as: t(4;11)(q21;q23)/MLL-AFF1 (n=72; 13%); other MLL+ 11q23 abnormalities (n=11; 2%); t(1;19)(q23;p13)/TCF3-PBX1 (#28; 5%); Ho/Tr (n=33; 6%); high hyperdiploidy (n=36; 7%); abnormal 14q32/IGH translocation (n=27; 5%); t(12;21)(p13;q22)/ETV6-RUNX1 (n=2; 0.4%); iAMP21 (n=3; 0.6%); other abnormalities (n=210; 39%); and no abnormality (n=120; 22%). The 315 informative T-ALL patients were classified as: t(10;14)(q24;q11)/TLX1 (n=64; 20%); other 14q11 or 7q34/TCR (n=31; 10%); t(5;14)(q35;q32)/TLX3 (n=29; 9%); t(10;11)(p12;q14)/PICALM-MLLT10 (n=14; 4%); deletion 1p32/SIL-TAL (n=18; 6%); MLL+ 11q23 abnormalities (n=6; 2%); other abnormalities (n=93; 30%); and no abnormalities (n=60; 19%). A complex karyotype was observed in 27/527 (5%) BCP-ALL and 21/298 (7%) T-ALL patients and a monosomal karyotype (as per Breems, JCO 2008) in 82/518 (16%) BCP-ALL and 26/286 (9%) T-ALL patients. In BCP-ALL, trends towards higher CIF and shorter EFS were observed in t(4;11) patients, with or without SCT censoring (HRs, 1.34 to 1.64; p values <0.10). Shorter EFS was observed in 3 subsets: 14q32 (HR, 2.10; p=0.002), Ho/Tr (HR, 1.45; p=0.10), and monosomal karyotype (HR, 1.42; p=0.029), but CIF were not different. This might be related to the older age of patients in these subsets (medians, 43y, 53y and 44y; p=0.029, <0.001 and <0.001, respectively) and worse treatment tolerance. For instance, higher incidences of non ALL-related deaths were observed in patients with 14q32 abnormalities or monosomal karyotype (p=0.031 and 0.067, respectively). Patients with high hyperdiploidy only tended to have lower CIF and longer EFS. Complex karyotype did not impact CIF and EFS, even after SCT censoring. Conversely, in T-ALL, complex karyotypes were associated with shorter EFS (HR, 2.20; p=0.004), even if the difference in CIF did not reach significance. A worse outcome was also observed in patients with t(10;11)(p12;q14)/PICALM-MLLT10 (HR, 2.45 and 2.14; p=0.016 and 0.021, for CIF and EFS respectively). A longer EFS was observed in patients with t(10;14)(q24;q11)/TLX1 (HR, 0.55; p=0.014), with a trend for lower CIF (HR, 0.59; p=0.070), while no inferior outcome was observed in t(5;14)(q35;q32)/TLX3 patients. Conclusion: These results show that, in the context of an intensified pediatric-inspired protocol designed for adult Ph-negative ALL patients, few cytogenetic subsets remained reliably predictive of response to therapy. Differences observed in EFS might partly be due to treatment-related mortality. Combining cytogenetics, molecular genetics and minimal residual disease monitoring could allow for better individual risk assessment (Beldjord, Blood 2014). Disclosures No relevant conflicts of interest to declare.

The t(9;11) Confers Good Prognosis in AML Patients Treated with Stem Cell Transplantation As Compared to Non-t(9;11) and Other Adverse-Risk Abnormalities

Abstract 2527 Background: In contrast to most translocations affecting the MLL gene, the t(9;11) is not associated with a markedly poor prognosis. Several studies revealed a very favorable outcome in the pediatric patient group. In adult AML, the t(9;11) has also been associated with superior survival, at least compared to other 11q23 abnormalities. Therefore, 11q23 rearrangements in adult AML are now often dichotomized into t(9;11) and non-t(9;11), with the former being included in the intermediate-risk group and the latter in the adverse-risk group. The proposed European Leukemia Net (ELN) cytogenetic reporting criteria reflect this division. We investigated whether the outcome of AML patients treated with allogeneic hematopoietic stem cell transplantation (HSCT) with t(9;11) remains significantly different from the rest of the adverse-risk cytogenetic group. Methods: Conventional cytogenetics and FISH data from diagnostic bone marrow of 110 adult AML patients treated with HSCT was reviewed and patients classified according to the recommendations of the European Leukemia Net and included 32 with favorable risk, 60 in the intermediate-risk group, and 18 in the adverse-risk group. FISH confirmed MLL rearrangement in cases with apparent 11q23 abnormalities. We compared outcome of patients with t(9;11) to the group of patients with adverse-risk cytogenetics that included all MLL-positive non-t(9;11) among other cytogenetic abnormalities classified adverse-risk. Our study included 62 (56%) patients treated in first remission (CR1), while most non-CR1 AML patients were treated with HSCT in CR2. Patients were between 14 and 57 years, with median age of 25 years. Results: Of the 110 AML patients, 9 (8%) had MLL gene rearrangement. Of these patients only 5 (4.5% of all patients) had t(9;11). When all patients with MLL rearrangement were considered, there was no significant difference between this group and the patients with adverse-risk cytogenetics in overall survival (OS) or event-free survival (EFS). In contrast, when only patients with the t(9;11) were considered, the t(9;11) patients had significantly longer OS (P=0.02) and EFS (P=0.03) as compared with patients with adverse cytogenetics including all non-t(9;11) MLL-rearranged cases. The outcome of MLL-positive non-t(9;11) patients was similar to the group with adverse-risk cytogenetics. MLL rearrangements in the non-t(9;11) group included t(4;11)(q21;q23), t(6;11)(q27;q23) and a variant t(6;11;7)(q27;q23;q11.2), as well as t(11;17)(q23;q25). The survival for patients with t(9;11) remained significantly longer even when only patients treated with HSCT in first remission were considered, although numbers were small. All five patients with t(9;11) were treated with HSCT in CR1. Conclusions: The data supports the conclusion that MLL-positive t(9;11) AML patients should be classified differently from the rest of the MLL-rearranged cases and should be considered as part of the intermediate-risk group. This classification separating the t(9;11) cases from the rest of the MLL-positive cases should be maintained even when patients are treated with allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

The Spectra of Chromosomal Aberrations In Acute Leukemia and Implications for the Development of Optimal Profiles of Leukemic Fusion Genes In Multiplex RT-PCR System.

Abstract 3362 Background: Cytogenetic aberrations have been identified as important prognostic parameters for acute leukemia, in addition to diagnosis and treatment. The aim of the current study was to investigate the spectrum of chromosomal abnormalities in East Asian (Korean) patients with acute leukemia. Based on the spectrum of genetic aberrations found in our study, we proposed a revised list of chromosomal aberrations for the development of optimal profiles of leukemic fusion genes in multiplex RT-PCR system. Patients and Method: We prospectively analyzed blood or bone marrow samples from 348 patients (260 adults, 88 children) with leukemia from 2006 to 2009 at the Chonnam National University Hwasun Hospital (Hwasun, Korea). Simultaneous analyses of conventional cytogenetics, FISH and a commercially available multiplex RT-PCR system (HemaVision) were performed. Result: Thirty-six types of chromosomal abnormalities were found in the total patient population. Twenty types of chromosomal abnormalities were detected in 42% of all patients by multiplex RT-PCR system and in 33% of all patients by conventional cytogenetics including FISH. Within the group of 213 AML patients, the common cytogenetic abnormalities were t(15;17)(q21;q22) (n=35), t(8;21)(q22;q22) (n=31), 11q23 abnormalities (n=7), inv(16)(p13;q22) (n=3), t(9;9)(q34:q34) (n=2), and the abnormalities t(9;22)(q34;q11), t(9;12)(q34;p13), t(3;21)(q26;q22), t(11;17)(q23;q21), t(12;22)(p13;q11) and t(16;21)(p11.2;q22.3) were each involved in one case. In the group of 104 ALL patients, the following fusion transcripts were detected: 21 t(9;22)(q34;q11), 15 t(12;21)(p13;q22), nine 11q23 abnormalities, four t(1;19)(q23;p13), with t(9;9)(q34:q34), t(9;12)(q34;p13) and del(1p34) each involved in one case. In addition, cryptic translocations were detected in 9% of patients with normal karyotypes or numerical aberrations by conventional cytogenetics. Among 53 patients showing negative results for multiplex RT-PCR, one child patient with early pre-B-ALL harbored MLL1/AF4 fusion genes and 5 patients had 3 types of clinically significant chromosomal aberrations such as t(3;3)(q21;q26.2), t(8;14)(p24.1;q32) and i(17)(q10). Conclusion: The current study demonstrated the spectrum and frequency of chromosomal abnormalities in Korean leukemia patients, which were slightly differed from previous studies including western and some Asian countries. These results may offer important implications in understanding characteristics of molecular pathophysiology, the development of a new RT-PCR system as well as revision of the current commercial available RT-PCR system. Disclosures: No relevant conflicts of interest to declare.

Mechanisms of Susceptibility to 11q23 MLL Gene Locus Rearrangements in CD34+ Cells Exposed to Etoposide.

Abstract 185 Exposure to the topoisomerase II inhibitor etoposide (VP16) is a significant risk factor in the development of t-MDS/AML. VP16 induces a variety of chromosomal lesions, with the most prominent ones involving the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23. The MLL gene is essential for the maintenance of adult hematopoietic stem cells (HSCs) and progenitors. It is not clear whether the high frequency of MLL gene rearrangements in VP16-treated patients represents increased susceptibility of this locus to VP16-induced damage or a growth advantage of MLL-rearranged hematopoietic progenitors. In the present study we investigated the following: i) the sequence of acquisition of chromosomal lesions in normal CD34+ cells following in vitro exposure to VP16; ii) the specific propensity for development of lesions in 11q23 MLL locus; iii) and the fate of these lesions over time. CD34+ cells were selected from peripheral blood stem cell samples obtained from normal donors (n=5); they were exposed to increasing doses of VP16 (0-40 μM) for 4 hours; washed and cultured in serum-free medium containing growth factors; and assessed for apoptosis, colony forming cell (CFC) assay and chromosomal damage. Chromosome painting was performed using whole chromosome probes for chromosomes 1, 5, 7, 11 and 21, covering 25.2% of total genomic DNA with >100 metaphases scored per dose per time point. Evaluations were performed after 72 hours of culture, representing the 1st cell division after VP16 exposure, and after 144 hours representing 3-4 cell divisions. A dose-dependant decrease in colony formation (71.52 ± 0.99% of controls with 5 mM VP16, and 33.62 ± 4.4%%, with 20 mM VP16 at 72h, p<0.001), and increase in apoptosis (9.12 ± 1.67% with 5 mM VP16, and 39.3 ± 5.06 with 20 mM VP16 at 72h, p<0.001) was observed. The percentage of aberrant metaphases at 1st division was 2.4% with 5 μM and 14.9% with 20 μM of VP16. Both stable aberrations (translocations) and unstable (dicentrics, fragments) aberrations were observed. The percentage of aberrant metaphases was reduced after 3-4 cell divisions (1.4% with 5 μM and 8.7% with 20 μM VP16), and only stable aberrations were present whereas unstable aberrations were eliminated. A higher frequency of aberrations was observed in chromosome 11 compared to the other chromosomes. A 1.5- to 4-fold increase in stable lesions of chromosome 11 relative to other chromosomes was seen at the 1st division and a 2- to 3-fold increase relative to other chromosomes was seen after 3-4 divisions. To assess whether the 11q23 MLL locus was specifically affected by VP16 treatment, we performed interphase FISH with a break-apart probe for the MLL gene (11q23). A break-apart probe for the MYC gene (8q24) was studied as a control. Cells were cultured for three weeks. In preliminary experiments we observed that a higher percentage of cells showed 11q23 abnormalities (9.3%) compared with 8q24 abnormalities (5.4%) at the 1st division. 11q23 abnormalities persisted at high levels in cells (9.9%) cultured for 3 weeks. In contrast, 8q24 abnormalities were markedly reduced (0.4%) after 3 weeks of culture, suggesting selective maintenance of MLL-rearranged cells in culture. In conclusion we show that in vitro exposure to VP16 results in a dose-dependent induction of both stable and unstable chromosomal aberrations in CD34+ hematopoietic progenitors. We observe persistence of stable chromosomal aberrations with particular predisposition to aberrations in chromosome 11. Our results suggest a selective predisposition to 11q23 MLL locus rearrangements that appears to be related to enhanced susceptibility to damage to this locus following VP16 exposure as well as selective maintenance of MLL rearranged cells over subsequent cell divisions. These findings are now being applied to understand the relationship between individual susceptibility to acquisition of VP16-induced 11q23 abnormalities in CD34+ cells and risk of development of t-MDS/AML. Disclosures: No relevant conflicts of interest to declare.