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We performed serial measurable residual disease (MRD) monitoring in bone marrow (BM) and peripheral blood (PB) samples of 155 intensively treated patients with RUNX1-RUNX1T1(+) AML, using a qRT-PC–based assay with a sensitivity of up to 10(−6). We assessed both reduction of RUNX1-RUNX1T1 transcript levels (TLs) and achievement of MRD negativity (MRD(−)) for impact on prognosis. Achievement of MR(2.5) (>2.5 log reduction) after treatment cycle 1 and achievement of MR(3.0) after treatment cycle 2 were significantly associated with a reduced risk of relapse (P = .034 and P = .028, respectively). After completion of therapy, achievement of MRD(−) in both BM and PB was an independent, favorable prognostic factor in cumulative incidence of relapse (4-year cumulative incidence relapse: BM, 17% vs 36%, P = .021; PB, 23% vs 55%, P = .001) and overall survival (4-year overall survival rate BM, 93% vs 70%, P = .007; PB, 87% vs 47%, P < .0001). Finally, during follow-up, serial qRT-PCR analyses allowed prediction of relapse in 77% of patients exceeding a cutoff value of 150 RUNX1-RUNX1T1 TLs in BM, and in 84% of patients exceeding a value of 50 RUNX1-RUNX1T1 TLs in PB. The KIT mutation was a significant factor predicting a lower CR rate and inferior outcome, but its prognostic impact was outweighed by RUNX1-RUNX1T1 TLs during treatment. Virtually all relapses occurred within 1 year after the end of treatment, with a very short latency from molecular to morphologic relapse, necessitating MRD assessment at short intervals during this time period. Based on our data, we propose a refined practical guideline for MRD assessment in RUNX1-RUNX1T1(+) AML.

t(8;21)(q22;q22)RUNX1-RUNX1T1 is a recurrent somatic lesion detected at diagnosis in approximately 12-15% of children with acute myeloid leukemia (AML). Children with this isolated translocation are usually considered at standard risk, but our last multicenter trial revealed a higher than expected cumulative incidence of relapse for these patients1. Genetic and epigenetic heterogeneity is emerging as a fundamental property of AML in the context of the clonal architecture dynamic evolution. In view of this observation, we hypothesized that within t(8;21) patients there may coexist a complex mosaic of cells containing combinations of the same genetic t(8,21) lesion together with different epigenetic variants, and that epigenetic complexity may play a crucial role in predisposing patients to relapse. The importance of the identification of molecular markers distinctive of t(8,21)-rearranged patients prone to develop relapse could be instrumental to improve their cure rate. We performed high throughput DNA methylation profiling (RRBS-seq) and integrated results with gene expression profiling (Affymetrix HTA 2.0) of 16 isolated t(8;21) AML samples collected at diagnosis, and analyzed data by comparing patients who did or did not experience relapse. We applied a logistic regression algorithm to identify differentially methylated regions (DMRs) considering a minimum change in methylation level of 25%. We validated results in a proteome context by reverse phase protein array (RPPA) in an independent cohort of 35 t(8;21) AML patients. DNA methylation profiling analysis identified 337 DMRs able to correctly predict t(8;21) patients who did relapse from those who did not. In particular, 23 DMRs (7%) were located at promoters, while most of them were equally distributed between intronic (48%) and exonic (45%) regions. Globally, we found hypomethylated DMRs being significantly enriched in relapsed patients, in particular in repetitive elements regions of the genome (LINE, SINE, DNA transposon: 38.9% vs 52.4%; p We then considered the role of methylation over gene expression and found a weak correlation between DMRs (mostly at promoters) and their associated gene levels (14.5% of DMRs with an inverse correlation r These data show that the methylation signature may be considered a novel, emerging diagnostic tool making possible to better stratifying t(8,21)-rearranged patients through the identification, already at diagnosis, of those who are prone to relapse . Preliminary data of functional analysis suggest that epigenome of t(8;21) blasts may control cell adhesion properties at bone marrow niche and treatment response, contributing to patients relapse. 1 Pession A, Blood. 2013;122(2):170-8. Disclosures No relevant conflicts of interest to declare.

CBF-AML is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion genes require a "second hit" to initiate leukemogenesis. Although compared with other AML subtypes patients with CBF-AML have a relatively favorable prognosis, still almost 40% of CBF-AML patients experience relapses of their disease. Mutation assessment of 84 leukemia- and/or cancer-associated genes using a customized targeted next-generation sequencing (NGS) approach was done on samples from 177 adults with CBF-AML [t(8;21), n=68; inv(16)/t(16;16), n=109]. We identified frequent mutations in the CCND1 and CCND2 genes as novel molecular alterations in AML with t(8;21). They were detected in 10 (15%) patients with t(8;21), making CCND1/2 mutations the third most frequently detected mutations in t(8;21) AML patients. In contrast, we found a single CCND2 mutation in only 1 (0.9%) patient with inv(16), and CCND1/2 mutations in only 11 of 1,426 non-CBF-AML patients (0.77%). Testing for CCND1 and CCND2 mutations using Sanger sequencing in additional 25 CBF-AML patients with t(8;21), for whom NGS analysis was not possible, identified 3 CCND1/2 mutated patients, thereby confirming the CCND1/2 mutation frequency in t(8;21) AML (15% in the NGS set, and 12% in Sanger sequencing set). All CCND2 mutations cluster around the highly conserved amino acid residue threonine 280 (Thr280), and encode a degradation-resistant CCND2 protein. Curiously, de novo germline CCND2 mutations affecting the identical amino acid residues Thr280 or Pro281 have been recently found to cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH) type 3, an extremely rare neurodegenerative disorder (Mirzaa et al., Nat Genet. 2014;46:510-5). The variant allele fractions of the detected CCND1/2 mutations were variable, suggesting that these mutations can represent both early and later mutational events. Interestingly, three of the 11 CCND1/2 mutated patients had two different CCND1/2 mutations, suggesting a dependence of their leukemia on this mutational event. The evaluation of mutations in other genes found in CBF-AML patients with CCND1/2 mutations did not reveal any statistically significant co-occurring or mutually exclusive mutations. The most frequent mutation co-occurring with CCND1/2 was ASXL2, detected in 4 of the 10 (40%) cases with t(8;21). Patients with CCND1/2 mutations presented with lower percentages of blood (P=0.02) and bone marrow (P=0.05) blasts than patients without these mutations, but did not differ significantly with regard to other pretreatment characteristics. No differences in the achievement of complete remission, disease-free or overall survival were observed between CCND1/2 mutated and CCND1/2 wild-type patients. Mechanistically, we show that Thr280Ala mutated CCND2 significantly increased cell growth (as assessed by TiterGlo assays and cell counts) in all experimental set-ups. Western blotting revealed an increase of retinoblastoma gene phosphorylation, indicating increased downstream signaling. Moreover, both propidium iodide mediated cell cycle analysis and immunofluorescence staining for phospho-histone H3 indicated an increased mitotic rate and a decreased G1 fraction. In summary, recurrent mutations in CCND1/2 are consistent with the currently known mutational landscape of t(8;21) AML, which is dominated by receptor tyrosine kinase, RAS, and chromatin remodeling gene mutations─all of which can result in targeting of CCND1/2. While CCND1 and CCND2 play a crucial role in the regulation of hematopoietic differentiation and cell proliferation, and their differential activation has been implicated in leukemogenesis, frequent mutations in these genes have not previously been reported in AML. The fact that no outcome difference was seen when comparing CCND1/2-mutated versus wild-type patients may be due to the relatively small sample size, or may possibly reflect the common use of cell cycle affecting high-dose cytarabine-based therapy in CBF-AML patients. Thus, the identification of CCND1/2 mutations as frequent mutational events in t(8;21) AML may provide further justification for cell cycle-directed therapy in this disease. Disclosures Stone: Merck: Consultancy; Karyopharm: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; Juno Therapeutics: Consultancy; ONO: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy; Celator: Consultancy; Agios: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Background: Acute myeloid leukemia (AML) with t(8;21)(q22;q22) results in the formation of the RUNX1-RUNX1T1 fusion transcript which can be used to monitor minimal residual disease (MRD) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Early identification of patients (pts) with a high risk of relapse will allow pre-emptive therapy including allogeneic hematopoietic cell transplantation (alloHCT). Recent studies in AML with NPM1 mutation or the CBFB-MYH11 gene fusion revealed that MRD persistence is significantly associated with a high risk of relapse. However, the prognostic impact of MRD assessment in RUNX1-RUNX1T1-positive AML is not well established. Aims: To assess the prognostic impact of qRT-PCR-based MRD monitoring in bone marrow (BM) of pts with t(8;21)/RUNX1-RUNX1T1-positive AML obtained at defined time-points (diagnosis, first and second cycle of chemotherapy, end of treatment). Methods: In total, 120 pts were included based on the availability of a diagnostic BM sample and at least two subsequent BM samples obtained during therapy and at the end of treatment; 106 pts were enrolled in one of six AMLSG treatment trials: AML HD93 (n=1), AML HD98A (NCT00146120; n=13), AMLSG 06-04 (NCT00151255; n=4), AMLSG 07-04 (NCT00151242; n=43), AMLSG 11-08 (NCT00850382; n=31), AMLSG 21-13 (NCT02013648; n=14); 14 pts were treated outside clinical trials. All pts received anthracycline- and cytarabine-based intensive induction followed by subsequent high-dose cytarabine consolidation cycles. For MRD assessment, qRT-PCR from BM specimens was performed using TaqMan technology; RUNX1-RUNX1T1 transcript levels (TL) were reported as the normalized value of RUNX1-RUNX1T1 per 106 transcripts of the housekeeping gene beta2-microglobulin. The maximum sensitivity of the assay was 10-6. Results: The median age of the pts was 47 years (yrs; range, 18-73 yrs); at the time of diagnosis there was a broad range of RUNX1-RUNX1T1 TL (18490 to 14440000) with a median of 227800. RUNX1-RUNX1T1 TL did not correlate with clinical features (age, WBC, platelets, LDH, BM blasts) or associated gene mutations such as KIT, FLT3-ITD/TKD, NRAS or ASXL2. However, pts with additional FLT3 mutation showed higher TL compared to wild-type pts (median, 412955 vs 219052). Cox regression analysis using RUNX1-RUNX1T1 TL as a log10 transformed continuous variable showed that higher RUNX1-RUNX1T1 TL were significantly associated with a higher cumulative incidence of relapse (CIR), inferior event-free survival (EFS) and shorter overall survival (OS) for the two time points "after first treatment cycle" and "at end of treatment" (CIR: HR, 1.84, p=0.001; HR, 1.60, p=0.03; EFS: HR, 1.59, p=0.01, HR, 1.74, p=0.01; OS: HR, 1.63, p=0.02, HR 2.13, p=0.009, respectively). In univariate analyses achievement of MRD negativity (n=35) at the end of treatment was significantly associated with a superior 4-yr OS (93% vs 67%; p=0.007) and 4-yr EFS (81% vs 61%; p=0.04) whereas achievement of MRD negativity after the first (1/85) and second (20/89) treatment cycle was low not reaching significance for any of the clinical endpoints. Separation of the RUNX1-RUNX1T1 TL according to quartiles of distribution showed significant differences in OS (p=0.04), and remission duration (p=0.006) "after first cycle" whereas "at end of treatment" significant differences were only found for OS (p=0.009). Finally, we evaluated the impact of concurrent KIT mutations on the kinetics of RUNX1-RUNX1T1 TL. Following the first treatment cycle, the median RUNX1-RUNX1T1 TL were significantly lower in the KIT wildtype group compared with the KIT mutated group (p=0.02); the same was true "at the end of treatment" (p=0.02). Conclusions: In our study, achievement of MRD negativity at the end of treatment was significantly associated with a better outcome in t(8;21)-positive AML. The fact that earlier time points did not allow the identification of pts with a high relapse risk is probably due to the high sensitivity of the qRT-PCR assay which is also reflected by the low number of pts achieving qRT-PCR negativity after first and second treatment cycle, respectively. Further analyses are ongoing including multivariable as well as molecular subgroup analyses. *These authors contributed equally to the work: MA, AC MA was supported by the Else-Kröner-Fresenius-Stiftung (EKFS). Disclosures Paschka: Celgene: Honoraria; Pfizer Pharma GmbH: Honoraria; Bristol-Myers Squibb: Honoraria; Medupdate GmbH: Honoraria; Novartis: Consultancy; ASTEX Pharmaceuticals: Consultancy. Lübbert:Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding. Fiedler:Amgen: Consultancy, Other: Travel, Patents & Royalties, Research Funding; Teva: Other: Travel; Kolltan: Research Funding; Ariad/Incyte: Consultancy; Novartis: Consultancy; Gilead: Other: Travel; GSO: Other: Travel; Pfizer: Research Funding. Heuser:Karyopharm Therapeutics Inc: Research Funding; Pfizer: Research Funding; Bayer Pharma AG: Research Funding; Celgene: Honoraria; Tetralogic: Research Funding; BerGenBio: Research Funding; Novartis: Consultancy, Research Funding. Schlenk:Pfizer: Honoraria, Research Funding; Amgen: Research Funding.

Cytogenetics is considered one of the most valuable prognostic determinants in acute myeloid leukemia (AML). However, many studies on which this assertion is based were limited by relatively small sample sizes or varying treatment approach, leading to conflicting data regarding the prognostic implications of specific cytogenetic abnormalities. The Medical Research Council (MRC) AML 10 trial, which included children and adults up to 55 years of age, not only affords the opportunity to determine the independent prognostic significance of pretreatment cytogenetics in the context of large patient groups receiving comparable therapy, but also to address their impact on the outcome of subsequent transplantation procedures performed in first complete remission (CR). On the basis of response to induction treatment, relapse risk, and overall survival, three prognostic groups could be defined by cytogenetic abnormalities detected at presentation in comparison with the outcome of patients with normal karyotype. AML associated with t(8;21), t(15;17) or inv(16) predicted a relatively favorable outcome. Whereas in patients lacking these favorable changes, the presence of a complex karyotype, −5, del(5q), −7, or abnormalities of 3q defined a group with relatively poor prognosis. The remaining group of patients including those with 11q23 abnormalities, +8, +21, +22, del(9q), del(7q) or other miscellaneous structural or numerical defects not encompassed by the favorable or adverse risk groups were found to have an intermediate prognosis. The presence of additional cytogenetic abnormalities did not modify the outcome of patients with favorable cytogenetics. Subgroup analysis demonstrated that the three cytogenetically defined prognostic groups retained their predictive value in the context of secondary as well as de novo AML, within the pediatric age group and furthermore were found to be a key determinant of outcome from autologous or allogeneic bone marrow transplantation (BMT) in first CR. This study highlights the importance of diagnostic cytogenetics as an independent prognostic factor in AML, providing the framework for a stratified treatment approach of this disease, which has been adopted in the current MRC AML 12 trial.

Although acute myeloid leukemia (AML) with t(8; 21) (q22; q22) is associated with a high complete remission (CR) rate and prolonged disease-free survival, treatment outcome is not universally favorable. Identifying factors that predict for treatment outcome might allow therapy to be optimized based on risk. AML with t(8; 21) has a distinctive immunophenotype, characterized by expression of the myeloid and stem cell antigens CD13, CD15, CD34, and HLADr, and frequent expression of the B-cell antigen CD19 and the neural cell adhesion molecule CD56, a natural killer cell/stem cell antigen. Because CD56 expression has been associated with both extramedullary leukemia and multidrug resistance, we sought to correlate CD56 expression with treatment outcome in AML with t(8; 21). Pretreatment leukemia cells from 29 adult de novo AML patients with t(8; 21) treated on Cancer and Leukemia Group B (CALGB) protocols were immunophenotyped by multiparameter flow cytometry as part of a prospective immunophenotyping study of adult AML (CALGB 8361). CD56 was expressed in 16 cases (55%). There was no correlation between CD56 expression and age, sex, white blood cell count, granulocyte count, the presence of additional cytogenetic abnormalities, or the presence of extramedullary disease at diagnosis. The CR rate to standard-dose cytarabine and daunorubicin was similar for cases with and without CD56 expression (88% v 92%; P = 1.0). Post-CR therapy included at least one course of high-dose cytarabine in 24 of 26 patients who achieved CR; numbers of courses administered were similar in cases with and without CD56 expression. Although post-CR therapy did not differ, CR duration was significantly shorter in cases with CD56 expression compared with those without (median, 8.7 months v not reached; P = .01), as was survival (median, 16.5 months v not reached; P = .008). We conclude that CD56 expression in AML with t(8; 21) is associated with significantly shorter CR duration and survival. Our results suggest that CD56 expression may be useful in stratifying therapy for this subtype of AML.

Acute myeloid leukemia (AML) is associated with various acquired genetic changes in hematopoietic progenitor cells. Core binding factor (CBF) AML which consists of AML with t(8;21)(q22;q22)/RUNX1-RUNXT1 and inversion 16 are identified in 13-15% of patients with de novo AML. In both subtypes, the cytogenetic rearrangements disrupt genes that encode subunit of core-binding factor, a transcription factor that is essential in regulating normal hematopoeisis. AML t(8;21) tend to occur in younger age group and display favorable prognosis with chances of attaining complete remission up to 80%. However, relapse rate may reach 30-40%. Recently, more publications reported variable outcome of this subset of leukemia. Therefore, we set the study to evaluate the outcome of our cohort of AML patients with t(8;21) diagnosed between January 2008 and February 2015. We performed retrospective review of all patients who have been diagnosed as AML t(8;21) from our department's database. The data were collected from the electronic hospital information system (eHis). All data were analyzed in this non-randomized study on 30th June 2015. A total of 39 out of 812 AML patients diagnosed between January 2008 and February 2015 were included in this study with mean follow-up duration of 25.3 months (3-89 months). These patients were diagnosed from bone marrow assessment according the WHO 2008 classification with presence of t(8;21)(q22;q22) karyotype or detectable RUNX1-RUNXT1 fusion transcript. Mean age was 34.6 (13-72 year-old). Mean presenting white blood cell count was 25.3 x10^9/L (4.0-95.0x 10^9/L). 30 patients had karyotyping done at diagnosis, and 18 had no additional cytogenetic abnormalities. 84.6 % received standard daunorubicin and cytarabine (3+7) as induction chemotherapy and the remaining patients received other modified anthracycline-cytarabine combination chemotherapy. 25 patients achieved complete morphological remission (CR) after induction. 20 patients had sustained morphological CR after completion of cytarabine-based consolidation protocol and 13 still in CR until their last follow-up. 7 patients relapsed morphologically (mean duration of last chemotherapy 5.43 months) and only 2 patients managed to achieve and maintain CR after salvage chemotherapy and hemopoeitic stem cell transplant (HSCT). Those who relapsed while on consolidation chemotherapy (5 patients) also have dismal outcome as 4 of them died from progression of disease despite salvage chemotherapy and only 1 still alive and in CR after salvage chemotherapy and HSCT. In our cohort, 14 patients were not in CR after induction and received salvage chemotherapy. 2 of them remained refractory and eventually died. 12 patients achieved CR after salvage chemotherapy, followed by HSCT in 10 patients. 6 out of these 10 patients remained in CR until their last follow-up. 1 patient relapsed after HSCT and the other 3 died of transplant-related complications. Extramedullary disease was seen in 5 young males (age 13-27 years, involving 2 paraspinal, 1 lacrimal gland and 1 extraconal). 2 achieved CR after induction, while the other 3 achieved CR after consolidation, but 2 of them relapsed. 4 patients undergone HSCT and 3 remained in CR while 1 died of transplant-related complication. 30 patients had molecular monitoring throughout their treatment course. The best molecular response was unquantifiable fusion transcript, seen in 18 patients with mean survival of 33.2 months. The mean survival of 12 patients who had any level of molecular positivity was 26.5 months. Relapse rate was 38.5%, with relapse duration 1-14 months (mean 4.62 months). The overall survival in relapse/refractory group was 19.2 months, compared with 77.2 months in the other group. The analysis of our AML cohort t(8;21) suggests a favourable outcome, with short remission duration of less than 6 months in 73.3% who relapsed. Extramedullary disease and induction failure are poor prognostic features and HSCT is a feasible treatment strategy for this cohort. Role of PCR negativity as best molecular response during the treatment course as a prognostic indicator needs further evaluation. Disclosures No relevant conflicts of interest to declare.

Background: The ASXL2 (Additional Sex comb-like 2) gene on chromosome 2p23.3 encodes an epigenetic regulator that is thought to act through histone modification and thereby regulating gene transcription in a context-dependent manner. Recently, ASXL2 mutations (ASXL2mut) were found with a high incidence (~23%) in a cohort of 110 pediatric or adult AML patients (pts) with t(8;21)(q22;q22) (Micol et al., Blood 2014). Aim: We assessed the frequency and prognostic impact of ASXL2mut in the context of other clinical and genetic factors in a large clinically well-defined cohort of intensively treated adults with t(8;21) AML. In addition, the stability of ASXL2 mutation status was analysed in a subset of pts at the time of relapse. Methods: Diagnostic samples from 204 adults with t(8;21);RUNX1/RUNX1T1 -positive AML [median age: 49 years, range: 18-82] were analysed for ASXL2mut in exon 11 and 12 using a combination of PCR-based amplification and subsequent direct DNA-sequencing. Paired samples at diagnosis and relapse were evaluated for the ASXL2 mutation status in a subset of 22 pts. Additional mutation analyses were performed for KIT, FLT3 (ITD and TKD), NRAS, and ASXL1 genes. All pts received intensive treatment either within AMLSG trials (n=155) or according to standard chemotherapy regimens. Results: Thirty four ASXL2mut were identified in 33 (16.2%) of the 204 pts. All mutations (frame-shifts, n=32; non-sense, n=2) created stop codons leading to premature protein truncation with loss of the terminal PHD domain; 27% of the mutations affected codon T740 in exon 12. At diagnosis, there was no significant difference between pts with ASXL2mut and ASXL2 wildtype (ASXL2wt) with respect to sex, WBC, haemoglobin, platelets, LDH serum levels, and circulating or bone marrow blasts. Of note, ASXL2mut were not associated with increasing age, a finding which is commonly observed for ASXL1 mutations in AML. In terms of secondary chromosome aberrations ASXL2mut were frequently associated with del(9q) (P=.006), whereas they never co-occurred with trisomy 8. There was no significant association between ASXL2mut and all other gene mutations analysed. Analysis for ASXL2 mutation status in 22 paired samples obtained at diagnosis and relapse showed a high stability since the mutation was still present in two pts at relapse whereas none of the remaining 20 ASXL2 wildtype cases acquired ASXL2mut. There was no difference in complete remission (CR) rate after double induction between pts with ASXL2mut (88%) and those with ASXL2wt (92%); the same was true when comparing pts with ASXL1 or ASXL2 mutations (ASXL1/2mut) as one group (93%) versus those with ASXL1/2 wildtype. Neither ASXL2mut nor ASXL1/2mut did impact the endpoints event-free-survival, cumulative incidence of relapse, relapse-free and overall survival. Conclusions: Beside KIT and NRAS, ASXL2 is among the most frequently mutated genes in t(8;21) AML. ASXL2mut did not impact achievement of CR or any survival endpoint. Nevertheless, the high incidence (16.2%) of ASXL2mut in t(8;21) AML and the exclusivity to this subgroup of core-binding factor AML implies a peculiar role of ASXL2 in the leukemogenesis of t(8;21) AML providing a basis for further studies. Disclosures Horst: MSD: Research Funding; Pfizer: Research Funding; Amgen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Boehringer Ingleheim: Research Funding. Schlenk:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Honoraria; Arog: Honoraria, Research Funding; Teva: Honoraria, Research Funding.

Thirty cases of newly diagnosed pediatric acute myeloblastic leukemia (AML) with French-American-British (FAB) M2 morphology were analyzed with cytogenetics and a comprehensive panel of monoclonal antibodies reactive with lymphoid-, natural killer (NK)-cell-, and myeloid- associated antigens. The t(8;21)(q22;q22), or t(8;21;V)(q22;q22;V), translocation was identified in 16 of the 30 cases. Cases with the t(8;21) did not differ significantly from the remaining M2 cases with respect to expression of CD11b, CD13, CD14, CD15, CD33, CD34, CD36, CD41a, CD42b, CDw65, TdT, or HLA-DR. Expression of the B-cell antigen CD19 was detected in 13 of the 16 t(8;21) cases (81%), but in only 1 of the 14 (7%) other M2 cases (P = .00006). Expression of the CD56 NK-cell antigen was also significantly more frequent among t(8;21) cases (63% v 14%; P = .01). Coexpression of CD19 and CD56 was found only in the t(8;21) group (9 of 16 cases, P = .0009). Furthermore, this phenotype was not found in 48 evaluable cases of de novo AML of the FAB M1, M3, M4, M5, or M7 subtypes. The 14 M2 AML cases lacking the t(8;21) commonly expressed CD2 (n = 5) or CD7 (n = 8). However, no case with the t(8;21) expressed either antigen (P = .01 and .0005, respectively). Thus, the t(8;21) biologic subgroup of pediatric M2 AML has distinct immunophenotypic characteristics that distinguish it from other types of de novo AML.

Thirty cases of newly diagnosed pediatric acute myeloblastic leukemia (AML) with French-American-British (FAB) M2 morphology were analyzed with cytogenetics and a comprehensive panel of monoclonal antibodies reactive with lymphoid-, natural killer (NK)-cell-, and myeloid- associated antigens. The t(8;21)(q22;q22), or t(8;21;V)(q22;q22;V), translocation was identified in 16 of the 30 cases. Cases with the t(8;21) did not differ significantly from the remaining M2 cases with respect to expression of CD11b, CD13, CD14, CD15, CD33, CD34, CD36, CD41a, CD42b, CDw65, TdT, or HLA-DR. Expression of the B-cell antigen CD19 was detected in 13 of the 16 t(8;21) cases (81%), but in only 1 of the 14 (7%) other M2 cases (P = .00006). Expression of the CD56 NK-cell antigen was also significantly more frequent among t(8;21) cases (63% v 14%; P = .01). Coexpression of CD19 and CD56 was found only in the t(8;21) group (9 of 16 cases, P = .0009). Furthermore, this phenotype was not found in 48 evaluable cases of de novo AML of the FAB M1, M3, M4, M5, or M7 subtypes. The 14 M2 AML cases lacking the t(8;21) commonly expressed CD2 (n = 5) or CD7 (n = 8). However, no case with the t(8;21) expressed either antigen (P = .01 and .0005, respectively). Thus, the t(8;21) biologic subgroup of pediatric M2 AML has distinct immunophenotypic characteristics that distinguish it from other types of de novo AML.

Twenty-three acute myelocytic leukemia (AML) patients with t(8;21) chromosomal abnormality, all classified as M2 (French-American-British [FAB] classification), were investigated. Blastic cells from all patients were positive for the stem cell-associated antigens, CD34 and HLA-DR, and the immature myeloid antigens, CD13 and CD33. The nonblastic leukemic cells expressed the more mature myeloid antigens, CD11b and CD15, with loss of the immature phenotype. The incidence of positivities for the stem cell-associated antigens, CD34 and HLA-DR, in t(8;21) AML cells was significantly higher in comparison with those in other AML showing granulocytic differentiation (M2 or M3). AML cells with t(8;21) also showed some phenotypic abnormalities. Frequent expression of CD19 was found in the blastic population of t(8;21) AML (18 of 23 cases) without other B-cell antigens and Ig gene rearrangements. CD19 expression was confirmed by immunocytochemistry and Northern blotting. The CD19+ blastic cells coexpressed both CD34 and HLA-DR. In addition, CD33+ cells among the blastic fraction in t(8;21) AML cells were fewer in number than in those of M2 or M3 AML without t(8;21). Our findings indicate that leukemic blasts of t(8;21) AML commonly express CD19 while preserving the stem cell-associated antigens, and differentiate into the granulocytic pathway with discordant maturation such as low CD33 expression.

Distinct forms of tyrosine kinase domain (TKD), juxtamembrane domain, exon 8, and internal tandem duplication (ITD) mutations of c-KIT, were observed in about 46% of core binding factor leukemia (CBFL) patients. To evaluate their prognostic significance, 67 adult patients with CBFL were analyzed to ascertain the c-KIT mutation status. In acute myeloid leukemia (AML) with t(8;21), the presence of c-KIT TKD mutation at codon 816 (TKD(816)) was associated with a high white blood cell count at diagnosis (median, 29.60 x 10(9)/L) and a higher incidence (33%) of extramedullary leukemia (EML) during the course of the disease. Data also showed that the TKD(816) mutated patients (n = 12) had a significantly higher incidence of relapse and a lower overall survival (OS) at 24 months, compared with the 17 c-KIT unmutated (c-KIT(-)) patients (90% vs 35.3%, P = .002; 25% vs 76.5%, P = .006, respectively). No difference in relapse incidence (P = .126) and OS (P = .474) was observed between the c-KIT mutated other than TKD(816) (n = 7) and the c-KIT(-) patients. These findings indicate that c-KIT TKD(816) mutation has a negative impact on the outcome of AML with t(8;21).

Abstract 2646 Poster Board II-622 Background: t(8;21)(q22;q22) acute myeloid leukemia (t(8;21) AML) show a high rate of complete remission (CR) and prolonged CR duration, especially following consolidation chemotherapy with high-dose cytarabine (HD-ARAC), and are thought to have a better prognosis than other AML patients. Nevertheless, only approximately 50% of patients were alive at 5 years. This suggests that some patients have more aggressive leukemic phenotypes and indicates the need for predictive molecular marker of relapse and treatment optimization with novel and/or more aggressive therapies such as stem cell transplantation.Recently, several groups reported that c-kit mutation (MutKIT) defined an unfavorable subgroup in t(8;21) AML. However MutKIT has been reported approximately 20% at diagnostic samples of core binding factor leukemia. It is possible that MutKIT are more high frequency at relapse sample of t(8;21) AML. In the present study, we analyzed samples collected at diagnosis and relapse to investigate the role of MutKIT s and Flt3 internal tandem duplication (ITD) in t(8;21) AML. Methods: We analyzed MutKIT and Flt3 ITD among 32 t(8;21) AML patients diagnosed between 1991 to 2009 at the Nippon Medical School. All of them were succeeded achieving CR, but 18 patients (56.3%) relapsed, became refractory chemotherapy, and poor prognosis. Exon 8 and 17 of MutKIT were analyzed by direct sequence and QProbe-system (ARKRAY, Inc. Kyoto, Japan). QProbe-system was high sensitivity mutation screening method, detected approximately 1% MutKIT using mutation specific guanine quenching probe (Leukemia Res 32 (2008) 1462–1467). FLT3 ITD was analyzed by PCR amplification. Results: Using direct sequence, MutKITs were found in 5 (18.5%) of the 27 patients at diagnosis (D816V: 3 patients AN822K: 2 patients), and 7 (46.6%) of 15 patients at relapse. Interestingly 3 patients were detected MutKIT at only relapse (D816V: 2 patients AN822K: 1 patient). All mutations found in exon 17 clustered within the A-loop. Next we analyzed to detected very slight amount of mutation by QProbe-system. N822K were newly found of the 3 patients at diagnosis in spite of negative by direct sequence Finally MutKIT were found 8 (29.6%) at diagnosis. Flt3 ITD were found in 2 (9.5%) of the 21 patents at diagnosis, and none of 15 patients at relapse. 2 patients with Flt3 ITD were not accompanied MutKIT. All of N822K newly found by QProbe-system and Flt3 ITD positive patients were relapsed but turn to mutation negative. To evaluate the importance of MutKIT as a predicted relapse, we analyzed the cumulative incidence of relapse (CIR) and relapse free survival (RFS) of these patients with Kaplan-Meier method. The CIR was higher for patients with MutKIT (p=0.040, Wild type c-kit (WtKIT): 45.8% vs MutKIT: 87.5%). In direct sequencing method, The RFS tend to be shorter for patient with MutKIT, but no significant differences (p=0.260; WtKIT: 30 months vs MutKIT: 11 months).On the other hand, in QProbe-system, the median RFS was shorter for patients with MutKIT (p=0.033; WtKIT: 64 months vs MutKIT: 11 months). In addition of FLT3-ITD analysis, median RFS was shorter for patients with MutKIT or FLT3 ITD (p=0.005; Wild type of both genes: 64 months vs MutKIT or FLT3 ITD: 10 months). Additionally, we compared overall survival (OS) of wild type of both genes with MutKIT or FLT3 ITD. The OS tend to be shorter for patient with MutKIT or FLT3 ITD, but no significant differences. Discussion: In this study, we confirmed the prognostic significance of MutKIT in patients with t(8;21) AML when we use the highly sensitive quenching probe method (QProbe-system). We showed that QProbe-system is quite useful in predicting the prognosis of patients with t(8;21) AML and in determining its therapeutic strategy including tyrosine kinase inhibitors and/or up-front stem cell transplantation. We also revealed the important role of MutKIT (46.7%) at relapse, but several questions were still remained. It is unknown for some patients how to gain MutKIT at only relapse and why mutations detected at diagnosis were disappeared at relapse. We need further study to clarify the function and commitment of MutKIT in relapse of t(8;21) AML. Disclosures: No relevant conflicts of interest to declare.

Introduction. Within the group of core binding factor (CBF) AML, the presence of the t(8;21)(q22;q22) confers a favorable prognosis based on high complete remission rates and high survival probabilities. However within this subgroup the presence of KIT mutations and in some studies specifically mutations at codon 816 in exon 17 have been associated with inferior event free survival, relapse free survival, cumulative incidence of relapse and overall survival. Dasatinib a dual SRC/ABL kinase inhibitor is an active agent already approved for the treatment of imatinib resistant or intolerant chronic myelogenous leukemia which has shown in vitro activity against KIT exon 17 mutations including the D816 imatinib resistant mutation. The aim of the present study was the investigation of the activity of dasatinib on cell proliferation and apoptosis of leukemic cell lines with or without KIT mutations. Materials and methods. The leukemic cell lines ME-1, NB4 and KASUMI were cultured in RPMI. Following RNA extraction RT-PCR was performed for the amplification of the extracellular (exon 8,9), transmembrane/juxtamembrane (exon 10,11) and tyrosine kinase 2 domains (exon 17,18) of c-Kit.Following sequencing only the KASUMI cell line derived from a t(8;21)(q22;q22) AML was found to bear the N822K KIT mutation at exon 17, also described in patients’samples. The KASUMI, the K562 cell line bearing the t(9;22) used as a positive control and the NB4 cell line without KIT mutations used as a negative control, were subsequently cultured under the presence of dasatinib at the concentrations of 1nM, 10nM, 100nM, 500 nM. Cell proliferation, was determined at 24, 48, 72 h using the Cell Proliferation Elisa, BrDU protocol and apoptosis was determined by the method of annexin using flow cytometry at the same time points. Results The BrDU value of K562 cells at 48h without the drug was 1.046 significantly higher compared to those of cells cultured under the presence of Dasatinib at 1nM, 10nM, 100nM, 500 nM (0.6485, 0,5647, 0,4770, 0.4755 respectively) (p Conclusion. Dasatinib is an effective suppressor of proliferation and inducer of apoptosis of the KASUMI cell line with the t(8;21)(q22;q22) and the N822K KIT mutation. These encouraging results need to be confirmed on patients’ cells with the view to integrate the drug in conventional chemotherapy regimens in future clinical trials.

AML1-MTG8 generated by t(8;21)(q22:q22) contributes to leukemic transformation but additional events are required for full leukemogenesis. Mutations in the receptor tyrosine kinase (RTK) including C-KIT and FLT3 genes appear to be the genetic events that cause acute myeloid leukemia (AML) harboring t(8;21) and are associated with unfavorable prognosis (Nanri et al. Leukemia19:1361;2005). On the other hand, the activating V617F mutation in the JAK2 cytoplasmic tyrosine kinase has been detected in a significant proportion of patients with myeloproliferative disorders. Although the same mutation has been identified in a small number of AML patients, it is likely that a relatively high incidence of JAK2-V617F mutation has been seen in t(8;21) AML patients. However, whether they are associated with other biological parameters and whether they influence clinical prognosis in patients with t(8;21) AML have not been determined. To clarify the biological and prognostic impact of the JAK2 mutation, we examined the clinical and prognostic relevance of the mutation. Of 45 patients with t(8;21) AML, mutations in the C-KIT and internal tandem duplications (ITD) in the FLT3 were observed in 18 (40%) and 3 (6.7%), respectively. We detected the JAK2-V617F mutation in 3 patients with t(8;21) AML (6.7%), which was consistent with previous studies. Although the occurrence of C-KIT and FLT3 mutations was mutually exclusive, one patient harboring JAK2 mutation also had a C-KIT second tyrosine kinase mutation and another one patient had a K-RAS mutation. Collectively, a total of 23 (51%) patients showed mutations in the RTK pathway. One patient carrying both C-KIT and JAK2 mutations did not respond to multiple induction chemotherapies. Another patient with the JAK2 and K-RAS mutations achieved a complete remission (CR) but later relapsed. Remaining one patient received allogeneic stem cell transplantation during first CR and continued CR. As we were limited to study a small number of mutated cases for a comparison of clinical and genetic features, we examined the clinical significance of C-KIT, FLT3 and JAK2 mutations as a collective group. A cumulative incidence of relapse (CIR) in 21 patients with RTK mutations was 77%, whereas CIR in 19 patients lacking mutations was 26% (P=0.0083). The 6-year relapse-free survival in patients with mutations was 20% compared to 54% in those without mutations (P=0.0595). These results suggest that the mutations in the JAK2, C-KIT and FLT3 could be an additional genetic events leading to the development of AML and are associated with the clinical outcome in patients with t(8;21) AML.