Intermediate Cytogenetic Risk Acute Myeloid Leukemia Research Articles

IL1R1 Expression Predicts the Benefit from Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia and Intermediate-Risk Cytogenetics

Introduction: Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy with variable clinical outcomes depending on baseline patient and disease characteristics. After achieving complete remission (CR) with induction chemotherapy, eligible patients are treated with consolidation chemotherapy or allogeneic hematopoietic stem cell transplantation (HSCT) to prevent relapse. In patients with intermediate-risk cytogenetics (IRC) without adverse-risk mutations, the decision for post-remission therapy is challenging because of the significant risks of morbidity and mortality associated with HSCT which can offset its benefit in preventing relapse. Therefore, novel biomarkers are needed to support the decision to recommend HSCT in first CR (CR1) in patients with IRC AML. Methods: We used the Leucegene AML prognostic cohort composed of patients diagnosed with de novo IRC AML and treated with intensive induction chemotherapy (N = 316) to identify biomarkers that are predictive for the benefit of HSCT in CR1. Whole-transcriptome sequencing was performed on diagnostic patients' samples. Gene expression data was normalized in transcripts per million (TPM) then log transformed and standardized into Z-scores. We used univariable and multivariable Cox proportional hazards (CPH) regression models for overall survival (OS) and relapse-free survival (RFS) to evaluate the association between gene expression and clinical outcomes. For multivariable analyses (MVA), we included age and white blood cell (WBC) count at diagnosis and NPM1, FLT3-ITD, DNMT3A, biallelic CEBPA, ASXL1 and RUNX1 mutations as covariables. To identify genes predictive of the benefit of HSCT in CR1, we evaluated interaction terms between gene expression and HSCT in CR1 as a time-dependent variable (HSCT-TD) in extended CPH regression models for OS and RFS. We dichotomized the expression of genes based on optimal cutoffs for p value and hazard ratio (HR) for OS and RFS while optimizing their clinical utility to predict the benefit of HSCT. Results: We identified IL1R1 as the top gene associated with RFS in MVA and with a significant interaction with HSCT-TD (p < 0.05). Based on an optimal cutoff of 2.0 TPM, 193 (61%) and 123 (39%) patients had low (< 2.0 TPM, IL1R1low) and high (≥ 2.0 TPM, IL1R1high) expression of IL1R1, respectively. Patients with IL1R1high were older (median age 59 years with IL1R1high vs 54 years with IL1R1low, p < 0.01), had a higher WBC count (44 vs 31 x 109/L, p = 0.06), and a higher frequency of myelomonocytic or monocytic differentiation (43% vs 25%, p < 0.01). FLT3-ITD (54% vs 33%, p < 0.01) and RUNX1 (18% vs 9%, p = 0.04) mutations were more frequent in patients with IL1R1high whereas biallelic CEBPA mutations (1% vs 6%, p=0.04) were more frequent in patients with IL1R1low. High expression of IL1R1 was associated with worse clinical outcomes. The CR rate was 74% and 85% in patients with IL1R1high and IL1R1low, respectively (p = 0.02). The 5-year OS rate was 10% in patients with IL1R1high vs 38% in patients with IL1R1low (HR 2.27, p < 0.01). The 5-year cumulative incidence of relapse (CIR) was 17% higher in patients with IL1R1high (76% vs 59%, p < 0.01). In MVA, high expression of IL1R1 was independently associated with OS (HR 1.80, p < 0.01) and RFS (HR 1.81, p < 0.01). HSCT in CR1 significantly improved OS in patients with IL1R1high (HR for HSCT-TD 0.26, p < 0.01), but not in patients with IL1R1low (HR 0.70, p = 0.17). With a 6-month landmark analysis, the 5-year OS rates were 67% vs 27% among patients with IL1R1high and 62% vs 54% among patients with IL1R1low in patients who underwent HSCT in CR1 or not, respectively (Figure 1). Specifically among patients without FLT3-ITD mutations, patients with IL1R1high benefited from HSCT in CR1 (HR 0.41, p = 0.04) whereas patients with IL1R1low did not (HR 1.03, p = 0.92). Among patients who have undergone HSCT in CR1, the 5-year post-HSCT OS was 60% vs 56% in patients with IL1R1high and IL1R1low, respectively (HR 0.85, p = 0.68), confirming that HSCT may abrogate the adverse impact of high IL1R1 expression. Conclusion: In patients with IRC AML, high expression of IL1R1 is independently associated with worse OS and RFS which may be overcome by HSCT in CR1. Among patients without FLT3-ITD mutations who account for ~60% of IRC AML, those with IL1R1high benefit from HSCT in CR1, as opposed to those with IL1R1low. IL1R1 expression may guide the decision to proceed with HSCT in CR1 in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Novel Antigens for Normal Karyotype Triple Mutated Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. AML is divided in several subgroups based on genetic alterations and prognosis varies among AML subgroups. Normal karyotype AML with NPM1, DNMT3A and FLT3(ITD) mutations (NK triple mutated AML) is associated with a long-term overall survival of ~20% and represents ~6% of AML cases (Papaemmanuil et al., NEJM, 2016). No curative chemotherapy for this subgroup of AML patients currently exists, highlighting the need to develop novel therapeutic strategies for this group of AML patients. Methods To identify novel cell surface antigens for NK triple mutated AML that could ultimately be targeted by immunotherapeutic approaches, we developed a method to map the surface proteome of primary human AML specimens based on biotinylation of cell surface proteins, affinity purification and mass spectrometry. To further enrich mass spectrometry datasets with surface proteins, we developed an algorithm that assesses the probability of a protein to be located on the cell surface based on annotations from various databases. Using this approach, the surface proteome of 100 primary human AML specimens was analysed, which included 12 NK triple mutated AML samples. Differential surfaceome analyses identified several surface proteins specifically expressed by NK triple mutated AML specimens. Expression analyses were performed using publicly available datasets to select proteins with limited expression in normal tissues and hematopoietic cells. Surface expression and AML subgroup specificity for these potential antigens was evaluated by flow cytometry using primary human AML specimens from various AML subgroups (n=76). Moreover, to assess the intra-specimen expression profile of these antigens, single cell RNA-sequencing of 15 primary human NK triple mutated AML specimens was performed. Finally, we evaluated the prognostic impact of antigen expression on overall survival in a subgroup of patients from the Leucegene cohort composed of patients diagnosed with de novo AML with intermediate risk cytogenetics who have been treated with intensive induction chemotherapy (n = 316). Results Surface proteome analysis of NK triple mutated AML specimens and hit filtering according to selection pipeline described above identified several promising NK triple mutated AML antigens. Surface expression analysis of these antigens by flow cytometry showed specificity of antigen expression for NK triple mutated AML specimens. Transcriptomic analyses using RNA sequencing data from primary human AML specimens of the Leucegene cohort (n=691) showed significantly higher antigen transcript levels in NK triple mutated AML specimens compared to samples from other AML subgroups. Moreover, multivariate transcriptome analysis comparing the impact of most frequent mutations in AML on antigen expression revealed that FLT3-ITD mutation is the only significant factor associated with antigen overexpression in AML samples. Single cell RNA sequencing data from NK triple mutated AML specimens revealed that these antigens are uniformly expressed across the vast majority of blast populations, including blast cells with an expression profile reminiscent of leukemic stem cells. Interestingly, in patients with intermediate cytogenetic risk AML, high antigen expression was significantly associated with worse overall survival. Patients with high antigen expression who proceeded to allogeneic hematopoietic stem cell transplantation (HSCT) in first complete remission also had a significantly lower 5-year overall survival rate post-HSCT compared to patients with low antigen expression. Conclusion We identified novel antigens for FLT3-ITD mutated and NK triple mutated AML, which show homogeneous expression in blast populations of AML samples including most primitive ones, making it ideal antigens for immunotherapeutic targeting. Patients with intermediate cytogenetic risk AML and high antigen expression show poor prognosis even after HSCT in first complete remission, highlighting the need to develop novel therapeutic strategies for these patients. Different immunotherapeutic approaches targeting these antigens are currently being developed by our group, with the hope of identifying novel therapeutic strategies for the treatment of NK triple mutated AML.

Gemtuzumab Ozogamicin Plus Standard Induction Chemotherapy Improves Outcomes in Intermediate Risk Cytogenetic Acute Myeloid Leukemia

Background: Gemtuzumab ozogamicin (GO) is a CD33-directed antibody-drug conjugate approved for the treatment of newly diagnosed (ND) and refractory/relapsed CD33-positive acute myeloid leukemia (AML). Meta-analysis of individual patient (pt) data from 5 randomized controlled trials (n=3325) confirmed a significant survival benefit of GO added to intensive induction chemotherapy for pts with AML without adverse cytogenetics. While favorable-risk ND-AML pts, specifically core-binding factor AML (CBF-AML), particularly benefited with a 5-year overall survival (OS) improvement of 20.7% (log rank p= 0.0006), pts with intermediate-risk AML also showed a significant 5-yr survival improvement of 5.7% (p=0.005) (Hills RK et al Lancet Oncol 2014: 15, 986). However, the smaller difference in these latter pts coupled with apprehension for risk of sinusoidal obstruction syndrome (SOS) following hematopoietic stem cell transplantation (HSCT) has led many clinicians to not use 7+3 plus GO in the intermediate-risk AML population. Aim/Objective: To perform a retrospective analysis of outcomes of 7+3 plus GO vs 7+3 in ND intermediate-risk cytogenetic AML pts treated at a single comprehensive cancer center. Methods: We studied the demographic, clinical and genetic data of intermediate risk ND-AML pts treated at Roswell Park Comprehensive Cancer Center (n=113) between 2015-2022. Conventional cytogenetics and fluorescence in situ hybridization (FISH) were performed. Genomic profiling was performed using next-generation sequencing methods (FoundationOne®CDx). Risk stratification was defined per the European LeukemiaNet (ELN)-2017 classification. 7+3 plus GO pts received at least one dose of GO 3 mg/m2 (up to one 4.5 mg vial) plus cytarabine 100 mg/m² continuous IV infusion on days 1-7, and daunorubicin 60 mg/m² IV on days 1-3 (7+3). Control pts received 7+3 alone. Prophylactic heparin was administered to prevent hepatic SOS based on institutional standards if not thrombocytopenic. Outcomes included response rate to induction, successful bridging to HSCT, relapse rate, and overall survival. Results: 113 intermediate-risk ND-AML pts were studied: 33 received 7+3 plus GO and 80 received 7+3. Median age was 57 vs. 59 years, respectively (range: 19-76 years). Moreover, there was no significant difference in the percentage of pts > 60 yrs (45% vs 48%). Gender was equally distributed. Pts treated with 7+3 plus GO had a significantly higher rate (p=0.019) of complete remission (CR) and complete remission with incomplete count recovery (CRi) (27/33, 82%) than 7+3 (44/80, 55%; Figure 1A). Among pts with available minimal residual disease (MRD) status detected by multiparameter flow cytometry, 100% achieved MRD-negative status after 7+3 plus GO (18/18) compared to 86% with 7+3 (12/14). Non-responders to 7+3 plus GO were enriched for ASXL1, DNMT3A, TET2, and TP53 mutations as compared to responders who harbored more FLT3, IDH1/2, KRAS/NRAS and RUNX1 mutations. Among pts achieving CR/CRi, 48% after 7+3 plus GO vs 30% after 7+3 (p=0.13) were successfully bridged to HSCT. Cumulative incidence of relapse after CR/CRi was significantly lower after 7+3 plus GO (11%) than 7+3 (43%; p=0.007). Finally, 7+3 plus GO pts demonstrated a superior, yet not statistically significant, median overall survival (OS) benefit (median OS not reached) compared to 7+3 alone (median OS = 35 months; Figure 1B). No pts receiving 7+3 plus GO developed SOS. Conclusions: Intermediate risk ND-AML pts treated with 7+3 plus GO at our academic center had significantly higher rates of MRD-negative CR and CRi, lower rates of relapse, and higher percentage of pts receiving HSCT as compared with 7+3 alone. Non-responders in the 7+3 plus GO group harbored adverse risk genetic mutations that likely dictated the failure to response. Although addition of GO was associated with a non-significant trend towards improved overall survival, longer follow-up is warranted to decipher an absolute survival advantage of this regimen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

5'-nucleotidase, cytosolic II genotype, and clinical outcome in patients with acute myeloid leukemia with intermediate-risk cytogenetics.

Acute myeloid leukemia (AML) is a complex disease, and its treatment needs to be adjusted to the risk, which is conferred by cytogenetics and molecular markers. Cytarabine is the main drug to treat AML, and it has been suggested that the genotype of cytarabine metabolizing enzymes may have a prognostic relevance in AML. Here we report the association between the 5'-nucleotidase, cytosolic II (NT5C2) rs10883841, cytidine deaminase (CDA) rs2072671 and rs532545 genotypes and the clinical outcome of 477 intermediate-risk cytogenetic AML patients receiving cytarabine-based chemotherapy. Patients younger than 50 years old with the NT5C2 rs10883841 AA genotype had lower overall survival (OS) (p: .003; HR 2.16, 95% CI 1.29-3.61) and lower disease-free survival (DFS) (p: .002; HR 2.45, 95% CI 1.41-4.27), associated to a higher relapse incidence (p: .010; HR 2.23, 95% CI 1.21-4.12). Interestingly, subgroup analysis showed that the negative effect of the NT5C2 rs10883841 AA genotype was detected in all subgroups except in patients with nucleophosmin mutation without high ratio FLT-3 internal tandem duplication. CDA polymorphisms were associated with the complete remission rate after induction chemotherapy, without influencing OS. Further studies are warranted to determine whether this pharmacogenomic approach may be helpful to individualize AML treatment.

Comparable outcomes of haploidentical transplant with TBF conditioning versus matched unrelated donor with fludarabine/busulfan conditioning for acute myeloid leukemia.

We compared transplant outcomes of 708 acute myeloid leukemia (AML) patients receiving haploidentical allogeneic hematopoietic-cell transplantation using thiotepa/busulfan/fludarabine (TBF) conditioning with posttransplant cyclophosphamide (ptCy), to 2083 patients receiving matched unrelated donor (MUD) transplantation using fludarabine/busulfan (FB) conditioning and in vivo T-cell depletion. For intermediate cytogenetic risk AML transplanted in first complete remission (CR1), multivariate analysis revealed that haplo-TBF significantly increased nonrelapse mortality (NRM) (HR 2.1; p = 0.0006) but did not affect relapse incidence (RI), leukemia-free survival (LFS), overall survival (OS), or graft-versus-host disease-free, relapse-free survival (GRFS). For high cytogenetic risk AML transplanted in CR1, haplo-TBF significantly increased NRM (HR = 2.7; p = 0.02), decreased RI (HR = 0.45; p = 0.03) but had no influence on LFS, OS, or GRFS. For AML transplanted in CR2, haplo-TBF significantly increased NRM (HR = 2.36; p = 0.008), decreased RI (HR = 0.38; p = 0.005), but had no influence on LFS, OS, or GRFS. Finally, for AML patients transplanted with active disease, haplo-TBF had no influence on transplant outcomes. In conclusion, compared to MUD-FB, haplo-TBF increased NRM, reduced RI in high-risk AML in CR, resulting in similar LFS, OS, and GRFS. These results comparing two different approaches support the use of a haploidentical family donor for high-risk AML patients lacking a matched sibling donor.

UGT1A1 genotype influences clinical outcome in patients with intermediate-risk acute myeloid leukemia treated with cytarabine-based chemotherapy.

The treatment of acute myeloid leukemia (AML) is adjusted according to cytogenetic risk factors and molecular markers. Cytarabine remains the main drug to treat AML, and several studies have explored the prognostic relevance of the genotype of cytarabine metabolizing enzymes in AML. Glucuronidation has been identified to be relevant in the cytarabine clearance, but there are still few data concerning the clinical impact of genetic polymorphisms known to condition the activity of UDP-glucuronosyl transferases in AML patients. Here we report the association between the UGT1A1 rs8175347 genotype and the clinical outcome of 455 intermediate-risk cytogenetic AML patients receiving cytarabine-based chemotherapy. Patients with the UGT1A1*28 homozygous variant (associated to a lower UGT1A1 activity) had a lower overall survival (OS) (25.8% vs. 45.5%; p: 0.004). Multivariate analysis confirmed this association (p: 0.008; HR: 1.79; 95% CI: 1.16-2.76). Subgroup analysis showed the negative effect of the UGT1A1*28 homozygous genotype on OS in women (14.8% vs. 52.7%; p: 0.001) but not in men. This lower OS was associated with longer neutropenia after consolidation chemotherapy and with higher mortality without previous relapse, suggesting an association between a low glucuronidation activity and mortal toxic events.

Outcomes of Autologous Hematopoietic Cell Transplantation Compared With Chemotherapy Consolidation Alone for Non–High-Risk Acute Myeloid Leukemia in First Complete Remission in a Minority-Rich Inner-City Cohort With Limited Access to Allografts

IntroductionIn the United States, autologous hematopoietic cell transplantation (autoHCT) has fallen out of favor over chemotherapy consolidation for non–high-risk acute myeloid leukemia (AML) when allogeneic hematopoietic cell transplantation (alloHCT) is unfeasible, which is common in racial minorities because of donor registry under-representation and socioeconomic challenges. We compared autoHCT consolidation outcomes with chemotherapy alone in a minority-rich cohort in the Bronx. Patients and MethodsWe identified adults with favorable or intermediate cytogenetic risk AML in first complete remission after induction at Montefiore Medical Center from 1999 to 2015, and analyzed 81 patients who received consolidation with ≥2 cycles of chemotherapy, of whom 28 received autoHCT. ResultsThe cohort predominantly consisted of ethnic/racial minorities (69%). Age, sex, race, presenting white cell count, and cytogenetic risk were similar between groups. The autoHCT group had longer relapse-free (RFS; 43 vs. 11 months; P = .003) and overall (OS) survival (not reached vs. 36 months; P = .043). Adjusted multivariable analysis showed significant benefit of autoHCT over chemotherapy alone for RFS (hazard ratio [HR], 0.53; 95% confidence interval [CI], 0.37-0.75; P < .001) and OS (HR, 0.61; 95% CI, 0.40-0.95; P = .027). ConclusionIn this inner-city non–high-risk AML cohort, autoHCT provided OS and RFS benefit compared with chemotherapy alone. AutoHCT might constitute a valuable option for ethnic/racial minorities affected by significant barriers to alloHCT, whereas integration of measurable residual disease can help select patients more likely to benefit.

High Expression of SPAG1 Is Associated with a Worse Clinical Outcome in Intermediate Risk Acute Myeloid Leukemia That Can be Partially Overcome By Hematopoietic Stem Cell Transplantation

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease with variable responses to therapy and clinical outcomes. Cytogenetics and molecular analyses help to stratify patients and to select therapy, especially regarding indication of hematopoietic stem cell transplant (HSCT) after achieving complete remission (CR). Patients in the intermediate cytogenetic risk category (~40%) represent a clinical dilemma regarding consolidation because of the high rate of relapse with chemotherapy alone and high rate of morbidity/mortality with HSCT. Consequently, we aimed to identify new prognostic markers to refine the risk stratification of this patients' subgroup and to identify which patients are most likely to benefit from HSCT.Methods: We analyzed RNA sequencing data of 263 specimens from patients with de novo AML treated with curative intent including 165 patients with intermediate risk cytogenetics. Data from 24 586 genes were normalized as RPKM values with logarithmic transformation and standardization. Cox proportional hazard models were used to assess the prognostic impact of gene expression (GE) on overall survival (OS) and relapse-free survival (RFS). We performed univariate analyses (UVA) and multivariate analyses (MVA) adjusted for age and white blood cell count (WBC) at diagnosis, mutations in NPM1, FLT3, CEBPA, RUNX1, ASXL1, TP53 and DNMT3A and HSCT as a time-dependent (TD) covariate. Interaction between GE and TD-HSCT was tested in MVA for OS and RFS. Genes with a significant interaction between GE and TD-HSCT (p < 0.10) were retained for further analyses. GE of candidate markers were dichotomized using a bioinformatic method assessing hazard ratios (HR) and p values for all potential cut-offs to identify the most optimal threshold. The markers were finally reassessed as dichotomic variables for association with covariates, CR rates, RFS and OS. All statistical tests were two-sided with p values < 0.05 considered significant.Results: We identified SPAG1 (Sperm Associated Antigen 1) as the gene with the highest HR for OS and RFS in the intermediate cytogenetic risk group. SPAG1 expression was dichotomized on the median RPKM value in the global cohort of 263 de novo AML specimens (RPKM cut-off 2.06). Using this cut-off, 79 patients had low expression of SPAG1 and 86 patients had high expression of SPAG1 in the intermediate cytogenetic risk cohort. Median age, sex, WBC at diagnosis and HSCT in CR1 rates were similar between the two groups. Patients with high expression of SPAG1 had a higher frequency of FLT3-ITD (51.2% vs 32.9%, p = 0.02) and DNMT3A mutations (51.2% vs 32.9%, p = 0.02). SPAG1-high patients were enriched in the NPM1/FLT3-ITD/DNMT3A triple positive mutation population (SPAG1-high 24/33 vs SPAG1-low 9/33, OR 3.00, p = 0.01). The frequencies of all other analyzed mutations were similar between both groups. CR rates did not differ between the two groups (SPAG1-high 77.9% vs SPAG1-low 79.7%, p = 0.77). In UVA with censorship at time of HSCT, SPAG1-high patients had worse OS (5-year estimates 14.2% vs 28.1%, HR 1.75, 95% CI 1.16-2.63, p < 0.01) and RFS (5-year estimates 9.3% vs 27.1%, HR 1.90, 95% CI 1.20-3.01, p < 0.01) (Figure 1). In MVA with censorship at time of HSCT, high expression of SPAG1 remained significantly associated with OS (HR 1.78, 95% CI 1.12-2.83, p = 0.01) and RFS (HR 2.34, 95% CI 1.38-3.96, p < 0.01). Furthermore, there was a significant interaction between SPAG1 GE and TD-HSCT (p = 0.09) in the RFS model. Importantly, SPAG1 had a lower prognostic impact for RFS in the model including TD-HSCT (HR 1.65, 95% CI 1.07-2.55, p = 0.02) compared with the model in which survival was censored at time of HSCT (HR 2.34, p < 0.01). High SPAG1 expression was also associated with worse OS in the TCGA AML dataset which is enriched in intermediate cytogenetic risk samples (p < 0.001).Conclusion: In patients with intermediate cytogenetic risk AML, high expression of SPAG1 is independently associated with worse OS and RFS. The prognostic impact of SPAG1 expression on RFS is lower when adjusted for TD-HSCT indicating that the adverse prognosis conferred by high expression of this gene may be partially overcome by HSCT in CR1. Consequently, SPAG1 expression might help identify AML patients with intermediate cytogenetic risk who are most likely to benefit from HSCT in CR1. These results need to be validated in other independent cohorts and prospective studies before implementation into clinics. [Display omitted] DisclosuresSauvageau:ExCellThera: Employment, Equity Ownership.

Allogeneic stem cell transplantation benefits for patients ≥ 60 years with acute myeloid leukemia and FLT3 internal tandem duplication: a study from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.

Intermediate-risk cytogenetic acute myeloid leukemia with an internal tandem duplication of FLT3 (FLT3-ITD) is associated with a high risk of relapse, and is now a standard indication for allogeneic stem cell transplantation. Nevertheless, most studies supporting this strategy have been performed in young patients. To address the benefit of allogeneic transplantation in the elderly, we made a selection from the European Society for Blood and Marrow Transplantation registry of de novo intermediate-risk cytogenetic acute myeloid leukemia harboring FLT3-ITD in patients aged 60 or over and transplanted from a related or unrelated donor between January 2000 and December 2015. Two hundred and ninety-one patients were identified. Most patients received a reduced-intensity conditioning (82%), while donors consisted of an unrelated donor in 161 (55%) patients. Two hundred and twelve patients received their transplantation in first remission, 37 in second remission and 42 in a more advanced stage of the disease. The 2-year leukemia-free survival rate was 56% in patients in first remission, 22% in those in second remission and 10% in patients with active disease, respectively (P<0.005). Non-relapse mortality for the entire cohort was 20%. In multivariate analysis, disease status at transplantation was the most powerful predictor of worse leukemia-free survival, graft-versus-host disease and relapse-free survival, and overall survival. In this elderly population, age was not associated with outcome. Based on the current results, allogeneic transplantation translates into a favorable outcome in fit patients ≥ 60 with FLT3-ITD acute myeloid leukemia in first remission, similarly to current treatment recommendations for younger patients.

Allogeneic Stem Cell Transplantation for Elderly Patients with Intermediate-Risk Cytogenetic Acute Myeloid Leukemia and Internal Tandem Duplication of FLT3 (FLT3-ITD); A Study from the Acute Leukemia Working Party (ALWP) of the European Society of Blood and Marrow Transplantation (EBMT)

Introduction: The presence of an internal tandem duplication of FLT3 (FLT3-ITD) confers a higher risk of relapse and is now a current indication of allogeneic stem cell transplantation (SCT) in patients with intermediate-risk cytogenetic acute myeloid leukemia (IRC-AML) in first complete remission (CR1). Most studies encouraging this strategy have been performed in patients below 60 year-old, after a myeloablative conditioning (MAC) and using a sibling donor. Because age remains associated with a worse outcome after SCT, we decided to analyse outcomes of SCT in patients aged 60 year-old or older with intermediate-risk AML and FLT3-ITD.Methods: Using the EBMT registry, we selected de novo acute myeloid leukemia (AML) harboring IRC-AML and FLT3-ITD in patients transplanted from a related or matched unrelated donor (9/10 or 10/10) between January 2000 and July 2014.Results: Two hundred and five patients have been allocated. Median age at the time of SCT was 64 (range, 60-75) year-old and median follow-up was 20 (range, 2-139) months. Ninety-four percent of the patients had a good performance status (Karnofsky at SCT ≥ 80%). Most patients had a normal karyotype at diagnosis (90% versus 10% with other intermediate-risk karyotype) and NPM1 status was reported in 131 patients out of which 100 (76%) were mutated. Thirty-four patients received a MAC, 142 had a reduced-intensity conditioning (RIC) and 29 a non-myeloablative conditioning (NMA). One hundred forty-six patients received their SCT in first remission (CR1), 24 in second remission (CR2) and 35 in more advanced stage of the disease, respectively. The 2-year leukemia-free survival (LFS) was 52% in patients in CR1, 17% in those in CR2 and 11% in patients with advanced disease, respectively (p<0.005). Similarly, the 2-year overall survival (OS) was 54% in CR1, 24% in CR2 and 11% in advanced disease, respectively (p<0.005). The 2-year non-relapse mortality (NRM) for the all cohort was 20%. The cumulative incidence of grade II to IV acute graft-versus-host disease (GvHD) was 24% and that of chronic GvHD was 30%.In multivariate analysis, disease status at SCT was the most powerful predictor of worse LFS and OS. Age, as a continuous variable, was not significantly associated with outcomes. Donor type (unrelated versus sibling donor) and donor CMV positivity correlated with worse OS and higher NRM. Next, we performed a second analysis focusing on patients transplanted in CR1. A multivariate analysis performed in this subgroup showed that age, as a continuous variable, did not translate into worse LFS, OS or NRM. Interval from diagnosis to CR1 was significantly associated with LFS, being 63% in patients achieving CR1 in less than 43 days and 45% in patients achieving CR1 in more than 43 days, respectively (p<0.005). NMA conditioning was associated with a trend to a worse OS.Conclusion: Allogeneic SCT in elderly (≥60 up to 75 year-old) patients with IRC-AML harboring the FLT3-ITD mutation, similar to current strategy in younger patients, appears as a good treatment strategy if performed in CR1, independently of age, with somewhat inferior outcome in transplants from unrelated donors. Nevertheless, indication for transplantation should still be evaluated taking into account donor type, co-morbidities and performance status. DisclosuresCraddock:Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Speakers Bureau; Sunesis: Honoraria; Johnson and Johnson: Consultancy. Russell:Therakos: Other: shares.

Exploring the Expression Profile of Long Non-Coding RNA (lncRNA) in Different Acute Myeloid Leukemia (AML) Subtypes: t(8;16)(p11;p13)/MYST3-Crebbp AML Harbors a Distinctive Lncrna Signature

Introduction: Long non-coding RNAs (lncRNAs) have recently emerged as important actors in the regulation of multiple cellular processes including cancer. Acute myeloid leukemia (AML) is a heterogeneous disease; most of the main cytogenetic AML subgroups harbor a specific gene expression profile. AML with translocation t(8;16)(p11;p13) (t(8;16) AML) is a subtype with specific clinical and biological characteristics including a distinctive gene (Camós et al, Cancer Research 2006) and microRNA (Díaz-Beyá et al, Leukemia 2013) expression profile. In this translocation, MYST3 on chromosome 8p11 fuses with CREBBP on chromosome 16p13.3. The MYST3-CREBBP fusion protein is able to interact with multiple transcription factors (TF) producing a disturbed transcriptional program. However, the lncRNA expression pattern of different cytogenetic AML subtypes, including t(8;16) AML, have not been described yet.Aims: To examine the expression profile of lncRNAs within different AML subtypes, and to characterize the expression pattern of lncRNAs in t(8;16) AML in comparison to other AML subtypes.Patients and Methods: 46 AML patients, 4 normal bone marrow (NBM) and 3 CD34+ NBM samples were included in the study. Samples included different AML subtypes: intermediate-risk cytogenetic AML (IR-AML, n=18), t(15;17) (APL, n=4), t(8;21) AML (n=4), inv(16) AML(n=2), t(6;9) AML (n=7), AML with monosomal karyotype (n=4), t(3;3) AML (n=1), t(9;11) AML (n=1) and t(8;16) AML (n=5). Within IR-AML patients with a different mutational profile: FLT3-ITD (n=7), NPM1 (n=5), CEBPA (n=7) and DNMT3A (n=6) were included. The lncRNA expression was studied using Affymetrix® Human Gene 2.1 ST platform which includes 9698 lncRNAs transcripts. The filtering and normalization of the array data was performed using oligo package from Bioconductor. Statistical analyses were performed with TiGR MultiExperiment Viewer, BRB tools and R. The Transcription factor Affinity Prediction Web Tool was used to determine the putative transcription factors binding to the differentially expressed lncRNAs promoters.Results: The hierarchical cluster analysis showed that all 4 NBM as well as all 3 CD34+ NBM clustered together according to their lncRNA expression. Interestingly, all 5 t(8;16) AML samples clustered together, as well as the 3 APL, the 7 t(6;9) AML and 5 out of 7 cases with CEBPA mutations. The specific lncRNA signature of APL was composed of 79 differentially expressed lncRNA and t(6;9) AML lncRNA signature comprised of 15 differentially expressed lncRNAs. When we focused on t(8;16) AML lncRNA profile, we identified an specific 113-lncRNA signature in the supervised analysis (Figure).Interestingly, when we analyzed which (TF) had motifs overrepresented in the promoters regions of the t(8;16) AML lncRNA signature, we identified GATA2 as the TF with significantly overrepresented motifs for GATA2 (p<0.001). Interestingly, levels of GATA2 were differentially expressed in t(8;16) AML samples in comparison with other AMLs samples (p<0.001). GATA2 has been described to interact with CREBBP, one of the partners involved in t(8;16) AML. Of note, 4 overexpressed lncRNAs of the signature (linc-HOXA11, HOXA11-AS, HOTTIP and NR_038120) were located in the HOXA genomic region, previously found upregulated in t(8:16) AML. Since several studies suggest an active crosstalk between microRNAs and lncRNAs, we also correlated the expression of these lncRNAs with the microRNA t(8;16) AML profile. We found significant correlation between linc-HOXA11 and miR-222* (R2 =0.996, p=0.003), HOXA11-AS and miR-let-7c (R2=0.994, p=0.006), HOTTIP and miR-196b*(R2=0.958, p=0.041), and NR_038120 with miR-486-3p (R2=0.999, p=0.0004) and miR-19a (R2=0.953, p=0.04).Conclusions: LncRNAs expression profile seems specific of several AML subtypes, including t(8;16) AML. Some of the lncRNAs of this distinctive signature in t(8;16) AML are located in the HOXA genomic region, and correlate with several of the characteristic microRNAs previously described in this entity. Interestingly, we have predicted in silico GATA2, which interacts with CREBBP, as the most significant TF that could potentially regulate this lncRNAs signature. Nonetheless, further investigation is warranted to determine the mechanisms leading to this lncRNA signature and to identify the specific targets of these lncRNAs.Río Hortega CM13/00205, FIS PI13/00999 DisclosuresNo relevant conflicts of interest to declare.

Prognostic impact of combined NPM1+/FLT3− genotype in patients with acute myeloid leukemia with intermediate risk cytogenetics stratified by age and treatment modalities

The prognostic impact of combined NPM1+/FLT3− genotype is not well defined in elderly patients with acute myeloid leukemia (AML), and in the setting of different treatments, such as cytotoxic chemotherapy (Chemo), hematopoietic cell transplantation (HCT), or hypomethylating agents (HMA). Eighty-two elderly (age >60 years) and 78 younger adults (age 18–60 years) with newly diagnosed intermediate-risk cytogenetic AML were classified according to the presence or absence of NPM1+/FLT3− genotype, and treatments (Chemo vs. HCT. vs. HMA). The estimated 3-year overall survivals (OS) in elderly (N=17) and younger adults (N=13) with NPM1+/FLT3− treated with Chemo were 59% and 64%, respectively (P=0.71). In the absence of NPM1+/FLT3−, younger adults had a superior OS when treated with HCT than with Chemo (P<0.0001), but elderly showed no survival advantage with HCT after adjustment for baseline covariates. Elderly patients lacking NPM1+/FLT3− had a comparable OS when treated with Chemo vs. HMA (P=0.79). Combined NPM1+/FLT3− is associated with a favorable prognosis irrespective of age in AML patients treated with Chemo. In the absence of NPM1+/FLT3− genotype, younger adults undergoing HCT have an improved survival, while elderly have comparable OS when treated with Chemo vs. HMA.

Allogeneic Hematopoietic Stem-Cell Transplantation (HSCT) in First Complete Remission Is Superior Compared to Chemotherapy/Autologous HSCT in Patients with Intermediate-Risk Cytogenetics Acute Myeloid Leukemia Lacking Mutations in NPM1, FLT3-ITD, and CEBPA: A Joint Study of AMLSG, Cetlam and Acute Leukemia Working Party of EBMT

Prognosis of acute myeloid leukemia (AML) is mainly determined by presenting cyto- and molecular genetics, as systematized in the recent European LeukemiaNet (ELN) risk classification. Moreover, mutational profiling is currently used to assign post-remission therapy, especially in the category of intermediate-risk cytogenetics (IR-AML), given the observed benefit of allogeneic hematopoietic stem-cell transplantation (alloHSCT) in first complete response in patients harboring high-risk molecular features such as FLT3-ITD and the lack of benefit in patients with a favorable genotype (i.e., mutated NPM1 and CEBPA biallelic mutation). Nonetheless, the prognosis for patients with an IR-AML lacking mutations of these three genes (“triple negative” AML) is uncertain, and optimal post-remission strategy for these bona fide intermediate-risk patients is mostly unknown.In order to elucidate if alloHSCT could improve the prognosis of this molecular subgroup, we analyzed the outcome in a large series of patients with “de novo”, IR-AML (70% harboring a normal karyotype), diagnosed between 2000 and 2013, lacking mutations of NPM1, FLT3-ITD, and CEBPA (only patients with biallelic mutations were excluded), who had achieved a first complete remission after 1 or 2 courses of standard induction therapy. These 630 patients (median age, 52 years, range, 18-72; 58% male) have been included in the database of three different cohorts, the cooperative AML groups AMLSG (n=239) and Spanish CETLAM (n=146), and the HSCT registry of the ALWP-EBMT (n=245). The prognostic impact of alloHSCT in CR1 was analyzed as a time-dependent variable in univariable (Mantel-Byar method, Simon-Makuch plots) and multivariable (Cox with time dependent-variable) analyses.After a median follow-up of 37 months, 3-year survival (OS), leukemia free-survival (LFS) and relapse incidence (RI) in the pooled two subgroups of AMLSG and CETLAM cooperative groups, regardless of post-remission therapy, was 53% [95% CI: 47-58], 36% [95% CI: 31-41], and 51% [95% CI=45-56], respectively (57±3%, 37.5±3%, and 44±4% in patients up to 60 year-old, respectively), without significant differences between both cohorts. In order to investigate the potential benefit of alloHSCT, we analyzed the entire group, including also patients from the ALWP-EBMT cohort. Remarkably, the outcome of patients from national cooperative groups AMLSG-CETLAM compared to registry ALWP-EBMT who received an alloHSCT in CR1 was not statistically different (3-year OS, LFS, and RI: 57 vs. 68%, p=0.13; 61 vs. 73%, p=0.08; and 22 vs. 19%, p=0.67). Patients who received an alloHSCT in CR1 (n=396) had an improved outcome compared to other post-remission options (high-dose cytarabine based chemotherapy, n=189; autoHSCT, n=45) with a higher OS (3-yr: 65 vs. 49%, p=7x10-5) and LFS (3-yr: 60 vs. 26%, p<10-13), and lower RI (3-yr: 23 vs. 72%, p<10-16). Moreover, post-remission option (alloHSCT in CR1 vs. other), as time-dependent variable, was the strongest prognostic factor in the multivariate analysis in terms of OS (HR=0.61, 0.46-0.8; p=0.0003), LFS (HR=0.49, 0.37-0.65; p<0.0001), and RI (HR=0.24, 0.17-0.34, p<0.001). Other independent prognostic factors identified were age for OS (HR=1.24, 1.11-1.40 -by 10 years-; p=0.0004) and LFS (HR: 1.21, 1.08-1.34; p=0.0007), presenting WBC at diagnosis for OS (HR=1.29, 1-1.67 –over median-; p=0.05), and female gender for OS (HR=0.74, 0.57-0.97; p=0.03). Interestingly, the outcome after alloHSCT did not differ depending on donor type (2-yr RI, LFS, and OS: 14 vs. 19%, p=0.97; 67 vs. 68%, p=0.42; 73 vs. 72%, p=0.95 after alloHSCT with matched sibling, n=200, or an unrelated donor, n=183, respectively).In conclusion, IR-AML patients with a triple negative genotype constitute a subgroup with a high risk of relapse after postremission therapy with high-dose cytarabine based chemotherapy or autoHSCT. This large study, involving patients from two cooperative groups and the EBMT registry, indicates that an alloHSCT in first CR is associated with a marked relapse reduction and survival benefit in the two cooperative group cohorts with prospective treatment data as well as in the whole cohort including the EBMT registry data. DisclosuresNo relevant conflicts of interest to declare.

BAALC-associated miR-3151 is an Independent Prognostic Factor in Young Patients With Intermediate-Risk Cytogenetic Acute Myeloid Leukemia

Introduction Patients with intermediate-risk cytogenetic AML (IR-AML) have a heterogeneous prognosis, and are currently further stratified based on determined gene mutations. However, the optimal post-remission therapy, especially in younger patients, is unclear. Recently, miR-3151, a novel microRNA (miRNA) located in intron 1 of the BAALC gene, has been identified. High miR-3151 expression –either alone or in combination with high BAALC levels– independently correlates with poor prognosis in patients ≥ 60 years with normal cytogenetics AML (CN-AML) (Eisfeld AK, et al. Blood 2012). However, the prognostic value of miR-3151 in younger patients (≤60 years) with IR-AML has not been examined. We hypothesized that miR-3151 expression could also be a prognostic marker in younger patients. Aim To analyze whether miR-3151 expression – either alone or in combination with BAALC – improved prognostic assessment in younger patients (up to 60 years) with IR-AML and to characterize in this subset of patients the miRNA signature associated with high miR-3151 expression. Methods Samples were available from two separate cohorts of patients with IR-AML who had received intensive therapy: a training set of 76 patients from a single institution and a validation set of 108 patients from several centers who had been treated within the CETLAM AML-2003 protocol. Information on NPM1, FLT3- ITD and CEBPA was available for both patient cohorts. The expression levels of 670 miRNAs had previously been analyzed in the 76 patients in the training set. In the present study, the expression of miR-3151 and BAALC was analyzed using TaqMan® MicroRNA Assay and TaqMan® Gene Expression Assay (Applied Biosystems), respectively. Expression levels of miR-3151 and BAALC –both alone and in combination – were correlated with patient outcome. Statistical analyses were performed with SPSS v.15.0.1, R software v.2.9.0 and TIGR MultiExperiment Viewerv4.0. Results In the training set, higher expression of miR-3151(dichotomized by its median value of normalized expression) correlated with a shorter 5-year overall survival (OS) (32%±17% vs. 61±17%, p=0.029) and 5-year leukemia-free survival (LFS) (29%±17% vs. 58±17%, p=0.036) in patients ≤ 60 yrs. When the analysis was restricted to patients with CN-AML, miR-3151 expression retained its prognostic significance (p= 0.016). In addition, increased BAALC expression was associated with shorter OS (28%±20% vs. 58±14%, p=0.054) and LFS (17%±18% vs. 55±14%, p=0.039). In the multivariate analysis for OS and LFS, including age, WBC, NPM1 mut, FLT3 -ITD, BAALC and miR-3151 expression levels as covariates, miR-3151 showed independent prognostic significance for OS (p=0.016; HR=2.52, 95% CI: 1.2-5.4),and a statistical trend for LFS (p=0.09). Patients with low expression of both miR-3151 and BAALC had better prognosis (OS: 66%±18% vs. 34±16%, p=0.027; LFS: 67%±20% vs. 27±16%, p=0.009).Interestingly, the combination of both miR-3151 and BAALC retained a significant prognostic value for LFS within the favorable molecular subgroup/ ELN favorable subgroup (patients harboring NPM1 mut without FLT3 -ITD or biallelic CEBPA mut; LFS: 44%±30% vs. 100%, p=0.017) and showed a trend in the unfavorable molecular subgroup/ELN Intermediate II OS: p=0.064 and LFS: p=0.072). In the validation set, miR-3151 overexpression confirmed its prognostic impact in patients in the univariate analysis for OS (45%±12% vs. 26±19%, p=0.039) and LFS (51%±14% vs. 30±24%, p=0.034) and in the multivariate analysis for OS (OS: p=0.038; HR 1.88, IC 95%: 1.06-3.34) and LFS (p= 0.014; HR 2.411 (1.198-4.855) Finally, a supervised analysis revealed that samples exhibiting high levels of miR-3151 expression had a distinctive miRNA signature including miR-509, miR-135a, miR-100*, miR-186*, let-7a* and miR-501. Conclusion miR-3151 is an independent prognostic marker in patients with IR-AML. The study of miR-3151 refines the molecular prognostic stratification of these patients and hence could be of help to guide therapy. Acknowledgements Marina Diaz-Beya is supported by Fundacion Espanola de Hematologia y Hemoterapia. This research is supported by Sociedad Espanola de Hematologia y Hemoterapia and by grants from Fondo de Investigaciones Sanitarias FIS-PI080158. Disclosures: No relevant conflicts of interest to declare.

BAALC-Associated Mir-3151 Is An Independent Prognostic Factor In Younger Patients With Intermediate-Risk Cytogenetic Acute Myeloid Leukemia

IntroductionPatients with intermediate-risk cytogenetic AML (IR-AML) have a heterogeneous prognosis, and are currently further stratified based on determined gene mutations. However, the optimal post-remission therapy, especially in younger patients, is unclear. Recently, miR-3151, a novel microRNA (miRNA) located in intron 1 of the BAALC gene, has been identified. High miR-3151 expression –either alone or in combination with high BAALClevels– independently correlates with poor prognosis in patients ≥ 60 years with normal cytogenetics AML (CN-AML) (Eisfeld AK, et al. Blood 2012). However, the prognostic value of miR-3151 in younger patients (≤60 years) with IR-AML has not been examined. We hypothesized that miR-3151 expression could also be a prognostic marker in younger patients. AimTo analyze whether miR-3151 expression – either alone or in combination with BAALC– improved prognostic assessment in younger patients (up to 60 years) with IR-AML and to characterize in this subset of patients the miRNA signature associated with high miR-3151 expression. MethodsSamples were available from two separate cohorts of patients with IR-AML who had received intensive therapy: a training set of 76 patients from a single institution and a validation set of 108 patients from several centers who had been treated within the CETLAM AML-2003 protocol. Information on NPM1, FLT3-ITD and CEBPA was available for both patient cohorts. The expression levels of 670 miRNAs had previously been analyzed in the 76 patients in the training set. In the present study, the expression of miR-3151 and BAALC was analyzed using TaqMan® MicroRNA Assay and TaqMan® Gene Expression Assay (Applied Biosystems), respectively. Expression levels of miR-3151 and BAALC –both alone and in combination – were correlated with patient outcome. Statistical analyses were performed with SPSS v.15.0.1, R software v.2.9.0 and TIGR MultiExperiment Viewerv4.0. ResultsIn the training set, higher expression of miR-3151(dichotomized by its median value of normalized expression) correlated with a shorter 5-year overall survival (OS) (32%±17% vs. 61±17%, p=0.029) and 5-year leukemia-free survival (LFS) (29%±17% vs. 58±17%, p=0.036) in patients ≤ 60 yrs. When the analysis was restricted to patients with CN-AML, miR-3151 expression retained its prognostic significance (p= 0.016). In addition, increased BAALCexpression was associated with shorter OS (28%±20% vs. 58±14%, p=0.054) and LFS (17%±18% vs. 55±14%, p=0.039).In the multivariate analysis for OS and LFS, including age, WBC, NPM1mut, FLT3-ITD, BAALC and miR-3151 expression levels as covariates, miR-3151 showed independent prognostic significance for OS (p=0.016; HR=2.52, 95% CI: 1.2-5.4),and a statistical trend for LFS (p=0.09).Patients with low expression of both miR-3151 and BAALC had better prognosis (OS: 66%±18% vs. 34±16%, p=0.027; LFS: 67%±20% vs. 27±16%, p=0.009).Interestingly, the combination of both miR-3151 and BAALC retained a significant prognostic value for LFS within the favorable molecular subgroup/ ELN favorable subgroup (patients harboring NPM1mut without FLT3-ITD or biallelic CEBPAmut; LFS: 44%±30% vs. 100%, p=0.017) and showed a trend in the unfavorable molecular subgroup/ELN Intermediate I&II subgroups (patients lacking both NPM1 and CEBPA mutations and/or harboring FLT3-ITD; OS: p=0.064 and LFS: p=0.072).In the validation set, miR-3151 overexpression confirmed its prognostic impact in patients in the univariate analysis for OS (45%±12% vs. 26±19%, p=0.039) and LFS (51%±14% vs. 30±24%, p=0.034) and in the multivariate analysis for OS (OS: p=0.038; HR 1.88, IC 95%: 1.06-3.34) and LFS (p= 0.014; HR 2.411 (1.198-4.855)Finally, a supervised analysis revealed that samples exhibiting high levels of miR-3151 expression had a distinctive miRNA signature including miR-509, miR-135a, miR-100*, miR-186*, let-7a* and miR-501. ConclusionmiR-3151 is an independent prognostic marker in patients with IR-AML. The study of miR-3151 refines the molecular prognostic stratification of these patients and hence could be of help to guide therapy. AcknowledgmentsMarina Díaz-Beyá is supported by Fundación Española de Hematologia y Hemoterapia. This research is supported by Sociedad Española de Hematologia y Hemoterapia and by grants from Fondo de Investigaciones Sanitarias FIS-PI080158. Disclosures:No relevant conflicts of interest to declare.

Duration Of Neutropenia Is Associated With Reduced Relapse Risk In Children With De Novo Acute Myeloid Leukemia: A Report From The Children's Oncology Group

BackgroundChildren receiving treatment for de novo acute myeloid leukemia (AML) commonly experience infections and prolonged duration of neutropenia. There is great variability in these outcomes and pharmacogenomics may provide one mechanism to explain this variability. We hypothesized that relapse outcomes may be inversely related to the degree of toxicity. ObjectivesTo describe the relationship between the number of sterile site infections and duration of neutropenia during the first four cycles of chemotherapy and the risk of recurrence and overall survival. MethodsAAML0531 was a Children's Oncology Group (COG) randomized phase 3 clinical trial that included 1022 children with de novo AML enrolled between August 2006 and June 2010. Inclusion criteria were age ≥ 1 month to ≤ 30 years. For this specific analysis, we focused on non-Down syndrome patients who completed at least 4 cycles of chemotherapy on protocol therapy without recurrence on therapy and who did not receive HSCT in first remission. Because high-risk patients were allocated to best allogeneic donor HSCT, we focused on favorable and intermediate risk cytogenetic groups. The treatment protocol consisted of 5 cycles of intensive chemotherapy; patients were randomized to receive or not receive gemtuzumab ozogamicin (GMTZ) during Induction I and Intensification II.The cumulative time to neutrophil recovery between start of Induction II and completion of cycle 4 (Intensification II) was examined; Induction I was not included since many centers did not wait for neutrophil recovery before starting Induction II. Relapsed risk and survival analyses were conducted from the end of Intensification II. Potential confounders explored for this analysis were gender, age, race, weight group, cytogenetic risk group and allocation to GMTZ. ResultsThere were 569 patients included; 279 (49.0%) were male and 210 (36.9%) had favorable risk cytogenetics. There were 1116 sterile site infections; 735 (65.9%) were Gram-positive bacteria. The median cumulative time with neutropenia was 96 (range 46 – 204) days. The Table illustrates that infections did not significantly influence the risk of relapse or overall survival. However, increased duration of neutropenia was associated with a lower risk of relapse both in univariate and multiple regression models after adjustment for African-American race, favorable risk cytogenetics and GMTZ allocation. ConclusionsLonger duration of neutropenia is associated with a reduced risk of relapse for children with favorable and intermediate cytogenetic risk AML. Toxicity may be mediated through pharmacogenomics which suggests that individualized chemotherapy dosing may be an effective strategy. Disclosure:No relevant conflicts of interest to declare.

MicroRNA expression at diagnosis adds relevant prognostic information to molecular categorization in patients with intermediate-risk cytogenetic acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease, and optimal treatment varies according to cytogenetic risk factors and molecular markers. Several studies have demonstrated the prognostic importance of microRNAs (miRNAs) in AML. Here we report a potential association between miRNA expression and clinical outcome in 238 intermediate-risk cytogenetic AML (IR-AML) patients from 16 institutions in the CETLAM cooperative group. We first profiled 670 miRNAs in a subset of 85 IR-AML patients from a single institution and identified 10 outcome-related miRNAs. We then validated these 10 miRNAs by individual assays in the total cohort and confirmed the prognostic impact of 4 miRNAs. High levels of miR-196b and miR-644 were independently associated with shorter overall survival, and low levels of miR-135a and miR-409-3p with a higher risk of relapse. Interestingly, miR-135a and miR-409-3p maintained their independent prognostic value within the unfavorable molecular subcategory (wild-type NPM1 and CEBPA and/or FLT3-ITD), and miR-644 retained its value within the favorable molecular subcategory. miR-409-3p, miR-135a, miR-196b and mir-644 arose as prognostic markers for IR-AML, both overall and within specific molecular subgroups.

Favorable outcome of patients with acute myeloid leukemia harboring a low-allelic burden FLT3-ITD mutation and concomitant NPM1 mutation: relevance to post-remission therapy

AbstractRisk associated to FLT3 internal tandem duplication (FLT3-ITD) in patients with acute myeloid leukemia (AML) may depend on mutational burden and its interaction with other mutations. We analyzed the effect of FLT3-ITD/FLT3 wild-type (FLT3wt) ratio depending on NPM1 mutation (NPM1mut) in 303 patients with intermediate-risk cytogenetics AML treated with intensive chemotherapy. Among NPM1mut patients, FLT3wt and low ratio (<0.5) subgroups showed similar overall survival, relapse risk, and leukemia-free survival, whereas high ratio (≥0.5) patients had a worse outcome. In NPM1wt AML, FLT3-ITD subgroups showed a comparable outcome, with higher risk of relapse and shortened overall survival than FLT3wt patients. Allogeneic stem cell transplantation in CR1 was associated with a reduced relapse risk in all molecular subgroups with the exception of NPM1mut AML with absent or low ratio FLT3-ITD. In conclusion, effect of FLT3 burden is modulated by NPM1 mutation, especially in patients with a low ratio.

Hematopoietic transplantation for acute myeloid leukemia with internal tandem duplication of FLT3 gene (FLT3/ITD)

Patients with acute myeloid leukemia (AML) traditionally classified as having an intermediate cytogenetic risk [mostly cytogenetically normal AML (CN-AML)] really include a significant proportion of cases with a poor outcome. This is based on the molecular findings at diagnosis, mainly the presence of internal tandem duplication in the FMS-like tyrosine kinase 3 gene(s) (FLT3/ITD). Optimal postremission therapy for these high-risk molecular cases is not well established; as the prognosis is adverse hematopoietic cell transplantation (HCT), mainly allogeneic HCT (allo-HCT), is the most widely accepted strategy. As a rule, patients with FLT3/ITD have a poor outcome with conventional chemotherapy alone. Only patients with an associated nucleophosmin 1 (NPM1) mutation and those with a low mutated-to-wild-type allelic ratio of FLT3/ITD have less unfavorable outcome. Most studies show an advantage of allo-HCT in first complete remission (CR1), with higher 3-5 year disease-free survival and lower relapse risk than with chemotherapy or autologous transplantation (auto-HCT). Regarding allo-HCT proceeding early after reaching CR1 seems to improve survival, rather than after several courses of consolidation chemotherapy. Patients with intermediate-risk cytogenetics AML and FLT3/ITD, especially NPM1-wild cases and those NPM1 mutated with a high allelic ratio, should proceed to allo-HCT if possible early after achieving CR1.

Overall Hydroxymethylation Levels As an Independent Prognostic Marker in Intermediate-Cytogenetic Risk Acute Myeloid Leukemia

AbstractAbstract 1375 Introduction:Acute myeloid leukemia (AML) is a highly heterogeneous disease, with diverse genetic and epigenetic variables determining sensitivity to current standard therapy. A disruption of the normal DNA methylation pattern, which can result in altered gene and microRNA (miRNAs) expression, has been observed in different AML subtypes. Hydroxymethylation of 5-methylcytosine (5-mC) has recently been described as an intermediate key step in the process of DNA demethylation. Nonetheless, the correlation of DNA methylation and hydroxymethylation levels with clinical and biological characteristics and clinical outcome in AML is mostly unknown. Aim:To investigate the prognostic impact of overall methylation and hydroxymethylation levels in patients with intermediate-cytogenetic risk AML (IR-AML) and to identify miRNAs correlated with methylation and hydroxymethylation in these patients. Patients and Methods:We have analyzed 86 IR-AML patients (median age, 53 [range, 17–74]; 52% males) who received intensive therapy from 1994 to 2009 in a single institution. The level of overall methylation and hydroxymethylation in total DNA was estimated after determining the percentage of 5-mC and hydroxymethylcytosine (5-hmC), using anti-5-mC and anti-5-hmC monoclonal antibodies (MethylFlash Methylated or Hydroxymethylated DNA Quantification Kit, Epigentek). The expression of 670 mature miRNAs was analyzed using TaqMan Human MicroRNA Arrays (Applied Biosystems). The statistical analysis was performed with SPSS version 15.0.1 and R software version 2.9.0. MaxStat package of R was used to determine the optimal cutoffs. Results:The univariate analysis for overall methylation showed that patients with lower levels of methylation (cutoff < percentile 75) had shorter overall survival (OS) than those with higher 5-mC levels (5-year OS: 30±6% vs 52±11%; p=0.03) and a trend for shorter leukemia-free survival (LFS)(p=0.06). Overall methylation levels did not show any correlation with clinical features at diagnosis or with gene mutations, including DNMT3A. Concerning hydroxymethylation, patients with lower 5-hmC levels had a worse prognosis than those with higher 5-hmC levels, with a lower complete response rate (79% vs. 96%; p=0.04), shorter OS (5-yr OS: 21± 6% vs. 55± 6%; p=0.008), and shorter LFS (5-yr LFS: 24±8% vs. 52 ±10%; p=0.03). Interestingly, when analyzed as a continuous variable, 5-hmC levels retained their prognostic value as a marker of response rate (T-test, p=0.007), OS (Cox, p=0.015), and LFS (Cox, p=0.041). Moreover, 5-hmC levels were inversely correlated with FLT3-ITD (p=0.001) and the FLT3-ITD/FLT3wild-type ratio (Pearson correlation:-0.6; p=0.01). The multivariate analyses, including the main clinical and biological variables, identified older age, wild-type NPM1, FLT3-ITD, and lower 5-hmC levels (HR=3.072; 95% CI: 1.096–3.917; p=0.025) as independent prognostic markers of shorter OS and wild-type NPM1, FLT3-ITD, and lower 5-hmC levels (HR=2.002, 95% CI: 1.032–3.881, p=0.040) as independent prognostic markers of shorter LFS. Of note, lower 5-hmC levels retained their value as a marker of worse prognosis in the subgroup of IR-AML patients with unfavorable molecular markers (wild-type NPM1 and CEBPA and/or FLT3-ITD; p= 0.037).Finally, we have identified a 3-miRNA signature (miR-378*, p=0.02; miR-493, p=0.02; and miR-181, p=0.02) associated with global methylation levels, and a 12-miRNA signature associated with hydroxymethylation, including miR-183* (p=0.001), miR-125a-3p (p= 0.01), miR-586 (p=0.02), and miR-513–3p (p=0.02). Conclusions:Hydroxymethylation levels appear as an independent prognostic factor in IR-AML and maintain their prognostic value in the subset of patients with unfavorable molecular markers. Moreover, methylation and hydroxymethylation are associated with specific miRNA profiles. Further studies are warranted to confirm the clinical impact of these findings and to clarify the underlying molecular mechanisms. Disclosures:No relevant conflicts of interest to declare.