Positive Acute Myeloid Leukemia Research Articles

Targeting the NRF2/HO-1 Antioxidant Pathway with Consequences for Notch Signaling in FLT3-ITD Positive AML: A Novel Therapeutic Approach

Approximately 30% of patients with acute myeloid leukemia (AML) harbor activating mutations in the fms-like tyrosine kinase receptor 3 (FLT3). Such mutations are associated with increased propensity to relapse and dramatically decreased survival. FLT3 tyrosine kinase inhibitors (TKI), such as quizartinib, have shown modest clinical effects as single agents, and resistance remains a significant clinical problem. In addition to constitutive activity of the FLT3 receptor, AML cells with an internal tandem duplication of FLT3 (FLT3-ITD) exhibit elevated levels of reactive oxygen species (ROS). Antioxidants such as heme-oxygenase 1 (HO-1) are often upregulated in conjunction with increased ROS levels. In addition to its role as an antioxidant, HO-1 also has known proliferative and anti-apoptotic functions in some cell types. A study in non-small cell lung carcinoma recently suggested that HO-1 inhibits the Notch pathway through binding the Notch receptor. As Notch signaling exerts pro-apoptotic effects in AML, targeting HO-1 may induce Notch signaling, representing a potential therapeutic approach.Our data show that HO-1 is particularly elevated in FLT3-ITD expressing AML cells. Interestingly, HO-1 protein was found to be further elevated in AML cells with acquired resistance to FLT3-directed TKI compared to TKI-sensitive cells. Importantly, knockdown of HO-1, or inhibition of HO-1 catalytic activity with zinc protoporphyrin (ZnPP), leads to decreased survival and proliferation, demonstrating a critical pro-survival role of HO-1. We have determined that expression of HO-1 is under the control of NRF2, a transcriptional regulator of the antioxidant response, in FLT3-ITD positive AML cells. Inhibition of NRF2 with brusatol resulted in increased sensitivity to quizartinib in TKI-sensitive cell lines. Further, brusatol restored sensitivity to low nanomolar concentrations of quizartinib in TKI-resistant cells. Together, our work shows that combined inhibition of FLT3 and NRF2 using quizartinib and brusatol, respectively, exhibits synergy in the context of FLT3-ITD AML.Given the reported interaction between HO-1 and Notch, we sought to determine whether this interaction occurs in AML. Using the TCGA AML database, we found significant co-occurrence of HMOX1 (HO-1) and NOTCH2 mRNA expression in 200 patient samples. In vitro immunoprecipitation of the Notch2 receptor revealed that HO-1 directly interacts with Notch2 in FLT3-ITD positive AML cells. Importantly, brusatol treatment resulted in activation of the Notch pathway as demonstrated by cleavage of the Notch receptor and increased transcript levels of HES1, a canonical downstream target of Notch signaling. Given that HO-1 interacts with Notch2 and is under NRF2 control, we propose that decreasing HO-1 levels with brusatol treatment will modulate the Notch pathway such that its anti-leukemic effects can be exerted.In conclusion, inhibition of HO-1, directly or through NRF2 inhibition, demonstrates synergistic effects with FLT3 inhibition in both TKI-sensitive and -resistant AML. Furthermore, NRF2 inhibition induces Notch activation, providing a potential mechanism for this synergistic combination. Our data suggest that antioxidant modulation is a potential therapeutic approach for FLT3-ITD positive AML. DisclosuresNo relevant conflicts of interest to declare.

Midostaurin in Combination with Intensive Induction and As Single Agent Maintenance Therapy after Consolidation Therapy with Allogeneic Hematopoietic Stem Cell Transplantation or High-Dose Cytarabine (NCT01477606)

Background: Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in roughly 25% of younger adult patients (pts) with acute myeloid leukemia (AML), implicating FLT3 as a potential target for kinase inhibitor therapy. The multi-targeted kinase inhibitor midostaurin shows potent activity against FLT3 as a single agent but also in combination with intensive chemotherapy.Aims: To evaluate the feasibility and efficacy of midostaurin in combination with intensive induction therapy and as single agent maintenance therapy after allogeneic hematopoietic stem cell transplantation (alloHSCT) or high-dose cytarabine (HIDAC).Methods: The study includes adult pts (age 18-70 years (yrs)) with newly diagnosed FLT3-ITD positive AML enrolled in the ongoing single-arm phase-II AMLSG 16-10 trial (NCT: NCT01477606). Pts with acute promyelocytic leukemia are not eligible. The presence of FLT3-ITD is analyzed within our diagnostic study AMLSG-BiO (NCT01252485) by Genescan-based fragment-length analysis (allelic ratio >0.05 required to be FLT3-ITD positive). Induction therapy consists of daunorubicin (60 mg/m², d1-3) and cytarabine (200 mg/m², continuously, d1-7); midostaurin 50 mg bid is applied from day 8 onwards until 48h before start of the next treatment cycle. A second cycle is optional. For consolidation therapy, pts proceed to alloHSCT as first priority; if alloHSCT is not feasible, pts receive three cycles of age-adapted HIDAC in combination with midostaurin from day 6 onwards. In all pts maintenance therapy for one year is intended. This report focuses on the first cohort of the study (n=149) recruited between June 2012 and April 2014 prior to the amendment increasing the sample size; the amendment to the study is active since October 2014.Results: At study entry patient characteristics were median age 54 years (range, 20-70, 34% ≥ 60 yrs); median white cell count (WBC) 48.4G/l (range 1.1-178G/l); karyotype, n=103 normal, n=3 t(6;9), n=2 t(9;11), n=20 intermediate-2 and n=7 high-risk according to ELN recommendations, n=14 missing; mutated NPM1 n=92 (62%). Data on response to first induction therapy were available in 147 pts; complete remission (CR) 58.5%, partial remission (PR) 20.4%, refractory disease (RD) 15% and death 6.1%. A second induction cycle was given in 34 pts. Overall response after induction therapy was CR 75% and death 7.5%. Adverse events 3°/4° reported during the first induction cycle were most frequently gastrointestinal (n=34) and infections (n=81). During induction therapy midostaurin was interrupted, dose-reduced or stopped in 55% of the pts. Overall 94 pts received an alloHSCT, 85 in first CR (n=65 age<60 yrs, n=20 age ≥60 yrs) and 9 pts after salvage outside the protocol or after relapse (n=70 from a matched unrelated and n=24 from a matched related donor). In pts receiving an alloHSCT within the protocol in median 2 chemotherapy cycles were applied before transplant (range 1-4) and the cumulative incidence of relapse and death at 12 months were 9.2% (SE 3.3%) and 19.5% (SE 4.8%). Maintenance therapy was started in 52 pts, 40 pts after alloHSCT and 12 pts after HIDAC. Only 4 adverse events 3°/4° were attributed to midostaurin. First analyses revealed a low cumulative incidence of relapse irrespective of the FLT3-ITD mutant to wildtype ratio (<0.5 versus ≥0.5) in patients proceeding to alloHSCT with 12% and 5% as well as for those after HIDAC consolidation with 28% and 29%, respectively.Conclusions: The addition of midostaurin to intensive induction therapy and as maintenance after alloHSCT or HIDAC is feasible and compared to historical data may be most effective in those patients with a high FLT3-ITD mutant to wildtype ratio. DisclosuresSchlenk:Novartis: Honoraria, Research Funding. Salwender:Celgene: Honoraria; Janssen Cilag: Honoraria; Bristol Meyer Sqibb: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Götze:Celgene Corp.: Honoraria; Novartis: Honoraria.

Allogeneic Stem Cell Transplantation Improves Survival in Patients with Acute Myeloid Leukemia Characterized by a High Allelic Ratio of Mutant FLT3-ITD

Allogeneic hematopoietic cell transplantation (alloHCT) as a postremission therapy in patients with FLT3-ITD–positive intermediate-risk acute myeloid leukemia (AML) remains controversial. FLT3-ITD mutations are heterogeneous with respect to allelic ratio, location, and length of the insertion, with a high mutant-to-wild-type ratio consistently associated with inferior prognosis. We retrospectively analyzed the role of alloHCT in first remission in relationship to the allelic ratio and presence or absence of nucleophosmin 1 mutations (NPM1) in the Study Alliance Leukemia AML2003 trial. FLT3-ITD mutations were detected in 209 patients and concomitant NPM1 mutations in 148 patients. Applying a predefined cutoff ratio of .8, AML was grouped into high- and low-ratio FLT3-ITD AML (HRFLT3-ITD and LRFLT3-ITD). Sixty-one patients (29%) were transplanted in first remission. Overall survival (OS) (HR, .3; 95% CI, .16 to .7; P = .004) and event-free survival (EFS) (HR, .4; 95% CI, .16 to .9; P = .02) were significantly increased in patients with HRFLT3-ITD AML who received alloHCT as consolidation treatment compared with patients who received consolidation chemotherapy. Patients with LRFLT3-ITD AML and wild-type NPM1 who received alloHCT in first remission had increased OS (HR, .3; 95% CI, .1 to .8; P = .02) and EFS (HR, .2; 95% CI, .1 to .8; P = .02), whereas alloHCT in first remission did not have a significant impact on OS and EFS in patients with LRFLT3-ITD AML and concomitant NPM1 mutation. In conclusion, our results provide additional evidence that alloHCT in first remission improves EFS and OS in patients with HRFLT3-ITD AML and in patients with LRFLT3-ITD AML and wild-type NPM1.

AB119. Induction of suppressor of cytokine signaling-3 in FLT3-ITD positive MV4-11 acute myeloid leukemia cells in response to 5-Azacytidine and Trichostatin A

Background and objective Suppressor of cytokine signaling-3 (SOCS-3) has been shown to be an important candidate in molecular therapeutic strategies in management of acute myeloid leukemia (AML), particularly in patients carrying FLT3-ITD mutation. SOCS-3 suppresses cytokine signalling by inhibiting the activity of Janus Kinase-2 (JAK-2), and by competing with signal transducer and activator of transcription (STAT) molecules that leads to underexpression. The study aims to determine the epigenetically silence genes in AML cells carrying a FLT3-ITD mutation and epigenetically expressed genes afer treatment with demethylating agent and histone deacetylase inhibitor.

CD97 expression influences migration and adhesion of FLT3-ITD positive acute myeloid leukemia cells and modulates the bone marrow stromal microenvironment

CD97 expression influences migration and adhesion of FLT3-ITD positive acute myeloid leukemia cells and modulates the bone marrow stromal microenvironment

Synergistic effect of sorafenib and cGvHD in patients with high-risk FLT3-ITD+AML allows long-term disease control after allogeneic transplantation.

The multikinase inhibitor sorafenib has shown a strong anti-leukemic effect in FMS-like tyrosine kinase 3 (FLT3)-mutated acute myeloid leukemia (AML); however, remission is often transient. To better understand the role of sorafenib, we performed a retrospective analysis of all patients who received sorafenib in combination with allogeneic hematopoietic stem cell transplantation (HSCT) at our center. Seventeen patients with FLT3-ITD positive AML were treated with sorafenib in combination with allogeneic HSCT. Seven patients received sorafenib therapy pre- and posttransplant, and 10 patients were given sorafenib only posttransplant. Median duration of sorafenib treatment was 13months (range 1-42); median dose was 600mg (range 100-1200). Fourteen patients (82%) achieved a complete remission (CR), while 5 patients (29%) eventually developed progressive disease. Developing chronic graft-versus-host disease (GvHD) had a strong protective influence on the risk of sorafenib resistance (p = 0.028, HR 0.08, 95% CI 0.01-0.76). In a total of 8 patients, sorafenib had to be stopped, paused or dose-reduced due to toxicity. In 5 patients with pronounced toxicity, we switched to an alternating dosing schedule with 1month on/1month off sorafenib. These patients subsequently remained in sustained complete molecular remission, with a median follow-up of 20months. Our data indicate that sorafenib can achieve high rates of sustained remission in high-risk patients treated in combination with HSCT.

Clinical significances of WT1 gene and AML1-ETO fusion gene expression for monitoring minimal residual disease in children with acute myeloid leukemia

Objective To explore the diagnostic value of WT1 gene and AML1-ETO fusion gene in the monitoring of minimal residual disease (MRD) and prediction of relapse in childhood acute myeloid leukemia (AML). Methods From May 2007 to March 2015, a total of 13 AML1-ETO fusion gene positive AML children and 15 leukemia fusion gene negative AML children who received initial treatment in Department of Pediatrics in Sichuan Provincial People's Hospital were enrolled as study objects, and divided into fusion gene positive group (n=13) and fusion gene negative group (n=15). Relative expression levels of WT1 gene and AML1-ETO fusion gene were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technique in bone marrow samples from children of two groups at the beginning of treatment and during follow-up period. Correlation of relative expression levels of WT1 gene and AML1-ETO fusion gene were analyzed by SPSS statistical software. The sensitivity, specificity, accuracy, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio of MRD predicting relapse were calculated and compared. Kaplan-Meier method was used to compare the relapse-free survival (RFS) rate between two groups and to assess MRD on the prediction of relapse. The study protocol was approved by the Ethical Review Board of investigation in Human Being of Sichuan Provincial People's Hospital.Informed consent was obtained from all participants. Results ①The relative expression levels of WT1 gene and AML1-ETO fusion gene were positively correlated in 83 bone marrow samples from 13 cases of fusion gene positive group (R2=0.874, P 0.05) at the time points of first diagnosis, first induction of remission, and relapse. ③After the first time induction of remission in the fusion gene positive group children, AML1-ETO quantitative levels as predictive relapse of diagnostic indicators were the highest in the sensitivity, accuracy, positive predictive value, negative predictive value as 85.7%, 76.9%, 75.0% and 80.0% respectively and negative likelihood ratio was minimum as 0.29. The specificity of WT1 gene in the fusion gene negative group was highest as 71.4%. ④After the first time induction of remission, there were 8 patients whose relative expression levels of AML1-ETO fusion gene were more than 0.01 in fusion gene positive group. Among these 8 cases, 6 patients relapsed and relapse rate was 75.0%. Relative expression levels of AML1-ETO fusion gene in other 5 cases were less than 0.01. Among the 5 cases, 1 patient relapsed and relapse rate was 20.0%. The difference of relapse rate between patents whose relative expression levels of AML1-ETO fusion gene were more and less than 0.01 was statistically significant (χ2=13.0, P=0.000). Up to the time of the follow-up , RFS rate of the patients with the relative expression level of AML1-ETO fusion gene more than 0.01 was significantly lower than that of patients with the relative expression level less than 0.01 (P=0.001). ⑤ After the first time induction of remission, there were 8 patients whose relative expression levels of WT1 gene were more than 0.01 in fusion gene negtive group. Among these 8 cases, 6 patients relapsed and relapse rate was 75.0%. Relative expression levels of WT1 gene in other 7 cases were less than 0.01. Among the 7 cases, 1 case relapsed and relapse rate was 14.3%. The difference of relapse rate between patients whose relative expression levels of WT1 gene were more and less than 0.01 was statistically significant (χ2=15.0, P=0.000). Up to the time of the follow-up , RFS rate of the patients with the relative expression level of WT1 gene more than 0.01 was significantly lower than that of patients with the relative expression level less than 0.01 (P=0.004). Conclusions AML1-ETO fusion gene is suitable for monitoring the MRD of AML children with fusion gene positive. WT1 gene is more suitable for detecting MRD and predicting relapse in AML children with fusion gene negative. It may indicate relapse when WT1 and AML1-ETO fusion gene relative expression levels significantly increased. Key words: Leukemia, myeloid, acute; gene, Wilms' tumor; AML1-ETO fusion gene, human; Neoplasm, residual; Polymerase chain reaction

ITD mutation burden for the prognosis in FLT3-ITD positive acute myeloid leukemia patients

目的探讨FLT3-ITD突变数量、重排碱基长度和比例与急性髓系白血病（AML）患者的总体生存（OS）时间及持续完全缓解时间（CRD）的关系。方法采用PCR-毛细管电泳法检测130例AML（除外M3）患者的FLT3-ITD突变。根据电泳峰型数量分为单或多个（片段）突变；FLT3-ITD突变比例为突变型峰面积/野生型和突变型峰面积之和；分析FLT3-ITD突变患者临床特征并随访患者预后。结果FLT3-ITD多个突变常见于60岁以上患者；FLT3-ITD单个突变患者初诊骨髓原始细胞比例高于多个突变患者（0.758对0.638，P=0.028）；FLT3-ITD突变数量不影响患者预后。FLT3-ITD突变重排碱基长度亦对患者预后无明显影响。FLT3-ITD突变比例<10％患者的OS时间和CRD与同期C-KIT突变的中危组AML患者相似，均显著长于突变比例≥10％患者（中位OS时间分别为未达到、未达到、9.9个月，P<0.05；中位CRD分别为未达到、未达到、6.7个月，P<0.05）。FLT3-ITD突变比例≥10%的AML患者中，FLT3-ITD突变伴随NPM1或CEBPA突变患者的中位CRD显著长于单纯FLT3-ITD突变的患者（25.0个月对5.1个月，P=0.003），但两组中位OS时间差异无统计学意义（11.4个月对8.0个月，P>0.05）。结论FLT3-ITD突变阳性的AML（除外M3）患者中，FLT3-ITD突变比例<10％的患者预后好于突变比例≥10％的患者。

High Risk of Hepatitis B Reactivation among Patients with Acute Myeloid Leukemia.

BackgroundHepatitis B virus (HBV) infections are common and associated with significant morbidity and mortality in cancer patients. However, the incidence and risk factors of HBV reactivation in patients with acute myeloid leukemia (AML) are rarely investigated.MethodsAML patients followed-up at the National Taiwan University Hospital between 2006 and 2012 were analyzed. The clinical characteristics and laboratory data were retrospectively reviewed.ResultsFour hundred and ninety patients comprising 265 men and 225 women were studied. The median age was 52 years (range, 18 - 94). Chronic HBV carriage was documented at the time of leukemia diagnosis in 57 (11.6%) patients. Forty-six (80.7%) of the 57 HBV carriers received prophylaxis with anti-HBV agents. Sixteen HBV carriers (28.1%) developed hepatitis B reactivation during or after chemotherapy, including 7 patients who had discontinued antiviral therapy. The incidence of hepatitis B reactivation among AML patients with HBV carriage was 9.5 per 100 person-years. Prophylaxis with anti-HBV agents significantly decreased the risk of hepatitis B reactivation among HBV carriers (13% vs. 61%, p<0.001). Four (2.8%) of 142 patients with initial positive anti-HBsAb and anti-HBcAb experienced hepatitis B reactivation and lost their protective anti-HBsAb. Multivariate analysis revealed that diabetes mellitus (p=0.008, odds ratio (OR) = 2.841, 95% confident interval (CI): 0.985-8.193) and carriage of HBsAg (p<0.001, OR=36.878, 95% CI: 11.770-115.547) were independent risk factors for hepatitis B reactivation in AML patients.ConclusionsHepatitis B reactivation is not uncommon in the HBsAg positive AML patients. Prophylaxis with anti-HBV agent significantly decreased the risk of hepatitis B reactivation.

Concentrative nucleoside transporter 3 as a prognostic indicator for favorable outcome of t(8;21)-positive acute myeloid leukemia patients after cytarabine-based chemotherapy.

Although acute myeloid leukemia (AML) exhibits diverse responses to chemotherapy, patients harboring the t(8;21) translocation are part of a favorable risk group. However, the reason why this subgroup is more responsive to cytarabine-based therapy has not been elucidated. In the present study, we analyzed expression levels of cytarabine metabolism-related genes in patients diagnosed with AML with or without t(8;21) and investigated their correlation with clinical outcomes after cytarabine-based therapy. Among the 8 genes studied, expression of the concentrative nucleoside transporter 3 (CNT3) gene was significantly higher in t(8;21)-positive patients compared to the others in the test population and the validation cohort (P<0.001 in Mann-Whitney U test; P<0.002 in Pearson's correlation analysis). Additionally, in both multivariate and univariate analyses, t(8;21)-positive patients categorized in a higher CNT3 expression tertile had longer disease-free survival [hazard ratio (HR), 0.117; 95% confidence interval (CI), 0.025-0.557; P=0.008] and overall survival (HR, 0.062; 95% CI, 0.007-0.521; P=0.010) compared to t(8;21)-positive patients in a lower CNT3 expression tertile. Notably, these trends did not occur in t(8;21)-negative patients. Our results demonstrate that CNT3 expression is associated with overall favorable outcomes and is predictive of clinical outcomes in AML patients with t(8;21). This suggests that CNT3 expression can be used to optimize treatment strategies for AML patients.

Clinical and laboratory characteristics of 12 Ph/BCR-ABL positive acute myeloid leukemia patients

目的探讨有利于Ph染色体和（或）BCR-ABL融合基因阳性急性髓系白血病（Ph/BCRABL+ AML）诊断的临床和实验室特征。方法收集2006年2月至2013年12月收治的12例Ph/BCR-ABL+ AML患者资料，以典型慢性髓性白血病急髓变（CML-MBC）患者为对照组，回顾性分析两者临床及实验室特征，并随访生存情况。结果12例患者中位年龄27.5岁，无或轻度脾脏肿大者10例（83.3%），FAB分型以M2和M4为主，中位外周血和骨髓嗜碱粒细胞比例、巨核细胞数低于典型CML-MBC患者。免疫表型均为髓系表达，表达CD34者8例（66.7%）。11例患者检测到t(9;22)，伴附加染色体异常5例（45.5%），其中1例为inv(16）。12例患者均检测到BCR-ABL融合基因，e1a2型3例（25.0%），余为b2a2/b3a2型，其中1例伴有CBFβ-MYH11表达。6例受检患者中2例存在AML常见突变，其中CEBPA突变1例，FLT3-TKD突变1例。诱导治疗完全缓解(CR) 7例（58.3%），7例接受化疗联合酪氨酸激酶抑制剂（TKI）者6例CR，3例接受单独化疗者1例CR。总体中位生存期16.5个月，异基因造血干细胞移植（allo-HSCT）组为33.5个月，高于非移植组（5.5个月）。结论e1a2型融合基因、与AML常见融合基因共表达、存在AML常见突变有利于Ph/BCR-ABL+ AML诊断；此类患者诱导缓解率低，生存期短，化疗联合TKI获得缓解后尽早行allo-HSCT是改善其生存的唯一有效途径。

Acute myeloid leukemia with monosomy 7, ectopic virus integration site-1 overexpression and central diabetes insipidus: A case report.

Central diabetes insipidus (DI) is a rare complication in patients with acute myeloid leukemia (AML), typically occurring in patients with abnormalities of chromosomes 3 or 7. The association between AML with monosomy 7 and DI has been described in a number of studies; however, DI has been rarely reported in cases of ectopic virus integration site-1 (EVI1)-positive AML with monosomy 7. The current study reports a case of AML with monosomy 7 and EVI1 overexpression, with central DI as the initial symptom. The patient was an 18-year-old female who presented with polyuria and polydipsia. Bone marrow aspiration revealed 83.5% myeloperoxidase-positive blasts without trilineage myelodysplasia. The karyotype was 45,XX,-7, and the patient presented monosomy 7 and EVI1 overexpression (-7/EVI1+) without 3q aberration. Treatment with induction therapy was unsuccessful. To the best of our knowledge, this is the second case of DI-AML with -7/EVI1+ and without a 3q aberration. The possible mechanisms associated with EVI1, monosomy 7 and DI were investigated.

Individualized Treatment Strategy with Small-Molecular Inhibitors in Acute Myeloid Leukemia with Concurrent FLT3-ITD and FLT3-TKD Mutation

FLT3 is a frequently mutated gene in acute myeloid leukemia that encodes a receptor tyrosine kinase. We report the case of a patient with FLT3-ITD positive secondary acute myeloid leukemia after treatment for breast cancer. Due to poor response to induction therapy and relapse before consolidation therapy we started a palliative treatment with the tyrosine kinase inhibitor sorafenib. After initial response clinical resistance occurred. Sequencing showed an additional FLT3-TKD mutation. Sunitinib effectively inhibited FLT3-ITD/TKD mutated cells in vitro and induced a reduction of blasts and prolonged survival in vivo. Individualized tyrosine kinase inhibitor therapy may prolong survival in selected patients with FLT3-ITD+ acute myeloid leukemia.

Restoration of PRG2 Expression by 5-Azacytidine Involves in Sensitivity of PKC-412 (Midostaurin) Resistant FLT3-ITD Positive Acute Myeloid Leukaemia Cells

Background: Tumor-suppressor genes Tumour suppressor genes (TSG) are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Transcriptional silencing of PRG2 gene could be involved in development and progression of cancers. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA de-methylation resulting in re-expression of silenced TSG. Midostaurin (PKC412) is a multitargeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in patients with AML. However, majority of AML patients in clinical trials developed resistance to PKC412. Methods: Resistant cells were developed by overexposure of MV4-11 cells to PKC-412 and treated with 5-Aza. Apoptosis and cytotoxicity of PKC-412 were determined using Annexin V and MTS assays, respectively. Gene expression profiling was performed using microarray followed by quantitative real-time PCR. STATs activity was examined using Western bloting and methylation of PRG2 was studied using pyrosequencing analysis. Results: The cytotoxic dose of PKC-412 on resistant cells was significantly higher than parental and MV4-11Rpkc+5-Aza cells (p=0.003). The resistant cells showed significant higher viability and lower apoptotic cells compared to other cells (p<0.001). Expression of PRG2 was more than 300 folds higher in MV4-11R-pkc+5-Aza cells compared to parental and resistant cells (p=0.001). STAT3 was activated in resistant cells. Methylation of PRG2 gene was significantly decreased in MV4-11R-pkc+5-Aza cells. Conclusion: The restoration of PRG2 expression induces sensitivity towards PKC-412 and an increased in cell death. Our findings suggest that PRG2 gene could play a role in the sensitivity to PKC-412 and serve as a target for treatment of AML patients resistant to PKC-412 tyrosine kinase inhibitor.

The GSK3β-Phosphorylation Sites in the DEK-Portion Are Indispensable for the Leukemogenic Potential of the DEK/CAN Fusion Protein in t(6;9)-Positive Acute Myeloid Leukemia

The t(6,9)(p23,q34) translocation occurs in 1-5% of adult patients with acute myeloid leukemia (AML). It is associated with a poor prognosis and defines a high risk group of AML in the WHO classification. The t(6;9) is in most of the cases the only cytogenetic aberration at diagnosis. The hallmark of t(6;9)-positive AML is the DEK/CAN fusion protein. DEK/CAN is a leukemogenic oncogene, but little is known about the molecular mechanism of DEK/CAN-induced leukemogenesis. The 165 kDa DEK/CAN phosphoprotein is encoded by a single transcript of 5.5Kb. The DEK portion of the DEK/CAN contains all the major functional domains of DEK mediating DNA-binding and multimerization. DEK increases life span of primary cells in culture by inhibiting cellular senescence and apoptosis. Post-translational modifications of DEK, mainly phosphorylation, influence the activity of DEK; unphosphorylated DEK has a higher affinity for DNA than the phosphorylated form, which in turn has a higher ability for multimerization. The main kinases that phosphorylate DEK are Glycogen synthase kinase 3 β (GSK3β) and Casein kinase 2 (CK2). The respective phosphorylation sites are conserved in the DEK portion of DEK/CAN. However, little is known about the role of phosphorylation for the biological functions of DEK/CAN. Therefore we generated several mutants of DEK and DEK/CAN by point-mutating the putative GSK3β-sites (ΔP1) from S to A and by deleting the CK2 sites in addition to these mutations (ΔP2). The reduction of S/T phosphorylation was confirmed by a ProQ staining and affinity chromatography on lysates of 293T cells expressing DEK, DEK/CAN and the respective ΔP1 or ΔP2 mutants. Further biological and biochemical consequences of these mutations for DEK and DEK/CAN were investigated in murine factor dependent 32D progenitor cells and in primary murine Sca1+/lin- hematopoietic stem cells (HSC), retrovirally or lentivirally transduced with DEK, DEK/CAN and/or their phosphorylation mutants ΔP1 or ΔP2, respectively.Here we report that the loss of the GSK3β- and CK2-phosphorylation sites did not interfere with the subnuclear localization of either DEK or DEK/CAN as revealed either by subnuclear fractionation experiments or by co-localization with native DEK/CAN in confocal laser scan microscopy assays on 32D cells co-expressing DEK/CAN and ΔP1-DEK/CAN or ΔP2-DEK/CAN. In contrast, the destruction of GSK3β-phosphorylation sites not only led to a loss of apoptosis inhibition by DEK and DEK/CAN upon factor withdrawal in 32D cells, but also abolished the increased self renewal potential of DEK/CAN-positive HSC. In fact DEK/CAN-positive HSCs significantly increased colony numbers in colony forming units spleen-day 12 (CFU-S12) assays as compared to empty vector controls, whereas ΔP1-DEK/CAN and ΔP2-DEK/CAN did not have any effect.In summary, our results suggest an important role of the GSK3β-phosphorylation for the DEK/CAN-induced leukemogenesis, which establishes the GSK3β-activity as a molecular target for therapeutic intervention in t(6;9)-positive AML. DisclosuresNo relevant conflicts of interest to declare.

FLT3 Mutation Increases Relapse Risk after Allogeneic Hematopoietic Cell Transplant for Acute Myeloid Leukemia in First or Second Complete Remission: A Center for International Blood and Marrow Transplant Research (CIBMTR) Analysis

Relapse after allogeneic hematopoietic cell transplant (HCT) remains a major cause of transplant failure. Patients (pts) with FLT3 mutated acute myeloid leukemia (AML) tend to have poor prognosis and are often referred for early HCT. We studied 511 patients to determine if FLT3 mutations impact transplant outcomes.Adults with a diagnosis of de-novo AML, with available FLT3 mutation status in first or second complete remission (CR) who underwent a human leukocyte antigen (HLA) matched sibling or unrelated donor (8/8 or 7/8) HCT after myeloablative (MAC), non-myeloablative (NMA), or reduced intensity conditioning (RIC) reported to the CIBMTR from 2008-2011 were included. Clinical outcomes following HCT for FLT3 positive and FLT3 negative AML pts were compared adjusting for significant patient-, disease-, and HCT-related variables.511 pts met the inclusion criteria: 158 (31%) with FLT3 mutations. Median follow-up of survivors was 37 months (range, 12-65). Patient characteristics are shown in Table 1. The FLT3 mutated group had significantly higher white blood cell (WBC) count at diagnosis, more pts. with normal cytogenetics and more cycles of pre-HCT consolidation. Univariate analysis (Table 2) showed increased probability of relapse after HCT (log-rank p=0.01) in the FLT3 positive group but no significant differences in overall survival (OS) (log-rank p=0.13). There were no statistical differences in non-relapse mortality (NRM), relapse-free survival (RFS), or the cumulative incidence of acute and chronic GVHD. In multivariate analysis performed excluding the confounder “WBC count at diagnosis” FLT3 mutation status had a statistically significant negative effect on risk of relapse (P ≤ 0.05), but no effect on overall mortality.We could not address the differential impact of varying FLT3 mutations or interaction of other molecular aberrations with FLT3, in addition we did not have information about post relapse interventions. Encouragingly, nearly 50% of patients with FLT3 positive AML who underwent allogeneic HCT in CR were long-term survivors after allogeneic HCT. Though associated with augmented risks of relapse, FLT3 mutation status does not adversely impact long-term survival. Pre-emptive strategies using FLT3 inhibitors after HCT may reduce relapse risk and should be studied prospectively in order to improve outcomes of these high-risk patients.Table 1:Patient CharacteristicsFLT3 Negative (N=353)FLT3 Positive (N=158)Median Age (Range)46 yrs (18-60)47 yrs(18-60)Median WBC count * x109/L (Range)10 (<1-344)34 (<1-305)Cytogenetics*Normal114 (32%)102 (65%)Other228 (65%)51 (32%)Missing11 (3%)5 (3%)Disease StatusCR1252 (71%)125 (79%)CR2101 (29%)33 (21%)Cycles of consolidations prior to HCT in CR1*086 (34%)24 (19%)171 (28%)49 (39%)≥255 (26%)40 (32%)Missing30 (12%)12 (10%)Donor Type8/8 Sibling150 (42%)67 (42%)8/8 Unrelated165 (47%)70 (44%)7/8 Unrelated38 (11%)21 (13%)Conditioning RegimenMAC302 (86%)136 (87%)NMA/RIC51 (14%)21 (13%)* p ≤ 0.05Table 2:Univariate Analysis of Outcomes at 3 yearsFLT3 Negative (N=353)FLT3 Positive (N=158)Relapse * (95% CI)28% (24-33)38% (30-45)Overall Survival (95% CI)55% (50-60)49% (40-57)* p ≤ 0.05 [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Randomized Trial of Busulfan with Cyclophosphamide Versus Busulfan with Fludarabine As Preparative Regimen to Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia: A Study from the Gruppo Italiano Trapianto Midollo Osseo (GITMO)

▪BackgroundThe combination of a myeloablative dose of intravenous (iv) busulfan with cyclophosphamide is the standard preparative regimen for allogeneic hematopoietic stem cell transplantation (HSCT) in acute myeloid leukemia (AML) patients. However, in patients older than 40 years, this conditioning can be associated to high non relapse mortality (NRM). A similar myeloablative dose of busulfan combined to fludarabine was found associated to a lower NRM in older AML (Alatrash, BBMT 2011).Patients and study designThe Gruppo Italiano Trapianto Midollo Osseo (GITMO) conducted a Phase III, randomized, multicenter, trial to compare the standard myeloablative combination of iv busulfan (Busilvex®, Pierre Fabre, Boulogne, France) at a dose of 0.8 mg/kg/6h over two hours infusion for 4 consecutive days (16 doses), for a total dose of 12.8 mg/kg, in combination with cyclophosphamide at the dose of 60 mg/kg/day for 2 consecutive days for a total dose of 120 mg/kg (BUCY2 arm) or fludarabine at the dose of 40 mg/m2/day for 4 consecutive days, for a total dose of 160 mg/m2 (BUFLU arm). Eligible were patients with a diagnosis of AML in 1st or 2nd complete remission (CR) with an age ≥40 and ≤ 65 years, and the availability of an HLA compatible sibling or unrelated donor as defined by molecular high-resolution typing (4 digits) of the HLA gene loci class I (HLA- A, B, and C) and class II (DRB1). Excluded were patients with a t(15;17) or PML/RARα positive APL or with a t(8;21)(q22;q22) or an inv(16) or t(16;16)(p13;q22) positive AML in 1st CR. The GvHD prophylaxis was based on conventional Cyclosporine A and Methotrexate. In case of unrelated donors, anti Thymocyte Globulin (Thymoglobuline®, Sanofi-Aventis) was given at a total dose of 5 mg/kg (or 7.5 mg/kg, in case of HLA acceptable disparity) (one antigen/allele disparity in class I, or one allele disparity in class II). The primary study end-point was the one-year NRM using an intent-to-treat analysis. The required sample size was calculated assuming that the one-year NRM would have been halved (from 25% to 12.5%) in the BUFLU arm. The cumulative incidence of NRM was estimated by considering relapse as a competing event. All outcomes were evaluated from the date of transplantation. The study was approved by the Institutional Review Boards of each center.ResultsFrom July 2008 to February 2013, 25 centers in Italy and 1 in Israel, enrolled 245 patients who were randomly assigned to BUCY2 (n=121) or BUFLU (n=124), stratified according to donor type and remission (1st vs. 2nd or more). The main clinical features (balanced between the randomization arms) were as follows: the median age was 50 years, 209 patients (85%) were in 1st and 36 (15%) in 2ndCR and the ELN risk subgroups were good (11%), intermediate-1 (46%), intermediate-2 (20%) and adverse (23%). The donor was a sibling related (n= 112, 46%) or matched unrelated (n= 133, 54%) while the stem cell graft was the peripheral blood (PB, n= 168, 69%) or the bone marrow (BM, n= 77, 31%). The overall survival rate in the BUCY2 and BUFLU arm was 71% vs. 78% at 1 year, 65% vs. 62 % at 2 years and 56% vs. 57% at 5 years, respectively (P=ns). A non-significant lower incidence of relapse was documented in the BUCY2 vs. the BUFLU arm being 20.7% vs. 24.2% at 1 year, 25.6% vs. 29% at 2 years and 28.9 vs. 32.3 at 5 years, respectively. On the contrary, at 1 year, the overall NRM in the BUCY2 arm was 17.4% vs. 7.3% in the BUFLU (Gray Test P=0.02). At 2 years and throughout the study, the same significantly different NRM was observed between study arms being respectively 18.2% vs. 8.9% and 19% vs. 9.7% (Gray Test P=0.03) (Figure 1). Causes of NRM in the BUCY2/BUFLU arms were: infections 8/6, organ failures 9/0, GvHD 5/3, hemorrhage 1/1, others 0/2. All in all, at 1, 2 and 5 years the leukemia free survival of the BUCY2 and BUFLU arm was similar being 62% vs. 69%, 56% vs. 62% and 50% vs. 56%, respectively (P=ns) (Figure 2). The number of patients with grade III-IV acute GvHD was higher in the BUCY2 arm (P= 0.02). There were no significant between-group differences in the incidence of chronic GvHD.ConclusionIn AML patients older than 40 years, the reduced toxicity conditioning with iv BUFLU significantly reduced the NRM compared to BUCY2. The increased incidence of leukemia relapse in the BUFLU arm was not associated with a detrimental effect on overall and leukemia free survival. (Funded by a grant from the Agenzia Italiana per il Farmaco (AIFA), ClinicalTrial.gov Identifier: NCT1191957). [Display omitted] [Display omitted] DisclosuresRambaldi:Pierre Fabre Pharma: Consultancy.

Transfer of Ex Vivo Expanded NK and γδT Cells from Untouched Posttransplant PBMCs to Clear Minimal Residual Disease in Acute Myeloid Leukemia

GMP-grade NK cell expansion for clinical purpose has been demonstrated feasible and safe. Here we share our pilot data on posttransplant immunotherapy with ex vivo expanded NK cells to treat minimal residual disease in a pediatric patient with posttransplant relapsed myeloid leukemia. Our patient, a 13 year old boy who underwent 2nd allogeneic stem cell transplantation (haploidentical stem cell transplantation from his mother) due to posttransplant relapsed acute myeloid leukemia. After the 2nd haploidentical stem cell transplantation (SCT) minimal residual disease (MRD) was detected by multiparameter flow cytometry and by two molecular markers CALM-AF10 fusion transcript and a NRAS-mutation. For posttransplant compassionate use immunotherapy by NK cell transfer, NK cells were expanded from untouched isolated PBMCs of the patient post 2nd haploidentical SCT. GMP-grade expansion of the NK cells was done under static conditions in our GMP-facility. Isolated PBMCs were pooled with 100Gy irradiated K562mb15 4-1BBL feeder cells (kindly provided by Dario Campana) in a proportion of 1:20 (NK to K562mb15 4-1BBL). PBMCs and K562mb15 4-1BBL were seeded in conventional cell culture flasks (175cm2) at a density of 1.1E6 cells/ml. Cell culture media contained RPMI1640 supplemented with 10% AB-human serum, 1% L-glutamine and 100IU Proleukine® IL2/ml. Cell culture was monitored daily for cell number, white blood cell differentiation, pH of the cell culture, glucose metabolism, lactate production and microbial sterility testing at the beginning and the end of the expansion period. The cell product was harvested on day 15-17. Fresh isolated PBMCs and the expanded NK cell product were characterized by flow cytometry. NK cells were expanded >1000 fold (3.1 and 3.4 log-fold) in 14-17 days. The product contained a total number of 9.8E9 and 19.9E9 cells, which was 328 and 665E6/kgBW. The expansion protocol supports NK and γδ T cell expansion whereas the number of αβ T cells stays stable. Cytotoxicity assay against various targets revealed excellent cellular cytotoxicity and antibody dependent cellular cytotoxicity. To prevent relapse in our patient with posttransplant MRD positivity, NK cells from the patient post 2nd haploidentical SCT were expanded for cellular immunotherapy. 2 weeks post 1st NK cell transfer (day +170) the patient achieved complete MRD response in the bone marrow. Unfortunately the patient showed detectable MRD one month later. Therefore another NK cell expansion and transfer was done. 2 weeks post 2nd NK cell transfer (day +232) the patient again achieved complete MRD response in the bone marrow and is in complete molecular remission ever since (day +340). The NK cell products were tolerated well. Transient coughing and temporary increase of temperature were registered. Both, in vitro and in vivo effect of the NK cell product were documented. Clinical use of expanded and activated NK cells and γδ T cells can induce molecular remission in posttransplant MRD positive acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Establishment of TRAIL-Resistance Kasumi-1 Cell Line and the Analysis of It different mRNA Expression Profile with the Original Kasumi-1 Cell Line

IntroductionTumor necrosis factor related apoptosis inducing ligand (TRAIL) can induce the apoptosis of many human leukemia cells while sparing of normal cells, but its resistance is also universal. Our previous study on apoptosis of t(8;21) positive acute myeloid leukemia cell line Kasumi-1 induced by rhTRAIL showed that the survival rate no longer decreased significantly when rsTRAIL reached a certain concentration which implied Kasumi-1 cells might have a resistant tendency to TRAIL. Then, we established a TRAIL-resistant Kasumi-1 cell line (Kasumi-1 TR) by intermittently escalating rsTRAIL concentration in culture media, and compared the mRNA expression profile with the original Kasumi-1 cell line by using Affymetrix Human Genome U133 Plus 2.0 Array.MethodsKasumi-1 TR cell line was established by intermittently treated Kasumi-1 cells with progressively escalating rsTRAIL concentration. Proliferation of leukemia cells were measured by CCK-8 assay, and rsTRAIL IC50 of cells and resistance index were calculated according to proliferation of cells treated with rsTRAIL at different concentrations. TRAIL and TRAIL receptors 1-4 on cells surface were detected by flow cytometry. Expression profiles of Kasumi-1 cells and Kasumi-1 TR cells were analyzed by Affymetrix Human Genome U133 Plus 2.0 Array to identify differentially expressed genes, and the search of genes possibly related with TRAIL-resistance were using by GO functional analysis and pathway enrichment analysis.Results1) Kasumi-1 TR cells proliferation was faster than that of Kasumi-1 cells(Fig 1A); 2) IC50 of 24 hours for Kasumi-1 cells was 756.833ng/ml (logIC50 2.879 ± 0.148), IC50 of 24 hours for Kasumi-1 TR cells was 1634646.005ng/ ml (logIC50 6.213 ± 0.637), the RI of 24h was 2159 (Fig 1B); IC50 of 48 hours for Kasumi-1 cells was 345.390ng/ml (logIC50 2.538 ± 0.153), IC50 of 48 hours for Kasumi-1 TR cells was 33642.641ng/ml (logIC50 is 4.257 ± 0.317), the RI for 48h was 97 (Fig 1C); 3) Cell surface expression of TRAIL and its receptors 1-4 had no difference between two cell lines(Fig 1D). 4) There were 1537 genes up regulated by more than 2 times while 487 genes down regulated by more than 2 times in Kasumi-1 TR cells compared with the original Kasumi-1 cells (Fig 1E). Of which BCL-2 family antiapoptotic gene BCL2 is increased by 3.153 times and BCL2A1 increased by 18.23 times, IFNAR1 involved in JAK/STAT pathway increased by 12.841 times and TRAIL death receptor TNFRSF10A down regulated by 3.256 times(Fig 1F).Conclusions: The Kasumi-1 cell line with rsTRAIL resistance (Kasumi-1 TR) is established, and its resistance may be associated with the up expression of BCL2, BCL2A1, IFNAR1 and down regulated expression of DR4.AcknowledgmentThis work was supported by grants from NSFC (30672415) and STCSM (054119528). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mutated Regions of Nucleophosmin 1 Elicit CD8+ T-Cell Responses in Patients with Acute Myeloid Leukemia

ObjectiveStimulating lymphocytes from healthy donors by DCs loaded with NPM1mut peptides in vitro in order to induce specific cellular immune responses against the peptides. And detecting the NPM1mut peptide specific T-cell in peripheral blood of patients with NPM1mut positive acute myeloid leukemia (AML), to provide a theoretical basis for immune therapy of AML.Methods1. NPM1 mutation detection: newly diagnosed patients of AML underwent gene mutations screening routinely.2. Samples collection: peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood collected from healthy donors with HLA-A* 0201 or A*1101 and NPM1mut positive AML patients in complete remission.3. Inducing differentiation of PBMCs into DCs: non-adherent cells were removed from PBMCs suspension, and then adherent cells were subsequently cultured for 5 days with GM-CSF and IL-4 to induce differentiation. Then cells were cultured in medium containing GM-CSF, IL-4, IL-6, IL-1β, TNF-α, and PGE2 for another 2 days to induce maturation.4. Generation of NPM1-specific cytotoxic T cells: DCs were pulsed with different NPM1 peptides followed by irradiation with 40 GY. The non-peptide pulsed DCs group was set as negative control. The above mentioned DCs were then co-incubated with their own PBMCs, maintained by IL-2 and IL-7. On day 7 of co-incubation, the same treated DCs as mentioned above were added again to restimulate lymphocytes.5. ELISPOT analysis and intracellular staining: peptide-stimulated T cells were tested by ELISPOT and intracellular staining method on day 7 and 14 . These two methods both detected the secretion of IFN-γ of cytotoxic T cells. T2 cells were used as target cells, and in terms of HLA-A*1101 positive donors, their own DCs were used as targets.Results1. In the Han Chinese population, the most common alleles of HLA-A loci were A* 1101, A*2402, and A*0201 allele.2. Through prediction of the aforementioned software, HLA-A*0201 restricted wild type NPM1 amino acid sequences (DLWQWRKSL), mutated NPM1-A/D amino acid sequences (AIQDLCLAV), and HLA-A*1101 restricted mutated NPM1-A/D amino acid sequences (AVEEVSLRK) were synthesized. There were no proper epitopes for either HLA-A*1101 restricted wild type NPM1 protein or HLA-A*2402 restricted wild type and mutated NPM1 protein.3. Expression of surface antigens of DCs on day 7 were as follows: CD14(2.6%), CD80(43.4%), CD83(7.8%), CD86(99.9%), HLA-DR(67.1%), which was consistent with DCs phenotype.4. About 10 days after DCs were co-incubated with their own PBMCs, the number of lymphocytes increased significantly, especially in the NPM1mut peptide pulsed group. On day 7 of co-incubation, ELISPOT analysis results of all samples were negative. However, 3 cases’ ELISPOT results were positive for the mutated peptide holes on day 14, in contrast, the results of the wild type peptide holes were negative. The NPM1mut peptide-specific T lymphocytes positive rate [=(average number of spots in mutated peptide holes-the average number of spots in negative control holes)/total lymphocytes per hole] was about 1/2500. Intracellular staining showed that in the aforementioned 3 cases, the proportion of CD8+IFN-γ+cells of the mutant peptide group was higher than either the wild type peptide group or negative control group, but there was no difference between the later two groups.5. 6 peripheral blood samples of patients with NPM1mut +AML were performed ELISPOT analysis, with only 1 case (16.7%) showing a approaching positive result.ConclusionsNPM1mut peptide pulsed DCs can stimulate their own PBMCs from healthy donors in vitro to produce mutated NPM1 specific T lymphocytes, and it is expected to be used as immune therapy of AML. The mutated NPM1 specific CTL in NPM1mut+AML patients are almost undetectable that indicates immune system have been comprised because of chemotherapy and disease, and can not responses to antigens efficiently. Perhaps AML patients can accept CTL transfusion from their relatives. Our study provide an experimental basis for cellular immunotherapy of AML. DisclosuresNo relevant conflicts of interest to declare.