APL Patients Research Articles

A Novel Suppressive Long Noncoding RNA within the IGF1R Gene Locus Is Imprinted in Acute Myelocytic Leukemia

Background：In acute myeloid leukemia (AML) cells, the insulin-like growth factor type I receptor (IGF1R) is one of the most abundantly phosphorylated receptor tyrosine kinases, promoting cell growth and therapeutic resistance through the PI3K/Akt signaling pathway. However, little is known regarding the molecular mechanisms underlying IGF1R gene dysregulation in AML. Long non-coding RNAs have been implicated in a variety of important biological processes by epigenetic mechanisms.Materials and methods：AML cell lines used in this study, KG-1, KG-1a were purchased from ATCC. The full length of IRAIN lncRNA was characterized by Marathon cDNA Amplification Kit. For qPCR, cDNA samples were amplified using CFX96TM real-time system (BIO-RAD) by SYBR PrimeScript™ RT-PCR Kit (TaKaRa). Both reverse transcription-associated trap (RAT) and chromatin conformation capture (3C) assays were used to examine the chromosome architecture of IGF1R and DNA-RNA interaction. The protocol of AMLpatients bone marrow and peripheral blood cell samples was approved by the Human Medical Ethical Review Committee from Jilin University First Hospital and informed consent was obtained from each AML patient and normal donor.Results：1. we discovered a novel intragenic 5366bp long noncoding RNA (lncRNA) within the IGF1R locus, named IRAIN, which is transcribed in an antisense direction from an intronic promoter.2. we demonstrated IRAIN was expressed exclusively from the paternal allele, with the maternal counterpart being silenced in normal donor and AML patients.3. we confirmed that IRAIN interacts with IGF1R chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop. Knockdown of IRAIN lncRNA with shRNA abolishes this intrachromosomal interaction.4. We demonstrated IRAIN was downregulated both in AML cell lines and high risk AML patients. Furthermore，the expression of IRAIN was dramatical higher in APL patients achieved molecular remission.Conclusion：Our results identify IRAIN as a new imprinted suppressive lncRNA involved in long range IGF1R DNA interactions in AML. As a putative tumor suppressor and a novel treatment target， further studies are needed to delineate the specific role of this newly identified lncRNA in the uregulation of the IGF pathway in AML. DisclosuresNo relevant conflicts of interest to declare.

Clinical Research on Hematological Malignancies Complicated with dic-a Single Center Report in China

Objective This study aimed to explore the most appropriate way of diagnosis and treatment among the patients with hematological malignancies complicated with disseminated intravascular coagulation (DIC) by evaluating the risk factors which would affect the prognosis and the efficiency of therapies.Methods The data of 162 definitely-diagnosed patients in West China Hospital between 2009 and 2013 has been collected and analyzed in our retrospective study. We have summarized the information on clinical features, laboratory indicators and interventions, which has been statistically analyzed to evaluate the effect on mortality.Results Elderly patients, AML patients accompanied with hyperleukocytosis, APL patients accompanied with differentiation syndrome combined deteriorated DIC symptoms, patients accompanied with infections, refractory/relapsed patients, patients with any of the following clinical symptoms such as gastrointestinal bleeding, intracranial hemorrhage, more than three sites of bleeding, acute renal dysfunction,acute respiratory dysfunction, acute left heart failure, MOF or shock, presented higher mortality (P<0.05).We found that there was difference in terms of mortality when we defined the cut-off value of PLT as 10×109/L (P<0.05). According to the ISTH criteria or Chinese guideline, the patients with abnormal PTAAPTTA AT-III, or ISTH score>6 had poorer prognosis. In our study, we tried to classify Fig < 1 g/L or > 4 g/L as a Fig abnormal group, the rest of the patients classified as a Fig normal group, then we found the abnormal Fig group had an increased mortality (P < 0.05). In addition, it had been found that patients who received standard chemotherapy had lower mortality (P < 0.05). With respect to non-APL patients, ones who took anti-fibrinolysis agents had similar mortality compared with the patients who didn’t (P>0.05).Conclusions Among the patients of hematological malignancies complicated with DIC, the patients with any of above clinical risk factors or abnormal laboratory indicators would have poorer prognosis. Moreover, the cut-off value of PLT should be dropped when we diagnose or evaluate this kind of patients’ prognosis. We put forward a new cut-off value of Fig as described above and it is valuable to evaluate the prognosis. The treatment of underlying diseases and substitute therapy act as basis of these patients, however, the adverse effects should be pay attention to as well as infections and acute heart failure. No benefit has been shown in the non-APL patients treated with anti-fibrinolytic agents. DisclosuresNo relevant conflicts of interest to declare.

Characteristics and treatment outcomes in 822 adult patients with acute myeloid leukemia:a single center experience

To investigate the characteristics and the short- or long-term treatment outcomes of the adult patients with acute myeloid leukemia (AML) in China. From 1999 to 2010, 822 adult cases with AML were enrolled, diagnosed and classified by the FAB and WHO criteria, respectively. The treatment outcomes and prognostic factors were analyzed retrospectively. In all patients with a median age of 38.5(15-83) years, acute monoblastic and monocytic leukemia (M5), AML with t(15;17)/PML-RARα (APL) and AML with t(8;21)/AML1-ETO(M2b) were the most common subtypes, accounting for 29.7%, 20.9% and 14.6% respectively. In APL patients, CR was achieved in 95.2%, with an early death (ED) rate of 4.8%. And the estimated overall survival (OS) and disease-free survival (DFS) at 5 year was 87.5% and 88.8%, respectively. Patients with other AML subtype (Non-APL) revealed a CR rate of 82.0%, ED of 4.3%, and estimated 5-year OS and DFS both of 48.8%. The OS rate of Non-APL patients at 3-year varied significantly (P<0.01) among three prognostic groups by cytogenetic risk stratification:favorable, 69.5%; intermediate, 52.8%; unfavorable, 29.8%. The prognostic factors for OS among Non-APL included age, cytogenetic abnormalities, courses of the median/high-dose cytarabine and allogeneic hematopoietic stem cell transplantation. When compared with the previous reports, the AML patients in our study were younger and showed a different subtype distribution. Treatment outcomes of APL and Non-APL were just the same as those in international leukemia centers. Chemotherapy by risk stratification, after diagnosis and classification according to the WHO criteria, is a key point to improve the outcomes in AML.

A HIF-1 network reveals characteristics of epithelial-mesenchymal transition in acute promyelocytic leukemia.

BackgroundAcute promyelocytic leukemia (APL) is a sub-type of acute myeloid leukemia (AML) characterized by a block of myeloid differentiation at the promyelocytic stage and the predominant t(15:17) chromosomal translocation. We have previously determined that cells from APL patients show increased expression of genes regulated by hypoxia-inducible transcription factors (HIFs) compared to normal promyelocytes. HIFs regulate crucial aspects of solid tumor progression and are currently being implicated in leukemogenesis.MethodsTo investigate the contribution of hypoxia-related signaling in APL compared to other AML sub-types, we reverse engineered a transcriptional network from gene expression profiles of AML patients’ samples, starting from a list of direct target genes of HIF-1. A HIF-1-dependent subnetwork of genes specifically dysregulated in APL was derived from the comparison between APL and other AMLs.ResultsInterestingly, this subnetwork shows a unique involvement of genes related to extracellular matrix interaction and cell migration, with decreased expression of genes involved in cell adhesion and increased expression of genes implicated in motility and invasion, thus unveiling the presence of characteristics of epithelial-mesenchymal transition (EMT). We observed that the genes of this subnetwork, whose dysregulation shows a peculiar pattern across different AML sub-types, distinguish malignant from normal promyelocytes, thus ruling out dependence on a myeloid developmental stage. Also, expression of these genes is reversed upon treatment of APL-derived NB4 cells with all-trans retinoic acid and cell differentiation.ConclusionsOur data suggest that pathways related to EMT-like processes can be implicated also in hematological malignancies besides solid tumors, and can identify specific AML sub-types.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0084-4) contains supplementary material, which is available to authorized users.

A Canadian consensus on the management of newly diagnosed and relapsed acute promyelocytic leukemia in adults.

The use of all-trans-retinoic acid (atra) and anthracyclines (with or without cytarabine) in the treatment of acute promyelocytic leukemia (apl) has dramatically changed the management and outcome of the disease over the past few decades. The addition of arsenic trioxide (ato) in the relapsed setting-and, more recently, in reduced-chemotherapy or chemotherapy-free approaches in the first-line setting-continues to improve treatment outcomes by reducing some of the toxicities associated with anthracycline-based approaches. Despite those successes, a high rate of early death from complications of coagulopathy remains the primary cause of treatment failure before treatment begins. In addition to that pressing issue, clarity is needed about the use of ato in the first-line setting and the role of hematopoietic stem-cell transplantation (hsct) in the relapsed setting. The aim for the present consensus was to provide guidance to health care professionals about strategies to reduce the early death rate, information on the indications for hsct and on the use of ato in induction and consolidation in low-to-intermediate-risk and high-risk apl patients.

Triple positive antiphospholipid antibody profile in outpatients with tests for lupus anticoagulants.

A triple positive antiphospholipid (aPL) antibody profile [two positive serum IgG aPL antibodies along with one positive functional plasma lupus anticoagulant (LAC) test result] is associated with an increased risk for thrombosis, whereas patients with single positive test results may have little to no increased risk. The frequency of triple positivity in outpatients with various combinations of LAC test results is unclear. We extracted from our database all LAC test results [dilute Russell viper venom times (dRVVT) and silica clotting times (SCT)] that had concomitant serum IgG aPL testing [both serum anti β2-glycoprotein I (anti-β2GPI) and anti-cardiolipin (aCL) antibodies]. There were 3195 patients without a prolonged prothrombin time. Double antibody positivity was found in 1% (31/2955) of those with normal functional LAC test results, in 16.0% (31/81) of those with a positive dRVVT, in 12.7% (10/79) of those with a positive SCT, and in 56.3% (45/80) of those with both tests positive (p<0.001). A triple positive aPL antibody profile was found in 28.3% (68/240) of those with at least one positive LAC test result. We conclude that 28% of patients with elevated LAC tests have a triple positive aPL antibody profile and patients with two positive LAC tests have a higher prevalence of a triple positive profile than do those with one positive LAC test result.

Arsenic trioxide induces oxidative stress, DNA damage, and mitochondrial pathway of apoptosis in human leukemia (HL-60) cells.

BackgroundAcute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), which accounts for approximately 10% of all acute myloid leukemia cases. It is a blood cancer that is formed by chromosomal mutation. Each year in the United States, APL affects about 1,500 patients of all age groups and causes approximately 1.2% of cancer deaths. Arsenic trioxide (ATO) has been used successfully for treatment of APL patients, and both induction and consolidated therapy have resulted in complete remission. Recently published studies from our laboratory have demonstrated that ATO pharmacology as an anti-leukemic drug is associated with cytotoxic and genotoxic effects in leukemia cells.MethodsIn the present study, we further investigated the detailed molecular mechanism of ATO-mediated intrinsic pathway of apoptosis; using HL-60 cells as a test model. Oxidative stress was assessed by spectrophotometric measurements of MDA and GSH levels while genotoxicity was determined by single cell gel electrophoresis (Comet assay). Apoptosis pathway was analyzed by Western blot analysis of Bax, Bcl2 and caspase 3 expression, as well as immunocytochemistry and confocal imaging of Bax and Cyt c translocation and mitochondrial membrane potential depolarization.ResultsATO significantly (p < 0.05) induces oxidative stress, DNA damage, and caspase 3 activityin HL-60 cells in a dose-dependent manner. It also activated the intrinsic pathway of apoptosis by significantly modulating (p < 0.05) the expression and translocation of apoptotic molecules and decreasing the mitochondrial membrane potential in leukemia cells.ConclusionTaken together, our research demonstrated that ATO induces mitochondrial pathway of apoptosis in HL-60 cells. This apoptotic signaling is modulated via oxidative stress, DNA damage, and change in mitochondrial membrane potential, translocation and upregulation of apoptotic proteins leading programmed cell death.

Review on Effectiveness of Primary Prophylaxis in aPLs with and without Risk Factors for Thrombosis: Efficacy and Safety.

Context. Antiphospholipid antibodies syndrome is an autoimmune disorder that is characterized by the association between presence of antiphospholipid antibodies and risk of thrombosis and/or pregnancy morbidity. Objectives. To systematically review the evidence for primary prophylaxis in patients with antiphospholipids antibodies syndrome or APS with or without other traditional risk factors of thrombosis when they did not have any thrombotic event yet. Methods. PubMed, the Cochrane Library, and Allied Health Literature were searched for studies that examined the efficacy and safety of primary prophylaxis in aPL patients from 1990 to February 2013. We examined literature looking at patients with aPLs with other risk factors for thrombosis and aPLs with no additional risk factors for thrombosis. Conclusion. We concluded that, in patients with aPLs, primary prophylaxes with HCQ and aspirin have been observed to reduce the frequency of thrombotic events in the case of asymptomatic aPL-positive patients with SLE. We also in this study concluded that LDA was effective in patients with autoimmune diseases. Independent cardiovascular risk factors include autoimmune defects such as SLE, rheumatoid arthritis, and atherosclerosis, where overall venous thrombosis will be induced by systemic inflammation. This review concludes that HCQ is an effective primary approach when compared to aspirin.

Successful treatment of high-risk acute promyelocytic leukemia in very elderly patients using all-trans retinoic acid plus reduced-dose idarubicin

To the Editor,Theintroductionofall-transretinoicacid(ATRA)therapyhasmarkedly improved the outcome for younger patients withacute promyelocytic leukemia (APL) [1, 2]; however, theoptimal treatment regimen for elderly APL patients remainsunclearbecausefewpatientsover70yearsofageareincludedin clinical trials [3]. Here, we report the successful use ofATRA plus reduced-dose idarubicin to treat nonagenarianhigh-risk APL patients.Patient 1 (Fig. 1a), a 91-year-old woman with a previoushistory of hypertension and cerebral infarction, was admittedto our hospital with fever and bleeding. Her Eastern Cooper-ativeOncologyGroupPerformanceStatus(ECOG-PS)was4.Laboratoryresultswereasfollows:hemoglobin(Hb),103g/l;white blood cell (WBC) count, 36.2×109/l; platelet (Plt)count, 51×109/l; serum lactate dehydrogenase (LDH),1,437 IU/l; prothrombin time-international normalized ratio(PT-INR),1.41;activatedpartialthromboplastintime(APTT),34.5 s; fibrinogen, 157 mg/dl; and D-dimer, 315.7 μg/dl.Polymerase chain reaction analysis of peripheral blood cellsidentified the PML-RARα fusion gene. The patient receivedATRA (45 mg/m

Beneficial Effect Of Anticoagulants In The Management Of Patients With Acute Promyelocytic Leukemia (APL): Results Of a Multicenter, Retrospective Epidemiologic Study Of The Disseminated Intravascular Coagulation Patients In Japan

BackgroundAcute promyelocytic leukemia (APL) is an uncommon subtype of acute myelogenous leukemia (AML) with a high mortality rate, mostly attributable to intracranial hemorrhage caused by disseminated intravascular coagulation (DIC). Almost all patients with APL develop DIC. The introduction of all-trans retinoic acid (ATRA) into the induction chemotherapy regimen has revolutionized the treatment of individuals with APL, with nearly 90% of newly diagnosed APL patients achieving complete remission and over 70% of patients surviving longer than 5 years. However, population-based studies have shown that the early death-rate during induction chemotherapy remains extremely high with nearly 30% incidence and the most common cause of death is associated with hemorrhage. Thus, development of a novel treatment strategy to alleviate abnormal coagulation in APL patients is urgently required.In Japan, the conventional therapies for coagulopathy, such as low molecular heparin (LMWH), danaparoid sodium, and synthetic protease inhibitors were aggressively administered in addition to blood transfusions in DIC patients with APL. In 2008, a new anticoagulant, recombinant human soluble thrombomodulin (rTM) was approved for the treatment of DIC in Japan. Therefore, we accessed the difference of clinical safety and beneficial effects among these anticoagulant treatments in DIC patients with APL, by evaluating data on the multicenter, retrospective epidemiologic study of the DIC patients. Patients and MethodsPatients with DIC associated with newly diagnosed APL were recruited from January 2000 through December 2012 in 3 Japanese hematology centers. APL was defined according to FAB classification and was confirmed by the presence of t(15;17) and/or the PML-RARα fusion gene. DIC was evaluated by the diagnostic criteria of the Japanese Ministry of Health and Welfare. The treatment algorithm for newly diagnosed APL patients was generally based on the protocol proposed by the Japan Adult Leukemia Study Group (JALSG). The concomitant use of fresh frozen plasma, platelets, and/or other anticoagulant drugs were generally based on the recommendation of the JALSG protocol as follows: Platelet concentrate and fresh frozen plasma were transfused to maintain platelet counts ³30 × 109/L and plasma fibrinogen levels ³150 mg/dL, respectively.Early death (death within 30 day from APL diagnosis), severe hemorrhagic events, and improvement of coagulopathy were analyzed between rTM and other anticoagulants treatment. The time to disappearance of hemorrhagic symptoms between the two groups were analyzed by the Kaplan-Meier method and the log-rank test. ResultsDIC developed at diagnosis in 64 APL patients. 35 patients with DIC were treated with rTM (rTM group) and 29 patients with DIC were treated with LMWH, danaparoid sodium, and/or synthetic protease inhibitors (control group). There were no significant differences in age nor gender between rTM group and control group. Number of patients treated with ATRA alone and ATRA plus chemotherapy were 7 and 28 in the rTM group, and 10 and 19 in the control group. Early death rate for the rTM group was significantly lower than that for the control group (14% vs. 38%, P=0.043). Two patients developed intracranial hemorrhagic early death in the control group. On the other hand, no severe hemorrhagic event and mortality was noted in the rTM group. The time to disappearance of hemorrhagic symptoms was shorter for the rTM group than the control group (P=0.021). ConclusionsThese findings suggest that supportive care with rTM in combination with ATRA and chemotherapy ameliorates coagulopathy and reduces the risk of hemorrhagic early deaths in patients with APL than other anticoagulants. Disclosures:No relevant conflicts of interest to declare.

Long-Term Outcome Of Acute Promyelocytic Leukemia (APL) With Lower Initial Leukocyte Counts By Using All-Trans Retinoic Acid (ATRA) Alone For Remission Induction Therapy: Japan Adult Leukemia Study Group (JALSG) APL97 Study

BackgroundATRA and anthracycline-based chemotherapy is a standard remission induction therapy for APL, leading to complete remission (CR) rate of 90% or more. (Asou et al, 2007; Ades et al, 2010; Avvisati et al, 2011). As reported in many APL studies including the JALSG study, risk adopted strategy according to initial leukocyte counts has demonstrated successful results. However, the long-term outcome of the patients with initial leukocyte counts < 3,000/µl received ATRA alone in the induction therapy and followed by post remission chemotherapies, remains to be elucidated. Furthermore, it is controversial whether concomitant chemotherapy is needed for such a very low risk group. In the JALSG-APL97 study, patients with initial leukocyte counts < 3,000/µl received ATRA alone until remission (Group AA), except for patients with leukocytosis during the ATRA therapy who received additional chemotherapy (Group AD). Here, we reported the long-term outcome of this study based on risk adopted therapy, particularly, focusing on the outcome of the very low risk group. MethodsThe treatment schedule of JALSG-APL97 study was initially reported by Asou et al. in 2007. In brief, patient groups were defined as: leukocytes < 3000/µl (Group A: ATRA alone), 3000/µl ≤ leukocytes < 10,000/µl (Group B: ATRA plus IDA/Ara-C: 2+5), and leukocytes ≥ 10,000/µl (Group C: ATRA plus IDA/Ara-C: 3+5). Patients who experienced leukocytosis received additional chemotherapy (Group D). After 3 courses of consolidation chemotherapy, patients achieved molecular CR were allocated to an intensive chemotherapy group or observation. The CR rate, overall survival (OS), event-free survival (EFS), and cumulative incidence of relapse (CIR) were analyzed for each group. ResultsTwo hundred and seventy-one newly diagnosed APL patients, ranging from 15 to 70 years of age, were evaluable. The number of patients in each group was 150 (A), 69 (B), 52 (C) and 70 (D), respectively. Of 150 patients in Group A, 83 achieved CR with ATRA alone (AA), and 67 were added chemotherapy due to leukocytosis during ATRA therapy (AD). In Group A, B, C and D, CR rates were 95.2%, 97.0%, 90.4% and 97.1%, respectively (P = 0.30); OS (Figure 1) 90.1%, 77.3%, 73.1% and 71.4%, respectively (P = 0.02); EFS (Figure 2) 71.1%, 63.6%, 55.7% and 64.3%, respectively (P = 0.26); CIR 23.5%, 27.6%, 34.5% and 20.4% (P = 0.51), respectively. Initial leukocyte counts in Group AA were significantly lower compared to those in Group AD (median leukocyte counts; 900/µl vs. 1,100/µl, P = 0.03). The median administration period of ATRA was similar between Group AA and AD (46 days vs. 43 days, P = 0.57). Differentiation syndrome was more frequent in Group AA (28.0% vs. 14.9%, P = 0.04). The CR rate and early death rate were not different between two groups (95.2% vs. 95.5%, P = 0.92 and 3.6% vs. 4.5%, P = 0.79, respectively). OS was significantly inferior (90.1% vs. 73.1%, P = 0.005) and non-relapse mortality after post-remission therapy was significantly higher in Group AD (5% vs. 16%, P = 0.04), compared to Group AA, while EFS was not different between two groups (71.1% vs. 65.7%, P = 0.33). The cumulative incidence of late relapse occurred more than 2 years after CR was significantly higher in Group AA compared to Group AD (17.5% vs. 3.9%, P = 0.04). [Display omitted] [Display omitted] ConclusionsRisk adopted therapy according to initial leukocyte counts is totally useful in this study as well as previous reports including us. OS was favorable in APL patients with initial leukocyte counts < 3,000/µl, achieved CR by using ATRA alone for remission induction therapy, whereas EFS in this group was still unsatisfactory in the long-term follow up. It could be explained by the high frequency of late relapse. Ades et al reported better long-term outcomes in patients concomitantly treated with ATRA and chemotherapy rather than in those treated with ATRA followed by chemotherapy in their APL patients with initial leukocyte counts < 5000/µL. However, very low risk patients (Group AA) could be put into the separate category, and therapeutic approaches to reduce the late relapse in this group should be discussed. Additionally, the genetic profile studies will provide us informative data in relation to initial leukocyte counts and leukocytosis during ATRA therapy. Disclosures:Kiyoi: Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Research Funding.

Utility and Impact Of Early t(15;17) Identification By Fluorescence In Situ Hybridization (FISH) In Clinical Decision Making For Patients With Acute Promyelocytic Leukemia (APL)

BackgroundAcute Promyelocytic Leukemia (APL) is a highly curable malignancy with most large co-operative group studies showing greater than 90% long term survival. Despite this, recent studies looking at survival in population-based studies suggest that approximately 30% of patients with APL die during induction. This has been confirmed in large population-based studies in Sweden and the US. A recent analysis of Surveillance, Epidemiology & End Results (SEER) data from 13 population-based cancer registries with 1400 APL patients in the US showed that 17% of all patients and 24% of patients > 55 years of age die within one month of diagnosis. Swedish registry data also showed that 29% of patients died within 30 days of diagnosis with most of them dying in the first two weeks. Early initiation of treatment is critical in improving outcomes. Early demonstration of t(15;17) will lead to more accurate decision making regarding treatment especially in the setting when patients are referred from rural areas or treated in academic institutions where confirmation is essential for research protocol purposes. The aim of this project was to validate earlier time frames for the Abbott Molecular Vysis LSI PML/RARA FISH probe (ASR 6-16 hrs). Materials and Methods15 patients (AML-M0, M1, leukocytosis, NHL, pregnancy loss & APL) were selected for validating various hybridization (hybe) times for FISH probe. After preparing the slides, they were chemically aged by treatment of 2XSSC at 73°C for 2 minutes. 2uL of probe (Abbott Molecular Vysis LSI PML/RARA) was applied to each sample after dehydrating with an ethanol series (70%, 85% & 90%) for 1 minute each. The slides were then placed in the Thermobrite for 2, 4, 6 hours and overnight to denature and hybridize. After each hybe the slides were treated with 0.4xSSC/0.3%NP-40 for 2 minutes at 73°C and later immersed in 0.2xSSC/0.3% NP-40 for 1 minute at room temp. 2uL of DAPI Antifade SS was added with a micropipette onto each hybridized area, and a coverslip was applied. 200 cells with robust signals were identified and calculated. Hybe efficiency was graded as acceptable (good), intermediate (okay) and unacceptable (poor). Expected normal signal pattern was two red and two green signals (2R2G), and the expected most common abnormal signal pattern was two fusion (yellow) signals, one red and one green (2F1R1G) and/or one fusion, one red and one green (1F1R1G). ResultsThe specificity of the probe ranged from 84% at two hours, 86% at four hours, 84% at 6 hours, and 87% for overnight hybe. The sensitivity increased from 79% at two hours, 80% at four hours, 81% at 6 hours, to 87% for overnight hybe, which was expected. Conclusion and DiscussionMajority of the APL patients who survive the induction phase are cured with a low chance of relapse. Early mortality continues to be a significant problem with deaths attributable to bleeding, differentiation syndrome and infection. Coagulopathy abnormalities are a hallmark of this disorder and can be rapidly corrected with early institution of treatment. Also, in patients with co-morbid conditions that would exclude the use of anthracyclines, arsenic is the treatment of choice and starting of arsenic is not recommended unless cytogenetic diagnosis is confirmed. The utility of early t(15;17) detection by FISH helps in initiating treatment early and may lead to improved outcomes. This can be easily achieved by shortening the timeframe for FISH results. Based on the validation studies the current recommendations are to read FISH results at the 4 hour incubation mark for a preliminary diagnosis and confirmation with overnight hybridization. Disclosures:Kota:Teva: Speakers Bureau; Ariad: Advisory board, Advisory board Other.

FLT3-ITD Internal Tandem Duplication Confers Poor Prognosis In Patients With Acute Promyelocytic Leukemia Treated With The AIDA Protocols. Long-Term Follow-Up Analysis

Although previous studies in APL have revealed that the FlT3-ITD mutation is associated with an elevated presenting WBC count, hypogranular variant (M3v) morphology and the short (bcr3) isoform of PML-RARA, the prognostic significance of FlT3 mutations in APL has not been firmly established. We report an update of these patients with a median follow-up of 9 years in which we observed that the presence at baseline of the FlT3-ITD mutation confers a very poor overall survival (OS). One hundred and forty-seven patients with newly diagnosed APL were observed and treated with the AIDA (73 patients) and AIDA2000 protocols (74 patients) at the Sapienza University of Rome during the period April 1993-October 2010. Diagnosis was initially established morphologically and subsequently confirmed in all cases by RT-PCR. The following clinical characteristics at diagnosis were analyzed according to the FlT3 status: age, sex, FAB classification, peripheral WBC and platelet count, hemoglobin, karyotype, PML/RAR isoform and relapse risk. For statistical analysis, the Wilcoxon-Mann-Whitney test was used to compare non-parametric series and the Fisher's exact test to compare categories. OS was estimated using the Kaplan-Meier method, whereas relapse-free considered as events relapse and death in CR. Thirty-three patients were identified as FlT3+, 19 were males (57%) and 14 were females; 27% had a variant type according to the FAB classification and 36% were classified as high-risk. Twenty-one FlT3+ patients (63%) presented the bcr3 transcript compared to 37/114 (32%) FlT3- patients (p=0.002). Eight FlT3+ patients (24%) experienced a differentiation syndrome compared to 14 (12%) in the negative cohort (p=0.02). After a median follow-up of 9 years (range 5-19), we could document a significant worse long-term outcome for FlT3+ patients: OS was 96% in the FlT3- cohort compared to 39% in the FlT3+ cohort (p=0.0001), relapse-free survival (RFS) 90% in FlT3- patients vs 30% in the FlT3+ ones (p=0.017), CIR at 9 years was 4% (95% CI, 1,279-6,238) for the FLT3-negative cohort and 60% (95% CI,22.632-81.236) for the FLT3-positive subset (p=0.0001). While this study confirms that there is no difference in response to induction with the AIDA schedule in FlT3+ APL patients, the longest follow-up so fare reported has allowed to demonstrate the significantly worse long-term outcome for this subset of APL, in terms of OS, RFS and DFS. Further studies aimed at investigating whether other clonal mutations may play a role in this unfavorable subset of patients are needed. Disclosures:No relevant conflicts of interest to declare.

Classification Of Acute Myelogenous Leukemia (AML) Based On Functionally Related Proteins Groups

BackgroundIn an era where the recognized heterogeneity of the pathophysiology of AML is increasing rapidly due to sequencing and other “omics” platforms, and where the number of available targeted therapeutics is also rapidly expanding, a means to individually match therapies to AML on a specific basis is needed. New targeted therapies modulate single pathways, so recognizing when a particular pathway is active is crucial, but mutations are rare within most of these pathways in AML. Furthermore pathway activation can arise by various means. Finally the interaction of multiple different activated pathways on the overall phenotype of a leukemic blast adds complexity. MethodsTo define the role of the pathway activation in AML we generated a custom made reverse phase protein array (RPPA) onto which were printed leukemia enriched (ficolled, CD3/CD19 depleted) cells from 511 newly diagnosed AML and 21 APL patients, including 49 with paired diagnosis and relapse samples. Both bone marrow (n=387) and peripheral blood (n=283) samples were used, with 140 cases having both. The RPPA was probed with 231 strictly validated antibodies (174 antibodies vs. total expression, 49 vs. phosphoproteins, 5 vs. cleaved forms, 3 vs. methylation sites). These proteins were divided on the basis of function into 21 Protein function groups (ProFnG) including: Apoptosis, Autophagy, Cell cycle, Creb, Cytoskeletal, Differentiation, Fli1, Hippo, Histone modifying, Hypoxia, Integrin & adhesion, MEK, P53, PI3K-AKT, PKC, SMAD, STAT, Transcription Ubiquitin and Wnt. ResultsEach ProFnGrp was analyzed to determine the optimal number of principal components (PrCp) and to identify noncontributory members, which were then discarded. Each PrCp (79 total) was then considered a variable and Hierarchical clustering was performed on continuous and binary signals (Sokal-Michener metric) (Figure 1). This defined 8 PrCp clusters and 8 patient groups(PatGp). Several categorical clinical features were unevenly associated with the PatGp including FAB (p=1.5e-12), WHO class (p=0.01), gender (p=0.03), Zubrod PS (p=0.005), antecedent hematological disorder (p=0.007), Cytogenetics (0.01), FLT3-ITD (p=0.003), Ras mutation (p=0.003). Likewise many continuous variables also correlated with PatGp: WBC (p=7e-14), %BM blast (p=2x10-16), % PB blast (p=2x10-16), Platelets (p=0.004), LDH (p=0.009), Albumin (p= 0.00001), Creatinine (p=0.007), CD13 (p=0.0002), CD33 (4.5xe-8), CD33 (1.1e-8). For all patients PatGp did not predict remission attainment. There was significant splay in overall survival and event free survival with median EFS durations ranging 52 , 100, 130 and 170 weeks ( p= 0.05) and in remission duration ranging from medians of 20- to 110 weeks. For intermediate cytogenetics Pat-Gps split into 4 with better outcomes (median OS 70-110 week, median RemDur 100-120 weeks) and 4 with worse outcomes (median OS 40-50 weeks, median RemDur 30-40 weeks). For unfavorable cytogenetics Pat-Gps also split into 4 with better outcomes (median OS 35-40 weeks, median RemDur 48-75 weeks) and 4 with worse outcomes (median OS 16-18 weeks, median RemDur 10-26 weeks). In a companion abstract we show the interaction between different protein functional groups. [Display omitted] ConclusionsAML could be classified based on protein functional groups. Although the classes were associated with cytogenetics the protein classifications gave prognostic information independent of cytogenetics and other traditional prognostic factors. Since this classification scheme is dependent on protein expression and functional activation states it may identify when particular pathways are being utilized and therefore be of use in triaging patients to targeted therapies. Disclosures:No relevant conflicts of interest to declare.

Arsenic Trioxide (ATO) Or ATRA For Consolidation Treatment Of Standard Risk Non Elderly Newly Diagnosed APL– Second Interim Analysis Of a Randomized Trial (APL 2006) By The French Belgian Swiss APL Group

▪ BackgroundATRA combined to anthracycline-based chemotherapy (CT) remains the classical treatment of newly diagnosed APL, but it is myelosuppressive and may be associated with long-term cardiac toxicity. Both ATO (Powell, Blood 2010) and ATRA (Sanz , Blood 2004) may allow to reduce the amount of CT and further diminish the relapse risk. In a randomized trial (APL 2006 trial), we compared for consolidation treatment ATO, ATRA and Ara C in standard risk APL ( ie with baseline WBC < 10G/L). MethodsIn this trial (started in Nov, 2006) newly diagnosed APL patients (pts) < 70 years with WBC < 10 G/L were randomized for consolidation between AraC, ATO and ATRA. The AraC group (standard group) received for induction: ATRA 45mg/m2/d until CR with Idarubicin (Ida) 12 mg/m2/dx3 and AraC 200mg/m2/dx7 started on day 3; first consolidation with the same CT course, second consolidation with Ida 9 mg/m2/dx3 and AraC 1g/m2/12h x4d ; maintenance during two years with intermittent ATRA 15d/ 3 months and continuous 6 MP + MTX,). The ATO and ATRA groups received the same treatment as the AraC group , but AraC was replaced respectively by ATO 0.15 mg/Kg/d d1 to 25 and ATRA 45 mg/m2/d d1 to 15 for both consolidation courses. We present here results of the second interim analysis, made at the reference date of 1st Jan 2012, in 349 pts aged < 70 years included in 78 centers before 2012. The primary endpoint was event free survival (EFS) at 2 years from CR achievement. Relapse, survival, side effects of the treatment and duration of hospitalization were secondary endpoints. ResultsPre-treatment characteristics were well balanced between the 3 consolidation groups. 347 pts (99.4 %) achieved CR, and 2 (0.5%) had early death. Overall, 3, 0, and 4 pts had relapsed and 5, 2, and 2 pts had died in CR in the AraC, ATO and ATRA consolidation groups, respectively. Two year EFS was 95%, 97.4% and 96.8% (p=NS) and 2 year OS was 96.6%, 97.4% and 99% (p=NS), in the AraC, ATO and ATRA consolidation groups, respectively. Median time to ANC >1 G/L and platelets > 50G/L after the first consolidation course was 24 and 25 days, 24 and 23 days, 17 and 20 days in the AraC, ATO and ATRA group, respectively (p<0.01). Similarly, time to ANC >1 G/L and platelets > 50G/L after the second consolidation course was 23 and 27 days, 19 and 18 days, 13 and 19 days in the AraC, ATO and ATRA group (p<0.01). The overall duration of hospitalization was 60.9, 63.1 and 33 days in in the AraC, ATO and ATRA groups, respectively (p<0.01). ConclusionVery high CR rates (close to 98-99%) are now obtained in standard risk APL on a very large multicenter basis using classical ATRA and anthracycline based CT combinations, with very few relapses. ATO or ATRA can replace AraC during consolidation cycles without increasing the relapse risk, and can possibly reduce the rate of deaths in CR (2 and 2 patients versus 5 patients, although the difference was NS). However Ida and ATO, when used concomitantly for consolidation cycles, proved as myelosuppressive as Ida-AraC cycles, while myelosuppression was reduced with Ida-ATRA consolidation courses. Disclosures:Off Label Use: ATO in the treatment of 1st Line APL.

The tumor suppressor gene DAPK2 is induced by the myeloid transcription factors PU.1 and C/EBPα during granulocytic differentiation but repressed by PML-RARα in APL

DAPK2 is a proapoptotic protein that is mostly expressed in the hematopoietic tissue. A detailed DAPK2 expression analysis in two large AML patient cohorts revealed particularly low DAPK2 mRNA levels in APL. DAPK2 levels were restored in APL patients undergoing ATRA therapy. PML-RARA is the predominant lesion in APL causing transcriptional repression of genes important for neutrophil differentiation. We found binding of PML-RARA and PU.1, a myeloid master regulator, to RARA and PU.1 binding sites in the DAPK2 promoter. Ectopic expression of PML-RARA in non-APL, as well as knocking down PU.1 in APL cells, resulted in a significant reduction of DAPK2 expression. Restoring DAPK2 expression in PU.1 knockdown APL cells partially rescued neutrophil differentiation, thereby identifying DAPK2 as a relevant PU.1 downstream effector. Moreover, low DAPK2 expression is also associated with C/EBPα-mutated AML patients, and we found C/EBPα-dependent regulation of DAPK2 during APL differentiation. In conclusion, we identified first inhibitory mechanisms responsible for the low DAPK2 expression in particular AML subtypes, and the regulation of DAPK2 by two myeloid transcription factors underlines its importance in neutrophil development.

MANAGEMENT OF ACUTE PROMYELOCYTIC LEUKEMIA IN THE ELDERLY

Unlike other forms of AML, APL is less frequently diagnosed in the elderly and has a relatively favourable outcome. Elderly patients with APL seem at least as responsive to therapy as do younger patients, but rates of response and survival are lower in this age setting owing to a higher incidence of early deaths and deaths in remission when conventional treatment with ATRA and chemotherapy is used. Elderly APL patients are more likely to present with low-risk features compared with younger patients, and this may explain the relative low risk of relapse reported in several clinical studies. Alternative approaches, such as arsenic trioxide and gentuzumab ozogamicin have been tested with success in this setting and could replace in the near future frontline conventional chemotherapy and ATRA.

Arsenic trioxide: Pharmacokinetics in acute promyelocytic leukemia (APL) patients.

7055 Background: Arsenic (As) has significantly increased survival for APL patients. As and its metabolites’ effects on cell proliferation, apoptosis, and methylation, especially long term, remain largely unknown. It is imperative to study the pharmacokinetics of As at therapeutic doses in order to limit untoward effects resulting from treatment. Arsenic trioxide (ATO), available as arsenous acid (iAsIII), is readily metabolized through sequential methyl group additions and electron reduction steps: iAsIII →MAsV →MAsIII →DMAsV →DMAsIII →TMAsVO →TMAsIII. iAsIII, MAsIII and DMAsIII are more biologically active and more toxic than pentavalent forms. The key enzyme involved is arsenic methyltransferase, and polymorphisms contribute to metabolic differences between individuals. Methods: Cancer patients not treated with ATO (controls) had one collection of blood and urine samples, while APL patients receiving therapeutic doses of ATO had collections immediately prior to and at 1, 2, 4, 6, and 24 hours, days 4, 8, 15, and 4 weeks after ATO-free interval. Total iAs (iAsIII+iAsV), MAs (MAsIII+MAsV) and DMAs (DMAsIII+DMAsV) were measured in plasma using hydride generation cryotrapping atomic absorption spectrometry, a sensitive automated method of arsine detection. The same As species were measured in urine and in exfoliated bladder cells isolated from urine. Results: We report data on ten control patients and six treated patients (ATO 0.15 mg/kg/day). Initial average As concentrations in treated patients (0.051 ng/ml) were similar at baseline to the controls (0.046 ng/ml). We observed that iAs is quickly metabolized from a peak average plasma concentration of 32 ng As/ml to a trough of 10 ng As/ml within six hours from infusion, remaining unchanged for at least 24 hours. MAs and DMAs concentrations begin to increase at six hours, and continue to rise by day 4 to an average concentration of 8.5 and 10.4 ng As/ml respectively, followed by decline to baseline 4 weeks after final ATO infusion. Conclusions: Treatment with ATO leads to the formation of MAs and DMAs whose long term toxicity in APL patients is poorly understood. Studies involving analysis of As metabolites are needed to assess possible long term toxicities on non-targeted organs.

Decreasing early mortality (30-day) in APL patients with use of streamlined treatment guidelines and support from core group of experts.

7091 Background: Recent evidence from population based studies in Brazil and US SEER data show that the early mortality (EM) in APL is around 30%. This is in contrast to observation in clinical trials where it is 5%. The common causes of death are hemorrhagic complications (HC), infection, differentiation syndrome (DS) and multi-organ failure. HC are unique to this condition due to DIC and HC are seen in upto 60% of patients. Hence, decreasing early deaths is a high priority at all leukemia treatment centers. We report our updated results showing that use of set of streamlined treatment guidelines along with support from experts decreases early deaths. Methods: At Georgia Regents University, between 7/2005 and 6/2009, 19 patients were diagnosed with APL. 7 patients (5 high-risk and 2 low-risk) died during induction resulting in an unusually high mortality rate of 37%. All patients who survived induction are still in remission at present. The high early death rate prompted us to develop a simple, 2 page treatment algorithm that focuses on quick diagnosis, prompt initiation of therapy, and proactive and aggressive management of all the major causes of death during induction. We also made our treatment protocol available to smaller treatment centers and helped the treating oncologists manage the patient during the first few days after diagnosis. Results: From 11/2010 to 12/2012, we treated 5 patients at GRU and helped manage 9 patients at 5 practices. Age range was 30-60 years. 4 patients were high-risk, 7 intermediate and three low-risk. There were no deaths during induction. Only 1 patient (8%) had HC and 4 had DS. Conclusions: While we recognize that this is a small cohort, our own experience and a similar approach pioneered by investigators in Brazil clearly shows this to be an effective model to decrease early deaths in APL. We believe our experience warrants large scale implementation of our protocol in an attempt to decrease early mortality in APL. We were awarded a 1.68 million grant by the Leukemia Lymphoma Society to implement this protocol in the states of Georgia and South Carolina with a catchment population of 15 million over a 3 year period.

Superficial vein thrombosis, thrombin generation and activated protein C resistance as predictors of thromboembolic events in lupus and antiphospholipid patients. A prospective cohort study

IntroductionPredicting thrombosis in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid antibodies (aPL) is still challenging. Our objective was to determine risk factors for thrombotic events including activated protein C (APC) resistance proven by a thrombin generation (TG) assay in patients with SLE and/or aPL. Materials and methodsWe performed a prospective cohort study in a French University Hospital and tertiary care center. Ninety-two consecutive patients with SLE and/or aPL without ongoing anticoagulant treatment were enrolled. The outcome was time to thrombotic event. We evaluated clinical and laboratory variables including APC sensitivity ratio (APCsr) determined by TG. An APCsr>90th percentile of a control population indicated APC resistance. ResultsPatients were followed-up for a median duration of 35months (inter-quartile range: 26 to 62; 320 patient-years). Thrombosis during follow-up occurred in 18 patients. In univariate analysis, together with history of hypertension, superficial vein thrombosis (SVT) and arterial thrombosis, patients with both aPL and APC resistance had an increased risk for incident thromboembolic events (HR, 3.67[95% confidence interval, 1.31 to 10.31]). In multivariate analysis, only history of hypertension (HR, 10.77 [95% confidence interval, 3.15 to 36.83]), SVT (HR, 7.45 [95% confidence interval, 2.25 to 24.66]) and arterial thrombosis (HR, 3.31 [95% confidence interval, 1.14 to 9.55]) remained independent risk factors. ConclusionsHistory of thrombosis including seemingly benign SVT have a higher predictive value for incident thrombotic events in SLE or aPL patients than APC resistance proven by TG.