Untreated Acute Promyelocytic Leukemia Research Articles

Prompt Initiation of Conventional Chemotherapy to Avoid Early Death in Patients with Newly Diagnosed Acute Promyelocytic Leukemia.

Objective Compared to prospective trials, the early death rate of newly diagnosed acute promyelocytic leukemia (APL) in the real-world clinical setting is higher. However, the early death rate was heterogeneous according to the reported institutes. Thus, the therapeutic approach at each institute may be important for preventing early death. This study evaluated the management strategy for untreated APL in our institute to avoid early death. Methods and Patients We identified consecutive 21 patients with untreated APL who received induction therapy including all-trans retinoic acid (ATRA) between July 2007 and December 2021 at the University of Tokyo Hospital. Results As therapeutic approaches, 16 patients (76%) received ATRA administration on the day of admission, and the remaining 5 received ATRA within 4 days from admission. Notably, all patients received conventional chemotherapy added to ATRA at a median of 1 day from admission (range: 0-9 days). As clinical outcomes, no patient died during induction therapy for untreated APL, and all achieved complete molecular remission. Conclusions Compared to the previous nationwide survey, a higher proportion of patients at our institute received conventional chemotherapy in addition to ATRA, and it was initiated more promptly, which may have helped prevent early death.

AML-278: Examination of Optimal Administration of Arsenic Trioxide in Continuous Renal Replacement Therapy During Acute Promyelocytic Leukemia Treatment

<h3>Background and Aim:</h3> Arsenic trioxide (ATO), a safe and effective treatment for acute promyelocytic leukemia (APL), has an effective plasma arsenic concentration (PAC) of 37.6–150 ng/mL. According to current recommendations, the ATO dosage should be reduced for patients receiving intermittent hemodialysis. However, there are no reports on proper ATO administration in patients receiving continuous renal replacement therapy (CRRT); the PAC during CRRT is unknown, and the optimal dosage for patients on CRRT has not been established. We evaluated the appropriate ATO dosage for patients on CRRT. <h3>Patients and Methods:</h3> We enrolled two APL patients undergoing CRRT during ATO therapy. We measured PAC over time to determine the optimal timing of administration required to achieve the therapeutic range for ATO. <h3>Results:</h3> Prior to APL treatment, both patients had normal renal function. The first case was a woman in her 30s with a recurrence of APL. She developed acute kidney injury owing to severe differentiation syndrome and disseminated intravascular coagulation, and she suffered from uncontrolled uremia and fluid volume. Thus, continuous hemodialysis (CHD) was initiated. Because of previous therapy, her PAC was 68 ng/mL at the start of CHD. We administered ATO (0.15 mg/kg) 18 hours after the start of CHD. Her PAC before ATO administration was 60 ng/mL. Immediately after ATO administration, her PAC was 80 ng/mL. However, owing to the redistribution of arsenic, the PAC gradually rose to 100 ng/mL. The second case was a male patient in his 30s who had untreated APL. He also developed acute kidney injury owing to differentiation syndrome. He initially received dialysis but soon required CHD. We administered ATO (0.15 mg/kg) before initiating CHD and at 13 and 39 hours after the start of CHD. His PAC was 110 ng/mL immediately prior to CHD; thereafter, ATO was administered, and his PAC ranged between 130 and 190 ng/mL. At 117 hours after the start of CHD, his PAC was 114 ng/mL, which was within the optimal range. <h3>Conclusions:</h3> PACs exceeded the effective range, even in the presence of CHD. Therefore, we suggest that patients undergoing CHD receive ATO every 48 to 72 hours.

Risk factors for early in-hospital death in patients who developed coagulopathy during induction therapy for acute promyelocytic leukemia: a nationwide analysis in Japan.

To prevent early death, management of coagulopathy is important in patients with untreated acute promyelocytic leukemia (APL). This study aimed to clarify factors associated with in-hospital death in patients with coagulopathy during induction therapy for APL. We retrospectively identified patients with newly diagnosed APL who received induction therapy including all-trans retinoic acid (ATRA) and developed coagulopathy, using a nationwide inpatient database in Japan. Of 1115 eligible patients, 175 (15%) died at a median of 13days (interquartile range, 7-30) after admission. In the multivariable analysis, compared with younger patients (aged < 40years), the occurrence of in-hospital death was significantly more common among older patients (aged ≥ 40 and < 60years: odds ratio = 2.58 [95% confidence interval: 1.29-5.19]; aged ≥ 60 and < 80years: 7.66 [3.89-15.10]; aged ≥ 80years: 16.83 [7.41-38.21]). Delayed initiation of ATRA and no conventional chemotherapy were significantly associated with in-hospital death (1.79 [1.16-2.76] and 2.40 [1.47-3.92], respectively). A total of 699 patients (63%) received anticoagulant therapies, but none of these was significantly associated with lower mortality. Although the present study was constrained by a lack of laboratory findings because of database limitations, the results showed that untreated patients with APL, especially the elderly, had a poor prognosis. Immediate administration of ATRA may reduce in-hospital mortality.

Association of PML-RARα Fusion mRNA Type With Pretreatment Hematologic Characteristics But Not Treatment Outcome in Acute Promyelocytic Leukemia: An Intergroup Molecular Study

AbstractIn each case of acute promyelocytic leukemia (APL) one of three PML-RARα mRNA types is produced, depending on the break/fusion site in the PML gene that is linked to a common RARα gene segment: a short (S)-form type, PML exon 3 RARα exon 3; a long (L)-form type, PML exon 6 RARα exon 3; or a variable (V)-form type, variably deleted PML exon 6 RARα exon 3. We evaluated whether PML-RARα mRNA type is associated with distinct pretreatment clinical characteristics and therapeutic outcome in previously untreated adult APL patients registered to protocol INT 0129 by the Eastern Cooperative Oncology Group, the Southwest Oncology Group, and the Cancer and Leukemia Group B. Of 279 clinically eligible cases, 230 were molecularly evaluable, and of these, 111 were randomized to receive remission induction therapy with all-trans retinoic acid (ATRA) and 119 with conventional chemotherapy. Nine cases not excluded by central pathology review were PML-RARα negative, and notably, none of five of these cases treated with ATRA achieved complete remission (CR). Among 221 PML-RARα–positive cases, there were 82 S-form cases (37%), 121 L-form cases (55%), and 18 V-form cases (8%). Before any antileukemic therapy, the S-form type, compared with the L-form type, was associated with higher values for the white blood cell (WBC) count (median 2,500/μL v 1,600/μL; P = .009), the percentage of blood blasts plus promyelocytes (median 29% v 8.5%; P = .03), and the absolute blood blasts plus promyelocytes (884/μL v 126/μL; P = .019). Also, an increased percentage of S-form versus L-form cases had the M3 variant phenotype, 24% v 12% (P = .036). There were no differences between S-form and L-form cases in either CR rate (79% v 69%; P = .14) or disease free survival distribution (multivariate analysis adjusting for the association of S-form type and higher WBC count; P = .40). We conclude that the S-form type is associated with previously-identified adverse risk WBC parameters but that the identification of the S-form or L-form type of PML-RARα mRNA, per se, does not predict clinical outcome or add to the value of an increased WBC count as a negative prognostic indicator in APL patients.

Association of PML-RARα Fusion mRNA Type With Pretreatment Hematologic Characteristics But Not Treatment Outcome in Acute Promyelocytic Leukemia: An Intergroup Molecular Study

In each case of acute promyelocytic leukemia (APL) one of three PML-RAR alpha mRNA types is produced, depending on the break/fusion site in the PML gene that is linked to a common RAR alpha gene segment: a short (S)-form type, PML exon 3 RAR alpha exon 3; a long (L)-form type, PML exon 6 RAR alpha exon 3; or a variable (V)-form type, variably deleted PML exon 6 RAR alpha exon 3. We evaluated whether PML-RAR alpha mRNA type is associated with distinct pretreatment clinical characteristics and therapeutic outcome in previously untreated adult APL patients registered to protocol INT 0129 by the Eastern Cooperative Oncology Group, the Southwest Oncology Group, and the Cancer and Leukemia Group B. Of 279 clinically eligible cases, 230 were molecularly evaluable, and of these, 111 were randomized to receive remission induction therapy with all-trans retinoic acid (ATRA) and 119 with conventional chemotherapy. Nine cases not excluded by central pathology review were PML-RAR alpha negative, and notably, none of five of these cases treated with ATRA achieved complete remission (CR). Among 221 PML-RAR alpha-positive cases, there were 82 S-form cases (37%), 121 L-form cases (55%), and 18 V-form cases (8%). Before any antileukemic therapy, the S-form type, compared with the L-form type, was associated with higher values for the white blood cell (WBC) count (median 2,500/microL v 1,600/microL; P = .009), the percentage of blood blasts plus promyelocytes (median 29% v 8.5%; P = .03), and the absolute blood blasts plus promyelocytes (884/microL v 126/microL; P = .019). Also, an increased percentage of S-form versus L-form cases had the M3 variant phenotype, 24% v 12% (P = .036). There were no differences between S-form and L-form cases in either CR rate (79% v 69%; P = .14) or disease free survival distribution (multivariate analysis adjusting for the association of S-form type and higher WBC count; P = .40). We conclude that the S-form type is associated with previously-identified adverse risk WBC parameters but that the identification of the S-form or L-form type of PML-RAR alpha mRNA, per se, does not predict clinical outcome or add to the value of an increased WBC count as a negative prognostic indicator in APL patients.

Primaquine phosphate induces the apoptosis of ATRA-resistant acute promyelocytic leukemia cells by inhibition of the NF-κB pathway.

As a subtype of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) is characterized by a chromosomal translocation, most of which result in the production of a PML-RAR alpha fusion protein. Although the overall survival rate of APL patients has improved dramatically due to all-trans retinoic acid (ATRA) treatment, ATRA-resistance remains a clinical challenge in the management of APL. Therefore, alternative agents should be considered for ATRA-resistant APL patients. Here, we report that antimalaria drug primaquine phosphate (PRQ) exhibits an anti-leukemia effect on both ATRA-sensitive cell line NB4 and ATRA-resistant APL cell lines, NB4-LR2, NB4-LR1, and NB4-MR2. Moreover, PRQ significantly inhibited primary colony formation of untreated or relapsed APL patients. Further study showed that PRQ could induce the apoptosis of APL cells by inhibiting NF-κB signaling pathway. The in vivo study showed that PRQ significantly inhibited NB4-LR2 xenograft tumors growth. These results suggest that PRQ is a potential therapeuticagentfor ATRA-resistant APL patients.

The Role of Prothrombinase Complex Assembly and in Situ Fibrin Deposition on the Surface of Acute Promyelocytic Leukemia Cells in Hemorrhage

Background: Despite dramatic improvement in treatment for acute promyelocytic leukemia (APL), early death due to hemorrhage remains a major obstacle to achieving a complete cure. In contrast to classical disseminated intravascular coagulation, APL-associated coagulopathy is characterized by rare microvascular fibrin thrombi. Thus, it is attractive to speculate whether other unknown mechanisms depleting coagulation factors and unrecognized fibrin-deposition location exist. Procoagulant activity associated with APL cells plays a direct role in bleeding complicationsin. We have shown that exposed phosphatidylserine (PS) on APL cells supports purified prothrombinase (Zhou J et al, JTH 2010) and fibrin preferentially deposits on promyelocytic chromatin from ETosis or apoptosis (Cao M et al, Blood 2017). However, relatively little is known about the PS-driven prothrombinase complex assembly and in situ fibrin deposition on APL cells.Aims: Our objectives were to determine how APL cells promote thrombin generation and modulate fibrin formation and distribution, as well as to explore the relationship between in situ fibrin deposition and consumptive hemorrhage in APL patients.Methods: Twenty-seven newly diagnosed APL patients were included. Fresh APL blasts were obtained from bone marrow specimens by centrifugation through Ficoll-Hypaque. Lactadherin was used as a probe for PS exposure on the fresh APL blasts and on an immortalized APL cell line (NB4). PS exposure and fluorescein-labeled FV/X binding were evaluated by flow cytometry. Thrombin generation was measured by modifed thrombin generation test. Fibrin production was quantified by turbidity. The distribution of PS, prothrombinase complex and in situ fibrin deposition were imaged by confocal microscopy. For the inhibition assay, APL cells were pre-treated with lactadherin, DNase I or anti-TF antibody for 10 min at 37 °C before incubation with plasma.Results: Thrombin generation and fibrin formation supported by NB4 and APL cells increased approximately 1.5-fold after exposure to daunorubicin and decreased 80% after treatment with all-trans retinoic acid (ATRA) or arsenic trioxide (ATO). Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure increased approximately 2.7-fold after treatment with daunorubicin, while ATRA and ATO initially led to a 70% reduction in PS exposure, which rose again on day 3 and 5 (P<0.001), respectively. Levels of externalized PS on APL cells paralleled levels of FV/FX binding, lag time, peak thrombin, endogenous thrombin potential and fibrin formation. Lactadherin inhibited the above parameters by approximately 80%, while anti-tissue factor antibody or DNase I produced no effect. Interestingly, confocal imaging showed that fibrin preferentially deposited on the surface of APL cells, which we defined as “in situ fibrin”. Untreated viable APL and NB4 cells displayed discrete or occasionally annular fibrin deposition on the membrane. Moreover, fibrin formation supported by apoptotic APL cells displayed the following progression: (i) patchy deposition, (ii) diffuse rim, (iii) “fibrin coat”, and (iv) network. The dynamic changes in fibrin formation paralleled the kinetics of PS exposure and prothrombinase assembly. Furthermore, initial percentage of PS-positive fresh APL cells was negatively correlated with plasma levels of fibrinogen and factor II, V, VIII, X in APL patients on admission (all P<0.01).Conclusion: PS-driven prothrombinase complex assembly and in situ fibrin deposition on the surface of APL cells consume massive coagulation factors, providing a novel explanation for consumptive hemorrhage in APL patients. Blockade of PS might be a novel therapeutic approach for preventing bleeding in APL via inhibiting invisible “in situ coagulation”, especially in high-risk APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genome‑wide profiling of lncRNA expression patterns in patients with acute promyelocytic leukemia with differentiation therapy.

Long non‑coding RNAs (lncRNAs) are crucial factors in acute promyelocytic leukemia (APL) cell differentiation. However, their expression patterns and regulatory functions during all‑trans‑retinoic acid (ATRA)‑induced APL differentiation remain to be fully elucidated. The profile of dysregulated lncRNAs between three bone marrow (BM) samples from patients with APL post‑induction and three BM samples from untreated matched controls was examined with the Human Transcriptome Array2.0. The dysregulated lncRNA expression of an additional 27APL BM samples was validated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis. The lncRNA functions were predicted through co‑expressed messenger RNA (mRNA) annotations. Co‑expressed lncRNA‑mRNA networks were constructed to analyze the functional pathways. In total, 825lncRNAs and 1,218mRNAs were dysregulated in the treated APL BM group, compared with the untreated APL BM group. The expression of 10 selected lncRNAs was verified by RT‑qPCR analysis. During APL differentiation, NONHSAT076891 was the most upregulated lncRNA, whereas TCONS_00022632‑XLOC_010933 was the most downregulated. Functional analysis revealed that several lncRNAs may exert activities in biological pathways associated with ATRA‑induced APL differentiation through cis and/or trans regulation of mRNAs. The findings of the present study assist in explaining the contributions of lncRNAs in APL myeloid differentiation and improve current knowledge on the potential mechanisms regarding dysregulated lncRNA expression in ATRA‑induced APL differentiation.

Final Analysis of the Australasian Leukaemia and Lymphoma Group (ALLG) APML4 Trial: All-Trans Retinoic Acid (ATRA), Intravenous Arsenic Trioxide (ATO) and Idarubicin (IDA) As Initial Therapy for Acute Promyelocytic Leukemia (APL)

▪BACKGROUNDThe combination of ATRA and anthracycline-based chemotherapy has traditionally been regarded as the gold standard for induction and consolidation in previously untreated APL patients (pts), with ATO usually reserved for relapse. Recent data in Sanz low-risk (LR) and intermediate-risk (IR) APL indicate ATRA + ATO is superior to ATRA + chemotherapy (N Engl J Med 2013), but the optimal therapy for high-risk (HiR) pts remains unclear. In an attempt to improve anti-leukemic efficacy whilst limiting reliance on anthracyclines for all risk categories of APL, the ALLG incorporated ATO into an ATRA + reduced chemotherapy backbone, and the results of an interim analysis with a median follow-up of 2 years (yr) have been published (Blood 120:1570, 2012). Herein, we report results of the protocol-specified final analysis, conducted when all surviving pts had been followed for at least 2 yr after completion of consolidation.METHODS As published, APML4 protocol treatment comprised: (i) induction with ATRA (45 mg/m2 d1-36), IDA (12 mg/m2 d2,4,6,8), ATO (0.15 mg/kg d9-36), prophylactic prednisone (1 mg/kg d1-10) and aggressive hemostatic support; (ii) consolidation with ATRA and ATO (continuous in cycle 1, intermittent in cycle 2); (iii) 2 yr oral maintenance with ATRA, 6-mercaptopurine and methotrexate. Between 2004-2009, 124 evaluable pts were accrued, and median follow-up by the censor-reversing Kaplan-Meier method in this final analysis is 4.2 yr.RESULTS Median age was 44 (3-78) yr, median white cell count was 2.4 x 109/L (0.1-85.8), and median platelet count 22 x 109/L (2-173). Risk groups were 33 LR, 67 IR, 23 HiR, 1 unknown. FLT3 mutations were present in 44%, and 11% had ≥ 2 additional cytogenetic abnormalities (2+ACA). There were 4 (3.2%) deaths during induction (myocardial infarction d1, cerebral hemorrhage d3 and d7, cerebral edema and seizures d30). Grade 3-4 non-hematological adverse events included differentiation syndrome in 14%, and frequent but reversible biochemical hepatic and gastrointestinal toxicity. Q-Tc prolongation > 500 msec occurred in 14%, but there were no cases of ventricular arrhythmias or torsades de pointe. Significant neurological and cutaneous toxicity were infrequent.After induction, 118 pts (95%) achieved hematological complete remission; 112 commenced consolidation, and all were in molecular remission after cycle 2. A total of 5 relapses and 3 deaths have occurred post-induction, but none of the deaths were attributable to protocol therapy. Severe adverse events were less common during the chemotherapy-free consolidation cycles, especially cycle 2. The frequency of grade 3-4 neutropenia was 62% in cycle 1 and 27% in cycle 2, but there was no grade 3-4 thrombocytopenia.The following table lists event-free survival (EFS), disease-free survival (DFS), overall survival (OS) and cumulative incidence of relapse (CIR):TableAll ptsLRIRHiR2 yr5 yr2 yr5 yr2 yr5 yr2 yr5 yrEFS92%90%97%97%93%89%83%83%DFS97%95%100%100%97%93%95%95%OS94%94%97%97%96%96%87%87%CIR3%5%0%0%3%7%5%5%In univariate analysis, 2+ACA predicted for inferior DFS (P = .04), whereas age > 70 was associated with increased risk of early death (ED, P = .02), inferior EFS (P = .0002), and inferior OS (P = .001). Neither Sanz risk category nor FLT3 mutation status was significantly correlated with these outcome endpoints (all P > .05). In multivariate analysis, the significant associations of 2+ACA (P = .04 for DFS) and age > 70 (P = .0002 for EFS, P = .005 for OS) were retained. In addition, Sanz risk category was correlated with EFS (P[trend] = .003) and OS (P[trend] = .02).When compared with our previous ALLG APML3 trial (ATRA + IDA for induction and consolidation without ATO; Haematologica 2012), treatment with APML4 was associated with substantial and statistically significant improvements (see Figure) in EFS (P = .002), DFS (P = .001) and OS (P = .02). The significance of trial assignment was retained in multivariate analysis when APML3 and APML4 data were combined.CONCLUSIONS APML4 is a potent anti-leukemic regimen with manageable toxicity and a low ED rate. EFS and OS remain impressive with mature follow-up, and were influenced primarily by advanced age and Sanz risk category. The CIR was 5%, and was only associated with 2+ACA. Our results support the inclusion of ATO in induction and consolidation as front-line therapy for APL whilst simultaneously limiting cumulative anthracycline exposure. [Display omitted] DisclosuresOff Label Use: Arsenic trioxide is currently only registered in the US, Europe and Australia for the treatment of relapsed APL. This report includes its use in the initial treatment of APL.

Implication of Tissue Factor Bearing Microvesicles in Hypercoagulable State in Acute Promyelocytic Leukemia

AbstractAbstract 4788 Introduction:Thrombosis is a common complication of patients with malignancies. Patients with hematological malignancy have a 28 fold increase risk to develop venous thromboembolism (VTE). A population-based cohort was used to determine the incidence and risk factors associated with development of venous thromboembolism (VTE) among Californians diagnosed with acute leukemia between 1993 to 1999. Principal outcomes were deep vein thrombosis in the lower and upper extremities, pulmonary embolism, and mortality. Among 5,394 cases with acute myelogenous leukemia (AML), the 2-year cumulative incidence of VTE was 281 (5.2%). Sixty-four% of the VTE events occurred within 3 months of AML diagnosis. The induction of hypercoagulable state mechanisms is not fully understood to date. Multifactorial aspects such as physic immobility, chemotherapy adverse effects or the overexpression of several procoagulant substances (cytokines, cysteine protease and tissue factor) by cancer cells are often provoked. Several studies strongly suggest that microvesicles (MVs) harboring tissue factor activity may have a primary role in VTE. MVs are small membrane vesicles shed from normal and/or tumor cells following activation or apoptosis. MVs may present TF and negatively charged phospholipids (PL) such as phosphatidylserine on their membrane. These elements are thought to be implicated in the procoagulant activity (PCA). Objectives:The aim of this study was to assess the capacity of untreated acute promyelocytic leukemia cells to shed procoagulant MVs. Methods:Acute promyelocytic leukemia (APL) cells lines (NB4 and HL-60) were cultured 48h in medium at 600,000 cells/mL. Cells and MVs were separated by filtrations (0.1–0.22–0.45–0.65μm). The PCA was assessed by thrombin generation assay. Alternatively, MVs were incubated with anti-TF antibodies (10μg/mL) or with annexin V (0,5μM to assess the contribution of TF and phospholipids to the PCA. Moreover HL-60 cells were incubated with HgCl2 which promote di-S bond formation (activation of TF). Results:NB4 cells and HL-60 cells can stimulate thrombin generation. HL60 cells reduced the lagtime 3.9-fold and increased the peak 1.6-fold in comparison to CTL and NB4 cells decreased the lagtime 10.9-fold and increased the peak 6.7-fold in comparison to CTL. No PCA was observed in HL-60 filtered with 0.65 μm membrane (no statistical difference in lagtime peak and ETP). By contrast, NB4 cells can support thrombin generation activity when filtered at different sizes. MVs of sizes <0.65 and <0.45 μm decreased the lagtime 4.2- and 3.8-fold, respectively and increased the peak of thrombin 4.6- and 4.1-fold, respectively. MVs of sizes lower than 0.22 and <0.1 μm reduced the lagtime 2.4- and 1.6-fold, respectively and increased the peak 2.3- and 1.4-fold, respectively. Thrombin generation activity of MVs of size <0.65 μm derived from NB4 cells wass abolished when anti-TF antibodies or annexin V were preincubated [Display omitted] [Display omitted] Discussion:NB4 cells and HL-60 cells have different PCA. NB4 cells have a higher procoagulant activity and most of the PCA is linked to MVs of size under 0.45 μm. NB4 cells spontaneously release different sized MVs which can support thrombin generation. By using an anti-TF function-blocking antibody (HTF-1) and annexin V which binds phosphatidylserine, we confirmed that the PCA of MVs is related to the expression of active TF and PL. HL-60 cells have a weaker procoagulant activity because TF is mostly present in an inactive form (activation of TF by reduction agent such as HgCl2 increased the PCA of HL-60 cells of +/− 35%). Moreover HL-60 cells do not produce MVs<0.65 μm associated with PCA. Conclusions:APL NB4 cells and HL-60 cells can stimulate thrombin generation. NB4 cells release MVs (of size <0.65 μm) whose procoagulant activity is mediated by TF and PL. These MVs could have a prognostic value for VTE in patient with APL. Disclosures:No relevant conflicts of interest to declare.

The Diterpenoid Lactone Andrographolide Induces Reactive Oxygen Species (ROS) Dependent Apoptosis in ATRA Sensitive and Resistant APL Cell Lines.

Abstract 2446Andrographolide is a crystalline diterpenoid lactone. It consists of an α-alkylidene- g-butyrolactone moiety and three hydroxyls at C-3, C-14 and C-19, which are responsible for its biological activities. It is the major bioactive ingredient of the medicinal plant Andrographis paniculata and it has been used in Asia for a variety of non-malignant conditions. We previously reported that Andrographolide results in mitochondrial-mediated apoptosis in lymphoma cell lines and fresh malignant cells from patients with lymphoma (Yang et al. Clin Cancer Res 2010:16:4755). Based on the mechanism of action in lymphoma and a prior report in APL (Manikam et al. J Pharm Pharmacol 2009:61:9), we hypothesized that andrographolide may have biological activity in acute promyelocytic leukemia (APL) an that this may be related to reactive oxygen species (ROS). We therefore investigated the effects of andrographolide on cell viability, apoptosis induction, mitochondrial membrane poential and signaling pathways in 3 APL cell lines, the ATRA sensitive line NB4 and the ATRA-resistant lines NB4–007/6 and NB4–306 and 3 samples from patients with APL. Methods:NB4 (ATRA sensitive cell line), NB4–007/6 and NB4–306 (ATRA resistant cell lines) were cultured in RPMI-1640 under standard conditions. Cell viability was measured using the trypan blue or propidium iodide exclusion method. Fresh leukemic cells were obtained from 3 patients after informed consent according to an NU IRB approved protocol. One had ATRA-resistant APL and 2 had de-novo untreated APL. We measured apoptosis by Annexin V-FITC by FACS. We measured mitochondrial membrane potential and cell differentiation by standard techniques. Results:Incubation with increasing concentrations of andrographolide demonstrates loss of cell viability as measured by MTT assay. The IC50 at 48 hours was 6uM for NB4–306, 6.5uM for NB4–007/6 and 9uM for NB4. Apoptosis by Annexin V/FACS demonstrated that at 48 hours there was increasing apoptosis in all 3 cell lines and that the ATRA-resistant cell lines NB4–007/6 and NB4–306 were significantly more sensitive to andrographolide than the ATRA sensitive cell line NB4 (p< 0.025). This was accompanied by PARP and caspase 3-cleavage. There was evidence of decrease in mitochondrial membrane potential, but no effect on differentiation as measured by CD11b expression by flow. We next interrogated signaling pathways and found that in the ATRA resistant line NB4–007/6 there was an increase in phosphorylation of the Forkhead box O transcription factors p-FOXO1 at Thr24 and up-regulation of FasL (which peaked at 6 hours) and p27Kip1. We also demonstrated that andrographolide caused N-acetyl L- cysteine (NAC) reversible down regulation of c-MYC (in the ATRA resistant lines) and p-AKT (T308) (in the ATRA sensitive line) expression. In fresh patient specimens (n=3) there was dose dependent increase in apoptosis at 48 hours (>70% at 10uM, 85% at 20uM). From prior reports and our own data we suspected that the effects of andrographolide were dependent on reactive oxygen species (ROS), and indeed apoptosis was completely inhibited by NAC. Conclusion:Taken together, these data suggest that andrographolide, a novel natural diterpenoid lactone with significant biological activity in cancer, may have activity in patients with ATRA-resistant APL by a mechanism of action that is distinct from ATRA. We believe that these data provide a compelling rationale to add this natural diterpenoid lactone to the clinical trial agenda in APL. Disclosures:No relevant conflicts of interest to declare.

Undiagnosed, Untreated Acute Promyelocytic Leukemia Presenting as a Suspicious Sudden Death*

Acute promyelocytic leukemia (APL) is a subtype of acute myelogenous leukemia frequently associated with clotting abnormalities and severe hemorrhagic diathesis. The disease is associated with a high incidence of early fatal hemorrhage. We report the sudden death of a 40-year-old male without significant medical history in which foul play had been initially suspected. A thorough postmortem investigation performed on the decedent lead to the diagnosis of APL. Cause of death was a cerebellar hematoma. Underlying APL should be considered in the differential diagnosis when unexplained bleeding is encountered in a decedent. This case emphasizes the value of routinely collecting bone marrow during an autopsy to enable accurate testing and diagnosis.

Phase II Study of All-Trans Retinoic Acid (ATRA), Arsenic Trioxide (ATO), with or without Gemtuzumab OzogamIcin (GO) for the Frontline Therapy of Patients with Acute Promyelocytic Leukemia (APL).

AbstractAbstract 1080 Background:The role of arsenic trioxide (ATO) in the frontline treatment of patients with acute promyelocytic leukemia (APL) remains unclear with a number of studies reporting high and durable responses with single agent ATO. We have conducted a trial combining all-trans-retinoic acid (ATRA) with ATO with or without gemtuzumab ozogamicin (GO) in patients with previously untreated APL. Patients and methods:From July 2002 to June 2010, 104 patients with newly diagnosed APL were treated with a combination of ATRA plus ATO in two studies. The first cohort of 47 patients received ATRA (45 mg/m2 daily) and ATO (0.15 mg/kg daily beginning on day 10 of ATRA). High-risk patients (White blood cell count [WBC] > 10 × 109/L) received GO 9 mg/m2 on the first day of induction. From July 2007, the second cohort of 57 patients received ATRA (45 mg/m2 daily) and ATO (0.15 mg/kg daily) concomitantly on day one of induction. They also received GO 9 mg/m2 on day 1, if high risk, and any time during induction if the WBC rose to > 30 × 109/L (and more recently if > 10 × 109/L). Monitoring for PML-RARA fusion gene using reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted after induction and throughout consolidation and follow up. The median age for the 104 patients was 46 years (range, 14–81). Their median presenting WBC was 2.7 × 109/L (0.4-131.4 × 109/L) and their median platelet count was 36 × 109/L (range, 7–261 × 109/L). Seventy three (70%) had low risk and 31 (30%) high risk disease (based only on the presentation WBC ≤ or > 10.0 × 109/L). Results:Overall, 102 patients (98%) achieved complete remission (CR) and 2 died during induction. With a median follow-up of 115 weeks (range, 4 to 397 weeks), 94 patients remain alive. The estimated 5-year survival rate was 88% and event-free survival 86%; only 5 of the patients achieving a CR (5%) have relapsed. The median overall survival, remission duration and event-free survival have not been reached (Figure). Thirty six patients have been alive and in remission for more than 3 years and 21 for more than 5 years. Two late deaths (beyond 300 weeks) occurred in CR from unrelated causes. Conclusion:The combination of ATRA and ATO (with or without GO) as initial therapy for APL is highly effective and safe; it can potentially substitute for chemotherapy containing regimens in high and low risk patients. [Display omitted] Disclosures:Ravandi:Cephalon: Honoraria, Research Funding, Speakers Bureau. Off Label Use: Off-label use of arsenic trioxide in frontline therapy of APL; off label use of gemtuzumab ozogamicin in APL. Verstovsek:Incyte Corporation: Research Funding.

Do Early Events Excluding Patients with Acute Promyelocytic Leukemia (APL) From Trial Enrollment Modify Treatment Result Evaluation? Real-Life Management of 100 Patients Referred to the University Hospital Saint-Louis Between 2000 and 2010.

AbstractAbstract 1083 Aim:Since combining differentiating agents and chemotherapy, acute promyelocytic leukemia (APL) is associated with a high cure rate. One remaining issue is the significant rates of early death and relapse observed in patients with high count APL (initial white blood cell count [WBC] ≥ 10.109/L). Early death rate might be underestimated in clinical trials, due to an unknown proportion of patients not registered because of initial severity. For this reason, we reviewed individual histories of all patients with APL referred to our institution during the last 10 years (09/2000-06/2010), with a special focus on admission in intensive care unit (ICU) and inclusion or non-inclusion in recruiting APL trials (European group APL-2000 and APL-2006), as well as long-term follow-up. Patients:A total of 100 patients with newly-diagnosed previously untreated APL, including 8 children, were admitted during this time period. Diagnosis was based on morphology and subsequently confirmed by the presence of the t(15;17) translocation and/or PML/RARA fusion transcript. Results:The rate of patients not enrolled within recruiting trials was 29% (n= 29). This rate was higher in children (n= 5/8, 62.5%) than in adults (n= 24/92, 26%) and remained stable during the two protocol periods (n=17/62, 27% for APL-2000; n= 12/38, 32% for APL-2006). Reasons for non-enrollment were inability to give informed consent in 10 patients (mechanical ventilation or neurological deficiency), physician's decision in 5 patients (2 very high leucocytosis, 1 severe infection, 2 severe liver dysfunction), and concomitant disease in 4 patients (2 HIV patients, 2 other cancers), refusal in 5 patients (including 1 Jehovah witness who eventually survived), and various administrative reasons in 5 patients. Non-enrolled patients had similar sex ratio (F/M=15/14 vs 35/36; p=.99), median age (40.5 [range, 4–79] vs 46 years [4-81]; p=.97), and frequency of additional chromosomal abnormalities (24% vs 28%; p=.80) than enrolled patients. Conversely, they had a higher rate of WBC ≥ 10.109/L (n=15/29 vs 22/71; p=.07) or ≥ 50.109/L (n=8/29 vs 5/71; p=.01), a lower rate of platelet count < 40.109/L (28/29 vs 46/71; p=.001), and a higher frequency of microgranular M3-variant subtype (11/29 vs 8/71; p=.004) and BCR3 PML-RARA isoform (14/25 vs 24/70; p=.09). Among the 29 non-enrolled patients, 24 nevertheless received the whole planned standard induction therapy, 2 received arsenic trioxide-based induction, and 3 early died before or the day after chemotherapy initiation. Ninety-nine patients were evaluable for response to induction (1 patient ongoing). Due to a higher early death rate (21% vs 3%; p=.007), the complete remission (CR) rate was lower in non-enrolled patients (79% vs 97%; p=.007). At 5 years, event-free survival (EFS) was estimated at 62% (95%CI, 37–79) vs 84% (95%CI, 72–91) (p=.02) and overall survival (OS) at 63% (95%CI, 36–81) vs 85% (95%CI, 72–93) (p=.03) in the non-enrolled and enrolled group, respectively. Once CR had been reached, non-enrolled patients displayed, however, a good post-CR outcome with 5-year remission duration at 78% and OS from CR at 80%. Of note, only one patient from this cohort died in first CR from a second neoplasia. Twenty-six patients (26%) were admitted in ICU for or during induction (13, 8, and 4 of them requiring mechanical ventilation, amine therapy, and dialysis, respectively). Chemotherapy was initiated in ICU in 19 of them. The rate of trial enrollment was 54% (n= 14/26) in ICU patients compared to 77 % (n= 57/74) in non-ICU patients (p=.04). Again, CR rate (p<.001), EFS (p=.004), and OS (p=.002) were significantly lower in ICU patients, but remission duration and OS from CR were very satisfactory in these patients despite their admission in ICU for or during induction (91% and 90% at 5 years, respectively). Conclusion:Even if this study only reports the experience of a large single center with potential patient selection, the observation that initial APL severity and/or need for ICU are associated with a lower trial enrollment rate suggests that early mortality might be underestimated in multicenter APL trials. Interestingly, patients who survive after early intensive care, including mechanical ventilation, may nevertheless receive an optimal induction and post-remission therapy and display the expected good outcome associated with APL. Disclosures:No relevant conflicts of interest to declare.

Arsenic trioxide improves event-free and overall survival for adults with acute promyelocytic leukemia: North American Leukemia Intergroup Study C9710

AbstractArsenic trioxide (As2O3) is a highly effective treatment for patients with relapsed acute promyelocytic leukemia (APL); its role as consolidation treatment for patients in first remission has not been defined. We randomized 481 patients (age ≥ 15 years) with untreated APL to either a standard induction regimen of tretinoin, cytarabine, and daunorubicin, followed by 2 courses of consolidation therapy with tretinoin plus daunorubicin, or to the same induction and consolidation regimen plus two 25-day courses of As2O3 consolidation immediately after induction. After consolidation, patients were randomly assigned to one year of maintenance therapy with either tretinoin alone or in combination with methotrexate and mercaptopurine. Ninety percent of patients on each arm achieved remission and were eligible to receive their assigned consolidation therapy. Event-free survival, the primary end point, was significantly better for patients assigned to receive As2O3 consolidation, 80% compared with 63% at 3 years (stratified log-rank test, P < .0001). Survival, a secondary end point, was better in the As2O3 arm, 86% compared with 81% at 3 years (P = .059). Disease-free survival, a secondary end point, was significantly better in the As2O3 arm, 90% compared with 70% at 3 years (P < .0001). The addition of As2O3 consolidation to standard induction and consolidation therapy significantly improves event-free and disease-free survival in adults with newly diagnosed APL. This trial was registered at clinicaltrials.gov (NCT00003934).

Interferon-γ Enhances PML Protein Expression in Acute Promyelocytic Leukemia Cells and Cooperates with All-Trans Retinoic Acid to Induce Maturation of NB4 and MR2 Cells

More than 90% patients with untreated acute promyelocytic leukemia(APL) obtain complete remission clinically by using All-trans retinoic acid(ATRA), a strong differentiation inducer. However, the rapid development of ATRA-resistance brings a new problem to the treatment of APL. Interferon(IFN), as an important cytokine, has broad biological activities. It can not only inhibit the growth of tumor cells, but also reverse the drug-resistance of chemotherapy. As proved by some research, the mechanisms of ATRA-resistance are probably related to lacking some important proteins which synthesized by Interferon-γ (IFN-γ). In order to explore a new way to solve the problem of ATRA-resistance in APL, we investigate the effect and mechanisms of IFN-γ in combination with ATRA on the proliferation/differentiation of NB4 cells(APL cell line with ATRA-sensitiveness) and MR2 cells(APL cell line with ATRA-resistance) respectively. ATRA, IFN-γ and IFN-γ in combination with ATRA were incubated with NB4 and MR2 cells respectively. The cell proliferation was tested by MTT assay, the cell differentiation was tested through light microscope, by NBT reduction test and flow cytometry(FCM). The results showed that ATRA could inhibit the growth of NB4 cells significantly (P<0.05), but it had no effect on the growth of MR2 cells (P>0.05). IFN-γ could inhibit the growth of both NB4 cells and MR2 cells slightly (P<0.05). Moreover, the growth inhibition effect of IFN-γ in combination with ATRA on NB4 and MR2 cells was obviously stronger than that of any single drug group (P<0.05). The results of cell morphology observation, NBT reduction test and CD11b antigen detection showed that ATRA could induce differentiation of NB4 cells significantly (P<0.05), but it had no effect on MR2 cells (P>0.05). Although IFN-γ alone had no effect on the differentiation of either NB4 or MR2 cells (P>0.05), it could augment the differentiation of NB4 cells induced by ATRA (P<0.05) and induce the differentiation of MR2 cells slightly (P<0.05) when it combined with ATRA. Furthermore, we have observed the expression of promyelocytic leukemia(PML) protein by indirect immune fluorescent test. The results showed that the number of fluorescent particles in both NB4 and MR2 cells' nucleus was increased significantly (P<0.05) when they were incubated with IFN-γ respectively, which indicated IFN-γ could induce the expression of PML protein, a tumor growth inhibitor. It can be seen that IFN-γ could augment the proliferation inhibition effect of ATRA on NB4 and MR2 cells through enhancing the expression of PML protein. Moreover, IFN-γ in combination with ATRA not only can strengthen the induction differentiation effect of ATRA on NB4 cells, but also can partially induce the maturation of MR2 cells with ATRA-resistance.

Proteome Analysis of the Proteins Associated with All-Trans Retinoic Acid Resistance in Acute Promyelocytic Leukemia Cells

Although 90% patients with untreated acute promyelocytic leukemia(APL) obtain complete remission because of the usage of all-trans retinoic acid(ATRA), patients with ATRA-resistance are increased gradually. ATRA-resistance has become one of the main causes which affect the long-term therapeutic efficacy of APL. The mechanisms of ATRA-resistance are complex, which probably involve the metabolism of ATRA, abnormal expression of cellular retinoic acid binding protein(CRABP) and P-glycoprotein(P-gp), mutation of RARα and aberration translocation of APL. However, in these previous researches, it was one or a few proteins but not the entirety proteins that were emphasized on the mechanisms of ATRA-resistance. Comparative proteomics can analyze the entire protein expression in cells in whole and has the superiority in screening the drug-resistance proteins differentially expressed. In order to investigate the mechanisms of ATRA-resistance in APL in whole, we compared and analyzed the protein expression profiles between MR2 cells(APL cell line with ATRA-resistance) and NB4 cells(APL cell line with ATRA-sensitiveness) by comparative proteomics. After the total proteins of MR2 cells and NB4 cells were extracted respectively, they were separated by two-dimensional electrophoresis(2-DE). The differences in proteome profile between MR2 cells and NB4 cells analyzed by ImageMaster™ 2D Platinum software. The average protein spots in 2-DE maps of MR2 and NB4 cells were 1160±51 and 1068±33 respectively. 8 protein spots were selected to be identified by Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS), in which the quantity of the protein differentially expressed was more than two times(≥2 or ≤0.5) between MR2 and NB4 cells' 2-DE map. They were all successfully identified and their definite information was obtained. Among them, 6 proteins were probably involved in the mechanisms of ATRA-resistance in APL and they were Cofilin-1, Elongation factor 1-beta (EF-1β), Tropomyosin isoform(TM), High mobility group protein B1(HMGB1), Ran-specific GTPase-activating protein (RanGAP1) and Galectin-1. Moreover, so far there was no related report on the roles of HMGB1, RanGAP1 and Galectin-1 in the mechanisms of ATRA-resistance in APL. These differential proteins identified provide the new clues for us to further elucidate the mechanisms of ATRA-resistance from multiple factor.

Proteome Analysis of Apoptotic MR2 Cells, Acute Promyelocytic Leukemia Cell Line with All-Trans Retinoic Acid Resistance, Induced by Arsenic Trioxide

Although all-trans retinoic acid(ATRA) provides complete remission in 90% patients with untreated acute promyelocytic leukemia(APL), it becomes ineffective to quite a few APL patients who have received ATRA before when their disease relapsed and used ATRA again. Arsenic trioxide(ATO) can make APL patients with ATRA-resistance obtain complete remission again by inducing APL cells apoptosis. However, the molecular mechanisms of apoptosis in ATRA-resistance APL cells induced by ATO remain unclear. For this reason, we take the apoptotic MR2 cells (APL cell line with ATRA-resistance) induced by ATO as a model, to screen and identify the proteins related with ATO-induced apoptosis by comparative proteomics. After MR2 cells were dyed with annexin V and PI staining, the percentage of the apoptotic MR2 cells induced by 1.0μmol/L ATO for 0h, 6h, 12h, 24h and 48h respectively was detected by Flow cytometry. The results showed that the majority of the apoptotic cells were in the earlier and later stage of apoptosis respectively, when MR2 cells were treated with ATO for 24 and 48 hours in sequence. The total proteins of MR2 cells of the control group, the earlier stages apoptosis group and the later stages apoptosis group were separated by two-dimensional electrophoresis(2-DE) respectively. Then, the differences in proteome profile among three groups were analyzed by ImageMaster™ 2D Platinum software. 14 protein pots were selected to be identified by Matrix-assisted laser desorption/ionization time of flight-mass spectrometry(MALDI-TOF-MS), in which the quantity of the protein differentially expressed was more than two times(≥2 or ≤0.5) among MR2-0h, MR2-24h and MR2-48h cells' 2-DE map. However, only 11 proteins were successfully identified and their definite information was obtained. Among them, there were 8 proteins that were all probably involved in the mechanisms of apoptosis in MR2 cells and they were Calreticulin(CRT), Heat shock 70 kDa protein(HSP70), High mobility group protein B1(HMGB1), Ran-specific GTPase-activating protein(RanGAP1), Elongation factor 1-beta(EF-1β), Beta-tubulin, Cofilin-1, and Prolyl 4-hydroxylase(P4H) respectively. CRT was probably related with the early stage of apoptosis in MR2 cells, while RanGAP1 and HSP70 might related with the late stage of apoptosis in MR2 cells. Moreover, so far there was no related report on the roles of CRT, HMGB1, RanGAP1, cofilin-1 and beta-tubulin in the mechanisms of APL cells apoptosis. These differential proteins identified provide the new clues for further researching the molecular mechanisms of apoptosis in the ATRA-resistance APL cells induced by ATO.

Impact of CRi on the Outcome of Elderly Patients with Untreated Acute Myeloid Leukemia (AML)

New international recommendations of response for treatment of AML include morphologic complete remission with incomplete blood count recovery (CRi). This response criteria was defined following evaluation of new drugs used for the treatment of AML in first relapse (Sievers et al., JCO 2001; 19:3244–3254). The objective of our study was to evaluate this criterion in elderly patients with AML who are in first line of treatment. Between 1995 and 2006, 454 patients aged 55 years or older with previously untreated acute non promyelocytic leukemia received a conventional anthracycline and cytarabine induction chemotherapy in our institution. Ages were between 55 and 85 years (median 65 years). Two hundred and fourty-eight patients achieved a complete response (CR) (55%), 37 patients achieved CRi (8%), 104 patients had persisting leukemia (23%), and 49 died during remission induction therapy (13%). Multilineage dysplasia, secondary AML and blasts expressing CD34 were significantly more frequent in patients achieving CRi than CR (58% versus 29%, p=0.001, 33% versus 15%, p=0.007, and 79% versus 54%, p=0.01, respectively). No favorable prognostic karyotype was found in the CRi group but cytogenetic distribution did not differ statistically between the two groups. All patients who reached CR or CRi were scheduled to receive consolidation. Only 24 patients (65%) in CRi actually received this consolidation chemotherapy course and 11 patients (30%) had intensification (intermediate-dose cytarabine and/or autologous stem cell transplantation) whereas for patients achieving CR, 233 (94%) and 214 patients (86%) received consolidation and intensification, respectively (p<0,001 for both). None of the patients in CRi received an allogenic stem cell transplantation whereas 18 (7%) of CR patients had one (p=0,2). Disease-free survival (DFS) and remission duration were significantly different between patients in CRi and CR, with a median of 4 and 12 months, and 5 and 9 months respectively (p<0,001 and 0,03). The median overall survival (OS) was also significantly lower for patients in CRi versus CR, respectively 8 and 23 months (p<0,001). By landmark analysis, there was no difference in OS between patients in CRi and a group of 98 patients with induction failure surviving at least 60 days (p=0,4). We also noted that OS was better, in the group of patients in CRi, for those who finally achieved CR criteria after 1 or more course of post-remission chemotherapy (median 16 months, against 7 months for patients still in CRi, p=0,03). Our results show that the CRi criterion is not equivalent to CR in elderly patients who received intensive chemotherapy as the first line treatment of AML. This should be kept in mind when the results of new agents used in this setting are compared to historical data.

All-Trans Retinoic Acid (ATRA) and Arsenic Trioxide (As2O3) Combination Therapy Induces High Rates of Durable Molecular Remission in Newly Diagnosed Acute Promyelocytic Leukemia (APL).

Background: Although ATRA and anthracyclines provide durable remissions in patients with untreated APL, we have reported that ATRA plus As2O3 may do the same while avoiding “chemotherapy.” However, follow-up of patients treated with ATRA plus As2O3 has been relatively limited, prompting this report.Methods: From 2/02 to 4/07, 67 patients with untreated APL were given ATRA 45 mg/m2 daily followed by As2O3 0.15 mg/kg IV 1-hr infusion daily starting on D10. Patients with leukocyte counts (WBC) > 10×109/L (low-risk) also received gemtuzumab ozogamicin (GO) 9 mg/m2 on D1 and/or idarubicin 12 mg/m2 on D1-4. Patients in complete remission (CR) received As2O3 0.15 mg/kg IV on D1-5 weekly for 4 weeks on and 4 weeks off and ATRA 45 mg/m2 daily for 2 weeks on and 2 weeks off (for 28 weeks). Polymerase chain reaction (PCR) testing for PML-RARα (sensitivity level, 10−4) was performed every 3 months from CR for at least 2 yrs. Patients with molecular relapse, defined by two sequential positive PCR tests for PML-RARα within 2 wks, received GO 9 mg/m2 once monthly for 3 months in addition to ATRA and As2O3 as in post-remission therapy. If the PCR results subsequently became negative, low-risk patients received no chemotherapy and high-risk patients received a single dose of GO.Results: The median patient age was 46 yrs (range, 14–81), and 30% were >60 yrs. Thirty-six percent of patients had WBC ≥10×109/L, 54% had coagulopathy, and 27% had Zubrod performance status (PS) > 1. The overall response rate was 91% (CR 90%, CRp 1%). The median time to response was 29 days (range, 19–70). Response rates were higher in patients with PS 0–1 (98% vs. 72%, p=.001), no coagulopathy (100% vs. 83%, p=.02), and LDH <1.5 × upper limit of normal (ULN)(97% vs. 83%, p=.046). The median follow-up in surviving patients is 25 months. Six patients died during induction; 1 died with central nervous system relapse; and 3 died in remission from other metastatic cancers (malignant melanoma, 1; breast, 1; and prostate, 1). The 2-yr survival rate was 84%. Survival rates were higher in patients with PS<1 (p=.0004), no coagulopathy (p=.01), and LDH <1.5×ULN (p=.02). The 2-yr failure-free survival (FFS) rate in responding patients was 92% (Sanz risk: low and intermediate 100%; high, 78%). Four patients relapsed (at 9, 9, 13, and 16 months); molecular relapse preceded hematologic relapse by 21, 23, 38, and 128 days, respectively. None of the remaining patients had evidence of molecular relapse. WBC ≥10×109/L (p=.006), LDH ≥1.5×ULN (p=.02), and high Sanz risk (p=.02) predicted relapse. Molecular remission rates are shown in Table.Time from CR (months)No. of pts in CR testedPCR negativeNegative, %05224345449864040100937359512333194151111100182222100242020100309910036881004833100Grade 3–4 nonhematologic toxicities were infections (n=18), neurologic (n=5), cardiac arrhythmias (n=4), APL differentiation syndrome (n=4), headache (n=3), renal failure (n=3); mucositis (n=1), rash (n=1), and transaminitis (n=1).Conclusions: ATRA plus As2O3 results in high rates of CR, molecular remission, FFS, and survival. PCR testing for PML-RARα accurately predicted relapse and should be performed in high-risk patients during the first year after CR.