Single Agent Arsenic Trioxide Research Articles

Early predictor for differentiation syndrome in newly diagnosed acute promyelocytic leukaemia patients treated with single-agent arsenic trioxide.

Differentiation syndrome (DS) is the second leading cause of death in acute promyelocytic leukaemia (APL) patients. Few studies have tested predictors of DS events. This study aimed to identify optimized predictorsofDS events related to APL. The data of 298 consecutive patients who were newly diagnosed with APL between December 2012 and June 2023 were retrospectively investigated. A systematic review of computer-based patient medical records was conducted to obtain clinical data, including baseline characteristics, routine blood examination findings, biochemical indices and clinical manifestations of DS. Among the 298 patients, 158 were classified into the no-DS group, while 140 had DS. Compared with those of patients without DS, the peripheral blast count, age, and WBC count at each time point were significantly different in patients with DS (P < 0.05 for all time points). Generalized linear mixed models (GLMMs) revealed that WBC Double (Coeff. 0.442, P = 0.000) and WBCPeak (Coeff. 0.879, P = 0.000) were independent risk factors for DS. The frequencies of clinical manifestations of unexplained fever (P = 0.003), dyspnoea (P = 0.002), weight gain of more than 5kg (P = 0.006), pleural effusion (P = 0.001), pulmonary infiltrates (P < 0.001), pericardial effusion (P = 0.002) and renal failure (P = 0.006) were considerably lower in moderate DS patients than in severe DS patients. The WBCDouble occurs earlier than the WBCpeak occurrence, so WBCDouble might be a new indicator of DS.

P538: TREATMENT PATTERNS AND CLINICAL OUTCOMES IN ACUTE PROMYELOCYTIC LEUKEMIA: REAL-WORLD DATA

Background: All-trans retinoid acid (ATRA) and arsenic trioxide (ATO) have changed the treatment paradigm of acute promyelocytic leukemia (APML). Current therapeutic strategies are based on the risk stratification at diagnosis and molecular response at the end of consolidation.We report our experience of treatment patterns and clinical outcomes of newly diagnosed APML patients. Aims: The primary objective of our study was to evaluate event-free survival in all risk categories of APML.Secondary objectives include disease-free survival, overall survival, rates of complete remission (CR) at the end of induction, rates of molecular CR at the end of consolidation. Methods: We included treatment-naïve APML patients age>14 years who were treated between May 2014 to May 2018.The diagnosis of APML was established by peripheral blood/bone marrow morphology and flow cytometry (FCM) and confirmed by fluorescent in situ hybridization (FISH) and reverse-transcriptase polymerase chain reaction (RT-PCR) for PML-RARA.The details of patients were retrieved from electronic medical records.Patients with low/intermediate risk were treated with ATO-ATRA based induction and consolidation,where as patients with high risk were treated with varying combination of ATO/ATRA/chemotherapy with or without maintenance. Results: We registered 149 patients during the study period.The median age was 37 years(range 15-72 years, male 56.6%, female-37%).93 patients (62.4%) were stratified as low risk and 56 patients (37.5%) as high risk based on Sanz’s score.The induction therapy for high risk APML was single agent ATO (42.8%),ATO/ATRA/anthracycline (26.7%),ATO/anthracycline (25%),and ATO/ATRA (5%).The consolidation for high risk APML includes ATRA/daunorubicin (50%),ATO/ATRA (26.8%),HIDAC (4%) and APL 2000 in one patient.All low-risk APML patients received ATO-ATRA in induction and consolidation.Anthracycline was added to three patients in induction to decease white cell count.The proportion of deaths that occurred in first week include 6.5% in low risk and 9% in high risk group.The median follow up was 42 months.The hematological CR at the end of induction was 85%.(128/149 patients,expired during induction-14,lost to follow up-7).The rate of end of consolidation PCR negativity was 83%.Differentiation syndrome occurred in 75% of patients.For the overall patient population, the 3-year probability for OS, EFS, DFS was 88.1% (95% C.I, 82.9 93.6), 75.9% (95% C.I, 68.9-83.5), 81.4% (95% C.I, 75-88.4) respectively. The 3-year survival probability for OS, EFS, and DFS for low risk APML was 90% (95% C.I, 84.1-96.4), 80.1% (95% C.I, 72-89.1), 84.5% (95% C.I, 76.9-92.7) respectively whereas for high risk APML was 84.7% (95% C.I, 75.3-95.2), 68.6% (95% C.I, 56.9-82.9), 76.1% (95% C.I-65-89) respectively.The total number of relapses were 11.The median time to relapse for low risk and high risk was 23months(18-32months) and 20months(range 5-39months) respectively. Image:Summary/Conclusion: Treatment with ATO and ATRA based regimen are effective in real world setting.The emphasis should be to reduce the early death rate due to bleeding and infectious complications.

Real World Data of Concurrent Arsenic Trioxide and All-Trans Retinoic Acid with Minimal Use of Anthracycline in the Treatment of Acute Promyelocytic Leukemia

▪In the setting of clinical trials, high cure rates have been reported for acute promyelocytic leukemia (APL) with the chemotherapy-free combination of arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) for low-intermediate risk disease, and with the addition of gemtuzumab ozogamicin or minimal anthracycline for high-risk disease. Despite clear in-vitro synergy between ATO and ATRA, most clinical trial protocols in APL have not used these drugs concurrently beyond induction. There is limited real world data on the use of chemotherapy-free combination of ATO and ATRA with minimal anthracycline use in the treatment of APL, especially in the high risk group. We did a retrospective analysis of the clinical outcomes of patients with newly diagnosed APL treated at our center from January 2015 to May 2020 using concurrent ATO, ATRA and minimal anthracycline in a risk stratified manner. The data was frozen and analyzed as on 1st June 2021. During the study period, 167 patients were diagnosed to have APL at our hospital. Of these, we excluded 5 patients who had presented with relapsed disease and 28 patients who were treated with single agent ATO. The remaining 134 patients were treated with a uniform protocol as summarized in Figure 1a. Figure 1b shows the KM plot for EFS (2 year: 90.8±2.5%) for these 134 patients. For analysis of regimen safety and efficacy, we excluded patients who wished to pursue treatment elsewhere within the first 1 week of treatment (n=7), and patients who had severe infections or life-threatening bleeding at presentation or before therapy initiation and subsequently died (n=4). Thus, a total of 123 patients with newly diagnosed APL were included for further analysis. The median age was 35 years (IQR: 24 to 45 years). Forty-six (37.4%) were females. The median duration of symptoms prior to admission was 14 days (IQR: 7 to 28 days). Sixty-one (49.6%) had high risk APL while the remaining 62 (50.4%) had low-intermediate risk APL. During induction, 28 (22.7%) patients had major bleeding while 5 (4%) patients developed major thrombosis. Sixty-seven (55%) patients had at least one documented infection during induction therapy. The median number of packed red cell concentrates, fresh frozen plasma, platelet rich concentrates and cryoprecipitates transfused during induction therapy was 5 (IQR: 3 to 6.5), 10 (IQR: 3 to 30), 40 (IQR: 23 to 55) and 6 (IQR: 0 to 18) respectively. Nineteen (15.4%) patients developed differentiation syndrome; all were treated with steroids, 8 required transient cessation of treatment and 6 required intensive care. Eight (6.5%) patients died during induction. During induction, grade 3 hepatotoxicity was noted in 8 (6.5%) patients, 10 (8.1%) patients had ATRA related headache or benign intracranial hypertension of which 5 required transient cessation of ATRA, while 11 (8.9%) patients had transient QTc prolongation of which 6 required transient cessation of ATO. Seventeen (13.8%) patients developed symptomatic sensory neuropathy requiring treatment. None required permanent discontinuation of therapy. At a median follow up of 854 days, there were 2 (1.6%) hematologic relapses and no deaths beyond induction therapy. The 2-year OS and EFS for the cohort (n=123) are 93.4% ± 2.3% and 91.6% ± 2.6% respectively (Figure 1c). Two (1.6%) patients have developed second malignancies. In a real world setting, concurrently administered ATO and ATRA with minimal anthracycline use results in excellent long term survival, even in the high risk group. Early deaths due to delayed presentation with infections and life threatening bleeding remain an unmet need for future research along with the need for strategies to reduce treatment-related toxicities. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Evolving Chemotherapy Free Regimens for Acute Promyelocytic Leukemia.

With the treatment advances over the last three decades, acute promyelocytic leukemia (APL) has evolved from being the most malignant form of acute leukemia to a leukemia with excellent long term survival rates. In the present review, we have summarized data leading to the development of the currently used treatment regimens for APL, which incorporate either none or minimal chemotherapeutic drugs. We have discussed the historical aspects of APL treatment along with the challenges associated with chemotherapy-based approaches and our experience with the use of single agent arsenic trioxide (ATO) which was one of the first successful, non-chemotherapy approaches used for APL. Subsequently, we have reviewed the data from major clinical trials in low-intermediate risk APL and high risk APL which guide the current clinical practice in APL management. With accumulating data on oral ATO, we postulate that the treatment for low-intermediate risk APL will be a completely oral ATO + ATRA regimen in the future. While for high-risk APL, we believe that minimal anthracycline use with ATO + ATRA might become the standard of care soon. A number of promising non-chemotherapy drugs with pre-clinical data would merit clinical testing in the high risk and relapsed setting, with potential to translate to a complete oral chemotherapy free combination regimen in combination with ATO and ATRA.

Analysis of gamma-glutamyltransferase in acute promyelocytic leukemia patients undergoing arsenic trioxide treatment

ABSTRACT Objectives The remarkable effect of arsenic trioxide (ATO) was verified, but elevated gamma-glutamyltransferase (GGT), aminotransferases (ALT and AST) are generally observed in acute promyelocytic leukemia (APL) patients undergoing ATO treatment. However, utilization of hepatoprotective agents or discontinuation of ATO may inhibit ATO efficacy. In order to maintain ATO effect from hepatoprotective agents’ influence so we investigate relationships between single elevation in GGT and hepatocellular injury in this study. Methods Correlation of GGT variation and leukocyte counts were analyzed in all 81 APL patients, correlations among liver enzymes (ALT, AST and GGT) were also analyzed in patients without prophylactic hepatoprotective agents. In following study, we take the clinical observation of changes in aminotransferases in patients with single elevation in GGT without hepatoprotective agents. Results The average elevated GGT in the WBC abnormal group was more than the normal group (53.86U/L vs. 31.03U/L, P = 0.008), a positive Pearson’s correlation of GGT variation and changed leukocyte counts in patients without prophylactic hepatoprotective agents. There are no significant correlation between aminotransferases (ALT and AST) and GGT but correlation between ALT and AST was statistically significant (R = 0.649, P = 0.000). For APL patients with single elevation in GGT, ALT and AST levels were normal throughout the ATO treatment without hepatoprotective agents. Conclusion Single elevation in GGT without elevated aminotransferases can’t be identified as hepatotoxicity, and the elevated levels of GGT are associated with increasing leukocyte counts. Continue single-agent ATO without prophylactic hepatoprotective agents is recommended in APL patients with single elevation in GGT, in order to maintain ATO effect.

Treatment Outcome of Newly Diagnosed Acute Promyelocytic Leukaemia by All-Trans Retinoic Acid followed by Arsenic Tri-Oxide

Background: Acute promyelocytic leukaemia (APL) is curable with carefully selected treatment protocol. Reduction of early fatal haemorrhage in initial phase of induction with cost effective combination of chemotherapeutic agents is considered to be the main challenge for successful outcome in APL. Objective: The study was conducted to see the effectiveness of the use of all-trans retinoic acid (ATRA) followed by single agent Arsenic tri oxide (ATO) in the induction phase of the management of APL. Methodology: This is an observational study and was conducted from October 2012 to September 2013, in department of Haematology and BMT of Dhaka Medical College Hospital, Dhaka, Bangladesh. Total 17 patients with PML/RARA positive APL were enrolled and all patients were treated with sequential use of ATRA in a dose of 45mg/m2 for initial 2-3 weeks up to differentiation and then ATO in a dose of 0.15mg/kg daily was used in induction up to 60 days after taking informed written consent. Outcome was drawn after consolidation of therapy. Results: The morphologic complete remission rate was 88.2%. Remission failure was in observed in 2 cases (11.8%) that died at day 12 and day 51 due to differentiation syndrome and arrhythmia respectively. No death observed within first 10 days of treatment initiation. Complications observed during management were weight gain (11, 64.7%), hyper leucocytosis (10, 58.8%), ATRA syndrome (9, 52.9%), headache (6, 35.3%), tachycardia (5, 29.4%), hypomagnesaemia (3, 17.7%) and supra ventricular tachycardia (1, 5.8%). Conclusion: Initial use of ATRA up to differentiation of promyelocytes before use of single agent ATO induction and consolidation can be a cost-effective alternative against newly diagnosed APL in reduction of early haemorrhagic death.

Time course of arsenic species in red blood cells of acute promyelocytic leukemia (APL) patients treated with single agent arsenic trioxide

ABSTRACTBackground:Arsenic trioxide (ATO) is widely applied to treat acute promyelocytic leukemia (APL). To elucidate metabolism and toxicity of arsenic, we analyzed time course of arsenic species in red blood cells (RBCs) of APL patients.Methods:Nine APL patients received ATO (0.16 mg/kg/day) through 18-h infusion. Blood was collected before daily administration (days 2 to 9), and at different time points on day 8. Inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were detected by HPLC-ICP-MS.Results:Arsenic species reached Cmax at 18 h on day 8. Arsenicals gradually accumulated during days 2 to 9, whereas their percentages remained almost constant. The general trend in red blood cells (RBCs) was iAs > MMA > DMA. MMA was consistently the predominant methylated arsenic metabolite in RBCs. iAs, MMA, and tAs (tAs = iAs + DMA + MMA) concentrations (P < 0.0001), MMA/DMA ratios (P = 0.0016) and iAs% (P = 0.0013) were higher in RBCs than in plasma.Conclusions:Time course of arsenic species reveal kinetic characteristic of ATO metabolites in RBCs. Arsenic species accumulated with administration frequency. Arsenic species in RBCs were remarkably different from those in plasma. Time course of arsenic species in RBCs is important in ATO clinical application.

Toward Candidate Proteomic Biomarkers in Clinical Monitoring of Acute Promyelocytic Leukemia Treatment with Arsenic Trioxide

The introduction of arsenic trioxide (ATO) in treatment of acute promyelocytic leukemia (APL) has resulted in high clinical complete remission (CR) rates over 90%. On the contrary, the risk for early death (ED) in APL patients treated with ATO continues to have a negative impact for optimization of APL therapeutics. There is an urgent need for precision medicine and biomarkers in clinical monitoring of ATO toxicity in APL, and ED in particular. This retrospective case series cohort proteomics study was conducted as a hypothesis generation effort and provides here several potential molecular leads on serum peptides expressed at different times after treatment with ATO in patients with APL. In 12 patients with a de novo APL diagnosis, and treated with single-agent ATO as frontline remission induction therapy, serum peptides were fractionated by weak cation exchange magnetic beads and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Ten peptides (m/z 2075.5, 2084.2, 2203.0, 2265.2, 2872.8, 2916.6, 3145.2, 3153.4, 3953.4, and 3964.8) were significantly downregulated in serum after ATO treatment. Among them, four peptides were identified as (1) Immunoglobulin heavy chain V-III region BUT, (2) RRP15-like protein, (3) filaggrin, and (4) protein SON isoform F. To the best of our knowledge, this is the first clinical oncology proteomic biomarker study with a view to future rational therapeutic monitoring of patients with APL in the course of single-agent ATO treatment and hematological CR.

Very Long Term Follow-up Data of Pediatric Acute Promyelocytic Leukemia Treated with Upfront Arsenic-Trioxide Based Regimens

Combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) is currently considered the standard of care in the management of acute promyelocytic leukemia (APL). While APL is considered a highly curable malignancy there is recognition that the outcome in pediatric patients is inferior to that reported in young adults. There have been concerns raised in the past on the potential long term side effects of the use of ATO, especially in a pediatric population. There is limited long term follow up data on the use of ATO in the pediatric population. At our center we have been using ATO based regimens to treat pediatric and adult patients with APL since 1998 and hence we undertook this retrospective analysis to evaluate the long term clinical outcomes and toxicity profile in the pediatric cohort.Data on all consecutive pediatric patients (age ≤18yrs) diagnosed with APL and treated in the Department of Haematology, Christian Medical College, Vellore, from January, 1998 to December, 2017 were included in this retrospective analysis. Of the total 73 patients with age ≤ 18yrs diagnosed during this period, 5 refused treatment and were discharged against medical advice. Treatment in the remaining 68 patients consisted of single agent ATO until 2015 (n=57), as reported previously by us (Mathews et al. Blood 2016). From 2015 combination of ATO, ATRA ± an anthracycline in induction and consolidation was administered in a risk adjusted manner (n=11). The median age was 2 years (range: 2-18) with equal gender distribution (50% each). Sixty two (91.2%) achieved complete hematologic remission (CR), 5 (7.4%) early deaths occurred from intracranial hemorrhage (n=3), neutropenic sepsis (n=1) and pulmonary thrombo-embolism (n=1), one patient did not achieve CR at the end of induction. The median time to CR was 45 days (range: 25- 62). Other acute ATO-related toxicities were low grade, transient and not associated with any mortality (transaminitis = 12 [17.6%]; ATRA like syndrome = 6 [8.8%]). With a median follow-up of 71 months, the 5 year OS and EFS of pediatric cohort (n=68) was 78.9±5.2% and 61.8±6.4% respectively (Fig 1). Among the 62 patients in CR, 21 (33.9%) relapsed at a median of 18 months (range: 5-126) from the initial diagnosis; 16 bone marrow, 3 bone marrow+CNS and 2 molecular relapses; an additional 2 patients died in remission (one viral encephalitis and another data not available). Nineteen out of 21 (90.5%) patients who relapsed received ATO based re-induction while 2 refused treatment and were discharged at request. Out of the 19 treated patients, all attained second CR. CR was consolidated with an autologous SCT (n=10) or ATO based chemotherapy (n=9). The OS and EFS of the 19 relapsed patients was 72.9±10.4% and 68±10.8% respectively.On long term follow up of this pediatric cohort (median follow up 71 months; 18 (26.5%) > 10 years follow up and 37 (54.5%) > 5 years follow up) there were no long term renal, hepatic, metabolic complications or second malignancies noted.Our results indicate the high efficacy and long term safety of ATO based regimens in the treatment of children with APL. Even among the relapse pediatric APL patients treated with upfront ATO, salvage chemotherapy with ATO based regimen followed by autologous stem cell transplantation is associated with excellent long term survival and is not associated with any major long term complications. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

282P - Efficacy and safety of single-agent arsenic trioxide (ATO) as an induction regimen in the treatment of newly diagnosed acute promyelocytic leukemia (APL): A prospective single arm phase II study from South India

282P - Efficacy and safety of single-agent arsenic trioxide (ATO) as an induction regimen in the treatment of newly diagnosed acute promyelocytic leukemia (APL): A prospective single arm phase II study from South India

Arsenic Trioxide Enhances the NK Cell Cytotoxicity Against Acute Promyelocytic Leukemia While Simultaneously Inhibiting Its Bio-Genesis

Natural killer cells (NK) contribute significantly to eradication of cancer cells, and there is increased interest in strategies to enhance it’s efficacy. Therapeutic agents used in the treatment of cancer can impact the immune system in a quantitative and qualitative manner. In this study, we evaluated the impact of arsenic trioxide (ATO) used in the management of acute promyelocytic leukemia (APL) on NK cell reconstitution and function. In patients with APL treated with single agent ATO, there was a significant delay in the reconstitution of circulating NK cells to reach median normal levels from the time of diagnosis (655 days for NK cells vs 145 and 265 days for T cells and B cells, respectively). In vitro experiments demonstrated that ATO significantly reduced the CD34 hematopoietic stem cell (HSC) differentiation to NK cells. Additional experimental data demonstrate that CD34+ sorted cells when exposed to ATO lead to a significant decrease in the expression of IKZF2, ETS1, and TOX transcription factors involved in NK cell differentiation and maturation. In contrast, exposure of NK cells and leukemic cells to low doses of ATO modulates NK cell receptors and malignant cell ligand profile in a direction that enhances NK cell mediated cytolytic activity. We have demonstrated that NK cytolytic activity toward NB4 cell line when exposed to ATO was significantly higher when compared with controls. We also validated this beneficial effect in a mouse model of APL were the median survival with ATO alone and ATO + NK was 44 days (range: 33–46) vs 54 days (range: 52–75). In conclusion, ATO has a differential quantitative and qualitative effect on NK cell activity. This information can potentially be exploited in the management of leukemia.

Difference in causes and prognostic factors of early death between cohorts with de novo and relapsed acute promyelocytic leukemia.

Early death (ED) remains the most critical issue in the current care of patients with acute promyelocytic leukemia (APL). Very limited data are available regarding ED in patients with relapsed APL. In this retrospective study, 285 de novo and 79 relapsed patients were included. All patients received single-agent arsenic trioxide as induction therapy. The differences in baseline clinical features, incidence, causes, and prognostic factors of ED were compared between the two patient cohorts. The relapse cohort exhibited a better overall condition than the de novo cohort upon hospital admission. The ED rate in the relapsed patients (24.1%) was somewhat higher than that in the de novo patients (17.9%), although the difference was not significant (P = 0.219). For both cohorts, hemorrhage was the main cause of ED, followed by differentiation syndrome, infection, and other causes. Increased serum creatinine level, older age, male sex, white blood cell (WBC) count > 10 × 109/L, and fibrinogen < 1g/L were independently risk factors for ED in the de novo patients, whereas WBC count > 10 × 109/L, elevated serum uric acid level, and D-dimer > 4mg/L were independent risk factors for ED in the relapsed patients. These data furnish clinically relevant information that might be useful for designing more appropriate risk-adapted treatment protocols aimed at reducing ED rate in patients with relapsed APL.

Shikonin potentiates the effect of arsenic trioxide against human hepatocellular carcinoma in vitro and in vivo.

Hepatocellular carcinoma (HCC) is a highly lethal malignancy mostly because of metastasis, recurrence and acquired resistance to conventional chemotherapy. Arsenic trioxide (ATO) is successfully used to treat hematological malignancies, and has been proven to trigger apoptosis in HCC cells. However, the phase II trial evaluating the efficacy and toxicity of ATO in patients with HCC showed that single-agent ATO is poorly active against HCC. Therefore, it is of great importance to develop effective chemosensitization agents to ATO. The aim of the present study was to determine whether shikonin (SHI), a natural product from the root of lithospermum erythrorhizon, could synergistically enhance the anti-HCC efficacy of ATO both in vitro and in vivo. We found that the combination of SHI and ATO exhibited synergistic anticancer efficacy and achieved greater selectivity between cancer cells and normal cells. By inducing intracellular oxidative stress, SHI potentiated ATO-induced DNA damage, followed by increased activation of endoplasmic reticulum stress. In addition, inhibition of ROS reversed the apoptosis induced by SHI and ATO, and recovered the activation of endoplasmic reticulum stress, which revealed the vital role of ROS in the synergism. Moreover, HepG2 xenograft tumor growth in nude mice was more effectively inhibited by combined treatment with SHI and ATO. These data suggest that the combination of SHI with ATO presents a promising therapeutic approach for the treatment of HCC.

Treatment of Acute Promyelocytic Leukemia with Single Agent Arsenic Trioxide: Experience from a Tertiary Care Center in India.

APML is a highly curable hematological malignancy which is treated with differentiation agents and chemotherapy. The morbidity caused by chemotherapy in the treatment of APML is a great cause of concern. We treated 12 patients with newly diagnosed APML with single agent arsenic trioxide between 2010 and 2014 irrespective of risk stratification. Out of 12 patient 2 patients died during induction. All the ten patients who completed induction, completed their consolidation and maintenance without any delays. One out of these ten patients relapsed 10months after treatment. The remaining nine patients (80%) are in molecular remission and are under regular follow up. The toxicity with arsenic was negligible and was very well tolerated. Hence arsenic trioxide as single agent can be offered as a standard alternative regimen to ATRA based chemotherapy in patients with economic constraints.

Role of NF-E2 Related Factor 2 (NRF2) on Chemotherapy Resistance in Acute Myeloid Leukemia (AML) and the Effect of Pharmacological Inhibition of NRF2

Although 70-80% of Acute Myeloid Leukemia (AML) patients achieve complete remission with the induction therapy consisting of a combination of Cytarabine (Ara-C) and Daunorubicin (Dnr), the overall survival is dismal with disease relapse, drug resistance and toxicities as the major causes of treatment failure. Single agent arsenic trioxide (ATO) used in acute promyelocytic leukemia (APL or AML-M3) is associated with relatively less toxicities which suggest the possibility of extending its use to elderly non M3-AML patients. But ATO trials in non-M3 AML were not encouraging enough to devise ATO as a better treatment option. NF-E2 related factor 2 (NRF2) has been recognised as one of the key molecules associated with resistance towards chemotherapeutic agents in cancer. Leukemic cells owing to their genomic instability and altered metabolism are known to maintain high reactive oxygen species (ROS) levels. NRF2 as a redox dependent transcription factor drives the expression of several antioxidant genes like NAD(P)H quinone oxidoreductase1 (NQO1), heme oxygenase1 (HMOX1), glutamate-cysteine ligase (GCL) and glutathione-S-transferases (GSTs) which coordinate to scavenge ROS. Although the role of NRF2 in resistance to cytarabine and daunorubicin has been reported previously, its role in ATO resistance in non M3-AML has not been addressed before. Also, the role of pharmacological inhibition of NRF2 in modulating resistance to ATO and other conventional therapeutic agents in AML has not been tested.To investigate whether NRF2 expression is associated with in-vitro sensitivity to chemotherapeutic drugs, NRF2 RNA expression in primary AML samples grouped according to their median Ara-C, Dnr and ATO IC50 (median IC50 6uM for Ara-C, 0.4µM for Dnr and 2.42µM for ATO) were compared. Primary AML samples with Ara-C, Dnr or ATO IC50 below median had significantly low NRF2 RNA expression compared to those above median (p=0.07, 0.004 and 0.01, respectively) (Fig1-A). Comparison of NRF2 expression in a subset of samples that were above median IC50 to both ATO and Dnr with those below median also showed similar trend. Flow cytometric evaluation of NRF2 expression in Ara-C, Dnr and ATO resistant AML cell lines (THP1 and U937) showed higher intracellular NRF2 levels (MFI=37.18 and 46.56) compared to sensitive cell lines (HL60 and MOLM13) (MFI=6.25 and 28.9). Treatment of resistant AML cell lines expressing high levels of NRF2 (THP1 and U937) with pharmacological inhibitor of NRF2 {Brusatol} followed by ATO (0.1-6uM), Ara-C (0.1uM-80uM) and Dnr (25nM-2000nM) improved their sensitivity to these drugs (Fig1-B) The effect of Brusatol in inhibiting NRF2 protein levels and its role in inducing apoptosis were also demonstrated by flow cytometry (Fig1-C). To further confirm the role of NRF2 in drug resistance, AML cell lines (MOLM13 and HL60) stably over expressing NRF2 was established. Overexpression was done using lentiviral expression system followed by blasticidin selection, further confirmed by immunoblot and quantitative real time PCR. The overexpressed cell lines showed increased resistance to ATO/ Dnr and Ara-C (Fig1-D) and up-regulation of NRF2 downstream targets compared to un-transduced cells (Fig1-E).Our results suggest that NRF2 plays a pivotal role in drug resistance and hence could be an ideal druggable target in AML, more so to the drugs that functions through ROS. Our data as well growing evidence from other malignancies suggest the possibility of using NRF2 inhibitors in combination with chemotherapeutic agents to combat drug resistance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Prospective Study on the Effectiveness of Arsenic Trioxide (ATO) in Remission Induction of Acute Promyelocytic Leukemia (APL)

Background: Arsenic Trioxide (ATO) as a single agent, has proven efficacy in inducing molecular remission in patients with Acute Promyelocytic Leukemia (APL). It is commonly used to treat relapsed APL. But there is Limited study on ATO in the management of newly diagnosed cases of APL. The concerned study was done to evaluate the effectiveness and outcome of ATO in remission induction of new cases of APL in the context of a limited resource hospital in Bangladesh.Methods: From March 2008 to March 2010, 20 patients with Promyelocytic Leukemia (PML) / Retinoic Acid Receptor ? (RAR ?) + newly diagnosed APL were enrolled. All patients were treated with a regimen of single-agent arsenic trioxide till remission at our center. After remission the regimen was administered on outpatient basis.Results: Overall 15 (75%) patients achieved complete hematological remission. 12 (80%) patients achieved molecular remission after induction phase and 3 (20%) after completion of consolidation phase. At a median follow up of 36 months (Range 25 -44 months), Disease Free Survival (DFS) and Overall Survival (OS) 86.6% and 85.3% respectively. Relatively young patients with long form of t (15;17) had shown good response with this response. However, the response is slower than All Trans Retinoic Acid (ATRA). Patients presenting with high White Blood Cell (WBC) count, low platelet count, variable form of t (15;17) are found to respond poorly. The toxicity profile, in the majority, was mild and reversible. Treatment cost was also reduced than that of conventional regimen.Conclusion: Single-agent arsenic trioxide as used in this study in the management of newly diagnosed cases of APL is safe, cost beneficent and is associated with durable responses. But additional interventions as combining ATRA with ATO would probably required in high risk cases.Chatt Maa Shi Hosp Med Coll J; Vol.14 (2); Jul 2015; Page 5-10

Nutlin-3 overcomes arsenic trioxide resistance and tumor metastasis mediated by mutant p53 in Hepatocellular Carcinoma.

BackgroundArsenic trioxide has been demonstrated as an effective anti-cancer drug against leukemia and solid tumors both in vitro and in vivo. However, recent phase II trials demonstrated that single agent arsenic trioxide was poorly effective against hepatocellular carcinoma (HCC), which might be due to drug resistance.MethodsMutation detection of p53 gene in arsenic trioxide resistant HCC cell lines was performed. The therapeutic effects of arsenic trioxide and Nutlin-3 on HCC were evaluated both in vitro and in vivo. A series of experiments including MTT, apoptosis assays, co-Immunoprecipitation, siRNA transfection, lentiviral infection, cell migration, invasion, and epithelial-mesenchy-mal transition (EMT) assays were performed to investigate the underlying mechanisms.ResultsThe acquisition of p53 mutation contributed to arsenic trioxide resistance and enhanced metastatic potential of HCC cells. Mutant p53 (Mutp53) silence could re-sensitize HCC resistant cells to arsenic trioxide and inhibit the metastatic activities, while mutp53 overexpression showed the opposite effects. Neither arsenic trioxide nor Nutlin-3 could exhibit obvious effects against arsenic trioxide resistant HCC cells, while combination of them showed significant effects. Nutlin-3 can not only increase the intracellular arsenicals through inhibition of p-gp but also promote the p73 activation and mutp53 degradation mediated by arsenic trioxide. In vivo experiments indicated that Nutlin-3 can potentiate the antitumor activities of arsenic trioxide in an orthotopic hepatic tumor model and inhibit the metastasis to lung.ConclusionsAcquisitions of p53 mutations contributed to the resistance of HCC to arsenic trioxide. Nutlin-3 could overcome arsenic trioxide resistance and inhibit tumor metastasis through p73 activation and promoting mutant p53 degradation mediated by arsenic trioxide.

NK Cell Mediated Cytotoxicity Against Malignant Promyelocytes Enhanced By Arsenic Trioxide: Potential Clinical Relevance

Abstract NK cells are primary effectors of the innate immune response against cells that have undergone malignant transformation. Several lines of evidence indicate that the expression level of NK ligands on leukemic cells affects the sensitivity of the leukemic cells to cytolytic activity by NK cells. Various agents have been evaluated for their ability to induce these ligands on leukemic cells to augment the NK cell mediated anti-leukemia effect. There is substantial evidence that has established the importance of the adaptive immune system in the treatment of acute promyelocytic leukemia (APL) (Rose Ann Padua et al. Nat Med 2003). While there is significant data which address the mechanisms of arsenic trioxide (ATO) on malignant promyelocytes, limited data is available of its effect on the innate and adaptive immune system. We undertook a series of experiments to address the impact of ATO on NK cell receptor and malignant promyelocyte ligand expression and its effect on NK cell mediated cytotoxicity. We also evaluated NK cell reconstitution in patients treated with ATO and the impact of KIR genotypes on relapse. We first evaluated the cytotoxic activity of NK92MI (NK cell line) against 5 different myeloid (K562, U937, HL60, UF1, NB4) and 2 lymphoid cell lines (Jurkat E6.1, SUP-B15) by CFSE/ 7AAD cytotoxicity assay. Target (T) cells (1x 105/100 µL/well) pre-treated with CFSE were co-cultured with effector NK cells (E) at a E:T ratio of 1:1, 2:1 and 5:1 for 5 hours at 37°C in 96 well plates. The percentage cytolytic activity of the NK cells was then calculated after adding 7AAD and acquired in FACS Calibur (Becton Dickinson, San Jose, CA, USA). Significant cytolytic activity was noted against K562 and NB4 cell lines. At the highest E:T ratio there was a median 22% cytolytic activity against NB4 (N=5). We observed that NB4 when treated overnight with 1µM ATO (&gt;99% viability retained after this exposure) significantly increased the cytotoxic effect of NK92MI cell line at all the E:T ratios as shown in figure 1A (n=5; P=0.0023). No other cell line showed a similar increase in cytotoxic effect following exposure to ATO at these concentrations (data not shown). We next evaluated the effect of exposure of NB4 cells to ATO at 1µM for 6 hours on NK ligand expression by flowcytometry. As shown in figure 1B there was a significant increase in activating ligand MICA/B in NB4 cell lines (n=3; P=0.016) which was not seen in any of the other cell lines. Similar significant increased expression of Nectin-2 (DNAM-1 ligand) and HLA Class I was seen. Exposure of NK92MI to ATO for 6 hours at 1uM (non cytotoxic dose:IC50-3.8uM) resulted in increased expression of activating receptors NKG2D, NKP30 and KIR2DS4 (figure 1C) and inhibitory receptor NKG2A and decrease in inhibitory receptors KIR3DL1/DL2. There were no changes in the expression of NKP46, KIR2DL1, KIR2DL2 and DNAM1 receptors. We undertook a prospective study to evaluate the pattern of NK (CD56+CD3-) reconstitution in patients with newly diagnosed APL treated at our center with a single agent ATO regimen (Mathews et al. JCO 2011). The mean NK cell counts in patients were below the 2SD deviation level of the normal range even after completion of therapy (approximately a year)(figure 1D). All other subsets evaluated (CD4, CD8, CD3, CD19, CD56+CD3+, CD4CD45RO) had returned to levels within the normal range by the end of consolidation therapy (approximately 3 months from diagnosis). KIR genotyping was done on 55 patients with APL who received treatment with single agent ATO based regimen. The median follow up of this cohort was 20 months and 14 cases relapsed following initial therapy. The presence or absence of 17 KIR genes was done by PCR-SSP method (KIR Typing kit, Miltenyi Biotech Inc, CA). There was no association with any specific genotype or haplotype with risk of relapse. In summary we have noted that there is up regulation of receptors on NK cells and ligands on malignant promyelocytes following exposure to ATO that favors NK cell mediated cytotoxicity. In-vitro we have demonstrated a significant increase in NK cell mediated cytolytic activity against malignant promyelocytes exposed to ATO even at relatively low E:T ratios. This could be an important mechanism by which ATO induces durable remissions in patients with APL. The delayed NK cell recovery following treatment with ATO raises the possibility of using NK cell therapy to augment the effect of ATO in the treatment of patients.Figure 1Figure 1. Disclosures: No relevant conflicts of interest to declare.

Patterns of Immune Reconstitution in Patients with Acute Promyelocytic Leukemia Treated with Single Agent Arsenic Trioxide and Its Impact On Time to Molecular Remission

AbstractAbstract ▪3552▪This icon denotes a clinically relevant abstractThere is a substantial data to suggest that immune response to acute promyelocytic leukemia (APL) contributes significantly towards achieving durable remission. The impact of arsenic trioxide (ATO) on immune reconstitution, when used to treat APL, has not been studied. Single agent ATO is able to induce complete molecular remission in practically all patients who complete an induction and consolidation course. However, while ATO is well tolerated approximately 20% of newly diagnosed cases will relapse with such a regimen. We undertook a prospective study to evaluate the pattern of immune reconstitution in patients with newly diagnosed APL treated at our center with a single agent ATO regimen.Peripheral blood samples were obtained from patients before treatment, on day 15 after starting ATO, post induction, pre consolidation, maintenance cycle 2 (6 months from diagnosis) and maintenance cycle 6 (10 months from diagnosis) for flow cytometry analysis. Briefly cells were labeled using a panel of monoclonal antibodies to CD3, CD4, CD8, CD56, CD16, CD19, CD45RO, CD45RA directly conjugated with FITC, PE, PerCP or APC followed by red cell lysis and cells were then washed and analyzed using FACS Calibur. Cells were gated using forward versus side scatter dot-plots. Lymphocyte subset percentages were calculated from the lymphocyte gate and absolute lymphocyte subset counts were calculated for the analysis.Analysis of lymphocyte subset reconstitution patterns have shown that all the subsets were below the normal range at diagnosis. Following treatment there was differential pattern of immune reconstitution in different lymphocyte subsets. The earliest recovery to normal range was seen in the CD8 subset (Figure 1) while the mean CD4 subset reached the normal range only at 3 months from diagnosis. A normal CD4 to CD8 ratio had not been reached even at the last follow up in this study (10 months from diagnosis). There was a significant delay in the immune reconstitution of NK cells (CD56+CD3-) and naïve T cells (CD4+CD45RA+). The pattern of immune reconstitution of some of the lymphocyte subsets that were evaluated is summarized in Figure 1.The number of relapses in this cohort was too few to make any association between relapse and the pattern of immune reconstitution. However, we had previously reported that RT-PCR positivity at the end of induction was a significant independent risk factor for subsequent relapses with this regimen. Among the 29 cases available for evaluation at the time of completion of induction and documentation of hematological remission 14 (48.2%) were RT-PCR negative. At this time point there was a significantly higher number of NK-T cells (CD56+CD3+) in those that were RT-PCR negative versus those that were positive (6.1±4.5 × 107/Lt Vs. 2.9±4.1 × 107/Lt; P=0.040). There was also a trend to lower CD3 and a lower CD56br/CD16dim subset in the cases that were RT-PCR positive at the end of induction.This study demonstrates that there is significant heterogeneity in the pattern of immune reconstitution in different lymphocyte subsets post treatment of APL with ATO. Modulation of immune recovery and response could potentially improve leukemia clearance and maintenance of remission. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Clinical, Cellular and Molecular Differences Between Newly Diagnosed and Relapsed Patients with Acute Promyelocytic Leukemia: Insights Into Mechanisms of Resistance

Abstract 1390We undertook a comprehensive prospective study to compare the clinical, cellular and molecular differences between newly diagnosed (ND) and relapsed (Rel) acute promyelocytic leukemia (APL) patients. 98 ND cases and 28 Rel cases (5 second relapses) were enrolled in this study. 85 (86.7%) of the ND cases were treated with a single agent arsenic trioxide (ATO) regimen. Five (17.8%) Rel cases were discharged against medical advice, the rest were treated with a combination of ATO, ATRA and chemotherapy and advised a SCT in molecular remission.Table 1 summarizes the major clinical differences between ND and Rel cases. Rel patients had a significantly lower WBC counts and higher platelet counts at diagnosis. Coagulopathy was less prominent in Rel cases as evident by the significantly lower number of cases with IC bleeds in this group and the significantly lower platelet rich concentrate requirement in induction (Table 1). Immunophenotyping of the malignant promyelocytes and blasts revealed a significantly increased CD34 expression and significantly reduced CD13 and CD38 expression in Rel cases (Figure 1A). Cytogenetic (CTG) analysis revealed that relapsed patients had a trend to greater proportion of cases with additional karyotypic abnormalities (Table 1).Evaluation of the malignant promyelocytes and blasts’ capacity to concentrate ATO intracellularly as shown in Figure 1B (level of ATO in 107 cells after 24 hour culture with 0.5μM ATO was measured using Atomic Absorption Spectrometry) was not significantly different. Similarly an in-vitro assay of cytotoxicity of ATO (IC-50 measured using standard MTT cell viability assay) was not significantly different between ND and Rel cases (median IC-50= 5.64 ± 3.8 vs. 4.59±4.5 μM; P= 0.28). We had previously noted a protective effect of stromal cell co-culture on the apoptotic action of ATO on malignant promyelocytes. We compared this effect in newly diagnosed and relapsed cases and noted that this protective effect was seen in both groups and was not significantly different (Figure 1C).A microarray expression profile on 8 randomly selected ND and 8 Rel cases was done. Total RNA extracted from BMMNCs with 90% promyelocytes + blasts and above (enriched when required) was used and microarray was performed with a standard one color Agilent–44K array platform. Differentially regulated genes were clustered using hierarchical clustering based on Pearson coefficient correlation, and 1744 genes were 2 fold and above differentially expressed between at relapse and at diagnosis (Figure 1D). 26 differentially expressed genes were validated by RQ-PCR using ABI Taqman assay system (data not shown). Differential gene list were classified based on functional category and pathways using Biointerpreter Software. The most prominent differentially regulated pathways were cell adhesion, cell survival, cell cycle, immune regulation, stem cell regulation, ubiquitinproteasome degradation system and ion transporters. The heat map of the one such prominent pathway (adhesion) is shown in the figure 1E.In conclusion, the Rel cases have significant clinical, immunophenotypic and molecular differences when compared to the newly diagnosed cases. The potential reason for relapse and poor prognosis in the Rel group appears to be multi-factorial. Exploring the pathways and genes differentially regulated between these two groups may provide insights in identifying potentially novel therapeutic targets. Preliminary in-vitro data from our centre, consistent with these observations, has shown that stromal cell adhesion mediates resistance to ATO and that inhibition of this by drugs such as bortezomib can overcome it. [Display omitted] Table 1Comparison of the baseline demographic data of newly diagnosed and relapsed acute promyelocytic leukemia patientsDemographic ParametersNewly Diagnosed N=98 N (%)/Median (Range)Relapsed N=28 N (%)/Median (Range)P-valueAge (years)28 (2-60)31 (8-54)0.997Sex: Male48 (49)25 (89.3)0.000WBC x 109/Lt14.5 (0.5-290)3.5 (0.5-24.90)0.000Platelet x 109/Lt17 (3-85)31.5 (6-248)0.006RT-PCR: bcr159 (60.2)21 (75)bcr22 (2)00.311bcr337 (37.8)7 (25)Lactate dehydrogenase (IU/Lt))748 (291-2700)554 (284-1155)0.006Fibrinogen mg%161 (25-689)163 (70-1122)0.975Additional Cytogenetic finding34 (36.6)11 (61.1)0.068IC Bleed in induction: Yes16 (16.3)0 (0%)0.022Platelet rich concentrates in induction (units)24 (0-85)16 (0-48)0.027 Disclosures:No relevant conflicts of interest to declare.