Relapsed Acute Promyelocytic Leukemia Research Articles

Biochemical risk factors and outcomes of acute promyelocytic leukemia patients with thrombotic events: a matched pair analysis.

Acute promyelocytic leukemia (APL) stands out as a distinctive form of acute leukemia, exhibiting a higher occurrence of thrombotic events when contrasted with other leukemia subtypes. Since thrombosis is a relatively rare but unfavorable condition with poor prognostic implications, it is crucial to determine the risk factors for thrombotic events in APL(thrombosis in large venous or arterial from onset to differentiation therapy in 30d). We performed a retrospective study involving 950 APL patients between January 2000 and October 2022, from which 123 were excluded by younger than 16years of age, 95 were excluded by incomplete data, and 6 were excluded by thrombosis related to CVC or PICC. A total of 23 APL patients with thrombosis for inclusion in our analysis were performed a 1:5 ratio matching based on sex (perfect match) and age (within 5years) to patients without thrombosis. These patients were continuously monitored in the outpatient department over a period of 5years. We meticulously examined clinical and laboratory data to pinpoint the risk factors related to thrombotic events in APL. Our primary clinical endpoints were all-cause mortality and achieving complete remission, while secondary clinical outcomes included APL relapse. Thrombotic events were observed in 2.4% (23/950) of APL patients. Compared to patients without thrombosis, patients with thrombosis had higher lactate dehydrogenase (LDH) [313 (223, 486) vs. 233 (188, 367) U/L, p = 0.020], higher indirect bilirubin [11.2 (7.4, 18.6) vs.8.3 (6.0, 10.7) umol/L, p = 0.004], higher creatinine [72 (62, 85) vs. 63 (54, 74) umol/L, p = 0.026], higher CD2 expression (65.2 vs. 15.2%, p < 0.001), higher CD15 expression (60.9 vs. 24.3%, p = 0.001), and PML/RARαisoforms (p < 0.001). Multivariate-logistic-regression analysis revealed several factors that were markedly related to thrombosis, including LDH (OR≈1.003, CIs≈1.000-1.006, p = 0.021), indirect bilirubin (OR≈1.084, CIs≈1.000-1.188, p = 0.043), CD2 expression positive (OR≈16.629, CIs≈4.001-62.832, p < 0.001), and CD15 expression positive (OR≈7.747, CIs≈2.005-29.941, p = 0.003). The S-type (OR≈0.012, CIs≈0.000-0.310, p = 0.008) and L-type (OR≈0.033, CIs≈0.002-0.609, p = 0.022) PML/RARα isoforms were negatively associated with thrombosis. Kaplan-Meier curves indicated that the survival rates were remarkably varied between APL patients with and without thrombosis (HR:21.34, p < 0.001). LDH and indirect bilirubin are variables significantly associated with thrombosis in APL, S-type and L-type PML/RARαisoforms exhibit a negative association with thrombotic events. The thrombotic events of APL can predict the subsequent survival of thrombosis. The findings of our study have the potential to facilitate early detection of thrombosis and enhance the prognosis for individuals with APL who develop thrombosis. Further validation of our findings will be essential through future prospective or multicenter studies.

Efficacy of Autologous and Allogeneic Hematopoietic Cell Transplantation in Adults with Acute Promyelocytic Leukemia: Results of a Systematic Review and Meta-Analysis

Despite therapeutic advances for acute promyelocytic leukemia (APL) with the emergence of all-trans retinoic acid, arsenic trioxide, and gemtuzumab-ozogamycin, approximately 10% of patients still experience disease relapse, typically occurring within 24 to 36 months following completion of front-line treatment. Traditionally, both allogeneic (allo) and autologous (auto) hematopoietic cell transplantation (HCT) have been considered reasonable treatment options for relapsed APL; however, no randomized controlled studies have been conducted comparing allo-HCT and auto-HCT in patients with relapsed APL. We performed a systematic review/meta-analysis to assess the totality of evidence pertaining to allo-HCT or auto-HCT in relapsed APL. Our search identified 1158 references, of which 23 met our inclusion criteria. While acknowledging the limitations of comparing these 2 treatment modalities indirectly, based on results from separate meta-analyses, it appears that pooled rates of event-free survival (71% versus 54%), progression-free survival (63% versus 43%), and overall survival (82% versus 58%) are higher after auto-HCT. This difference can be explained in part by the higher risk of pooled nonrelapse mortality (NRM) in patients undergoing allo-HCT (29% versus 5%), owing to inherent risks associated with this modality. In the absence of a randomized prospective clinical trial comparing allo-HCT and auto-HCT, our results show that both modalities are acceptable in patients with relapsed APL. The higher pooled NRM rate with allo-HCT is an important consideration when choosing this option. Additionally, the comparable pooled relapse rate for auto-HCT and allo-HCT (24% versus 23%) provides a rationale for evaluating post-HCT consolidative strategies to mitigate this risk.

Recommendations on Minimal Residual Disease Monitoring in Acute Promyelocytic Leukemia Treated with Combination of All- Trans Retinoic Acid and Arsenic Trioxide

Background All- trans retinoic acid (ATRA)-arsenic trioxide (ATO) combination therapy significantly reduces acute promyelocytic leukemia (APL) relapse compared to ATRA combined with chemotherapy, leading to a cure rate of 80-90% in APL patients. The ideal frequency and duration of minimal residual disease (MRD) monitoring needs to be identified in the era of ATO. Methods A total of 1072 APL patients were enrolled in this study, comprising 855 from the APL2012 trial (NCT01987297) and 217 from our center between January 2018 to June 2022 (APL2018 study). The APL2018 protocol was adjusted for consolidation and maintenance therapy based on APL2012 (Chen et. al. PNAS 2021): In consolidation therapy, low-, intermediate-, and high-risk patients were performed following the ATO-group scheme of corresponding risk stratification in the APL2012 study. From December 2019, chemotherapy was no longer administered to high-risk group during consolidation phase. In maintenance treatment, ATO was replaced with oral arsenic realgar-indigo naturalis formula, and methotrexate was removed. RT-PCR or RQ-PCR was routinely used for MRD monitoring, detecting PML::RARA fusion genes in bone marrow before each consolidation and maintenance therapy. MRD testing was conducted every 3 months within the first year, every 6 months within the second year, and annually from 3 to 5 years after treatment. Results A total of 993 patients achieved complete remission (CR) after induction therapy. The negative MRD rates were observed in 61.7% of patients before 1 st consolidation , 95.4% before 2 nd consolidation, 99.3% before 3 rd consolidation, and 99.5% before maintenance therapy , respectively. The median time to achieve negative MRD was 57 (22-301) days with no significant difference between relapsed and non-relapsed patients (p=0.166). A total of 34 patients (30 in the APL2012 trial and 4 in the APL2018 study) relapsed, including 5 at low risk, 9 at intermediate risk, and 20 at high risk. The median recurrence time was 13.5 (3.5-49.0) months after achieving CR, with 94% (32/34) relapsing within 3 years of achieving CR. After induction remission, the risk of relapse decreased with time (44% occurred within the first year, 29% within the second year, 21% within the third year, and only 6% after three years, Figure 1). The median follow-up for the 179 evaluable patients after achieving CR was 39.7 (3.5 -64.6) months in the APL2018 group. The estimated 5-year disease-free survival (DFS) rates were 95.8% (95% CI 92.8-98.9). Prior to the 1 st, 2 nd, and 3 rd consolidation treatment, and before maintenance therapy, the estimated 5-year DFS rates for MRD positive and negative patients were 92.4% (95% CI 86.1-99.2) versus 97.8% (95% CI 94.8-100), p=0.100; 57.1% (95% CI 30.1-100) versus 97.3% (95% CI 94.7-100), p&amp;lt;0.001; 0 versus 96.5% (95% CI 93.1-100), p&amp;lt;0.001; 0 versus 96.8% (95% CI 94.0-99.6), p&amp;lt;0.001, respectively. The results of the APL2012 study were similar to those of APL2018. In the APL2018 study, the estimated 5-year cumulative incidence of relapse (CIR) was 2.51% (95% CI 2.48-2.54), which was 0%, 1.16% (95% CI 1.13-1.19), and 7.83% (95% CI 7.44-8.22) in the low-risk, intermediate-risk, and high-risk patients, respectively (p=0.057). The estimated 5-year CIR for the low to intermediate risk group was 0.74% (95% CI 0.73-0.75), which was statistically different from high-risk patients (p=0.019). The estimated 5-year CIR for MRD positive and negative patients prior to the 1 st and 2 nd consolidation treatments was 4.92% (95% CI 4.76-5.08) versus 1.10% (95% CI 1.08-1.12), p=0.194; 14.29% (95% CI 10.30-18.28) versus 2.08% (95% CI 2.05-2.11), p=0.010, respectively. Conclusion Our results show that MRD after one course of consolidation treatment, rather than before, is a crucial predictive factor for APL relapse. Patients with positive MRD after 1 st consolidation therapy have a high risk of relapse. ATRA-ATO based treatment has significantly reduced APL relapse, so the value of frequent MRD monitoring may be limited. It is recommended to implement an optimized MRD monitoring for risk stratification. For low to intermediate-risk patients and those with negative MRD after a consolidation treatment, the frequency and duration of MRD testing can be minimized. For high-risk patients with sustained MRD negativity for more than 3 years, MRD testing can be considered for discontinuation.

Cytoreductive Chemotherapy in Induction Therapy Plays a Key Role in the Prognosis of Patients with Low-Risk Acute Promyelocytic Leukemia

Introduction: Despite the success for low-risk acute promyelocytic leukemia (APL) in the all-trans retinoic acid (ATRA) plus arsenicals era, several important clinical issues continue to account for treatment failure including early death (ED) and disease relapse. The present study was conducted to explore the potential role of cytoreduction during induction therapy on prognosis. Methods: All consecutive low-risk APL patients who received ATRA plus arsenic for induction and consolidation therapy at the Peking University People's Hospital, Peking University Institute of Hematology, between February 2014 and September 2021, were analyzed. Kaplan-Meier was used to estimate relapse rates and OS. Exploration of factors associated with relapse was based on Cox proportional hazards regression model. Results: Here 282 patients were diagnosed with low-risk APL and received ATRA plus arsenic as an induction and consolidation therapy. During induction therapy, six patients died early. Nine patients were withdrawn after induction therapy. Of the 282 patients evaluable for OS, the 5-year OS was 97.9%. Of the 267 patients evaluable for relapse, 14 patients (5.2%) relapsed throughout follow-up, with the 5-year cumulative relapse incidence of 5.9%. The median time to relapse, including hematological, molecular and extramedullary relapse, was 18.5 (IQR 12.25-24.5, range 6.2-52.0) months after hematological CR. After induction therapy, all 276 evaluable patients (100%) achieved hematological CR. Cytoreduction therapy, including hydroxycarbamide, anthracyclines and cytarabine during induction therapy was administrated based on the 2018 Chinese APL guidelines. Given the unavailability and inconveniences of intravenous chemotherapy at some stage in the COVID-19 pandemic, our center explored the routine of oral etoposide as cytoreductive therapy from January 2020. Etoposide was administered to patients with a WBC count of 3.2-9.8×10 9/L at diagnosis. The cumulative mean dose of etoposide was 706.5 mg (range, 150-1700 mg) during the induction therapy. In terms of different cytoreduction therapies, 86 patients were administered with hydroxycarbamide (30.5%), 113 with anthracyclines or cytarabine (40.1%), 31 with etoposide (11.0%), and 52 with no cytoreductive therapy (18.4%) during induction therapy. We previously explored that promyelocytic leukemia retinoic acid receptor alpha (PML-RARA) transcript level of 6.5% or more after induction therapy was associated with a subsequent risk of relapse. Compared to patients with no cytoreduction therapy or only hydroxycarbamide during induction therapy, the incidence of PML-RARA transcript levels of ≥6.5% was significantly decreased at the end of induction therapy in those who received anthracyclines/cytarabine and etoposide (27.5% vs. 26.2% vs. 15.5% vs. 0%, P = 0.003, Table 1). Notably, 2-year cumulative relapse rates were significantly lower in the anthracyclines/cytarabine treatment group and the etoposide treatment group (3.962% vs. 8.861% vs. 1.887% vs. 0%, P = 0.040, Table 1). The hydroxycarbamide treatment group did not decrease the relapse rate compared to the no cytoreduction group (11.4% vs. 5.9%, P = 0.289). To estimate RFS, the hydroxycarbamide treatment group and the no cytoreduction treatment group were merged to create a single group (the H/N group, 5-year RFS, 90.391%). Compared with the H/N group, the anthracyclines/cytarabine treatment group and the etoposide treatment group showed improved 2-year RFS (93.064% vs. 98.113% vs. 100.0%, P = 0.046, Figure 1). Multivariate Cox regression analysis revealed that myeloblasts in bone marrow at diagnosis, Leukocytosis during induction therapy, PML-RARA of ≥6.5%, and anthracyclines/cytarabine cytoreductive treatment were associated with a subsequent risk of relapse. Conclusions: In summary, the present study indicated that cytoreductive chemotherapy had a dual effect of both cytoreduction and prevention of relapse in low-risk APL. Oral etoposide as cytoreduction therapy combined with oral ATRA plus RIF is expected to be a regimen with low relapse, good tolerance and great convenience. A prospective, randomized, controlled, interventional study on oral etoposide versus intravenous daunorubicin for cytoreductive chemotherapy in induction therapy in patients with low-risk APL is now going on (NCT05832320).

An Entirely Oral Regimen of Oral-Arsenic Trioxide/All-Trans Retinoic Acid/Ascorbic Acid in Newly-Diagnosed Acute Promyelocytic Leukaemia (APL): Updated Results of an Ongoing Multicentre Trial

Background: Pure oral arsenic trioxide (oral-ATO) solution (Arsenol ®) was first formulated in Hong Kong. It achieves a bioavailability comparable with that of intravenous ATO (i.v.-ATO). Oral-ATO combined with all-trans retinoic acid (ATRA) and ascorbic acid (the AAA regimen) combined with chemotherapy is highly efficacious in relapsed and newly-diagnosed APL. To reduce treatment-related toxicities and long-term risks of second primary cancers, a chemotherapy-free approach is increasingly favoured. Aims: The objectives of this prospective multicentre study in newly diagnosed APL patients were: 1. to evaluate the efficacy and safety of an entirely oral AAA-induction in a risk-adapted strategy; 2. to evaluate the molecular responses during oral-AAA-based induction/consolidation/maintenance. Methods: Newly-diagnosed APL patients were stratified by presentation leucocyte count into standard-risk (≤ 10 x 10 9/L) and high-risk (&amp;gt; 10 x 10 9/L) groups. Standard-risk patients and high-risk patients ≥ 65 years old received AAA induction comprising oral-ATO (Arsenol ®, Jacobson Pharma Corporation, Hong Kong) (10 mg/day, or 0.16 mg/kg in patients &amp;lt; 18 years old, days 1-42), ATRA (45 mg/m 2/day, or 25 mg/m 2/day in patients &amp;lt;18 years old, in 2 divided doses, days 1-42), and oral ascorbic acid (1 g/day, or 15mg/kg/day in patients &amp;lt;18 years old, days 1-42). High-risk patients &amp;lt; 65 years old received in addition daunorubicin (50 mg/m 2/day i.v. for 3 days). On reaching first complete remission (CR1), consolidation with AAA (days 1-14) every 28 days for 2 cycles was given, followed by maintenance with AAA (days 1-14) every 8 weeks for 12 cycles. Real-time quantitative polymerase chain reaction for PML:: RARA with a sensitivity of 10 -5 was performed (weekly during induction, every 4 weeks during consolidation, every 8 weeks during maintenance and thereafter every 12 weeks for 2 years). For molecular response, the normalized copy number ratio was defined as PML::RARA copy number/ ABL copy number. Primary outcomes were overall survival (OS, time from presentation to death or last follow-up), and relapse-free survival (RFS, time from CR1 to molecular or haematological relapse, death or last follow-up), and safety (according to the Common Toxicity Criteria for Adverse Events (AE) version 5.0). The secondary outcome was molecular response. Data were censored on July 22, 2023. Results: Between January 1, 2018 to July 22, 2023, 5 paediatric/adolescent patients (male, N=2; female, N=3) at a median age of 12 (3-15) years and 116 adult (&amp;gt; 18 years old) patients (male, N=47 ; female, N=69) at a median age of 49 (19-91) years were accrued (standard risk: paediatric/adolescent, N=3; adults, N=87; high-risk: paediatric/adolescent, N=2; adults, N=29). Seven patients (all high-risk adults) died at presentation before induction (intracranial haemorrhage, N=6; severe APL differentiation syndrome, DS, with leucocyte &amp;gt;100 x10 9/L, N=1). One-hundred and fourteen patients received oral-AAA-based induction (AAA alone, N=90; AAA+daunorubicin, N=24), all achieving CR1. For PML:: RARA normalized copy number, all treated patients at week 8 achieved a ratio of &amp;lt;0.01; and on completion of AAA maintenance, all evaluable patients (N=56) achieved a ratio of &amp;lt;0.0001. The median follow-up was 29 (IQR: 8-47) months, with 56 patients (49%) having completed 2 years of AAA maintenance. There was only one relapse, occurring in an adult patient 12 months after completion of AAA maintenance. Molecular analysis showed a PML B2 domain A216V mutation that conferred resistance to ATO. One adult patient died of an unrelated gastrointestinal bleeding while in CR1. The 3-year OS was 99.1% (paediatric/adolescent: 100%; adult: 99%). The 3-year RFS was 97.9% (paediatric/adolescent: 100%; adult: 97.9%). The most common non-haematological AEs (all grade 1-2) were transaminitis (N=54, 47.3%) and headache (N=32, 28%) Notably, no cardiotoxicity (arrhythmias/cardiac failure) was observed. APL-DS occurred in 67 patients (58.7%) after initiation of AAA, all responding fully to intravenous dexamethasone. There were no treatment discontinuations. Conclusion: The use of an entirely oral AAA-based induction in a risk-adapted strategy that minimized chemotherapy was highly effective and safe in newly-diagnosed APL of all risk categories and age groups. Early deaths remained an obstacle to realizing a cure-for-all in APL.

MiR-603 promotes cell proliferation and differentiation by targeting TrkB in acute promyelocytic leukemia.

Arsenic trioxide (ATO) treatment effectively prolongs the overall survival of patients with acute promyelocytic leukemia (APL). Mutations in the oncogene PML::RARA were found in patients with ATO-resistant and relapsed APL. However, some relapsed patients do not have such mutations. Here, we performed microarray analysis of samples from newly diagnosed and relapsed APL, and found different microRNA (miRNA) expression patterns between these two groups. Among the differentially expressed miRNAs, miR-603 was expressed at the lowest level in relapsed patients. The expression of miR-603 and its predicted target tropomyosin-related kinase B (TrkB) were determined by PCR and Western blot. Proliferation was measured using an MTT assay, while apoptosis, cell cycle and CD11b expression were analyzed using flow cytometry. In APL patients, the expression of miR-603 was negatively correlated with that of TrkB. miR-603 directly targeted TrkB and downregulated TrkB expression in the APL cell line NB4. miR-603 increased cell proliferation by promoting the differentiation and inhibiting the apoptosis of NB4 cells. This study shows that the miR-603/ TrkB axis may be a potent therapeutic target for relapsed APL.

Targeting DHODH reveals therapeutic opportunities in ATRA-resistant acute promyelocytic leukemia

Although all-trans retinoic acid (ATRA)-induced differentiation has transformed acute promyelocytic leukemia (APL) from the most fatal to the most curable hematological disease, resistance to ATRA in high-risk APL patients remains a clinical challenge. In this paper, we discovered that dihydroorotate dehydrogenase (DHODH) inhibition overcame ATRA resistance. 416, a potent DHODH inhibitor previously obtained in our group, inhibited the occurrence of APL in cells and model mice. Excitingly, 416 effectively overcame ATRA resistance in vitro and in vivo by inducing apoptosis and differentiation. Further mechanistic studies showed that PML/RARα lost the regulation of Bcl-2 and c-Myc in NB4-R1 cells, which probably contributed to ATRA resistance. Notably, 416 maintained its Bcl-2 and c-Myc down-regulation effect in NB4-R1 cells and overcome ATRA resistance by inhibiting DHODH. In conclusion, our study highlights the potential of 416 for APL therapy and overcoming ATRA resistance, supporting the further development of DHODH inhibitors for clinical use in refractory and relapsed APL.

Allogeneic Hematopoietic Cell Transplantation for Patients with Relapsed Acute Promyelocytic Leukemia

Although autologous hematopoietic cell transplantation (HCT) is an established therapy for patients with relapsed acute promyelocytic leukemia (APL) after returning to complete remission (CR), the role of allogeneic HCT remains unclear for treating relapsed APL. This study aimed to investigate allogeneic HCT outcomes in patients with relapsed APL, focusing particularly on those who underwent transplantation in non-CR and those who had relapsed after prior autologous HCT. We retrospectively analyzed Japanese nationwide transplantation registry data of patients with relapsed APL age ≥16 years who underwent allogeneic HCT between 2006 and 2020. A total of 195 patients were eligible for this analysis, including 69 who underwent transplantation in non-CR and 55 who relapsed after prior autologous HCT. The median duration of follow-up for survivors was 5.4 years. Multivariate analysis revealed that both non-CR at transplantation (hazard ratio [HR], 1.74; 95% confidence interval [CI], 1.12 to 2.71; P=.014) and prior autologous HCT (HR, 2.10; 95% CI, 1.28 to 3.44; P=.013) were associated with higher risks of overall mortality. The 5-year overall survival (OS) rates for patients who underwent transplantation in CR and non-CR were 58% and 39%, respectively (P=.085), if they did not have a history of prior autologous HCT. In the patients who had relapsed after prior autologous HCT, the 5-year OS rate was 47% for those who underwent allogeneic HCT in CR and 6% for those who did so in non-CR (P=.001). Allogeneic HCT still provides an opportunity for long-term survival for certain patients with relapsed APL for whom autologous HCT is unlikely to be effective. The dismal outcome of those with prior autologous HCT who underwent allogeneic HCT in non-CR poses a significant therapeutic challenge.

Treatment for relapsed acute promyelocytic leukemia.

The advent of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has significantly improved the outcomes of acute promyelocytic leukemia (APL); nevertheless, a small fraction of patients still experience relapse. Due to the infrequency of APL relapse coupled with the rapid change in the therapeutic landscape, there are limited available data regarding the treatment of relapsed APL. In this situation, however, ATO-based therapy has been shown to result in high rates of hematological and molecular complete remission (CR). Autologous hematopoietic cell transplantation (HCT) is considered the postremission therapy of choice when patients achieve molecular CR, whereas recent studies have suggested that molecular CR is not prerequisite for the success of autologous HCT. Allogeneic HCT can be reserved for selected patients, i.e., those who cannot achieve CR and those who relapse after autologous HCT, because of high toxicities and the expectation of highly favorable outcomes with autologous HCT during CR. For patients who are ineligible for HCT, prolonged administration of ATRA + ATO would be a viable option. To further refine the therapy for patients with relapsed APL, it is imperative to aggregate clinical data of patients who relapse after the ATRA + ATO frontline therapy within the framework of national and international collaboration.

Treatment for relapsed acute promyelocytic leukemia: what is the best post-remission treatment?

BackgroundArsenic trioxide (ATO) is the standard treatment for relapsed acute promyelocytic leukemia (APL). However, consensus on post-remission therapies is still lacking.MethodsWe evaluated 52 patients who experienced relapse following initial treatment of APL between 2000 and 2019 at Catholic Hematology Hospital. Among them, 41 patients received reinduction treatment, 30 with ATO-based regimen, whereas 11 with conventional intensive chemotherapy (IC).ResultsThe ATO reinduction group showed a significantly higher second molecular complete remission (mCR2) rate, superior neutrophil and platelet recovery, and a lower infection rate than the IC reinduction group. No significant differences were observed in survival outcomes after post-remission treatment among the ATO-based (N=19), autologous (N=12), and allogeneic (N=6) hematopoietic stem cell transplantation (HSCT) groups. In the ATO-based and autologous HSCT groups, among patients with mCR2 after ATO reinduction, nine and five patients experienced a second relapse, respectively (50.7% vs. 41.7%, P=0.878). Among these patients, seven received salvage allogeneic HSCT; six remained alive. The other seven patients received ATO without HSCT. Five died from disease progression, and two survived and have been in mCR2 since.ConclusionPost-remission treatment outcomes of patients with relapsed APL were not significantly different, regardless of the treatment option, suggesting the feasibility of ATO-based treatment without HSCT in mCR2. Allogeneic HSCT may be an effective salvage treatment modality for patients with a second relapse. Owing to a few cases of relapsed APL, multicenter prospective studies may help elucidate the efficacy of each post-remission treatment.

Relapse Analysis and Resistance Mutations of PML-Rara Fusion Gene in Acute Promyelocytic Leukemia Patients Treated with All-Trans Retinoic Acid and Arsenic

The clinical manifestations, management, and prognosis of acute promyelocytic leukemia (APL) are distinctive in acute myeloid leukemia (AML). Administration of all-trans retinoic acid (ATRA) and arsenic agents have greatly improved the outcome of APL from the most lethal to the most curable subtype AML. Unfortunately, relapse is still observed. And in the case of combined medication with ATRA and arsenic, the resistance mutation spectrum of PML-RARA is far from being revealed. We retrospectively analyzed 40 APL patients that admitted to our hospital from Jan. 2013 to Jul. 2020. There were 21 males and 19 females, aged 6-65 years (median age 31.5), and follow-up time was 3-210 months (median time 58.85 months).There were 3 cases that developed secondary tumors, although APL remission has been achieved. One developed refractory AML with KMT2A-MLLT3 fusion during the consolidation treatment one year after the diagnosis of APL. One case relapsed with APL 5 years after the initial diagnosis, and she developed refractory AML with RUNX1-MECOM fusion 3.5 years more after enduring multiple relapses and treatment courses. Another case developed AML-M2 3.5 years after the initial diagnosis of APL, and achieved sustained remission again through allogeneic hematopoietic stem cell transplantation (allo-HSCT).A total of 8 cases came to our hospital at the initial onset of APL, and 1 came during remission. All these cases got sustained remission through combined triple therapy (ATRA, arsenic, and chemotherapy), with the follow-up time of 3 to 37 months (median 20.5 months). No PML-RARA resistant mutation was detected in these 9 cases.A total of 28 cases came to our hospital after APL relapse. The time from APL onset to the first relapse was 9-189 months (median 34 months). PML-RARA resistance mutations were detected in 9 cases, including 8 cases that carry single RARA mutations each, and 1 case that relapsed with multiple extramedullary infiltrations carry quadruple mutations (PML S214L-A216T and RARA R276W-H298N) (Figure 1a and b). A total of 4 cases underwent allo-HSCT after APL relapse. One of them relapsed 6 months after allo-HSCT, and the other 3 got sustained remission with the follow-up time of 47, 39, and 52 months, respectively.There were 2 cases relapsed after maintaining sustained remission for more than 5 years. One was a 58-year-old male who achieved sustained remission after 2.5 years of combined triple therapy. However, APL recurred 16 years after the primary diagnosis. He achieved remission again and now underwent the consolidation course of combined triple therapy. Another was a 23-year-old male who achieved sustained remission after combined arsenic and chemotherapy, but APL relapsed 75 months after the primary diagnosis. He was resistant to combined triple therapy and achieved remission through allo-HSCT. We failed to retrieve archived specimens at the first onset of APL for genomic variation comparison with the second onset. Therefore, we could not determine whether these two cases were relapsed from the original APL or developed another APL.Although we have achieved a high remission rate and long-term survival for APL through combined triple therapy, relapse and secondary tumors still occur in some cases. Especially for cases that endure heavy treatment, genomic toxicity may promote the oncogenesis of secondary tumors. Also, APL may recur over 5 years after the primary diagnosis and sustained remission. Whether there were inherited predisposing factors in these cases is worthy of further investigation.The incidence of PML and RARA resistant mutations in cases resistance for ATRA and arsenic is not very high. This may be related to the combination use of chemotherapeutic drugs and suggests underlying resistance mechanisms. Although both ATRA and arsenic were used in most cases, the incidence of ATRA resistance mutations is significantly higher than that of arsenic, which might attribute to the sparse interaction sites of arsenic and PML.A better combination strategy of arsenic, ATRA, with or without chemotherapy is worthy of further investigation to reduce the incidence of secondary tumors and relapse. Adhering to a systematically planed treatment course is essential for acquiring rapid remission and reduction of relapse. Treatment compliance should be paid attention to guaranteed, especially during the oral administration courses outside the hospital. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single Cell Analyses Reveal a Non-Canonical EZH2 Activity As a Main Driver of Retinoic Acid Resistance in PLZF/Rara Leukemia

Resistance to treatment is due to the heterogeneity of the tumor which contains a subset of cancer cells that escape treatment and are responsible for the relapse. Acute Promyelocytic Leukemia (APL), the M3 subtype of AML, is a good model to illustrate these problematics. Indeed, APL driven by oncogenic fusion proteins such PML/RARA t(15;17) or PLZF/RARA t(11;17) behave differently to differentiation therapeutics. Both APLs differentiate in vivo upon Retinoic Acid (RA) treatment; however, while PML/RARA APL patients exhibit partial or complete remission, PLZF/RARA APL patients remain clinically resistant. In the present study we aim to decipher the transcriptional and epigenetic networks that is linked to t(11;17) APL resistance towards RA.We took advantage of the PLZF/RARA RA resistant murine APL model to catch relapse-initiating cell features and their vulnerabilities. By developing an integrative single-cell multi-omics analysis (scRNA-seq and scATAC-seq), we uncovered transcriptional and chromatin heterogeneity of the PLZF/RARA APL blasts. We highlighted a subset of cells insensitive to RA-induced differentiation with a strong DNA repair signature (“Rep” cluster) and exhibiting a fine tuned transcriptional network targeting the histone methyltransferase Ezh2. To validate the function of Ezh2 in APL physiology, we combined epigenomic studies with RA-treated and non-RA-treated bone marrow transplantation experiments. We revealed high Ezh2 activity that marks the relapse of RA-treated APL. However, targeting Ezh2 methyltransferase activity was not sufficient to achieve disease cure and, suggests an independent methyltransferase Ezh2 activity linked to RA resistance.These findings demonstrate the power of single-cell multi-omics integration to highlight path to sensitize therapy-resistant leukemia cells. In addition, our study uncovers a dual role of Ezh2 in APL and suggests that targeting non-canonical Ezh2 activity could be a new promising therapeutic approach for RA resistant APL. DisclosuresIwama: Nissan Chemical Corporation: Research Funding.

Autologous hematopoietic cell transplantation during second or subsequent complete remission of acute promyelocytic leukemia: a prognostic factor analysis.

Autologous hematopoietic cell transplantation (HCT) is an effective therapy for patients with relapsed acute promyelocytic leukemia (APL). However, it remains unclear whether this procedure is equally effective for certain groups of patients. To address this question, we analyzed 296 patients with APL who had undergone autologous HCT during second or subsequent complete remission (CR2+) between 2006 and 2019. Among them, 24 patients were ≥65 years old, and 17 underwent autologous HCT during third or subsequent CR. Of the 286 patients whose measurable residual disease (MRD) data were available, 21 showed detectable MRD. The 5-year probabilities of relapse-free survival (RFS), overall survival, relapse, and nonrelapse mortality for the entire cohort were 85%, 88%, 9%, and 6%, respectively. The multivariate analysis revealed that the duration of first CR ( < or ≥2 years) was the sole factor associated with RFS (P = 0.002), but even those with CR1 duration <2 years showed a 5-year RFS of 76%. The other factors such as age, disease status, and MRD status were not predictive for the survival outcomes. Our findings demonstrate very favorable long-term results when autologous HCT is conducted during CR2 + across the various subgroups of patients with relapsed APL.

Haploidentical Hematopoietic Stem Cell Transplantation With Reduced Intensity Conditioning, T-Cell Replete Grafts and Post-Transplant Cyclophosphamide In Pediatrics

Hematopoietic stem cell transplants (HSCT) may be curative in the treatment of many hematological diseases. However, most patients do not have an HLA- matched sibling donor and an unrelated donor search may be a long process, during which the patients' disease may relapse or progress. Haploidentical transplants with in vitro T-cell depletion have been used in Pediatrics. However, it requires specialized laboratory infra-structure to manipulate the graft, increases the chances of graft failure and relapse. In vivo T-cell depletion can be achieved with the use of post-transplant cyclophosphamide, as reported by the Johns Hopkins and other groups. The results in adults are very promising and comparable to unrelated double cord blood transplants. Children, however, have not been included in any of the reported protocols. Our objective is to report the institutional experience with five consecutive children who received Haploidentical HSCT with reduced intensity conditioning, T-cell replete grafts and post transplant cyclophosphamide. MethodsChildren with clear indication to be treated with HSCT and no HLA-matched related or unrelated donor available were offered compassionate haploidentical transplants. Conditioning comprised cyclophosphamide 14.5 mg/kg/day on days -6 and -5, fludarabine 30 mg/m2/day for five consecutive days starting on day -6, and 2Gy total body irradiation given in a single fraction on day -1. Bone marrow was harvested from donors and infused into recipients on day 0. The target nucleated cell dose was 5 x10(8) mononuclear marrow cells/kg (i.e., peripheral blood count was substracted from the marrow cell count). There was no graft manipulation. GVHD prophylaxis consisted of cyclophosphamide 50 mg/kg IV on days +3 and +4 with 140% Mesna divided on hours 0, 4, 8 and 12 after each dose, Mycophenolate mofetil 15 mg/kg po every 8 hours (maximum 3 g/day) from day +5 to day +35, and tacrolimus from day + 5 to day +180 to keep levels between 5 and 10ng/ml. Prophylactic antimicrobial therapy was started on day +1 and included fluconazole and acyclovir. ResultsFive children with AML and T-ALL (three in CR2; one induction failure and one 4th relapse) were offered haploidentical transplants as the first HSCT. They had a median age of 10 years (4-16) and weight 41 kg (14-67), 2 girls. One graft was a peripheral blood stem cell collection per physician preference. The median total nucleated cell count was 12x10(8)/kg (10-27) with 10x10(6) CD34/kg (5-15). Mucositis was mild in three patients and none of them had sinusoidal obstruction syndrome. All patients engrafted after a median 14 days (13-17) but one of them, in 4th relapse of acute promyelocytic leukemia (APL), rejected the graft. Two of four evaluable patients had mild acute graft versus host disease (GVHD), and one mild chronic GVHD. The patient with APL died of two graft rejections on day+125, one T-ALL relapsed on day+180 and three children are disease-free with excellent clinical status one month to 1.7 years after transplant. We conclude that haploidentical HSCT with post-transplant cyclophosphamide is feasible to treat children without an available matched related or unrelated donor and should be prospectively studied. Disclosures:No relevant conflicts of interest to declare.

Resource Utilization Using Micro-Costing Method and Cost Effectiveness Analysis of Treatment of Acute Promyelocytic Leukemia with Generic Arsenic-Trioxide

We have previously reported on the challenges of treating AML in India, the major challenge being the cost of treatment (Chepsy et al. BJH 2015). With conventional treatment of AML it is recognized that the major part of this cost is not related to the cost of the individual chemotherapeutic drugs, but rather from the high cost of supportive care to take patients through repeated cycles of prolonged neutropenia induced by conventional chemotherapy. In contrast with arsenic trioxide (ATO) based regimens in acute promyelocytic leukemia (APL), when expensive patented ‘innovator’ ATO was used, the reported 3 year direct pharmacy cost of drugs was higher with an ATO+ATRA regime compared with a conventional ATRA+chemotherapy regimen (Euro 46,700 Vs 6,700), though the cost of supportive care was a third of conventional therapy (Kruse M et al. PLoS One. 2015). There is limited data on the cost effectiveness of using generic ATO, as available in India, in the treatment of APL (10 mg vial costs US $7 in India versus US $ 676 in North America)In a single center, retrospective study, 30 newly diagnosed APL (ND-APL) patients, treated with ATO and 16 relapsed APL (R-APL) patients, treated on a phase II trial with a ATO based regimen followed by an autologous stem cell transplant (regimens as previously reported by us; Mathews et al. Blood 2012 and Mathews et al. Blood 2016 abstract) were evaluated. In the absence of patients being treated at our center with a conventional ATRA+chemotherapy regimen (APL-CT) the cost of treatment for APL with such a regimen was assumed to be similar to the cost that would be incurred to treat a non-M3 AML patient at our center with a conventional AML chemotherapy schedule. Incremental cost effectiveness ratio was calculated using weighted costs with weighing factor obtained from survival data for 60 patients from our database for APL ATO group and from the AIDA 2000 trial for APL-CT group.A three phase micro-costing method (figure 1) used four cost centers involved in providing patient care: (i) chemotherapy administration (ii) supportive care (iii) treatment of complications (iv) laboratory services. For each of these cost centers there were four cost drivers: (i) drugs / reagents (ii) personnel (iii) ward (iv) instruments / overheads. The actual cost of cost drivers was found out by micro-costing and summed up to obtain the unit cost of each cost center (m). Each time a patient used a cost center this unit cost would be incurred. Next, the frequency of utilization (n) of each cost center was found out from the patient database and multiplied by the unit cost of the cost center to obtain the total cost of treatment in each protocol (m x n) (For illustration and international comparison all cost were converted to US$).In ND-APL patients, the mean cost of induction and consolidation was $5078.5±1581 and $902±212 respectively while in APL-CT it was $9812.1±3239 and $11803.1±4872 respectively (exchange rate 1 US$=61.87 Indian rupees). Our data illustrated that APL-CT group incurred higher costs mainly due to the greater duration of in-patient care that was required in this group and since they had higher rate of infectious complications (Table 1). During maintenance phase, in ND-APL, there were no in-patient stays and the entire chemotherapy was given on outpatient basis and the average cost was $2074±146 and the cost of following up these patients for an additional three years was $704.6±289. The mean lifetime cost of treatment of ND-APL using ATO was $8759.2±2078 while using APL-CT was $24393±5528. In R-APL patients, the total cost of re-induction and consolidation was $3498±1220 and $2274±372 respectively. The mean cost of an autologous-SCT was $4318±159. The average lifetime cost of treatment of relapsed APL patients using ATO based regimen followed by an autologous SCT was $12269.7±4915. The weighted cost per living for ATO and CT was estimated to be $12426 and $28330 respectively. The incremental cost effectiveness ratio showed ATO to be cheaper by $13359 for each life saved compared to conventional chemotherapy.ATO based regimen in ND-APL and R-APL are associated with significant reduction in resource utilization and costs of treatment and is also cost-effective as compared to conventional ATRA+chemotherapy. With the world still debating the best treatment for APL, generic ATO based regimen offers a significant opportunity for resource constrained countries to treat all patients with a diagnosis of APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Stromal-Cells Downregulate MiR-23a-5p Levels in Myeloid Leukemic Cells to Activate Protective-Autophagy Against Chemotherapeutic Agents

Role of stromal micro-environment in drug resistance has been extensively reported for several cancers. We have demonstrated earlier that there is significant microenvironment mediated drug resistance (EMDR) to arsenic trioxide (ATO) in acute promyelocytic leukemia (APL) and that this was predominantly driven by up-regulation of the NF-kB pathway in the malignant cell (Ganesan S et al. Leukemia 2016). In our current study we have probed the molecular mechanism of EM-DR in further detail. Towards this we evaluated the potential role played by miRNA in EMDR to ATO in APL. Using NGS based small RNA sequencing we identified hsa-miR-23a-5p to be significantly downregulated (adjusted P value <0.01; figure 1a) in NB4 cells upon co-culture with HS-5 stromal cells. We noted that this miRNA is negatively regulated by NF-kB pathway and inversely correlated with NF-kB status in relapsed APL patients (n=8; matched samples). These results were consistent with our earlier reported observations that NF-kB pathway is dysregulated and enhances drug resistance to ATO. Further, we observed that low expression of miR-23a-5p observed in the relapsed patient's samples showed a trend towards an increased association with subsequent second relapse.We also observed miR-23a-5p mimics were able to restore the sensitivity of NB4 cells to ATO in the presence of stromal cells (figure 1b), while NB4/GFP-MAD cells (NF-kB inhibited cell line showing high expression of miR-23a-5p ) were not protected from ATO on stromal co-culture. Looking for the targets of miR-23a-5p , TargetScan analysis data demonstrated a clustering of targets involving toll like receptor genes (TLR) and autophagy pathway genes (TLRs - TLR2, TLR3, TLR4 and TLR5 and autophagy pathway genes - AMBRA1, ATG13, ATG9 and ATG2 ). Previously reported data clearly demonstrated the ability of TLR genes to activate autophagy (Delgado MA. et al. EMBO J 2008). Based on this data we evaluated the role of autophagy in EM-DR. We noted that there was an upregulation of autophagy pathway in leukemic cells upon co-culture with stroma (where miR-23a-5p was downregulated). Re-introducing miR-23a-5p in the leukemic cells targeted autophagy pathway genes which was validated through western blot (figure 1c). We also observed that inhibiting autophagy by chemical inhibitors (bafilomycin A1 and hydroxychloroquine) along with ATO was able to overcome EM-DR to ATO (figure 1d). The in-vivo efficacy of combining ATO along with hydroxychloroquine was further validated in an APL mouse model where we were able to demonstrate a significant decrease in tumor burden on day20 when compared to controls and significantly prolonged the survival of APL mice treated with the combination in comparison to either drug alone.Similar results were obtained with AML cells, where the stromal cells conferred a protective effect over U937 cells against cytarabine (AraC) and daunorubicin (Dnr). The downregulation of miR-23a-5p was also observed in U937 cells upon co-culture. We also observed that re-introducing miR-23a-5p in these cells restored sensitivity to AraC and Dnr. Our results thus illustrates a complex molecular cross-talk between stromal cells and leukemic cells in protecting against the cytotoxic effects of various chemotherapeutic agents in myeloid leukemia. This data along with that reported earlier by us illustrates multiple levels of regulation of the NF-kB pathway and resistance to chemotherapeutic drugs by stromal cell co-culture. Our work thus suggests that miR-23a-5p mimics or autophagy-inhibitors could potentially be effective adjuvants along with chemotherapy/ATO in the treatment of myeloid malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Arsenic trioxide therapy predisposes to herpes zoster reactivation despite minimally myelosuppressive therapy

Acute Promyelocytic Leukemia (APL) is a unique subtype of acute myeloid leukemia that is highly responsive to minimally myelosuppressive therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). We and others have observed a higher than expected incidence of herpes zoster reactivation in APL patients treated with ATO. Memorial Sloan Kettering Cancer Center (MSKCC) has been using ATO since 1997 in all relapsed APL patients, and more recently has included it in our front-line APL regimens. Here we present a retrospective analysis of the factors contributing to herpes zoster reactivation among APL patients.

Characteristics of arsenic species in cerebrospinal fluid (CSF) of acute promyelocytic leukaemia (APL) patients treated with arsenic trioxide plus mannitol.

Arsenic speciation in cerebrospinal fluid (CSF) is critical for treatment/prevention of central nervous system (CNS) relapse in acute promyelocytic leukaemia (APL) patients treated with arsenic trioxide (ATO). Previous study showed low total arsenic level in CSF of APL patients. Mannitol infusion was applied to improve blood-brain barrier (BBB) permeability for arsenic. Arsenite (AsIII ), monomethylarsonic acid (MMAV ), dimethylarsinic acid (DMAV ), and arsenate (AsV ) in CSF and plasma were analysed by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The profile and concentration of arsenic species in CSF from APL patients administered ATO alone and in combination with mannitol were compared. The overall distribution trend of arsenic species in CSF was AsIII , DMAV > MMAV > AsV . Arsenicals accumulated in CSF with administration frequency. The permeability of BBB for AsIII was higher than that for MMAV and DMAV . Arsenic concentration in CSF was much lower than that in plasma. There were significantly higher arsenic species concentrations in CSF of APL patients treated with mannitol than that without mannitol. Mannitol infusion significantly increased AsIII penetration into CSF, which was beneficial to optimize efficacy in APL patients with CNS relapse.

Role of Hematopoietic Stem Cell Transplantation in Acute Promyelocytic Leukemia.

The indication of hematopoietic stem cell transplantation (HSCT) in acute promyelocytic leukemia (APL) has evolved historically from a widespread use in front-line therapy during the pre-ATRA era to a virtual rejection of this indication for patients treated with modern treatments. HSCT in first complete remission could only be considered for an extremely small fraction of patients with persistent MRD at the end of consolidation or for those who relapse. In the pre-ATO era, relapsed patients were usually treated with readministration of ATRA and chemotherapy as salvage therapy, generally containing high-dose cytarabine and an anthracycline, followed by further post-remission chemotherapy and/or HSCT. ATO-based regimens are presently regarded as the first option for relapsed APL. The selection of the most appropriate post-remission treatment option for patients in second CR (CR2), as well as the modality of HSCT when indicated, depends on several variables, such as pre-transplant molecular status, duration of first remission, age, and donor availability. Although with a moderate level of evidence, based on recent retrospective studies, autologous HSCT would be at present the preferred option for consolidation for patients in molecular CR2. Allogeneic HSCT could be considered in patients with a very early relapse or those beyond CR2. Nevertheless, the superiority of HSCT as consolidation over other alternatives without transplantation has recently been questioned in some studies, which justify a prospective controlled study to resolve this still controversial issue.

Management of relapse in acute promyelocytic leukaemia treated with up-front arsenic trioxide-based regimens.

SummaryThe standard of care for patients with acute promyelocytic leukaemia (APL) relapsing after front‐line treatment with arsenic trioxide (ATO)‐based regimens remains to be defined. A total of 67 patients who relapsed after receiving ATO‐based up‐front therapy and were also salvaged using an ATO‐based regimen were evaluated. The median (range) age of patients was 28 (4–54) years. While 63/67 (94%) achieved a second molecular remission (MR) after salvage therapy, three (4·5%) died during salvage therapy. An autologous stem cell transplant (auto‐SCT) was offered to all patients who achieved MR, 35/63 (55·6%) opted for auto‐SCT the rest were administered an ATO + all‐trans retinoic acid maintenance regimen. The mean (SD) 5‐year Kaplan–Meier estimate of overall survival and event‐free survival of those who received auto‐SCT versus those who did not was 90·3 (5·3)% versus 58·6 (10·4)% (P = 0·004), and 87·1 (6·0)% versus 47·7 (10·3)% (P = 0·001) respectively. On multivariate analysis, failure to consolidate MR with an auto‐SCT was associated with a significantly increased risk of relapse [hazard ratio (HR) 4·91, 95% confidence interval (CI) 1·56–15·41; P = 0·006]. MR induction with ATO‐based regimens followed by an auto‐SCT in children and young adults with relapsed APL who were treated with front‐line ATO‐based regimens was associated with excellent long‐term survival.