Arsenic in the treatment of newly diagnosed acute promyelocytic leukemia: current status and future research direction

Abstract

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia. In past decades, intensive studies on the biology and treatment of this disease have resulted in a remarkably thorough understanding of its pathogenesis and improvement of treatment outcomes. In particular, the introduction of all-trans retinoic acid to conventional chemotherapy improved dramatically the remission and survival rates of APL patients and consequently became the major treatment modality for it. In the last decade, the groundbreaking development of arsenic further improved the survival rate of APL patients. As the most active agent in APL, arsenic directly degrades the PML-RARα fusion transcript, leading to the differentiation and apoptosis of leukemia cells and the potential eradication of APL leukemia-initiating cells (LICs), thus making the disease a potentially curable type of leukemia. More notably, the recent development of oral arsenic compounds may further enhance not only clinical outcomes but also the convenience of patients, which may dramatically change the APL clinical scenario in the near future.

Keywords

acute promyelocytic leukemia / arsenic / all-trans retinoic acid / survival

Introduction

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia with a distinctive balanced reciprocal translocation between chromosomes 15 and 17, generating a fusion transcript promyelocyte (PML) and retinoic acid receptor-α (RAR-α) genes (PML-RARα) [1,2]. Intensive studies on the biology and treatment of this disease in the past decades have resulted in a remarkably thorough understanding of its pathogenesis and much improved treatment outcomes [3]. In the 1980s, the Shanghai Institute of Hematology’s pilot study on the treatment of the disease with all-trans retinoic acid (ATRA) demonstrated that leukemic promyelocytes have the unique capability to undergo differentiation with exposure to ATRA [4]. The clinical complete remission rate (CR) dramatically increased up to 90%. Subsequent clinical trials established the strategy of combined ATRA and conventional chemotherapy mostly involving anthracyclines which increases not only the CR rate but also long-term survival in terms of disease-free survival (DFS) and overall survival (OS) up to 70%-80% [5-7]. However, despite the success of contemporary strategies using ATRA plus anthracycline-based chemotherapy leading to long-term survival in an overwhelming majority of patients with newly diagnosed APL, there still remains a sizable proportion of patients who will eventually relapse. Further, there are also some potential long-term sequelae such as second malignancies including myelodysplastic syndromes and delayed cardiomyopathy associated with chemotherapy [8]. In the last decade, the notable development of arsenic use in the clinical treatment of APL and its molecular mechanism may be considered as a secondary milestone in the field of APL [9-12]. In the current study, the newly discovered mechanism of arsenic, its clinical use, and its potential future research direction in the development process are presented and discussed.

Clinical use of arsenic in APL

Arsenic as a single agent for APL

In a dose-dependent manner, arsenic trioxide (As2O3) induces differentiation and causes the apoptosis of promyelocytes in APL [9]. As a single agent, it is effective in inducing remission in patients with relapsed APL, and it has also been proven to be equally effective in newly diagnosed APL [11]. Most initial published studies on both relapsed and newly diagnosed APL treated with single-agent As2O3 have limited follow-ups, thus making the assessment of its long-term outcome in terms of both survival and toxicities difficult [11-13].

In 2006, an Indian group reported 72 newly diagnosed APL patients treated with single-agent As2O3 [13]. As2O3 was administered initially until CR or a maximum of 60-75 days. Following a 4-week interval, for those in CR, single-agent As2O3 was administered for another 4 weeks as a consolidation course. Then after a second 4-week interval, for those continuing to remain in CR, single-agent As2O3 was administered 10 days in a month for 6 months. Complete hematologic remission was achieved in 86.1% of the subjects. With a median follow-up of 25 months (8-92 months), the three-year event-free survival (EFS), DFS, and OS were 74.9%±5.6%, 87.2%±4.9%, and 86.1%±4.08%, respectively. Patients with a white blood cell (WBC) count lower than 5 × 10⁹/L and a platelet count higher than 20 × 109/L at diagnosis (n = 22) have an excellent prognosis with this regimen (EFS, OS, and DFS of 100%). The toxicity profile in the majority was mild and reversible. In the group’s latest update report, a total of 13 cases had relapse without additional toxicities observed [14]. With a median follow-up of 60 months, the five-year EFS, DFS, and OS were 69%±5.5%, 80%±5.2%, and 74.2%±5.2%, respectively. Again, the report confirmed that for the patients in the good risk group, the OS remained 100% over the follow-up period. Further, Ghavamzadeh et al. reported the result of the treatment of newly diagnosed patients with only two cycles of single-agent As2O3, but the DFS at 2 years was only 63.7% [15].

The largest series of APL that received As2O3 as monotherapy was reported by Zhang et al. as a retrospective analysis in 242 newly diagnosed or relapsed patients [16]. The remission rate was 87.9% for 136 untreated patients, and the relapse rate at 3 years after CR was 26.7%. The five- and seven-year OS rates were 92.0% and 76.7%, respectively. Overall, the arsenic treatment was well tolerated with mild side effects. The most interesting finding in the report is that the central nervous system leukemia (CNSL) occurring quite frequently in relapsed patients is accompanied with extramedullary residual leukemia or relapse. The determination of arsenic concentration in cerebrospinal fluid indicated that arsenic cannot penetrate the blood-brain barrier effectively. This suggests that an intrathecal prophylaxis should be given to reduce the incidence of CNSL at least in high-risk or relapsed patients.

The above data clearly demonstrated that the use of single-agent As2O3 for newly diagnosed APL is feasible compared with conventional ATRA-based or chemotherapy regimens. In the low-risk group, the long-term result was at least comparable with that of conventional ATRA+ chemotherapy, whereas additional interventions would probably be required in high-risk patients. Moreover, only two cycles of arsenic were found to be inadequate in the control of the disease.

Arsenic combined with ATRA for newly diagnosed APL

As the most active agent in APL, arsenic can degrade the PML-RARα fusion transcript directly, leading to the apparent differentiation and apoptosis of leukemia cells which may also synergize with ATRA in eradicating the APL in animal models [17-19].

Early combination therapy with ATRA and arsenic is limited to relapse and refractory patients [20]. Subsequently, a series of studies has been carried out in newly diagnosed patients administered with combined As2O3 and ATRA and intensive chemotherapy, minimal chemotherapy, or without chemotherapy [21-25]. In a small-scale pilot randomized study in the Shanghai Institute of Hematology, we compared the results of combined ATRA and As2O3 as induction in 21 newly diagnosed APL with ATRA (n = 20) or arsenic (n = 20) monotherapy [21]. Overall, 20 of the 21 patients obtained CR which was comparable to those of ATRA (19/20) and arsenic (18/20). Notably, the combination therapy led to a more rapid response (median of 25 days for the combination group vs. 40 and 31 days for ATRA and arsenic, respectively). With a limited follow-up of 18 months (8-30), none of the CR patients in the combination group relapsed, whereas 7 out of 37 patients in the monotherapy group relapsed (P<0.05). Subsequently, we started a new single-arm study by introduction of ATRA/arsenic and chemotherapy as induction, followed by a three-cycle chemotherapy consolidation, and then five cycles of sequential use of ATRA, arsenic, and low-dose chemotherapy [22]. In a total of 85 patients enrolled from 2001, 80 patients (94%) obtained CR, and only 3 patients experienced relapse. This resulted in a five-year EFS and OS for all patients at 89.2%±3.4% and 91.7%±3.0%, respectively. The five-year relapse-free survival (RFS) and OS for patients who achieved CR were 94.8%±2.5% and 97.4%±1.8%, respectively.

The MD Anderson Cancer Center group introduced a different regimen that combines ATRA and arsenic as induction and post-remission therapy for a total of five cycles [23]. Modest amounts of chemotherapy or gemtuzumab ozogamicin (GO) were given during the induction and post-remission therapy only for patients who presented with a high-risk disease. A total of 82 patients with APL were treated accordingly. The first 65 patients received ATRA and As2O3 (beginning on day 10 of ATRA), whereas high-risk patients received GO on the first day. The second cohort of 17 patients received ATRA and As2O3 concomitantly on day 1 and GO on day 1 if high risk or if their WBC increased to more than 30 × 10⁹/L during induction. Overall, 74 patients achieved CR, and 1 achieved CR without platelet recovery after the induction. With a median follow-up of 99 weeks (2-282), 3 patients experienced relapse at 39, 52, and 53 weeks, and the other 3 died in CR at 14, 21, and 71 weeks all from the second malignancy. Overall, the three-year OS was 85% [24].

More recently, the North American Leukemia Intergroup Study C9710 reported the role of ATRA plus arsenic as a consolidation treatment for patients in first CR [25]. In their randomized study, a total of 481 untreated APL patients received a standard induction of ATRA, cytarabine, and daunorubicin, followed by two courses of consolidation therapy with ATRA plus daunorubicin or two 25-day courses of ATRA+ As2O3 consolidation immediately after induction. Ninety percent of the patients on each arm achieved CR, and the three-year EFS was significantly better for those patients who received receive As2O3 consolidation (80% vs. 63%, P<0.0001). The survival rate also tended to be better in the As2O3 arm at 86% compared with 81% at 3 years (P = 0.059). Further, the three-year DFS was significantly better in the As2O3 arm (90% vs. 70%, P<0.0001).

Compared with the single arsenic regimen, the above data demonstrated that the combination of ATRA and arsenic further improved the long-term survival in newly diagnosed APL patients. Therefore, it should be considered as the mainstream treatment modality for the arsenic-based regimen. We may also conclude that the upfront treatment with arsenic either as induction or consolidation significantly improved the outcome. Therefore, clinicians have gradually adopted the use of combined ATRA and arsenic, along with the addition of limited doses of anthracycline, in the treatment of newly diagnosed APL. This is especially useful for those who are unable to tolerate anthracyclines due to heart disease, maximal cumulative doses of anthracyclines, older adults, and/or decline in chemotherapy [8,26].

Mechanism of arsenic and synergy with ATRA in the treatment for APL

Arsenic induces differentiation and apoptosis of leukemia cells by targeting PML-RARα

In our previous study, we demonstrated that As2O3 exerts a dose-dependent dual effect on APL cells, which in turn triggers apoptosis at high concentrations (0.5-2 × 10^-6 M) and induces partial differentiation at low concentrations (0.1-0.5 × 10^-6 M) [10]. The apoptosis of APL cells was associated with a series of events such as the collapse of mitochondrial transmembrane potential in a thiol-dependent manner, activation of caspases, and modulation of PML. All these in vitro data reflect well the clinical response of APL to arsenic, which is associated with incomplete cell differentiation and induction of apoptosis with caspase activation in leukemic cells.

Further molecular analysis demonstrated that As2O3 induces a significant modulation of the PML staining pattern which includes reaggregation of PML-nuclear body (NB) antigens, recruitment of PML-RARα proteins onto NBs, and degradation of PML-RARα. Arsenic-associated PML relocation leads to sumolyation at K160, which is necessary for 11S proteasome recruitment and subsequent arsenic-induced degradation of PML-RARα [27-29]. More recently, our group demonstrated the direct binding of arsenic to the cysteine residues of zinc fingers within the RBCC domain of PML-RARα and PML by MALDI-TOF mass spectrometry, which induces a conformational change in the protein domain. This led to PML oligomerization and an increase in the interaction with the small ubiquitin-like protein modifier-conjugating enzyme UBC9, which in turn enhanced sumolytion and subsequently degraded the PML-RARα protein. This was the first time that PML was identified as a direct target of As2O3, providing new insights into the drug’s mechanism of action and its specificity for APL [30].

In mice, the combination of ATRA and arsenic causes a rapid disappearance of APL cells and the curing of leukemia [18,19]. ATRA and arsenic cooperate to induce PML-RARα degradation. ATRA targets the RARα moiety of the PML-RARα oncoprotein, whereas arsenic targets its PML part which may synergize a more rapid degradation of PML-RARα. This supports the use of ATRA and arsenic combination therapy in clinical scenarios [31].

Arsenic synergy with ATRA targeting APL leukemia cells and potentially leukemia stem cell or LICs

Leukemia stem cells or leukemia-initiating cells (LICs) are a minority of leukemic cells that can self-renew and are resistant to conventional treatment such as chemotherapy. LICs are believed to be associated with the development of clinical refractoriness and relapses [32]. Several studies have shown that PML-RARα confers immortalization and self-renewal properties to APL leukemic cells and can initiate APL in mice models, raising the possibility that targeting PML-RARα may be important to the differentiation block and to progenitor immortalization events [33,34].

Several recent studies have compared the fate of LIC upon ex vivo or in vivo treatment with ATRA or arsenic [35-38]. ATRA at a lower dose required for clinical induction in vivo was found adequate for initiating differentiation, but LIC clearance is strictly observed only at high concentrations in a sharp dose-dependent manner [35]. In the clinical setting, relapses can appear in patients with ATRA plasma concentrations while still triggering differentiation, thus providing evidence that ATRA induction of differentiation reflects the reversal of PML-RARα-dependent transcriptional repression, but LIC clearance is the consequence of PM-RARα catabolism. Meanwhile, arsenic is more potent and specific [36].

An initial in vitro study showed that arsenic, rather than ATRA, can inhibit the leukemia stem cells derived from APL cell lines [37]. De Thé’s group demonstrated well in their elaborate experiments that arsenic is critical in the eradication of APL LIC in mice models [38]. The addition of Bortezomib, a proteasome inhibitor which inhibits the proteolysis process, counteracts the arsenic effect dramatically. These data suggested that differentiation has a major role in the debulking of APL cells, whereas PML-RARα degradation seems to be the primary basis for APL eradication by rapidly clearing LIC, which may result in APL eradication in the murine APL model [35]. This observation is critically important in that it does not only echo the improved long-term clinical outcome that results from the use of combined ATRA and arsenic but also serves as the key evidence supporting the clinical use of such combined treatment modality as a potential cure regimen for APL [38].

The effects of arsenic on LICs are further supported by another line of experiments carried out by Ito et al. in the study of the chronic myeloid leukemia (CML) mice model using Pml^+/+ and Pml^-/- BM cells transduced with p210bcr-abl and then cultured on stromal cells to enrich the LICs, followed by the subsequent transplantation mice model [39]. The authors demonstrated that Pml^-/- LICs failed to generate CML-like disease contrary to Pml^+/+ LICs, indicating that Pml-deficiency impaired LIC maintenance. More interestingly, the authors also demonstrated that As2O3 treatment can decrease Pml expression reversibly in hematopoietic stem cell (HSC) compartment, which results in the dramatic attenuation of colony-formation capability in vitro and impaired HSC quiescence in vivo. However, As2O3 did not affect Pml^-/- HSCs, indicating that the effect is Pml dependent. Subsequently, long-term culture-initiating cell assays revealed the remarkable inhibitory effect of As2O3 on LIC maintenance, and arsenic followed by Ara-C exposure significantly increased the efficacy of Ara-C-mediated induction of apoptosis, resulting in the eradication of LICs. The present study further highlighted the importance of arsenic targeting LIC and provided critical laboratory evidence on the role of arsenic in the treatment of CML and other leukemia types.

Toxicity of Arsenic

Despite the high cure rates of ATRA and As2O3 treatment, induction mortality at around 5%-9% remains a key problem in APL [8]. The most common causes of death were hemorrhage, infection, and the differentiation syndrome (DS) or retinoic acid syndrome. DS is characterized by respiratory distress, unexplained fever, weight gain, interstitial pulmonary infiltrates, and pleural or pericardial effusions [40,41]. Further, DS is often associated with the development of hyperleukocytosis and pulmonary edema. DS is reported in up to 30% of APL patients who receive ATRA induction therapy as well as arsenic therapy which is not observed during consolidation or maintenance therapy with ATRA and/or As2O3. This implies that APL cells play a crucial role in the development of DS [8,40,41]. In our pilot study, combined induction with ATRA and arsenic for newly diagnosed APL did not increase the frequency of hyperleukocytosis and DS compared with ATRA or arsenic monotherapy [21].

Aside from DS, at the doses used to induce remission in APL, As2O3 is not associated with the toxicities commonly seen with chemotherapy. However, it presents unique toxicity profiles such as liver toxicities, skin rashes, and cardiac toxicities causing QT prolongation [42,43]. Recently, Kojima et al. reported the role of arsenic methylation in arsenic-induced oxidative DNA damage associated with cancer development related to arsenic exposure and other toxicities [44]. The clinical use of As2O3 is metabolized to mono- and dimethylated arsenicals. The formation of reactive oxygen species by arsenic trioxide can initiate a cascade of intracellular signaling events which leads to the apoptosis of APL cells and also arsenic-associated cell transformation.

In our previous studies, up to 80% of patients experienced grade I or II liver toxicities. For most patients, supportive care and a temporary reduction in infusion dose by 50% were sufficient for the recovery of liver function. Only a few patients required suspension of arsenic treatment [22]. We also followed up those patients who received a total of six cycles of arsenic treatment for their long-term toxicity. In 33 eligible patients, there were no abnormal ECGs and echocardiograms. Serum tumor markers screening revealed a slight increase of the two indicators in two patients, but these returned to normal in the subsequent follow-up. Up to the last follow-up with a median of six years, no secondary malignancies were observed. Meanwhile, our data demonstrated that when the patients received arsenic treatment, their plasma arsenic concentration was increased to 49.3±13.0 ng/mL, and their urine arsenic concentration was also highly increased to 330.2±232.1 ng/mL. For long-term survivors, their plasma and urine arsenic concentrations decreased to 26.4±2.1 and 10.4±7.4 ng/mL, respectively, which were slightly higher than those for the healthy controls (18.4±6.0 and 4.2±1.6 ng/ml). Notably, the plasma and urine arsenic levels in long-term survivors were within the normal or safe level according to various standards of the Ministry of Health of China, the US Agency for Toxic Substances and Disease Registry, and the Trace Element Reference Values in the Human Tissues project of Europe [22].

Based on the preceding data, we may conclude that arsenic combined with ATRA does not exaggerate DS. With five cycles of arsenic, arsenic treatment is safe in terms of reasonable tolerance and long-term toxicities. However, due to the limited number of patients involved and limited follow-ups done in the current study, long-term toxicities (median of 10 years or even longer) such as arsenic-associated cancer (cancers of the skin, lung, urinary bladder, and potentially the kidney) remain a key concern and should be monitored closely [45]. Therefore, the accumulation of more clinical data is clearly warranted. On the other hand, clinical studies that better define the optimal dose and cycles of arsenic in APL patients may further reduce the potential toxicities associated with arsenic.

New arsenic formula

Aside from the conventional intravenous As2O3, several studies have focused on the development of oral arsenic formula. If these newly emerged arsenic compounds are fully proven to be safe and effective, they may dramatically change the future clinical scenario for APL patients in terms of convenience and hospitalization costs [46-50].

As4S4

Realgar, an ore composed mainly of tetra-arsenic tetra-sulfide (As4S4), is commonly used as a traditional medicine. In 2002, Lu et al. from Beijing University reported on the oral preparation of highly purified crystalline As4S4 [46]. In their study, they tested the compounds as a single agent in 23 patients. CR was achieved in 14 of 16 patients with newly diagnosed disease and 5 of 7 patients with hematologic relapse. The estimated DFS rates for 1 and 3 years were 86.1% and 76.6%, respectively, with a median follow-up of 13.5 months (2-40 months) in the newly diagnosed group. Overall, As4S4 was well tolerated with only moderate side effects such as asymptomatic prolongation of corrected QT interval, transient elevation in liver enzyme levels, rashes, and mild gastrointestinal discomfort. Neither myelosuppression nor appreciable long-term side effects were found.

Meanwhile, an independent group in China also developed a Realgar-based formula, the Realgar-Indigo naturalis formula (RIF), in which mined ore realgar is the principal element, whereas Indigo naturalis, Salvia miltiorrhiza, and Radix psudostellariae are adjuvant components that assist the effects of realgar [47,48]. In an initial phase II randomized double-blinded trial compared with ATRA as induction therapy for newly diagnosed APL, RIF was given starting from 0.75 to 1.25 g tid and increased to a daily dose at 7.5 g/d if well tolerated by the patient. Oral RIF was given until remission was documented or for a maximum of 60 days [47]. The CR rate was 96.7% for the RIF group compared with 94.9% for the ATRA group, with the median days to CR comparable in the two groups (49 vs. 44). In the RIF group, 40% of the patients experienced side effects, mostly gastric intestinal discomfort, skin rashes, and an abnormal hepatic enzyme. In a larger retrospective analysis of patients with newly diagnosed APL treated with RIF as induction and post-remission therapy, the five-year overall survival rate was 86% [48]. Based on promising data, a national multiple-center randomized trial was initiated in China to compare As2O3 and RIF directly both as induction and maintenance therapy in newly diagnosed patients.

Oral arsenic trioxide

Interestingly, a group from Hong Kong University developed an oral solution of As2O3 which gives a similar bioavailability but lower peak plasma arsenic concentrations compared with the conventional intravenous As2O3 [49]. In their updated report of 17 relapsed patients aged 50 to 78 years receiving oral arsenic at 10 mg/d as reinduction therapy, 16 patients achieved CR and 1 died of sepsis [50]. After CR, all patients received maintenance therapy comprising oral As2O3 (10 mg/d) and ATRA (45 mg/m2/d) for 2 weeks every 2 months for a total of 2 years. At follow-up, 1 patient died of relapse and another due to tuberculosis at 16 and 12 months, respectively. All other patients were alive with continuous remission. More importantly, the authors monitored closely the QT prolongation and demonstrated that oral arsenic may have a much limited effect on QT prolongation. In more than 600 weeks of oral As2O3 administered in five years, no ventricular tachyarrhythmias were observed which may occur more frequently with intravenous arsenic. Based on all these data, we suggest that oral arsenic may represent a major advancement in arsenic therapy, making long-term therapy feasible and facilitating clinical trials.

Future research direction

Advancements in the treatment of APL in the past decade dramatically improved the long-term outcome of the disease. However, the optimization of the therapy is further sought in succeeding years [3].

First, the combination of ATRA and As2O3 has emerged as a potentially effective regimen for newly diagnosed APL. It can be used as a platform for further clinical trials. For example, in low-risk patients, the effect of the combination may be tested, which aims to decrease or eliminate chemotherapy and to limit the cycles of arsenic. Large-scale clinical trials are warranted because the goal is to reduce potential toxicities while maintaining the curable effectiveness. More importantly, this is the first time that a concept of combined molecular targeting therapy without chemotherapy and with minimal side effects is tested in order to cure a potentially lethal leukemia. For those patients with high-risk disease or poor responders at molecular levels, the effect of the addition of cytotoxic treatment such as anthracycline-based and/or or gemtuzumab ozogamicin treatment should be tested.

Second, the upfront use of arsenic has been demonstrated clearly in a single-arm or randomized study, and different strategies to incorporate arsenic in the protocol (induction+ consolidation vs. consolidation vs. induction+ maintenance) have been found, with a large variance in the total arsenic dose (from 2 cycles to a total of 12 cycles) given in different protocols. Further clinical trials to define clearly the optimal strategy, particularly the minimum cycles required for long-term disease control with limited toxicities, are warranted (Table 1).

Third, the notable development of oral arsenic formulas which may potentially replace intravenous ones in the near future may not only enhance patients’ treatment convenience but also decrease the toxicity of intravenous As2O3. Head-to-head comparisons in clinical trials to test these new formulas are currently underway, and new results may be expected in the coming years which will establish the long-term safety and effectiveness of the combined method. Although it is still too early to make any conclusion, we may still envision out-patient-based oral ATRA and arsenic treatment with minimal or even without cytotoxic-based chemotherapy as the potential mainstream treatment modality for APL in the near future.

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