Introduction

Until a decade ago, the remission induction therapy of acute promyelocytic leukemia (APL) was the most difficult one among that of acute leukemia, and it required enormous efforts from both the patients and medical staff to get a complete remission (CR). This was especially true before sufficient platelet transfusions became routinely available through the use of blood cell separators. The management of excessive hemorrhaging due to disseminated intravascular coagulation and fibrinolysis was quite difficult at that time. Besides, hemorrhaging became exacerbated after the start of chemotherapy due to the destruction of leukemia cells, and many patients died of hemorrhaging before reaching CR. The introduction of all-trans retinoic acid (ATRA) in the late 1980s has dramatically changed these situations and marked a major advance in the treatment of APL.^1,2,3,4 With ATRA-based induction therapy, around 90% of newly diagnosed patients with APL now achieve CR and over 70% of patients are curable with subsequent postremission chemotherapy, with or without ATRA.^{5,6,7,8,9,10,11,12,13,14,15}

The treatment with ATRA was initially called a differentiation therapy, which is still true as an observed phenomenon. Subsequent analysis of the differentiation, however, has elucidated its mechanism, and now the ATRA treatment is regarded as a molecular-targeted therapy aimed at the pathogenetic molecule of this leukemia, that is, PML/RAR. Thus, ATRA has turned to be the first successful molecular-targeted drug in the history of cancer therapy.

The treatment outcomes of other acute leukemias have probably reached their maximal limit during the past decade as far as currently available cytotoxic drugs are used. To make a major breakthrough for further increase of the cure rate of these diseases, targeting therapy on leukemogenic molecules is presently the most promising approach and should be developed further. In this review, we will discuss the treatment of APL with special emphasis on the strategy toward further increase of cure rate, and how to utilize the experience of ATRA therapy in molecular targeted therapy, not only for other types of leukemia but for other cancers in future as well.

Remission induction therapy of APL

Beyond question, ATRA has become the first choice drug for newly diagnosed patients with APL from a medical, economical and patient-quality-of-life point of view.^16,17 ATRA, with or without cytotoxic drugs, is now able to induce over 90% CR in these patients. Some early studies mainly using ATRA alone failed to obtain such high CR rates in newly diagnosed APL patients. For example, the North American Intergroup study reported only a 72% CR rate in the ATRA arm compared to 69% CR in the chemotherapy arm.⁷ The low CR rates in the early to mid-1990s plausibly came from the inexperience of hematology oncologists in the hitherto unfamiliar differentiation therapy. Hematology oncologists at that time tended to discontinue ATRA when confronted with an increasing number of peripheral blood leukocytes during the administration of ATRA. They judged the treatment as a failure based on their experience of chemotherapy with cytotoxic drugs. If the same patients were treated by the same oncologists in 2003, CR rates would definitely be higher by around 20% because the oncologists have accumulated considerable experience and knowledge as to how to handle the initial ATRA-induced leukocytosis and complications.

To control the leukocytosis and consequent retinoic acid (RA) syndrome, concomitant use of cytotoxic drugs such as anthracyclines and cytarabine (Ara-C) is indispensable. Since leukemia cells from patients with APL are particularly sensitive to anthracyclines (perhaps because of significantly lower P-glycoprotein expression and other resistance markers in APL cells compared to other subtypes of acute myeloid leukemia (AML)^18,19,20), anthracyclines may be more important than Ara-C. In fact, shortly before the ATRA era, Gruppo Italiano per le Malattie Ematologiche dell'Adulto (GIMEMA) compared idarubicin (IDR) alone to IDR+Ara-C in the remission induction therapy of 257 patients with newly diagnosed APL in a prospective randomized study. Overall, 76% of the patients in the IDA arm alone and 67% of patients in the IDR+Ara-C arm achieved CR, and event-free survival (EFS) rates were 35 and 23%, respectively (P=0.0352).²¹ The results suggest that monotherapy with IDR favorably influences the EFS of patients with newly diagnosed APL. However, the dose of IDR in the monotherapy arm was 72 mg/m² compared to 40 mg/m² in the combination arm. Therefore, patients in the monotherapy arm received 1.8 times as much anthracycline as in the combination arm.

Do all patients need cytotoxic drugs to obtain CR?

Whether all patients with APL require concomitant use of cytotoxic drugs is still controversial. Many patients with low initial leukocyte counts achieve CR with ATRA alone. For example, among 369 newly diagnosed patients with APL (median leukocyte counts, 2000/l) in the APL92 study of the Japan Adult Leukemia Study Group (JALSG), 223 patients (60%) received daily oral ATRA alone according to the study protocol which indicated to give ATRA 45 mg/m² alone if patients' initial leukocyte counts were <3000/l. According to this protocol, 126 patients (57%) continued ATRA alone and 119 (94%) of them obtained CR, and 97 patients (43%) received additional chemotherapy (daunorubicin+behenoyl cytarabine) at the time when their blasts and promyelocytes increased >1000/l and 85 (88%) of them achieved CR. Thus, 126 (34%) of 369 patients were able to achieve CR with ATRA alone, and these patients belonged to the good prognosis group for survival.^9,15 Obviously potentially carcinogenic and organotoxic cytotoxic drugs should best be avoided whenever possible. However, if initial leukocytes are >3000–5000/l, cytotoxic drugs should be concomitantly added to ATRA. If patients have >10 000/l leukocytes, concomitant use of more intensive chemotherapy is indispensable, because a level of >10 000/l leukocytes is shown to be an independent adverse prognostic factor in the JALSG APL92^9,15 and the British Medical Research Council (MRC) APL studies.¹⁰

GIMEMA⁵ and the Spanish PETHEMA group¹³ use ATRA and IDR (AIDA) for all patients regardless of leukocyte counts. The European APL Group compared two induction schedules in 208 newly diagnosed APL patients of age 65 years and with 5000 initial leukocytes in a prospective randomized study. The ATRA followed by chemotherapy group received daily ATRA and DNR+Ara-C if the leukocytes increased to >6000, >10 000, or >15 000/l by days 5, 10 and 15 of ATRA treatment, respectively, and the ATRA+chemotherapy group received the same combination of ATRA and DNR+Ara-C starting on day 3 of ATRA treatment. Relapse at 2 years was estimated at 6% in the ATRA+chemotherapy group vs 16% in the ATRA followed by chemotherapy group (P=0.04), and EFS at 2 years was estimated 84 vs 77%, respectively (P=0.1).¹¹ The finding suggests that early addition of chemotherapy to ATRA can reduce the incidence of relapse in APL. They also recommend simultaneous administration of ATRA and chemotherapy in patients with high initial leukocyte counts to reduce the risk of severe ATRA syndrome.

MRC reported no benefit from ATRA among APL patients presenting with a high initial leukocyte count (>10 000/l). The predicted 5-year overall survival (OS) of patients receiving daily ATRA on the first day of chemotherapy until CR (extended ATRA) was 43% and the same for that of patients receiving a 5-day course of ATRA before commencing chemotherapy (short ATRA). In patients with a lower leukocyte count (<10 000/l), however, extended ATRA gave better OS: 80 and 57%, respectively (P=0.0025).¹⁰ In addition, the European APL Group observed that even patients with initial leukocyte counts >10 000/l were benefiting from the combination of ATRA and chemotherapy compared with the chemotherapy alone group in the APL91 randomized trial. The predicted 4-year EFS was 50% in the ATRA group and 15% in the chemotherapy group (P=0.04) among patients presenting with initial leukocyte counts >10 000/l.¹² JALSG also noted a beneficial effect of ATRA in combination with chemotherapy in patients with initial leukocyte counts >10 000/l compared with the same cohort of historical patients in their group before the ATRA era. Predicted 5-year OS of 93 patients with 10 000/l initial leukocyte counts receiving ATRA in the APL 92 study was 60%, while that of 37 patients receiving chemotherapy alone in the AML87 and AML89 studies was 37% (P=0.0445) (unpublished data). Thus, ATRA also appears beneficial in APL patients with a high leukocyte count.

Concomitant use of more intensive chemotherapy will probably improve the prognosis of patients with higher leukocyte counts even with currently available drugs. For example, the German AML Cooperative Group (GACC) employs intensified double induction therapy including high-dose Ara-C and mitoxantrone in combination with ATRA, although patients are of age 60 years and the follow-up period is too short to permit definitive conclusions. The early results show a CR rate of 92% and a 2-year relapse-free survival of 96%.¹⁴ Thus, ATRA+intensified chemotherapy seems to give better results regardless of leukocyte counts in younger patients who can tolerate intensive chemotherapy.

Can we induce a 100% CR rate in newly diagnosed APL?

So, with accumulating experience, the next question is whether CR can be obtained in almost all newly diagnosed patients with APL. The answer will probably be no. In multicenter studies, there are about 5–10% early deaths within 1 month after the start of ATRA treatment with or without chemotherapy.^{5,6,7,8,9,10,11,12,13,14,15,17} The cause of death is mainly intracranial bleeding. Even in individuals without APL, intracranial bleeding occurs more frequently in elderly people. Thus, elderly APL patients would be more prone to intracranial bleeding. Among 369 consecutively registered, newly diagnosed APL patients in the JALSG APL92 study, only one out of 72 patients of age <30 years failed to achieve CR, while six (32%) out of 19 patients of age 70 years failed to do so chiefly due to early death by hemorrhaging in the cranium or other sites such as the lungs.¹⁵ Younger patients can generally overcome the APL-associated coagulopathy, but not elderly patients.

Whether there is an intrinsic ATRA resistance in APL was extensively reviewed in the latest issue of this journal.²² We believe that hematology oncologists in major centers will be able to induce CR by ATRA-based regimens in almost 100% of APL patients, if patients do not have high leukocyte counts nor pre-existing coagulopathy. We assume that this is also true even in reportedly ATRA-resistant subtypes such as APL with t(11;17) or PLZF/RAR, if experienced hematology oncologists use cytotoxic drugs and/or granulocyte colony-stimulating factors in addition to ATRA,^23,24,25 although such subtypes are too rare to make firm conclusions.

If some studies report more than 95% of CR and claim that their regimens are superior, the regimens themselves are probably not the reason for the high CR rate. As discussed above, if only patients with good prognostic factors, such as those of a younger age and without pre-existing coagulopathy, are registered or analyzed, current regimens employed in most multicenter study groups would probably produce nearly 100% CR. The problem is whether patients are diagnosed early before the APL-associated coagulopathy develops. Thus, if community doctors are well educated on APL and diagnose this leukemia at its early stage, the CR rate will plausibly increase. In APL, the number of leukocytes at diagnosis is rather low in the majority of patients. The median numbers of leukocytes are around 2500/l at diagnosis in most multicenter studies. The reason why leukopenia rather than leukocytosis is common in APL is not well understood. One reason is that APL is diagnosed earlier than other types of AML, because APL patients often manifest hemorrhagic diatheses due to the coagulopathy and thus patients visit doctors rather early. This explanation would be supported by the fact that, even in other types of AML, leukopenia is a common laboratory finding preceding relapse. Therefore, if patients notice purpura or any abnormal bleeding tendency and have prompt access to community doctors, they can be diagnosed as having APL at its early stage by competent doctors and then immediately referred to hematology oncologists. Thus, the CR rate and consequently cure rate of this leukemia will increase. Otherwise, the remission rate could probably not surpass 95% in multicenter studies.

Coagulopathy during remission induction phase

GIMEMA assessed the clinical effectiveness of ATRA on the incidence of early hemorrhagic deaths and on APL-associated coagulopathy in a total of 622 consecutive patients with APL treated within the group during 1989–1997. Of those, 499 were treated with IDR plus ATRA (Study A) and 123 with IDR alone (Study B). Deaths occurring within 10 days of starting treatment were 4% in Study A and 7% in Study B (P=0.09), with 3 and 4% due to hemorrhaging. Overall induction mortality was 8 and 16%, respectively (P<0.003). In Study A, days with platelet counts 20 000/l or with fibrinogen 1 g/l were reduced by about 30%, the hemorrhagic score by 50% and the consumption of blood products by about 40%, and fewer patients were treated with antihemorrhagic drugs (39 vs 61%; P<0.001). Early deaths were influenced by a blast count >30 000/l at diagnosis (P<0.001) in both studies.²⁶ This analysis indicated that even ATRA could not prevent early fatal hemorrhages within 10 days in the advanced APL, although a substantial clinical improvement was evident in terms of reduction of the severity of bleeding symptoms, blood product consumption and overall induction mortality when ATRA was combined with IDR.

The coagulopathy in APL is mainly related to the tumor burden in patients' bodies. Thus, in patients with fewer numbers of leukocytes at the start of ATRA therapy, coagulopathy seldom occurs. The bulk of evidence clearly shows that ATRA has a profound impact on the hemostatic system including thrombomodulin²⁷ and annexin II,²⁸ thus leading to rapid resolution of the APL-associated coagulopathy.²⁹ ATRA has an effect on the leukemia cell functions in hemostasis, which are considered major pathogenetic determinants for the coagulopathy. These anticoagulant effects on tumor cells occur together with the drug's anticoagulation effects on normal endothelial and monocytic cells.²⁹ In some patients with a high number of leukocytes, however, the coagulopathy already exists before they reach oncology centers, and in these patients even ATRA therapy cannot rescue life-threatening hemorrhaging. Additionally, no or minor purpura at diagnosis is a significant independent favorable prognostic factor for achieving CR in the JALSG APL92 study.⁹ Therefore, it is very important for APL to be diagnosed at its early stage and have ATRA started as soon as possible.

RA syndrome during remission induction phase

Another problem during the remission induction phase is the RA syndrome.^30,31,32 The incidence of this syndrome ranges from 6 to 31%, and was apparently higher when ATRA alone was used in remission induction therapy at the early ATRA era.¹⁷ Since the RA syndrome was not reported when ATRA was used in postremission therapy in APL as well as in other diseases, it is evidently associated with the increase of neutrophils due to their differentiation by ATRA. Physiologically, around 10–50 times more neutrophils are said to be present extravascularly and thus, even if some patients show no increased numbers of peripheral leukocytes at the presentation of RA syndrome, this does not exclude the association between the increase of neutrophils and this syndrome, because leukocytosis eventually appears even in cases without leukocytosis at presentation. The median peak leukocyte counts in patients who developed the syndrome were 36 400/l (range, 6300–82 300/l) in one report³⁰ and 31 000/l (range, 6800–72 000/l) in another.³² Of note, RA syndrome is also observed during the remission induction therapy with arsenic trioxide (As₂O₃) due to a similar mechanism.³³

The European APL Group analyzed cases of RA syndrome in newly diagnosed APL. Out of 413 patients, 64 (15%) experienced ATRA syndrome during induction therapy. Clinical signs developed after a median of 7 days (range, 0–35 days). In two patients, they were already present before the start of ATRA. A total of 60 patients received chemotherapy in addition to ATRA, 58 also received high-dose dexamethasone, and ATRA was stopped when clinical signs developed in 30 patients. In total, 86% of patients who experienced RA syndrome achieved CR, as compared to 94% of patients who had no RA syndrome (P=0.07) and 1.2% died of RA syndrome. None of the patients who received ATRA for maintenance had RA syndrome recurrence. No significant predictive factors of ATRA syndrome, including pretreatment leukocyte counts, could be found. Kaplan–Meier estimates of relapse, EFS and survival at 2 years were 32, 63 and 68% in patients who had RA syndrome as compared with 15, 77 and 80% in patients who had no ATRA syndrome (P=0.05, 0.003, and 0.03), respectively. In a stepwise Cox model, RA syndrome remained predictive for EFS and survival.³¹

The USA Intergroup also examined the incidence, clinical course and outcome of patients with newly diagnosed APL who developed RA syndrome. In total, 44 (26%) of 167 patients developed the syndrome at a median of 11 days of ATRA (range, 2–47 days). The median leukocyte count was 1450/l at diagnosis and was 31 000/l at the time the syndrome developed. ATRA was discontinued in 36 (82%) of the 44 patients, and continued in eight patients, with subsequent resolution of the syndrome in seven of the eight. ATRA was resumed in 19 (53%) of the 36 patients in whom ATRA was stopped and discontinued in 17 (47%). The syndrome recurred in three of those 19 patients, with one death attributable to a resumption of the drug. Two deaths were definitely attributable to the syndrome. None of their patients receiving ATRA as maintenance developed the syndrome.³²

Currently, hematology oncologists are well aware of RA syndrome and know how to handle this – practically the sole, serious, adverse event of ATRA therapy.³⁴ Prevention of rapid development of leukocytosis by concomitant use of cytotoxic drugs is most important. Prophylactic use of corticosteroids is recommended by Australian investigators for patients whose leukocyte counts rose above 10 000/l,³⁵ but is controversial because the number of patients studied was too small and no prospective randomized trial has been conducted and also because adverse effects of corticosteroids such as diabetes mellitus may occur, especially in elderly patients. Early recognition of clinical signs of this syndrome followed by prophylactic or therapeutic use of corticosteroids is necessary. Fever, coughs, dyspnea and elevation of C-reactive protein should promptly be followed by evaluation with chest X-ray film. If the film shows any suspicious signs of RA syndrome, sufficient doses of methylprednisolone or dexamethasone should immediately be administered without hesitation. ATRA should temporarily be withdrawn. By these practices, the mortality rate from RA syndrome has currently been reduced from up to 28% in the early days of the ATRA era to less than a few percent.^17,34

Postremission consolidation chemotherapy

Most multicenter study groups give two to three courses of consolidation therapy with cytotoxic drugs for patients achieving CR. This approach is justified based on the therapeutic experience in AML. In the just-closed JALSG APL97 study, newly diagnosed APL patients received ATRA 45 mg/m² daily alone until CR if initial leukocyte counts were <3000/l; ATRA daily+IDR 12 mg/m² 2 days and Ara-C 80 mg/m² 5 days if they were between 3000 and 10 000/l; ATRA daily+IDR 12 mg/m² 3 days and Ara-C 100 mg/m² 5 days if they were >10 000/l; and additional IDR 12 mg/m² 2 days and Ara-C 80 mg/m² 5 days if peripheral blasts and promyelocytes exceeded >1000/l during the above therapy. In a preliminary analysis of 256 patients, 95% of the patients obtained CR. Around 50% of these CR patients already showed no detectable PML/RAR by RT-PCR at this stage. After three courses of intensive consolidation chemotherapy, all 220 patients tested showed no detectable PML/RAR. MRC,¹⁰ PETHEMA,¹³ GACG¹⁴ and GIMEMA³⁶ also report similar figures, but 2–10% of MRD were detectable after two to three courses of consolidation chemotherapy. Thus, consolidation chemotherapy eliminates detectable minimal residual disease (MRD) in a majority of patients. The prognosis of patients without detectable MRD seems to be better than those with it, as theoretically expected and actually reported by some.³⁷ Thus, two to three courses of intensive consolidation chemotherapy are indispensable for the cure of APL. Whether patients who have shown no detectable PML/RAR after the initial induction therapy need further consolidation chemotherapy or not remains to be clarified prospectively.

Maintenance/intensification therapy after consolidation therapy

Two large randomized studies suggest that maintenance therapy with ATRA is useful. The North American Intergroup study reported a significant benefit from daily maintenance with ATRA compared to observation. The estimated 3-year, disease-free survival (DFS) were 65 and 40%, respectively (P<0.001).⁷ The European APL93 study showed that patients receiving ATRA for 15 days every 3 months plus daily 6-mercaptopurine (6MP) and weekly methotrexate (MTX) had a lower relapse rate compared to patients treated with ATRA alone, chemotherapy alone and observation. The 2-year relapse rate was 11% in patients randomized to continuous maintenance chemotherapy and 27% in patients randomized to no chemotherapy (P=0.0002) and 13% in patients randomized to intermittent ATRA and 25% in patients randomized to no ATRA (P=0.02), although the follow-up period is too short to substantiate such low relapse rate.¹¹

Confirmatory randomized studies to see whether patients benefit from maintenance therapy with ATRA and/or low-dose chemotherapy are currently in progress. The North American Intergroup assigns patients in CR to either ATRA every other week with 6MP and MTX or to ATRA alone. GIMEMA assigns patients in CR to the same three maintenance regimens as those of the European APL Group or to observation. The GIMEMA study in particular will give valuable information on the effect of ATRA maintenance in addition to the group's rather intensive remission induction therapy with ATRA and chemotherapy (AIDA) and subsequent consolidation chemotherapy.

One problem of ATRA maintenance will be unambiguous resistance to ATRA if patients relapse during the ATRA maintenance. Most of them will possess mutations in PML/RAR gene.^22,38,39 These patients will hardly respond to new retinoids or modified ATRA preparations and thus OS may not be different from patients without ATRA maintenance, who will possibly obtain second CR by the readministration of ATRA, new retinoids, or modified ATRA preparations with or without chemotherapy. The above confirmatory studies by the North American Intergroup and GIMEMA will hopefully answer this question.

The JALSG APL97 study randomizes RT-PCR-negative patients for PML/RAR to either further intensification chemotherapy or observation after the three courses of consolidation therapy. This will also answer whether or not chemotherapy is beneficial as a maintenance therapy, as well as whether or not negative RT-PCR after three courses of intensive consolidation therapy signals the time for discontinuation of further therapy in APL.

New retinoids and liposomal formulation of ATRA

A new synthetic retinoid, Am80 (4[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carbamoyl] benzoic acid), is approximately 10 times more potent than ATRA as an in vitro differentiation inducer in NB-4 and HL-60 cells, is chemically more stable to light, heat and oxidation than ATRA, has a low affinity for cellular RA binding protein (CRABP) and does not bind to RA receptor (RAR)-.^40,41 Am80 would therefore be expected to have therapeutic effectiveness in patients with ATRA-resistant APL with increased CRABP,^22,38 and have less adverse drug reactions related to RAR-, which is the major RAR in the dermal epithelium. Japanese investigators treated APL patients who had relapsed from ATRA-induced CR with this drug. Of 24 patients, 14 (58%) achieved CR with 6 mg/m²/day Am80 orally alone. The interval from the last ATRA therapy was not different between CR and failure cases. The clinical response was well correlated with the in vitro response to Am80 in patients examined. Adverse events included RA syndrome, hyperleukocytosis, xerosis, cheilitis, hypertriglyceridemia and hypercholesterolemia but generally milder than those of ATRA, which all patients had received previously. Additionally, plasma levels of Am80 were not different on day 1 and day 21 during the daily Am80 administration, contrary to the plasma levels of ATRA, which decline considerably during daily administration.⁴² Thus, Am80 is effective in APL relapsed from ATRA-induced CR. Besides, patients who have achieved a second CR with Am80 enjoy a good prognosis with subsequent chemotherapy or stem cell transplantation (SCT). Of 14 CR patients, six underwent sibling or unrelated HLA-matched allogeneic SCT and four are alive, and four of eight patients who did not receive SCT have also survived without relapse for >4 years. MRD by RT-PCR was undetectable in all living patients.⁴³ Am80 is under the process of registration in Japan and a randomized study against ATRA will be conducted soon by JALSG.

USA Investigators evaluated an intravenous liposomal formulation of ATRA in 69 patients with APL: 32 new diagnoses; 35 relapses and two oral ATRA failures. Liposomal ATRA (90 mg/m²) was administered every other day and induced CR in 62, 70, and 20% in newly diagnosed, first relapses (ATRA naive or off oral ATRA 1 year), or relapses (second or subsequent relapse or first relapses off oral ATRA <1 year), respectively. The 1-year survival was 62, 56 and 20%, respectively. Toxicity was generally mild with, most commonly, headaches, but 26% of patients developed RA syndrome.^44,45 Liposomal ATRA is effective and provides a reliable dosage of ATRA for patients with APL who are unable to swallow or absorb medications and can induce molecular remissions without chemotherapy in several patients.

Investigators from the Memorial Sloan-Kettering Cancer Center (MSKCC) conducted a dose-ranging study of 9-cis retinoic acid (9-cis RA), a retinoid receptor 'pan agonist', in patients with relapsed and newly diagnosed APL. The daily dose ranged from 30 to 230 mg/m²/day orally. Four out of 12 relapsed patients and four out of five newly diagnosed patients achieved CR. The drug was generally well tolerated; headaches and dry skin being the most common adverse reactions. Thus, 9-cis RA is also an effective agent for remission induction in patients with retinoid-sensitive APL.⁴⁶

Arsenic compounds

In the early 1970s, investigators in Harbin, China, specializing in the integration of Traditional Chinese Medicine with the Western Medicine, started to use arsenic compounds in APL, and reported a successful remission induction therapy with the intravenously administered arsenic compound, Ailing No. 1, mostly consisting of As₂O₃, in a reliably large number of patients with APL.^47,48,49 As their reports were published in Chinese (and mainly in traditional Chinese) medical journals, this fact was unknown even in China until a report appeared in Blood in 1998 by the investigators from Shanghai, China,⁵⁰ who previously surprised the world with the first successful treatment with ATRA in 1988.¹ They confirmed the effectiveness of As₂O₃ in APL and reported CR in 14 of 15 relapsed patients with APL. Soon USA investigators reported CR in 11 of 12 patients with APL who had relapsed after extensive prior therapy.⁵¹ As₂O₃ reportedly induces the expression of the proenzymes of caspase 2 and caspase 3 and the activation of both caspase 1 and caspase 3, and the clinical response is associated with incomplete cytodifferentiation and the induction of apoptosis with caspase activation in leukemia cells.⁵¹

Although USA as well as Chinese investigators reported relatively mild adverse effects of As₂O₃, including rash, lightheadedness, fatigue and musculoskeletal pain, Japanese investigators soon cautioned against the systematical occurrence of the prolongation of QTc intervals with occasional ventricular tachycardia, based on their careful continuous telemetric monitoring of electrocardiograms while using the arsenic formulation which the USA FDA approved.⁵² This caution was confirmed by several subsequent reports of sudden death and torsades de pointes.^53,54 Thus, As₂O₃ should be recognized as having considerable toxicities^55,56 and should be reserved for the treatment of patients with relapsed disease after ATRA therapy. Additionally, Chinese investigators cautioned against the use of As₂O₃ for newly diagnosed APL, reporting that the treatment resulted in hepatic toxicity in seven of 11 newly diagnosed cases, including two deaths, in contrast to the mild liver dysfunction in one-third of the relapsed patients.⁵⁷ Since molecular CR is obtained in a relatively high proportion of patients, As₂O₃ will be an ideal drug for relapsed patients with mutations in the PML/RAR gene and thus having little chance of response to a second challenge with ATRA. Careful electrocardiogram monitoring, however, is mandatory.^56,58

Recently, other Chinese investigators from Peking reported that orally administered tetra-arsenic tetra-sulfide (As₄S₄) was also effective on APL, regardless of disease stage, and 80% of 129 patients obtained CR including molecular CR. Treatment with As₄S₄ was well tolerated with only moderate side effects, including asymptomatic prolongation of QTc interval, transient elevation of liver enzyme, rash and mild gastrointestinal discomfort. Pharmacokinetic studies showed that oral arsenics displayed rapid but very incomplete absorption and large oral doses were needed to attain adequate plasma levels.⁵⁹

Other new agents effective on APL

APL cells generally express abundant CD33 antigen on their cell surface. Gemtuzumab ozogamicin (Mylotarg™, CMA-676) is a conjugate of humanized anti-CD33 monoclonal antibody and a cytotoxic antibiotic, calicheamycin.⁶⁰ This drug induced a prolonged molecular remission in advanced APL after the treatment with ATRA, chemotherapy, As₂O₃ and autologous SCT.⁶¹ Investigators from the MD Anderson Cancer Center administered gemtuzumab ozogamycin and daily ATRA to 19 patients with untreated APL, three of whom also received IDR because of a high leukocyte count, and obtained a CR rate of 84%. CR patients received eight additional courses of gemtuzumab ozogamycin every 4–5 weeks along with ATRA, and all 12 patients tested became PCR-negative for RML/RAR.⁶² Although a randomized trial will be needed comparing this combination with other regimens such as ATRA and IDR, gemtuzumab ozogamycin appears active in APL.

Investigators from MSKCC studied another humanized CD33 monoclonal antibody, HuM195, in patients with APL. After attaining CR with ATRA and/or chemotherapy, patients received HuM195 twice weekly for 3 weeks. Patients received 6 monthly courses of maintenance with two doses of HuM195. Of 27 patients, 25 treated in first remission had positive MRD determined by RT-PCR for RML/RAR before HuM195 treatment. Of the 22 patients evaluable, 11 became MRD negative after HuM195 treatment without additional therapy. Among patients who received ATRA alone as induction, eight out of 18 patients had negative MRD after HuM195 treatment but before chemotherapy. Of 27 patients, 25 (93%) with newly diagnosed APL remain in clinical CR for 7+ to 58+ months.⁶³ The data suggest that HuM195 has activity against MRD in APL.

Investigators from MSKCC also reported a successful treatment of APL with sodium phenylbutyrate, an inhibitor of histone deacetylase.⁶⁴ Acetylation of DNA-associated histones is reportedly linked to activation of gene transcription, whereas histone deacetylation is associated with transcriptional repression. Recent studies have shown that inhibitors of histone deacetylases can relieve the transcriptional repression block caused by the products of certain oncogenes.⁶⁵ The patient proved clinically resistant to the treatment with ATRA alone. However, 23 days after sodium phenylbutyrate was added to ATRA, leukemia cells had disappeared from her bone marrow, and she achieved a clinical and cytogenetic CR shortly thereafter. With a second treatment course, analysis for MRD by RT-PCR proved negative. Thus, treatment with an inhibitor of histone deacetylase induces histone hyperacetylation in target cells and may restore sensitivity to the antileukemia effects of ATRA in APL.⁶⁴ However so far, no other successful report has followed on from the same investigators regarding this interesting drug.

Alterations of FLT3 gene, including internal tandem duplication (ITD) and the D835 mutation, are the most frequent gene alterations in AML. Japanese investigators found FLT3/ITD in 20% of 74 newly diagnosed patients with APL and reported that the presence of FLT3/ITD was associated with high peripheral leukocyte counts, high LDH levels and low fibrinogen concentration.⁶⁶ Italian investigators found FLT3/ITD in 37% and the D835 mutation in 8% of 90 newly diagnosed APL patients. The presence of ITDs was strongly associated with a high leukocyte count, M3 variant and the short PML/RAR isoform, and with a trend towards inferior outcomes in terms of DFS and relapse.⁶⁷ Therefore, molecular-targeted drugs, such as SU5416, SU5614⁶⁸ and PKC4512⁶⁹ against these altered FLT genes, may have therapeutic benefits on APL with the alteration and should be further investigated clinically.

Extramedullary leukemia and its management

Extramedullary leukemia used to be rather rare in APL. For example, one review article on extramedullary leukemia published in 1995 cites 154 well-documented cases in AML but no APL among them.⁷⁰ A growing body of evidence, however, shows that an extramedullary relapse of APL is frequently observed among patients who have been induced into CR by ATRA regimens, including some hitherto rare relapse sites such as the external auditory canal.^{71,72,73,74,75} There are a couple of explanations why extramedullary relapse is more common in patients treated with ATRA. First, ATRA reportedly upgrades the transcription of some leukocyte adhesion molecule genes, and thus ATRA-treated leukemia cells may show increased expression of adhesion molecules.⁷⁶ Second, the doses of cytotoxic drugs in the ATRA-based regimens are less intensive than those of chemotherapy-alone regimens and thus some leukemia cells in sanctuaries may escape the effect of cytotoxic drugs as well as of ATRA. Third, we may tend to observe more cases of extramedullary relapse just because the number of long-term survivors has increased since the introduction of ATRA. The third possibility is supported by the occurrence of secondary myelodysplasia/AML after the successful therapy for APL.⁷⁷

GIMEMA analyzed the risk of developing extramedullary relapse in APL patients enrolled into two consecutive studies of the group: chemotherapy alone (LAP0389) vs ATRA plus chemotherapy (AIDA). When all relapse types were taken into account, 51% of 184 patients and 18% of 740 patients who attained hematological CR underwent relapse in the LAP0389 and AIDA studies, respectively (P<0.0001). Extramedullary relapse was documented in five (5%) of 94 and 16 (12%) of 131 patients, respectively (P=0.08). Hematological and/or molecular relapse was diagnosed concomitantly in all, but two, patients with extramedullary relapse in the AIDA study. For all patients in the LAP0389 and AIDA series, the probability of extramedullary relapse was 3 and 4.5%, respectively (P=0.79), while the probability of central nervous system (CNS) involvement was 0.6 and 2% (P=0.28). No significant differences were found with regard to mean leukocyte counts and PML/RAR junction types in comparing patients with extramedullary relapse and hematological relapse.⁷⁴ Thus, from their analysis, APL patients receiving ATRA in addition to chemotherapy have no increased risk of developing extramedullary relapse as compared with those treated with chemotherapy alone. However, in the JALSG AML87 and AML89 studies (chemotherapy alone) and the APL 92 study (ATRA+chemotherapy), 37 (45%) of 82 patients and 121 (36%) of 333 patients who achieved CR had relapse, respectively. Extramedullary relapse was absent from all of 37 patients with relapsing APL in the former, and present in 10 (8%) of 121 patients in the latter (P=0.063) (unpublished data). Of note, in the JALSG AML studies, at least one dose of intrathecal MTX, Ara-C and prednisolone is routinely given for the prophylaxis of CNS leukemia during consolidation therapy. Although the CNS relapse was seen only in the APL 92 study, the incidence was not significantly higher than that of previous two studies (P=0.148). Since relapses at auditory canal are noteworthy and rarely reported previously,^73,75 it is quite possible that ATRA itself may contribute to these extramedullary relapses or that the therapeutic concentration of RA may not reach leukemia cells in these sanctuaries. Careful observation of extramedullary relapses as well as sufficient doses of cytotoxic drugs will be necessary in patients with APL who have been induced into CR by ATRA-based therapy.

SCT in APL

Although there is no randomized study, allogeneic as well as autologous SCT is no longer regarded as the treatment of choice as postremission therapy in patients with APL in first CR, because the outcome of current ATRA-based therapy is excellent and over 70% of newly diagnosed APL patients are curable.¹⁷ Knowing the higher incidence of therapy-related mortality in allogeneic SCT, it is quite reasonable to reserve this curable but potentially life-threatening therapeutic modality until time of relapse in APL. For patients who have relapsed from ATRA-based therapy, SCT will be the treatment of choice, if they have HLA-identical family or unrelated donors. The European Blood and Marrow Transplantation Group reports 58% OS for APL patients in second CR undergoing allogeneic SCT, while 77% OS with 20% treatment-related mortality for APL patients in first CR undergoing allogeneic SCT.⁷⁸ Thus, knowing the unavoidable SCT-related mortality and the >50% OS of patients undergoing SCT in second CR, it is justifiable to recommend allogeneic SCT not in the first CR but in the second or later CR in APL patients.¹⁷

Strategy for the further increase of cure rate in APL

The cure rate of APL has now approached that of childhood acute lymphoblastic leukemia (ALL) which is mainly attained by intensive chemotherapy. The therapeutic concept of childhood ALL, however, is not quite applicable nor successful in adult ALL, especially in elderly patients, and the outcome of adult ALL is still poor in spite of the fact that intensive chemotherapy regimens are used. Taking all these considerations into account, we can say that APL is currently the most successfully treated leukemia where the highest cure rate has been achieved among leukemias of all ages.

To increase the cure rate further, the following strategies will be needed (Table 1). First, detection and diagnosis of APL at its early stage is very important, hopefully before the appearance of APL-associated coagulopathy. To achieve this, knowledge and diagnostic recognition of this leukemia by general community doctors should be improved. Second, in induction therapy, ATRA plus concomitant chemotherapy is needed for patients with an initial leukocyte count >3000/l, and ATRA plus more intensive chemotherapy for patients with a high leukocyte count >10 000/l. Third, two or three courses of intensive consolidation chemotherapy are indispensable. Fourth, if MRD is negative by RT-PCR for PML/RAR after the consolidation therapy, probably no further therapy will be needed, although the results of currently on-going studies are awaited. The role of additional consolidation therapy at this stage with As₂O₃, Am80, CMA676 or other new agents should be clarified in randomized studies. If MRD is positive, ATRA with 6MP and MTX will be the treatment of choice as maintenance therapy. Alternatively, As₂O₃, Am80, CMA676 or other new agents may be tried at this time. Fifth, prophylactic use of intrathecal MTX and Ara-C should be implemented during the consolidation phase, as some multicenter study groups have already incorporated it into their protocols. Sixth, if patients relapse hematologically or even molecularly, As₂O₃ will be the treatment of choice under careful electrocardiogram monitoring for the prolongation of QTc interval and for the appearance of arrhythmia. Am80, liposomal ATRA, CMA676 or ATRA in combination with cytotoxic drugs may be used at this stage or later. Readministration of ATRA, however, theoretically implies another relapse in the future, even if it successfully induces the second CR, and therefore other drugs may be theoretically better if cure is the objective. Seventh, if HLA-identical family donors or DNA-identical unrelated donors are available, allogeneic SCT would be the treatment of choice for relapsed patients of age <50 years, after they have obtained second CR or if their leukemia cells have been greatly reduced by reinduction therapy. If only HLA-identical but DNA-nonidentical unrelated donors are available, SCT would better be postponed till the second hematological or molecular relapse. At this time, the third CR will possibly be obtained or leukemia cells will be greatly reduced by As₂O₃, Am80, liposomal ATRA, CMA676 or ATRA in combination with cytotoxic drugs. The role of autologous SCT in this stage should be clarified by prospective comparison.

Table 1 - Strategy for the further increase of cure rate in APL.

Full table

We hope that the above-mentioned strategies will further increase the CR and, consequently, cure rates of patients with APL but, naturally, most of the strategies should be verified by ongoing or upcoming randomized studies.

Lessons learned from ATRA treatment in APL

Recent development of molecular biology has clearly revealed that cancer is caused by abnormality of the genes. Among human cancer, leukemia has been studied most extensively because investigators are able to obtain a sufficient number of cancer cells from patients upon their informed consent. Since the successful treatment with ATRA in late 1980s, APL has been the most extensively studied leukemia, and the molecular mechanism of its leukemogenesis is well clarified.

Although the discovery was rather by chance, the astonishing effect of ATRA as a differentiation inducer has changed the once most-difficult-to-treat APL into the most-easy-to-treat acute leukemia. While the phenomenon is definitely differentiation, ATRA has turned out to be the first successful molecular-targeted drug in the history of cancer therapy. ATRA works on the pathogenic molecule, PML/RAR, and degrades its differentiation suppressing ability.^{79,80,81,82,83} Quite recently, hematology oncologists have also experienced another successful molecule-targeting drug, imatinib mesylate, working on the pathogenic molecule, BCR/ABL, of chronic myeloid leukemia.⁸⁴ Both drugs are quite effective and have only a few adverse reactions. It is quite understandable that a drug working on a stipulated pathogenic molecule has a few side effects on other molecules. With these few adverse effects, ATRA has reduced average medical costs of a Japanese APL patient by about 11 000 US dollars during 2 months after the first admission, and saved about 10 million US dollars annually in Japan.⁸⁵ Thus, molecule-targeting drugs are ideal ones from a medical, economical and patient-quality-of-life point of view.

However, we have learned that ATRA alone is not sufficient to obtain a high cure rate. The experience of the Shanghai groups tells that around 20% of APL patients are curable by ATRA alone (Wang ZY, personal communication). Therefore, to obtain a higher cure rate, the use of chemotherapy during remission induction and consolidation therapy is indispensable. The experience of ATRA should be exploited in the treatment of CML with imatinib as well as of other cancer, in which newly developed molecule-targeting drugs have just started to show their promising effects. In talking about molecule-targeting therapy, however, we should not forget the fact that such amazing effects as those of ATRA and imatinib are obtained because they work on the pathogenic molecules. Molecular-targeted drugs working on nonpathogenic molecules may not produce such remarkable effects as ATRA and imatinib. In this sense, drugs specifically working on pathogenic molecules should be developed further not only for leukemia but for other cancers, based on further elucidation of molecular mechanisms of their tumorigenesis.

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Acknowledgements

We thank all members of JALSG for their cooperation in the JALSG studies, and Ms Rena Okano for her excellent assistance in the preparation of manuscript.