APL cells are morphologically different from normal promyelocytes. These cells are larger and typically have creased, folded, bilobed, or kidney-shaped nuclei with heavy azurophilic granules and bundles of Auer rods (faggots). There are several main morphologic variants of APL. (a) The most common variant is the hypergranular or typical form, which accounts for approximately 75% of cases. The cytoplasm of these promyelocytes typically contains densely packed, bright-pink, reddish-blue, or dark-purple granules and frequently contains Auer rods, which are clumps of granular material containing lysosomes, peroxidase, lysosomal enzymes, and large crystalline inclusions. Bundles of Auer rods, called faggot cells, are also seen occasionally. (b) The microgranular (hypogranular or agranular; M3v) variant accounts for approximately 25% of cases³²⁾. These cells have bilobed nuclei and fine, dusky granules in the cytoplasm; Auer rods are rare. (c) The hyperbasophilic microgranular subtype has an increased nucleocytoplasmic ratio and strongly basophilic cytoplasm with cytoplasmic budding that mimics micromegakaryocytes. There are few granules and no Auer rods. (d) PLZF-RAR alpha (M3r) variants have regular, condensed chromatin in the nucleus. There are fewer granules, and Auer rods are rare compared with the hypergranular subtype.

Compared with other subtypes of AML, APL has distinctive immunophenotypic features such as frequent expression of CD13, CD33, and CD19; low or negative expression of CD34; and rare expression of CD117, HLA-DR, and CD11b. Unlike normal promyelocytes, APL cells express abnormally low levels of CD15³³⁾. Fifteen percent of APL cases express the CD56 antigen at diagnosis, and in some studies, this has been associated with worse outcome³¹⁾.

The pattern of hypogranular variants of APL is more heterogeneous, with a higher percentage of cells expressing the T-cell antigen CD2, the stem cell marker CD34, HLA-DR, and CD56. CD19 has also been correlated with the M3v form. CD34 expression was suggested as a reliable marker to distinguish between M3v and classic APL. Hyperbasophilic APL expresses HLA-DR, CD33, CD13, HLA-DR, CD34, CD2, and CD9.

Identification of the specific genetic lesion in APL cells with t(15;17)(q22;q12);PML-RARA is feasible at the chromosome, DNA, RNA, and protein level using conventional karyotyping, fluorescence in situ hybridization (FISH) for PML/RARA, Southern blot, reverse-transcriptase polymerase chain reaction (RT-PCR) for PML-RARA RNA, and anti-PML monoclonal antibodies. In general, cytogenetic, FISH, and molecular analyses are attempted simultaneously. Karyotyping is highly specific and is an essential part of standard work-up, but it has the following disadvantages: good-quality metaphases are required, it is time-consuming, and cryptic rearrangements leading to PML-RARA will be missed (false negatives). However, rare molecular subtypes of APL, such as t(11;17), t(5;17), and other additional coexistent cytogenetic abnormalities can be detected. FISH is less expensive, highly specific, faster than conventional cytogenetics, and can usually be completed within 24 h; however, it does not provide information about the PML-RARA isoform detected and therefore cannot be used for the molecular monitoring of residual disease. In contrast, RT-PCR provides information on the PML breakpoint location (bcr 1, bcr 2, and bcr 3) in addition to the advantages of FISH. This is the current "gold standard" for confirming the diagnosis of APL and is very useful for assessing response and follow-up monitoring of minimal residual disease (MRD). However, RT-PCR has a long turn-around time (approximately 2 days) and can have both false positives (contamination artifacts) and false negatives (due to poor RNA yield). RT-PCR should be performed at presentation to precisely characterize the amplification target, even in patients with APL confirmed by cytogenetics and/or FISH. Southern blot is a highly specific DNA-based method; however, it is now rarely used for APL diagnosis because it is laborious. Finally, indirect immunofluorescence or immunohistochemistry using anti-PML monoclonal antibodies is an easy method that allows for the rapid identification of the characteristic microgranular nuclear distribution of the PML protein in approximately 2 h, but is not widely used. However, sometimes the staining pattern of non-APL cases can be difficult to interpret.

After the introduction of ATRA in APL therapy, Huang et al³⁴⁾ were the first to report on the effectiveness of tretinoin for patients with APL in the late 1980s. In the early trials, tretinoin was administered as a single agent to induce remission followed by consolidation chemotherapy, but several studies showed that ATRA-induced remissions were short-lived and had no effects on survival³⁵⁾, suggesting the need to add chemotherapy during consolidation to obtain prolonged survival. More recent studies have shown that patients receiving ATRA followed by chemotherapy had significantly better outcomes than patients treated with chemotherapy alone. Furthermore, the European APL group showed improved outcomes in patients treated with simultaneous ATRA and chemotherapy than with sequential use³⁶⁾. Since then, there have been several other multicenter trials based on the same concept, and simultaneous administration of ATRA and anthracycline-based chemotherapy is currently considered the standard induction treatment for newly diagnosed patients. This combination therapy gives a better quality of complete remission (CR) with rates of 90-95% and is more effective in controlling ATRA-induced leukocytosis in newly diagnosed patients with APL.

The type of chemotherapy that is more advantageous for improving CR rates with ATRA is not clearly defined. Concerning the role of cytarabine, a randomized study was defined the patients treated with DNR+cytarabine had a better outcome than those treated with DNR alone³⁹⁾. Another group observed that cytarabine gave a benefit to patients with WBC counts greater than 10,000/mm³ and was associated with better outcome³⁸⁾. Therefore, this issue remains controversial. However, many clinicians still use ARA-C in combination with ATRA and anthracyclines. For anthracycline, most clinicians prefer IDA to DNR, but there is no prospective data showing any advantage for comparing IDA versus DNR. Possible therapeutic options for APL are summarized in Table 1.

Limited studies have reported therapeutic results using combinations of ATRA and anthracycline-based chemotherapy in children with APL3-¹³⁾. In general, these studies showed improved outcome, with CR rates above 90% and a disease-free survival rate of 77%. These outcomes in children are comparable with those reported in adult patients. However, pseudotumor cerebri are observed more frequently in children than in adults during ATRA treatment, and a high cumulative dose of anthracycline resulted in increasing late cardiac toxicity in children. To decrease these risks, some groups have used a reduced dose of ATRA (e.g., 25 mg/m² instead of 45 mg/m²), and a decreased incidence of pseudotumor cerebri with excellent results was reported at an ATRA dose of 25 mg/m²/d than with 45 mg/m²/d in children and adolescents with APL3-⁶⁾. More prospective, randomized, large clinical trials are needed in children with APL.

Most newly diagnosed APL patients achieve CR with ATRA-based induction chemotherapy followed by a complete molecular remission after consolidation therapy. Resistance to primary therapy in APL is extremely rare or absent, and this situation causes suspicion of incorrect diagnosis³⁹⁾. The most frequent cause of induction failure is early death due to fatal cerebral hemorrhage. APL coagulopathy will clearly be improved by the addition of ATRA. The outcome of clinical trials in pediatric patients with APL treated with ATRA and anthracycline-based chemotherapy is summarized in Table 2.

ATO has proven to be another effective agent for APL therapy. The mechanism of action for ATO, which differs from that of ATRA, includes the induction of partial differentiation at low concentrations and apoptosis at higher concentrations in APL cells. Since ATO has been shown to induce remission in the treatment of relapsed APL, with observed remission rates of more than 80%14, ¹⁵⁾, several trials have explored the role of this agent in front-line therapy16-²¹⁾. Previous studies have shown that ATO with or without ATRA is equally effective in inducing remission in newly diagnosed patients with APL, and a CR rate of more than 85% and 5-year EFS and OS of more than 90% were reported16-²¹⁾. Zhou et al²¹⁾ reported an 89.5% CR rate, an 83.9% 5-year OS, and a 72.7% EFS in 19 children with newly diagnosed APL treated with single-agent ATO. These results demonstrate the high efficacy and minimal toxicity of ATRA+ATO treatment for newly diagnosed APL in long-term follow up, suggesting a new potential front-line therapy for de novo APL patients.

Despite these promising results, the current recommendation for induction therapy in newly diagnosed APL is the standard approach with ATRA+chemotherapy. It might appear reasonable to consider the possibility of a randomized study comparing conventional treatment using ATRA+chemotherapy with ATO alone or ATRA+ATO to minimize the chemotherapy in patients with good or intermediate-risk disease.

An initial response to induction therapy can be determined with bone marrow aspiration and biopsy performed after the patient has recovered their absolute neutrophil count (>1,000/mm³) and platelet count (>100,000/mm³). However, morphologic, cytogenetic, and molecular follow up should be interpreted with caution in patients with APL during and soon after induction because the morphologic features of bone marrow receiving ATRA-containing regimens show a relatively hypercellular pattern. This reflects the residual differentiation of leukemic cells, and the persistence of atypical promyelocytes is occasionally detectable several weeks after the start of induction therapy with ATRA. Such features may be misleading as resistant disease. In these cases, ATRA-based treatment should be continued to obtain terminal differentiation of blasts and achieve hematological remission. To summarize, early morphologic, cytogenetic, and molecular assessments at the end of induction have no value for therapeutic decision making, and such assessments should wait until blasts have terminally differentiated and hematological remission has been achieved.

The treatment strategy, including the type of consolidation and drug combination for post-remission therapy, is more controversial. Nevertheless, there is a consensus on the administration of at least 2-3 courses of anthracycline-based chemotherapy with or without Ara-C³⁹⁾. More than 90-99% of patients achieved molecular remission after receiving 2-3 cycles of anthracycline-based chemotherapy⁴⁰⁾. A small fraction of patients have persistent molecular disease at the end of consolidation and is needed to required additional, more intensive treatments, including hematopoietic stem cell transplantation (HSCT) when feasible.

As in induction, the role of cytarabine also remains controversial in consolidation. A randomized clinical trial reported comparable CR rates for ATRA+idarubicin (IDA) and ATRA+daunorubicin (DNR+cytarabine regardless of differences in risk group²⁶⁾. However, there is a trend in favor of cytarabine administration for high-risk of relapse patients (initial WBC count >10,000/mm³) on the basis of published results, which have demonstrated an improved antileukemic effect for the cytarabine-included regimens and a reduced relapse rate in high-risk patients40-⁴²⁾. In summary, the combination chemotherapeutic drugs including cytarabine is recommended for patients with an initial WBC count >10,000/mm³ who are at major risk of relapse.

Although the benefit provided by the addition of ATRA to chemotherapy for consolidation is not confirmed, the GIMEMA and PETHEMA groups showed a statistically significant reduction in relapse risk by adding ATRA at the standard dose (45 mg/m²/d for adults and 25 mg/m²/d for children) in conjunction with 3 courses of consolidation chemotherapy for 15 days24, ⁴²⁾.

It is not certain whether there is a benefit provided by the addition of ATO for post-remission therapy. Significantly better EFS and OS were reported with the use of ATO as post-remission treatment before the standard consolidation regimen with 2 more courses of ATRA +DNR or ATO⁴³⁾. This result provides further support for the use of ATO for purpose of eliminating of chemotherapy in consolidation.

The potential use of maintenance treatment is an unresolved question in APL patients with molecular-negative disease at the end of consolidation. European APL group conducted the advantage of ATRA for maintenance therapy in APL and showed a lower relapse rate by combination of intermittent ATRA, methotrexate (MTX) and 6-mercaptopurine (6MP)⁴⁴⁾. But, more recent report by a Japanese study showed no any benefit of ATRA in maintenance therapy⁴⁵⁾ Studies to determine the role of ATRA-based maintenance therapy is still ongoing. The recommended schedule is to administer ATRA for 15 days at intervals of 3 months for 2 years. A therapeutic algorithm for newly diagnosed patients is proposed in Fig. 3.

Unlike the assessment of response after induction therapy, molecular assessment of response after the completion of consolidation can be clinically relevant and is important to determine the risk of relapse, because the achievement of molecular remission at this time is a major treatment objective in APL patients. RT-PCR of PML-RARα in bone marrow with relatively low sensitivity, detecting 1 leukemic cell in 10³ to 10⁴ cells, is usually used to detect molecular remission. Patients who test PML-RARα positive after consolidation have poor prognosis and a potential risk of rapid progression to overt relapse. To rule out false positives, BM is repeated for MRD assessment within 2 weeks. More aggressive salvage therapy with ATO and/or chemotherapy and allogeneic HSCT should be considered for these patients. In contrast, patients who test negative for PML-RARα after consolidation have a low risk of relapse and should undergo maintenance therapy. We need to consider how often molecular monitoring after consolidation therapy is appropriate. It is reasonable to determine the frequency of monitoring for MRD according to the initial risk of relapse. Conversion to PCR positivity for PML-RARα during remission is highly predictive of subsequent hematologic relapse and underlines the prognostic value of MRD.

A potentially life-threatening complication called differentiation syndrome (DS), formerly known as retinoic acid syndrome, occurs in 2.5-30% of newly diagnosed APL patients receiving ATRA treatment. This syndrome has also been described in patients with relapsed APL who received induction therapy with ATO. DS usually develops during induction therapy with ATRA and/or ATO, typically during the first 30 days of therapy. It is present neither during consolidation or maintenance therapy using 2 drugs, nor during ATRA treatment in non-APL malignancies. Leukemic APL cells might play a fundamental role in the development of DS. The clinical symptoms of DS are unexplained fever, cough, dyspnea, peripheral edema, weight gain, pleural fluid retention, interstitial pulmonary infiltrates, hypotension, pericardial effusion, and acute renal failure. This is often mistaken for fluid overload with pleural effusion or pneumonia, congestive heart failure, and diffuse alveolar hemorrhage. The syndrome usually develops within 2 weeks of therapy and is commonly associated with a rapidly rising leukocyte count. The probable risk factors for DS are a high leukocyte count and a rapidly increasing leukocyte count. This syndrome is associated with increased cytokine release from differentiating myeloid cells and modulation of adhesion molecules on the surface of aggregated APL blasts and vascular endothelial cells as a result of exposure to ATRA, which results in capillary leak and increased adherence to capillary endothelium. Steroid rapidly reduces these aggregations of APL cells. If DS is suspected or definitive, specific therapy with dexamethasone 0.3 mg/kg/dose twice daily in children (10 mg twice daily in adults) should be started immediately because this syndrome can be potentially life threatening, with a mortality rate as high as 9%⁴⁶⁾. Early introduction of chemotherapy has significantly reduced this incidence of DS. A temporary discontinuation of ATRA and/or ATO is indicated in severe case of DS. ATRA can be restarted in most cases once the syndrome has resolved.

Another complication of induction therapy with ATRA is pseudotumor cerebri (PTC), which is more commonly observed in children with APL than in adults, and occurs in 5-15% of those enrolled in clinical trials5, ⁶⁾. It is characterized by increased intracranial pressure, severe headache, nausea, vomiting, and in severe cases by vision disturbances and papilledema. The mechanism of CNS toxicity by ATRA is not clear, but an excess of retinol is postulated to lead to a sustained increase in the choroidal secretion of cerebrospinal fluid or a decrease in its absorption by the arachnoid villi. Treatment of PTC is a temporary discontinuation or dose reduction of ATRA and the administration of dexamethasone, osmotic diuretics, and analgesics. To decrease these risks, some groups have used a reduced dose of ATRA (e.g., 25 mg/m² instead of 45 mg/m²), and a decreased incidence of PTC with excellent results has been reported at an ATRA dose of 25 mg/m²/d than with 45 mg/m²/d in children and adolescents with APL⁴⁾.