INTRODUCTION

Acute promyelocytic leukemia (APL), which accounts for approximately 10% of all acute myeloid leukemia (AML) cases, is defined by the t(15;17) chromosomal translocation, which fuses the promyelocytic leukemia protein (PML) gene located on chromosome 15q22 to the retinoic acid receptor α (RARα) gene on chromosome 17q21 (1). The resulting PML-RARα fusion protein plays a key role in leukemogenesis by blocking neutrophilic differentiation at the promyelocyte stage. APL cells undergo terminal differentiation upon treatment with all-trans retinoic acid (ATRA) both in vitro and in vivo (2), making ATRA the first-line drug for inducing complete remission in APL patients (3, 4, 5). However, a significant percentage (20 to 30%) of patients relapse after initial remission and subsequently develop resistance to ATRA treatment (4, 5). The clinical outcome of those patients is quite poor, as no effective therapy is available for ATRA-resistant APL. Recently, arsenic trioxide (As2O3), an active component of antileukemic drugs in traditional Chinese medicine, was found to induce complete remission in both ATRA-sensitive and -resistant APL patients (6, 7, 8). The antileukemic mechanism of As2O3 is under active investigation. Studies have shown that at micromolar concentrations As2O3 triggers apoptosis of APL cells in association with the down-regulation of bcl-2 protein (9, 10, 11, 12, 13, 14), whereas at lower concentrations it induces partial differentiation (6). In APL cells, As2O3 rapidly induces a dramatic reorganization of APL-specific PML or PML/RARα-associated microparticulate structures into fewer larger subnuclear spots followed by a progressive degradation of PML/RARα protein (9, 10, 11, 14, 15, 16). Limited data is available addressing the pathology of As2O3 treated APL and whether the antileukemic mechanism of As2O3 is primarily cytolysis or through stimulation of cell differentiation. In this report, we examined the pathology of five refractory APL patients treated with As2O3 and correlated this with the cytogenetic and molecular findings.

MATERIALS AND METHODS

Clinical Protocol and Patients

The APL patients studied were all enrolled in a phase I/II trial of intravenous infusion of As2O3 for treatment of relapsed APL (Washington University Protocol #98–0185). An informed consent was obtained from all the patients. Patients with relapsed or primary refractory APL following conventional chemotherapy and ATRA who were not candidates for HLA-matched sibling bone marrow/stem cell transplantation were eligible for study entry. The diagnosis of all five patients in this report was based on FAB-AML criteria and flow cytometric immunophenotyping, and was further confirmed by cytogenetic analysis or by reverse transcription polymerase chain reaction (RT-PCR) assay that detected PML-RARα fusion transcripts. Three of the five cases were further morphologically classified as the microgranular variant of APL (M3v). The patients’ demographic and clinical data are shown in Table 1.

TABLE 1 Demographic and Clinical Data of Patients with APL before Arsenic Trioxide Therapy
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Treatment with Arsenic Trioxide

ACS reagent grade arsenic trioxide (As2O3) was obtained from Sigma Chemical Co. (St. Louis, MO) and was administrated through continuous intravenous infusion for 28 days. Patient 1 was started As2O3 at 10 mg daily (0.08 mg/kg/day) for the first 11 days and 50 mg daily (0.4 mg/kg/day) for the additional 17 days, and the total As2O3 administered was 550 mg. The remaining four patients were treated with lower doses of As2O3, with the dosage based on actual body weight, starting at 0.1 mg/kg/day (Table 2).

TABLE 2 Summary of Clinical, Morphologic, Cytogenetic, and Molecular Responses of the Patients to Arsenic Trioxide Therapy
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Morphologic, Immunophenotypic, Cytogenetic, and Molecular Evaluation

EDTA-decalcified, formalin-fixed bone marrow core biopsy specimens were stained with hematoxylin-and-eosin and chloroacetate esterase (Leder) stains. Wright-Giemsa stained bone marrow aspirates and peripheral blood smears before and after therapy for each patient were reviewed. Immunophenotyping was performed by two-color flow cytometry using Ficoll-Paque purified cells stained with phycoerythrin (PE)-labeled anti-CD 33 monoclonal antibody and fluorescein isothiocyanate (FITC)-labeled anti-CD11b monoclonal antibody (Coulter, Hialeah, FL) on Coulter EPICSTM XL-MCL instruments. Cytogenetic studies on metaphase spreads were performed according to standard techniques. Four of the five patients showed the characteristic t(15;17); one showed a complex three way translocation involving chromosomes 2, 15, and 17; and four patients showed additional cytogenetic abnormalities (Table 2). RT-PCR detection of PML/RARα fusion mRNA was performed according to Miller, et al. (17). Morphologic, cytogenetic, and molecular evaluations were made before the therapy and after the completion of each cycle of As2O3 treatment.

RESULTS

Peripheral Blood Findings

Three of the five patients developed significant leukocytosis during the first month of As2O3 treatment (Table 3). For example, two weeks after initiation of the treatment patient 1 showed an increase in white blood cell count from the pretreatment levels of 5000/mm3 to 180,000/mm3, which necessitated leukopheresis. During the first two weeks of As2O3 treatment, the APL cells exhibited little maturation. However, with continuation of the therapy, the APL cells began to mature, showing significantly decreased nuclear-cytoplasmic ratio and condensed chromatin by the end of the first month (Fig. 1).

TABLE 3 Changes of Peripheral White Blood Cell Counts Before and After Treatment with As2O3
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FIGURE 1
figure 1

As2O3 treatment induces maturation of acute promyelocytic leukemic cells. Peripheral blood smears of patient 1 before (A) and 3 weeks after (B) As2O3 treatment (Wright-Giemsa stain; original magnification, 250×).

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Bone Marrow Findings

After one cycle of As2O3 treatment, all the patients showed some degree of morphologic response. Two patients achieved morphologic remission after the first cycle of therapy, and the other two patients also showed morphologic remission following two cycles of therapy. All marrowed showed a myeloid predominance and were normocellular or hypercellular. The APL cells differentiated to cells with a significantly lower N/C ratio, condensed chromatin, and sparsely granular cytoplasm, which closely resemble APL cells treated with ATRA (Fig. 2). With continuation of therapy, these “myelocyte-like” cells eventually disappeared and normal trilineage hematopoiesis resumed. In addition to the “myelocyte-like” cells, two other distinctive morphologic findings were seen in arsenic-treated patients. The first was bone marrow necrosis. Patient 1 showed geographic marrow necrosis alternating maturing granulocyte hyperplasia after the treatment of high doses of As2O3 (0.4 mg/kg/day) (Fig. 3). This may be a dose-dependent effect as marrow necrosis was not seen in patients treated with lower doses of As2O3 (0.1 mg/kg/day). A second unusual effect of arsenic was an altered appearance of regenerating non-neoplastic myeloid progenitors. After the second cycle of As2O3 therapy, bone marrows from two of the patients showed sheets of Leder positive atypical promyelocytes with oval to round nuclear contours, prominent nucleoli, and abundant granular cytoplasm (Fig. 4). At the same time, normal trilineage hematopoiesis was seen in the adjacent marrow tissue, and both the cytogenetic analysis and RT-PCR for PML-RARα performed on the same bone marrow aspirate were negative. Furthermore, with continuous arsenic therapy, a subsequent bone marrow biopsy obtained one month later was unremarkable without these atypical promyelocytes.

FIGURE 2
figure 2

The bone marrow biopsies of patient 3 before and after the first cycle of As2O3 treatment. A, high-power view of bone marrow core section before As2O3 treatment showing Leder negative myeloblasts and dysplastic promyelocytes with high nucleocytoplasmic ratios, irregular nuclear contours and fine chromatin (Leder stain; original magnification, 250×); B, high-power view of bone marrow core section after the first cycle of As2O3 treatment, showing cells with slightly condensed chromatin and decreased nucleocytoplasmic ratio and occasional nucleoli, suggesting slight myeloid maturation (Leder stain; original magnification, 250×).

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FIGURE 3
figure 3

Bone marrow biopsy of patient 1 one month after treatment with high doses of As2O3. The marrow showed hypercellular areas showing trilineage hematopoiesis alternating with areas of necrosis. A, high-power view of hypercellular areas showing nearly normal hematopoiesis (Leder stain; original magnification, 250×); and B, high-power view of necrotic areas showing largely ghost cells, cellular debris, and occasional degenerating myeloid elements (Leder stain; original magnification, 150×).

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FIGURE 4
figure 4

The bone marrow biopsy of patient 4 following two cycles of As2O3 treatment. A, bone marrow core section showing sheets of atypical promyelocytes with smooth round or oval nuclear contours, frequent prominent nucleoli, and abundant Leder positive granular cytoplasm (Leder stain; original magnification, 250×); B, bone marrow aspirate showing a promyelocyte (center) with abundant granulated cytoplasm (Wright-Giemsa stain; original magnification, 250×).

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Flow Cytometric Findings

Flow cytometric analysis was performed in patient 1 and patient 5 after completion of the first cycle of therapy and showed significantly increased numbers of myeloid elements that expressed CD11b, which is expressed at high levels on more mature granulocytic and monocytic cells, along with decreased expression of CD13 and CD33. However, predominant CD13 and CD33 expression with diminished expression of CD11b was also observed during relapse in patient 3 two months after completion of the third cycle of the treatment. On the forward scatter versus side scatter plots, the APL cells showed a dramatic shift from the typical blast gate to more mature myeloid cells following As2O3 treatment (Fig. 5).

FIGURE 5
figure 5

The flow cytometric analysis and the bone marrow biopsies of patient 5 before and after the first cycle of As2O3 treatment. A, forward versus side scatter plot of the flow cytometric analysis of a pretreatment bone marrow aspirate showing large cells with light scattering properties of myeloblasts in gate B. B, forward versus side scatter plot of the flow cytometric analysis of a bone marrow aspirate after first cycle of As2O3 treatment showing a predominant population of cells compatible with myelocytes in gate C.

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Clinical Follow-Up

A summary of the morphologic cytogenetic, molecular, and clinical findings following therapy is given in Table 2. After one cycle of As2O3 treatment, patient 1 achieved morphologic remission although the cytogenetics remained positive for t(15;17). However, a bone marrow biopsy at four mo posttherapy showed relapse of APL and the patient expired one month later. An autopsy was not performed. The cause of death was suspected to be intracranial bleeding. Patient 2 developed fatal pulmonary hemorrhage in the setting of pneumonia and thrombocytopenia after 14 days of As2O3 treatment. Patient 3 successfully achieved molecular remission by the end of the third cycle of As2O3 therapy and remained disease free for approximately two mo after discontinuation of treatment before experiencing leukemic relapse. He died one month later of refractory respiratory failure. Patient 4 achieved molecular remission following two cycles of treatment and remains in molecular remission following completion of three cycles of As2O3 therapy. After the second cycle of As2O3 therapy, patient 5 is in morphologic and cytogenetic remission, although the RT-PCR assay performed on a recent bone marrow aspirate was positive for PML-RARα fusion transcripts.

DISCUSSION

In this report, we evaluated the pathologic, cytogenetic, and molecular findings in five relapsed APL patients who were treated with As2O3 therapy. Our data has confirmed earlier reports that clinical and molecular remission can be achieved with As2O3 treatment in some ATRA-refractory APL patients (6, 7, 8). Our main focus in this study, however, was to better define the pathology of arsenic treatment of APL.

In contrast to conventional cytotoxic chemotherapy that causes myelosuppression, As2O3 at low doses (0.1 mg/kg/day) had no myelosuppressive effect, and instead induced significant leukocytosis in three of the five patients during the first 2 to 4 weeks of induction. One distinctive aspect of As2O3 treatment was the replacement of the myeloblasts and dysplastic promyelocytes by cells showing slightly shrunken nuclei, condensed chromatin, and significantly increased amounts of cytoplasm containing sparse neutrophilic granules. On flow cytometric analysis, these cells exhibited a marked increase both in cellular size and granularity, along with increased expression of CD11b (an antigen expressed on mature myeloid cells), but with decreased expression of CD33, which is typically expressed in early or intermediately differentiated myeloid cells. Although the morphology and immunophenotype does not correspond precisely with any normal stage of myeloid differentiation, the cells most closely resemble hypogranulated myelocytes. Cells with a similar immunophenotype were previously described as “intermediate cells” in APL patients treated with ATRA therapy (3). These atypical myelocytes still carry the t(15;17) translocation and therefore represent partially differentiated APL cells. However, after the completion of two cycles of As2O3 treatment, these atypical myelocytes disappeared in two patients and normal trilineage hematopoiesis resumed with no detectable t(15;17) translocation or PML-RARa fusion transcripts. Similar findings have also been reported by Chen et al. (6). Interestingly, significant dyserythropoiesis commonly observed in patients with arsenic intoxication was not seen in those APL patients treated with As2O3. It appears that at higher doses arsenic can also trigger cell death, as treatment with high doses of As2O3 (0.4 mg/kg/day) (patient 1) resulted in significant bone marrow necrosis. Interestingly, the same phenomenon has also reported in patients treated with ATRA used at standard doses (18).

One finding observed in this study was the appearance of sheets of atypical promyelocytes in the bone marrow following therapy that did not represent residual disease. After two cycles of As2O3 therapy, patient 4 showed sheets of these cells, although both the cytogenetic analysis and RT-PCR for PML/RARα performed on the same marrow aspirate were negative. The presence of frequent prominent nucleoli in these atypical promyelocytes raised the possibility of persistent APL. However, unlike usual dysplastic APL cells that often exhibit irregular nuclear contours or bilobed nuclei, these atypical promyelocytes had smooth oval to round nuclear contours and abundant granular cytoplasm. Furthermore, on the subsequent bone marrow biopsy one month later these cells were not seen and there was no evidence of leukemia. All the hematologic and clinical parameters were normal, and the cytogenetics as well as molecular analysis remain negative. Similar atypical promyelocytes were also seen on the bone marrow of patient 3 after the third cycle of As2O3 therapy when the patient was in molecular remission based on the negative result of RT-PCR for PML/RARα.

Although several clinical studies in China (6, 7) as well as in the United States (8) have proved the clinical effectiveness of As2O3 therapy for both ATRA-sensitive and –resistant APL, its antileukemic mechanism is still not fully understood. Both in vitro and in vivo studies by Chen et al. (6) suggested two mechanisms of action for As2O3. At low concentrations, As2O3 induces partial differentiation of APL cells, whereas at high concentrations this drug appears to trigger apoptosis. This suggested mode of As2O3 action appears supported by our morphologic findings on patient 1, showing alternating hypercellularity and necrosis on the bone marrow after treatment with higher doses of As2O3. Most in vitro studies (9, 10, 11, 12, 13, 14, 15, 16) suggest that the action of As2O3 is primarily mediated through triggering apoptosis of APL cells, in association with down-regulation of bcl-2 protein and degradation of PML/RARα. As previously reported (19), our immunofluorescent staining on one case in this series showed that As2O3 induces rapid reorganization of dispersed microparticulate pattern of PML nuclear structures into two to four large aggregates (data not shown). Whether this reorganization of PML structures is critical for responsiveness to As2O3 remains to be seen.