Clinical and laboratory features of seven patients with acute myeloid leukemia (AML)-M2/M3 and elevated myeloblasts and abnormal promyelocytes

Background There is limited information on a special subtype of Acute myeloid leukemia (AML) characterized by >20% myeloblasts and >20% abnormal promyelocytes in bone marrow and peripheral blood. Objective The objective of the present investigation was to explore the clinical and laboratory features of seven patients with AML-M2/M3. Method We retrospectively assessed cell morphology, cytochemistry, immunophenotype, cytogenetics, and clinical features of seven patients with this rare subtype of AML. Results All seven cases had thrombocytopenia, coagulation abnormalities, >20% myeloblasts and abnormal promyelocytes. The PML/RARα fusion gene was present in six patients and two patients presented a mixed PML/RARα and AML1/ETO genotype. Five cases achieved CR and two cases did not achieve remission and one case transform into AML-M2 after CR1. Conclusions The clinical and laboratory features of seven patients with AML-M2/M3 are demonstrated in the present study, providing information on the FAB sub-classification.


Clinical information
The current study was approved by the Department of Hematology of the First Affiliated Hospital of Guangzhou Medical University. Patients were enrolled and treated from Jan. 2008 to Jun. 2012. Study eligibility criteria included availability of bone marrow histology and cytogenetic information at the time of referral to the Department of Hematology. Acute myeloid leukemia was diagnosed according to the World Health Organization criteria [1,2,8]. During the study period, seven patients, aged 8 to 76, were newly diagnosed with AML-M2/M3 based on >30% myeloblasts and >20% abnormal promyelocytes as well as predominant myeloperoxidase (MPO or POX) and naphthol AS-D chloroacetate esterase (AS-DCE, or specific esterase, SE) positive status. Bone marrow smears, PB smears, and detailed morphological, immunochemical, and cytogenetic analyses of BM biopsy tissue were conducted for each patient. The myeloid lineage was assessed using antibodies against CD9, CD11b, CD13, CD15, CD33, CD34, CD38, CD45, CD56, CD64, CD117, HLA-DR, and cMPO. The lymphocyte T cell lineage was assessed using antibodies against CD2, CD3, CD5 and CD7, while the B cell lineage was assessed using antibodies against CD19, CD20, CD22, and CD79a.

Flow cytometry
The flow cytometer of COULTER EPICS XL type with 488 nm excitation light source was used in the present investigation, and System II software was employed for analysis. After calibration with Fluorescent Microspheres and adjustment Fluorescence compensation of the machine, three-color fluorescent staining of the same type sample was done to serve as negative control to exclude nonspecific fluorescence staining. According to the degree of expression of CD45 and particle size of SSC, the cells were divided into granulocytes, monocytes, lymphocytes, immature lymphocyte populations and immature myeloid cells group, red blood cells and debris cells. After analysis of each cell group, naive cells were determined. The expression of antigen was analyzed by two-dimensional phenotypic spectrum.
Case 3: A 57-year-old female presented with mucocutaneous hemorrhage and fever In BM smears, the myeloid lineage was hyperactive, with 39.5% myeloblasts and 49% abnormal promyelocytes.

Case 7: A 30-year-old female presented with bone pain and fever
Bone marrow smears revealed 49% myeloblasts and 13% abnormal promyelocytes, while the cells in the PB smear were 14% myeloblasts and 13% promyelocytes. The BM cells harbored both the PML/RARа fusion gene, S type, and the AML1/ETO fusion gene. Immunophenotyping by FCM revealed two groups of blast cells. The SSC lower cell group was approximately 44.9% of the total and this population was 13.5% CD11b(+), 90.8% CD13

Chemotherapy
Five of the six cases were treated by standard chemotherapy [17,18] (daunorubicin + cytarabine, DA) combined with all-trans retinoic acid (ATRA) or arsenic trioxide (ATO). The other case (case 6) was first treated with ATRA, compound realgar, and natural indigo tablets (CRINT), and then given reduced dose DA [19]. After the initial treatment, 5 cases achieved complete remission (CR), including one case with recurrence after three months that transformed into AML-M2 (Case 6). The average time to achieve CR was 37 days. Two cases did not achieved remission (including one death). After stem cell transplantation, patients with AML-M2/M3 might have prolonged event-free survival (Table 5).

Discussion
The FAB classification scheme (1976) divided AML into subtypes based on the morphology of BM or PB cells, while the current World Health Organization (WHO) classification system incorporates morphology, cytogenetics, molecular genetics, and immunological markers for the diagnosis of AML subtypes [2,5,8]. Acute myeloid leukemia is divided into two subtypes with different treatments and prognoses, AML-M2 and APL (AML-M3), depending on the magnitude of the increase in myeloblasts or abnormal promyelocytes [3][4][5]. Here, we provide evidence for a mixed phenotype (AML-M2/M3) with the characteristics of both AML-M2 and AML-M3. The clonal and temporal relationship between APL and the secondary AML subtype are usually obscure [20][21][22][23]. In fact, a chimeric M3:M2 case of acute promyelocytic leukemia was recently presented [24].
The diagnosis of AML-M2/M3 requires all available information, including morphology, cytochemistry, immunophenotyping, genetics, and clinical features [2,8]. In particular, BM cell morphology and the presence of the PML/RARа and/or AML1/ETO fusion gene define this subtype, while the immunophenotype is not sufficiently distinct from previously defined subtypes for diagnosis.
In BM and PB smears, all cases showed a simultaneous increase in myeloblasts and abnormal promyelocytes. Some promyelocytes contained dust-like or sparsely distributed granules. Promyelocyte nuclei were kidney-shape or even bi-lobed with inconspicuous to prominent nucleoli. Some promyelocytes also contained Aure rods.   These morphological characteristics are distinct from the typical abnormal promyelocytic cells observed in AML-M3 [1][2][3].
It is uncertain whether the two cases with both fusion genes harbor two genetic rearrangements within the same clone or two independent clones with different rearrangements. Unfortunately, as this was a retrospective study, no further analysis was possible and there was no information on how the various FACS and molecular assays were performed. Further studies are needed to examine the frequency of this double genetic rearrangement in AML-M2/M3.
Many of the clinical characteristics of AML-M2/M3 are common to other subtypes of leukemia, including anemia, fever, hemorrhage, and infiltration. However, this group of patients was prone to DIC, cerebral hemorrhage, and other serious complications, with high mortality rates. Moreover, patients showed a trend for recurrence and transformation into AML-M2 [3,4]. Although some clinical procedures including transplantation of stem cell has been proposed in the present investigation, the suggestion was preliminary, not original and might not be general for all similar cases. Anyhow, we analyzed the treatment effects of ATRA, ATO, or CRINT combined with the DA regimen on seven patients with AML-M2/M3, whose clinical and laboratory features were analyzed scientifically and the data revealed that there were elevated myeloblasts and abnormal promyelocytes. Because up to now, there has been no uncontested criterion for the FAB sub-classification of AML, analyzing particular cases of AML without scientific genaralization of considered particular cases still is of some significance. Thus, it remains to be investigated in FAB sub-classification of AML and its clinical treatment. In addition, further studies are needed to examine the frequency of this double genetic rearrangement in AML-M2/M3.
At present, there are no guidelines for the treatment of this new subtype of AML. All of the patients were treated with ATRA, ATO, or CRINT combined with the DA regimen and five achieved complete temporary remission. Our initial observations suggest that hematopoietic stem cell transplantation may be beneficial for long-term survival. Therefore, the significance of the present study was that the patients with AML could be treated with ATRA, ATO, or CRINT combined with the DA regimen, and hematopoietic stem cell transplantation might be beneficial for long-term survival, possibly providing data for the guidelines for the FAB sub-classification and treatment of this new subtype of AML.
In the present study, we identified a novel entity of AML according to FAB classification and examined its clinical characteristics. Although the "gray-zone" entity  -, these antibodies were expressed in less than 10% of cells.
of each of the FAB sub-classification has been recognized since the first proposal of FAB classification in 1976, unfortunately, this issue was not endowed with clear solution. Later, in a "proposed revised criteria for the classification of AML" by the FAB cooperative group in 1985 [28], the potential fermentation concerning discrimination between M2 and M3 has been asserted. The group noted that M2 was diagnosed when mature myeloid cells were over 10%, even though "few" promyelocytes or later cells were present, indicating that M2 does not contain cases with "massive" promyelocytes. Therefore, according to the revised FAB criteria, the presented cases were not totally consistent with M2. In contrast, blasts percentage >30% did not exclude M3 diagnosis. Indeed, myeloblasts occupy >30% in about 10% of M3 cases [29]. Microscopic features of promyelocytes in the presented cases including sparse granules and bilobed nucleus were characteristics of AML M3 variant. Thus, we regarded the cases as AML-M2/M3, which remains to be further explored. Anyhow, the present investigation added new information for the analysis of AML-M2/M3. In conclusion, the clinical and laboratory features of seven patients with AML-M2/M3 are demonstrated in the present study, providing information on the FAB sub-classification.