The Sino-U.S. Shanghai Leukemia Cooperative Group (School of Public Health, Fudan University) diagnosed 623 patients (Median age is 51 years old, and ranged from 18 to 88 years, the percentage of males were 56.5%) with AML between June 2003 and December 2010, and conducted follow-ups to determine their survival time. Of these patients, 9 (1.4%) were diagnosed with AMKL. These patients were diagnosed with AMKL according to the 2008 WHO classification criteria (4) as follows: i) The bone marrow aspirate exhibited a blast cell infiltrate comprising ≥20% of all cells, and >50% of the blast cells were identified as megakaryoblasts; ii) the expression of CD41, CD42 and/or CD61 was positive, as demonstrated by flow cytometry with monoclonal or polyclonal platelet-specific antibodies; and iii) bone marrow aspiration was frequently accompanied by MF, and in cases with fibrosis, a bone marrow biopsy was required, and the cell of origin was required to be identified as part of the megakaryocyte lineage. This was indicated by positive immunocytochemical staining for platelet-specific antigens, including factor VIII, CD41, CD42 and CD61. Patients were be diagnosed with AMKL if they met at least one of the diagnostic criteria. In the present study, all the patients with AMKL were administered one, two or more intravenous (IV) courses of a standard induction regimen that was comprised of 60 mg/m² daunorubicin or 12 mg/m² idarubicin per day for 3 consecutive days, plus 200 mg/m² cytarabine that was continuously administered by IV infusion for 7 days. Following one course of a standard induction, a number of patients had no response and succumbed shortly after. Additionally, a number of patients had complete remission or partial remission and continued the regimen, receiving two or more courses of a standard regimen.

i) If the percentage of blast cells was >20% in the bone marrow of nucleated cells, and cell morphology was demonstrated to be megakaryoblasts, as demonstrated using a bone marrow smear, the diagnosis was AMKL. For this diagnosis, the results of flow cytometry or immunocytochemical staining were required to increase the accuracy of the diagnosis. ii) If the bone marrow aspiration could not indicate a diagnosis of AMKL, detection methods of flow cytometry and immunocytochemical staining were vital, and the final diagnosis was frequently determined by positive platelet-specific antigens. iii) If the bone marrow aspiration diagnosis was not successful due to MF, a bone marrow biopsy was the primary test method, and the final diagnosis was determined by immunocytochemical staining for factor VIII, CD41, CD42 or CD61.

Bone marrow aspirates and biopsies were obtained from the posterior iliac crest. Bone marrow smears were conducted by obtaining 0.2 ml marrow aspirate from each patient, in order to perform cytomorphological classification and cytochemical staining, including myeloperoxidase, periodic acid-Schiff, α naphthol-acetate esterase, Sudan black B and non-specific esterase staining, according to the International Committee for Standardization in Hematology (7). Bone marrow cellularity of the aspirate and biopsy were examined by routine light microscopy (×100 magnification) and assessed with the following grading: Definite hypercellularity, normal cellularity, moderate hypocellularity and severe hypocellularity (8).

An additional 3 ml bone marrow aspirate was used for flow cytometry analysis. Mononuclear cells (MNCs) were isolated from those samples using lymphocyte separation medium (Ficoll^®-paque Plus; GE Healthcare Life Sciences, Chicago, IL, USA). The cells were suspended in PBS (GE HealthCare Life Sciences) with 3% bovine serum albumin (A8020; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and was adjusted to 1×10⁶ MNCs/ml and an aliquot of 100 µl was labeled with 5 or 20 µl fluorescein conjugated monoclonal antibodies. The applied monoclonal antibodies were directed against the following surface antigens: CD34 PE (550761, 1:5), CD45 PerCP (564105, 1:20), human leukocyte antigen DR related antigen (HLA-DR APC, 560896, 1:5), platelet-specific glycoproteins, CD41 FITC (555469, 1:5) and CD61 PE (555754, 1:5), B-cell antigens, CD10 PE (561002, 1:20) and CD19 FITC (560994, 1:5), T-cell antigens (CD2 PE (555327, 1: 20), CD3 APC (555335, 1:5), CD4 APC-CY7 (561839, 1:20) and CD7 FITC (555360, 1:5), myeloid antigens, CD13 APC (561698, 1:20), CD14 PE-CY7 (560919, 1:20), CD15 FITC (555401, 1:5), CD33 PE (555450, 1:5), CD64 APC (561189, 1:20), CD117 PE (561682, 1:20) and myeloperoxidase (556035, 1:20, BD Biosciences). All monoclonal antibodies were obtained from BD Biosciences (San Jose, CA, USA). Subsequently, the cell suspension was incubated with these antibodies in the dark for 15 min at 4°C, then washed in PBS with 5% EDTA and centrifuged (200 × g) for 5 min at 4°C three times. Following this, the cells were suspended in 500 µl PBS for further use. The immunophenotyping was performed with the BD FACS Canton flow cytometer using FCS Express 3.0 software (De Novo Software, Glendale, CA, USA). Gating was set with CD45 and 90° light-scatter parameters in order to exclude erythrocytes, platelets and subcellular debris. A total of 1×10⁴ cells was acquired and the percentage of cells expressing the marker was calculated.

The samples from the bone marrow biopsy were fixed in B5 stationary liquid (10% formaldehyde) (mercuric chloride 6.0 g, anhydrous sodium acetate 1.25 g, distilled water 90 ml and formaldehyde 10 ml) for 2 h at room temperature, followed by processing in 70% absolute ethyl alcohol, decalcification in Decal (EDTA bisodic salt) for 2.5 h at room temperature and ordinary processing with paraffin embedding. The paraffin-embedded samples were placed on slides (Dako ChernMate Capillary Gap slides; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) and stored in a warm incubator at 56°C. After 2 h, the samples were rinsed with water and washed with dimethylbenzene, and then, washed by graded concentration of absolute ethyl alcohol (100, 95, 90, 80 and 70%). Then, the samples were sealed in 3% H₂O₂ for 10 min at room temperature and washed in distilled water, three times at room temperature. Subsequently, the samples underwent optimized antigen retrieval procedures, the 0.01 M sodium citrate buffer (pH 6.0) (C1010; Beijing Solarbio Science & Technology Co., Ltd.) was heated in microwave oven to 95°C, and kept the samples in boiled sodium citrate buffer for 15 min. After that, it was cooled at room temperature. Then the samples were washed in distilled water for 3 min, two times at room temperature, and in PBS for 5 min, three times at room temperature, and were sealed with 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc., MA, USA) for 20 min at room temperature. The applied monoclonal antibodies (Dako; Agilent Technologies, Inc.) were against factor VIII (ab236284, 1:200, Abcam, Cambridge, MA, USA), CD42 (ab134087, 1:250, Abcam) and CD61 (ab7166, 1:250, Abcam), and were incubated with the samples overnight at 4°C. Subsequently, the sections were washed in Tris-buffered saline (TBS) three times for 10 min each at room temperature, and then incubated with biotinylated rabbit anti-goat secondary antibody (1:250; Vector Laboratories, Inc., Burlingame, CA, USA) for 60 min at room temperature, followed by washing with TBS three times for 10 min each. Following this, the sections were processed with avidin-biotin complex (TA-015-BB, Thermo Fisher Scientific Inc.) reagent for 30 min at room temperature, incubated in 3,3′diaminobenzidine solution (710008, Sera Care Inc, MA, USA) for 30 min in the dark and counterstained with hematoxylin (0701; Beijing Solarbio Science & Technology Co., Ltd.). Subsequently, the sections were dehydrated in an ascending graded series of absolute ethyl alcohols (70, 80, 90, 95 and 100%), cleared in xylene and cover-slipped with neutral balsam. These samples were observed in a light microscope (Nikon 801; ×60 and ×100) and the imaging Software NIS-Elements F 3.00, SP7 (Build 547) (both Nikon Corporation, Tokyo, Japan). The stain intensity was classified as -, +, ++, +++. -, No staining is observed. + positive staining is observed in less than 25% cells. ++ positive staining in 25–49% cells. +++ extensive positive staining in more than 50% cells.

The Gomori silver impregnation staining kit (G1800; Beijing Solarbio Science & Technology Co., Ltd.) were used. The stationary process, paraffin embedding and dehydration procedure were the same as described previously. The oxidizing agent Gomori was added to the samples for 5 min at room temperature, washed with running water for 30 sec. Subsequently, the samples were bleached by oxalic acid solution for 2 min, then washed by running water for 2 min, by distilled water for 1 min, all the process was performed at room temperature. After that, Samples were stained with ammonium ferric sulfate solution (5%) for 5 min, washed by tap water for 1 min, by distilled water for 2 min. Silver ammonia solution was used to stain samples for 3 min, then distilled water was used to wash the solution for 1 min. Reducing agent of Gomori was added to the samples for 1 min and washed by running water for 10 min at room temperature. Subsequently, the sections were dehydrated in an ascending graded series of absolute ethyl alcohols (70, 80, 90, 95 and 100%), cleared in xylene and cover-slipped with a neutral balsam.

MF was graded according to the European Consensus Grading system (9) as follows: MF-0, scattered linear reticulin with no intersections; MF-1, loose network of reticulin with numerous intersections, particularly in perivascular areas; MF-2, diffuse and dense increases in reticulin with extensive intersections; and occasionally with only focal bundles of collagen and/or focal osteosclerosis MF-3, diffuse and dense increases in reticulin with extensive intersections, with coarse bundles of collagen, often associated with significant osteosclerosis.

Blood samples were obtained from the posterior superior iliac spine by bone marrow aspiration and collected in a heparinized tube. Blood samples (1 ml) were cultured in a culture dish containing 6 ml RPMI-1640 (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), 1% antibiotics and 100 µl 10 mg/ml phytohemagglutinin for each patient. After 24 h at 37°C, 50 µl colchicine (0.1 mg/ml) was added in culture dish and incubated at 37°C for 60 min. Subsequently, the samples were centrifuged at 200 × g for 10 min at room temperature, the supernatant was removed and then 5 ml 0.075 M KCl was added. Following this, the samples were incubated for 20 min at 37°C, followed by centrifugation at 200 × g for 10 min at room temperature. The supernatant was discarded and cells were resuspended in 1 ml cold fixative (methanol: Acetic acid, 3:1), incubated for 0.5 h at room temperature, and washed three times at 200 × g for 5 min each at room temperature. Finally, cells were suspended in 3 drops of the fixative. Slides were prepared by dropping a suspension of the pellet onto clean glass slides and dried at 37°C for 10 min. Giemsa staining was performed for 30 min at room temperature, then it was examined under a light microscope (×100 magnification), followed by G-banding for analysis. Primarily, 20–30 good-quality (The mitotic phase was complete and independent and the chromosome of intact cells was observed) metaphases were screened. Chromosome abnormalities were described according to the standard of The International System for Human Cytogenetic Nomenclature (10).

Total DNA was isolated from bone marrow MNCs using the QIAamp DNA Blood Mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocols. DNA quality was assessed using a Bio-Rad Experion electrophoretogram instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The purity of the DNA was determined by measuring the optical density of the 260/280 ratio using a NanoDrop spectrophotometer. DNA samples were then stored at −20°C for further use. All DNA samples were amplified in the C1000 Touch™ Thermal Cycler (Bio-Rad Laboratories, Inc.) using the Takara Premix Taq™ HS polymerase chain reaction (PCR) kit, UNG plus (Takara Bio, Inc., Otsu, Japan). In this system, a 50-µl reaction mixture contained the following components: 0.25 µl Takara TaqHS DNA polymerase, 5 µl 10X PCR buffer for UNG plus, 4 µl dU plus dNTP mixture, 0.5 µl UNG, 1 µl each 20 pmol/µl primer, 2 µl genomic DNA (200 ng) and 34.25 µl diethyl pyrocarbonate-treated water. The mixture was amplified at 94°C for 4 min and subsequently subjected to 36 cycles of 94°C for 45 sec, 60°C for 45 sec and 72°C for 45 sec, and then extended at 72°C for 8 min. Patients and controls were genotyped by a DNA tetra-primer Amplification Refractory Mutation Screening (ARMS) assay (11), a method that uses 2 primer pairs to amplify the normal and mutant sequences plus a positive control band specifically in a single reaction. Primers included mismatches to maximize discrimination of the two alleles (in lowercase) and mutant/wild-type-specific bases (underlined). The PCR primers were as follows: Forward outer (FO), 5′-TCCTCAGAACGTTGATGGCAG-3′; reverse outer (RO), 5′-ATTGCTTTCCTTTTTCACAAGAT-3′; forward wild-type-specific (Fwt), 5′-GCATTTGGTTTTAAATTATGGAGTATaTG-3′, and reverse-mutant-specific (Rmt), 5′-GTTTTACTTACTCTCGTCTCCACAaAA-3′ (The subsequent amplicons were electrophoresed on a 1.5% agarose gel and visualized following staining with ethidium bromide. The samples were visualized with a gel imaging system (Tanon 2500R; Tanon Science & Technology Co., Ltd. Shanghai, China).

Among all the patients with AMKL, the white blood cell (WBC) count of 3 patients was normal, while 4 patients had a low WBC count and 2 patients had an increased WBC count. A total of 8 patients exhibited different degrees of anemia, 6 patients exhibited elevated lactate dehydrogenase levels, 6 patients exhibited hyperpyrexia and 6 patients exhibited bleeding from the skin or the mucosa. Peripheral blasts were observed in 6 patients, with a range of 2–30% (Table I).

The group consisted of 3 patients who exhibited marked splenomegaly. Additionally, patient no. 4 underwent splenectomy at diagnosis. A total of 2 patients exhibited superficial lymphadenopathy, 1 patient exhibited hepatomegaly (patient no. 6 underwent splenectomy due to trauma at diagnosis) and 1 patient exhibited sternal tenderness (Table I).

All patients were treated with cytarabine and daunorubicin/idarubicin. A total of 4 patients (patient nos. 3–6) achieved complete remission, 1 patient (patient no. 7) achieved partial remission and the remaining 4 patients exhibited no response. The median overall survival time of all patients was 6 months (range, 1.1–24.0 months).

On the bone marrow smears, irregular forms of megakaryoblasts, pseudopodia or flocculence were frequently observed around the edge of the cytoplasm, which was occasionally accompanied by platelet aggregation (Fig. 1). Patient nos. 3 and 6 exhibited a notable increase in megakaryoblasts, as demonstrated by bone marrow aspiration, and the percentage of megakaryoblasts was 24 and 25%, respectively. For the other patients, it was not possible to accurately identify the cells as megakaryoblasts by observing cell morphology, therefore, the percentage of megakaryoblasts was reported as 0%. The bone marrow cellularity of these 2 patients (patient nos. 3 and 6) was moderately hypocellular or hypercellular, respectively (Table II). The percentage of bone marrow blasts in 2 patients (patient nos. 4 and 9) was <5%, while it was >30% in 4 patients and it ranged from 20–30% in 3 patients (Table II).

The bone marrow cellularity was also evaluated with a bone marrow biopsy. A total of 6 patients exhibited moderate or severe hypocellular bone marrow cellularity. The bone marrow cellularity of patient nos. 1, 2, 6 and 8 was severely, moderately, severely and moderately hypocellular, respectively, as demonstrated by bone marrow biopsy (Table III); however, the cellularity was hypercellular or moderate hypercellular, as indicated by bone marrow aspiration (Table II). The cellularity of the other patients was consistent between the results of the bone marrow aspiration and bone marrow biopsy. Gomori silver staining demonstrated that 7 patients had moderate or marked MF, and the remaining 2 patients were negative for the condition. Additionally, 7 patients exhibited the expression of platelet-specific antigens, including factor VIII, CD61, CD41 or CD42, as demonstrated by immunohistochemical staining (Fig. 2; Table III).

Immunophenotypic analysis was performed on all patients. The blast cells of 6 patients primarily expressed myeloid-specific antigens, including CD13 and CD33. A total of 3 patients primarily expressed platelet-specific antigens, including CD41 and CD61. The blast cell percentages of patient no. 4 and patient no. 9 were <20% by bone marrow smear analysis; however, the percentage of CD41- and CD61-positive cells was >90%, as demonstrated by flow cytometry. In 5 patients, the percentage of CD34-positive cells was >20% (Table IV).

A total of 6 patients exhibited an abnormal karyotype. Additionally, in 5 patients (5/6 patients) complex karyotypes were observed. The karyotypes of patient nos. 3 and 5 contained the translocation t(9;22)(q34;11.2) (Table V).

Primers FO and RO flanked the JAK2 exon 12 and generated a control 463-bp band in all patients. Primers Fwt and RO produced a 229-bp wild-type-specific product, and primers FO and Rmt generated a 279-bp mutant-specific product. Patient no. 6 was positive for the JAK2V617F mutation (Fig. 3).