A 50-year-old male farmer complained of fatigue and chest tightness with no obvious cause since March 20, 2019. After resting, his condition improved slightly, but then gradually worsened. The patient visited a local hospital for diagnosis on April 2, 2019. Peripheral blood film (PBF) showed white blood cells (WBC) of 5.6x109/L, red blood cells (RBC) of 1.43x1012/L, hemoglobin (Hb) of 53.2 g/L, and platelets (PLT) of 8x109/L. The attending physician recommended a transfer to a tertiary hospital for diagnosis and treatment. The patient was transferred to our hospital on April 4, 2019. Physical examinations showed enlarged bilateral submandibular lymph nodes without hepatosplenomegaly. PBF indicated WBC of 4.20x109/L with 43% neutrophils, 26% lymphocytes, 26% monocytes, and 5% blasts. PBF also revealed RBC of 1.12x1012/L with anisocytosis, Hb of 41 g/L, and PLT of 6x109/L. Bone marrow (BM) aspirates revealed a significant increase in bone marrow cellularity; 18% were myeloblasts with regular or irregular nuclei, fine chromatin, visible nucleoli, and reduced blue plasma. Nuclear-cytoplasmic dyssynchrony, nuclear malformations, binuclear, and Pelger malformed granulocytes accounted for 13% of the granulocytes, and 3% abnormal eosinophils were observed. Nucleated red blood cells were significantly reduced and accounted for 1.5%. Petal-nucleated red blood cells and anisocytosis were also observed; 18% of the aspirates were monocytes. Two granular megakaryocytes were observed on the whole slide, and the number of platelets was significantly reduced. Results from the bone marrow biopsy showed hypercellularity of the bone marrow tissue, with an increase in immature cells. Immunophenotyping of bone marrow cells showed 5.65% blasts, expressing CD34, CD117, CD13, HLA-DR, lack of CD7, CD10, CD38, CD33, CD11C, CD11b, CD64, CD15, CD19, CD123, CD56, CD36, CDCR4, CD14, CD300e, CD4, and CD2 (Supplementary Figure S1).
This study was approved by the Ethics Committee of the Traditional Chinese Medical Hospital of Langfang City, China.
Bone marrow nucleated cells (1-3x106/mL) were cultured in Gibco bone marrow cell culture media (USA) for 48 h and then analyzed by G-banding based on an international system for Human Cytogenomic Nomenclature (ISCN 2016). Karyotype analysis demonstrated 46,XY,inv(16)(p13.1q22)[10] (Figure 1). Fluorescence in situ hybridization (FISH) was performed using the Vysis dual-color separation CBFbeta probe (Abbott, USA), green fluorescent labeled 3'CBFbeta (16q22) probe, and the red fluorescent-labeled 5'CBFbeta probe. The rearrangement of the CBFbeta gene fusion was detected on chr16 (Figure 2).
RT-PCR was used to detect the following gene fusions based on a previous study. All of these genes were negative: BCR-ABL, SIL-TAL1, E2A-HLF, TEL-AML1, MLL-AF4, E2A-PBX1, AML1-ETO, MLL-AF9, PML-RARalpha, PLZF-RARalpha, STAT5b-RARalpha, MLL-AF6, MLL-AF10, MLL-ELL, MLL-ENL, NPM-MLF1, TEL-PDGFRB, FIP1L1-PDGFRA, AML1-MDS1/EVI1, DEK-CAN, TEL-ABL, ETV6-PDGFRA, NUP98-HoxA13, NUP98-HoxC11, NUP98-HoxD13, NUP98-HoxA9, NUP98- HoxA11, NUP98- PMX1, TEL-JAK2, MLL-AF17, MLL- AF1q, MLL- AF1p, MLL-AFX, MLL-SEPT6, (NPM, FIP1L1, PRKAR1A, NUMA1)-RARalpha, NPM-ALK, SET-CAN, TLS-ERG, and AML1-MTG16. WT1 was 11.7%. A rare fusion transcript of CBFbeta/MYH11 was detected, and electrophoresis of the PCR amplified product showed a positive band between 200-300bp (Figure 3A). Additional analysis of the PCR amplified product by capillary sequencing was performed. The sequence was as follows: TTTCAGAATTTTGAAGGCTCCCATGATTCTGAATGGAGTCTGTGTTATCTGGAAAGGCTGGATTGATCTCCAAAGACTGGATGGTATGGGCTGTCTGGAGTTTGATGAGGAGCGAGCCCAGCTTCACGAGTATGAGACGGAACTGGAAGACGAGCGAAAGCAACGTGCCCTGGCAGCTGCAGCAAAGAAGAAGCTGGAAGGGGACCTCTTCTAAA.
G denoted the end of the CBFbeta exon4 and C denoted the beginning of the MYH11 exon34 according to NM_002474.3 and CCDS_10565.1. This confirmed that CBFbeta exon4 and MYH11 exon34 were fused to form the fusion transcript. Comparing the CCDS sequence of the NCBI database, the CBFbeta/MYH11 fusion transcript belonged to a rare type I fusion transcript (Figure 3B). The patient was then diagnosed with inv(16)(p13.1q22); CBFbeta/MYH11 (type I) AML. Next-generation sequencing (NGS) analysis was used to detect additional prognostic mutational genes, including FLT3, NPM1, KIT, CEBPA, DNMT3A, IDH1, IDH2, TET2, EZH2, AML1, ASXL1, PHF6, TP53, SF3B1, SRSF2, U2AF1, ZRSR2, NRAS, CBL, SETBP1, ETV6, and JAK2. No mutations in these genes were observed.
The patient underwent chemotherapy with decitabine (DAC), daunorubicin (DNR), and cytarabine (Ara-C) (DAC: 50 mg qodx3; DNR: 40 mg d1-2, 60 mg d3; Ara-C: 200 mg d1-4, 150 mg d5, 200 mg d6-7). In addition, supportive treatment including heart, liver, and stomach protection, antiemetics, RBC, and platelet transfusions, was administered. On the 24th day after chemotherapy, BM cellularity was reduced, hence, supportive treatment was continued. On the 41st day, the cellular composition of the BM aspirates consisted of 10% myeloblasts, 11% monoblasts and pre-monocytes, and 2% abnormal eosinophils. The patient was administered a second cycle of chemotherapy with DAC, homoharringtonine (HHT), Ara-C, and etoposide (VP-16) (DAC: 50 mg qodx3; HHT: 2mg d1-5, 3mg d6-7; Ara-C: 150mg d1-7; VP16 0.1g, d1-7). In addition, supportive treatment was also administered. Seventy-five days after the initial chemotherapy, BM morphology returned to normal, and chromosomes were 46,XY(20). FISH showed CBFbeta gene separation and rearrangement. Chemotherapy was continued, and 272 days after the initial chemotherapy, CBFbeta gene separation and rearrangement was negative as determined by FISH, and the CBFbeta/MYH11 fusion transcript showed positive. After 487 days of the initial chemotherapy, bone marrow morphology showed complete response (CR), with 46,XY(20) chromosomes. FISH was negative for CBFbeta gene separation and rearrangement. However, the CBFbeta/MYH11 fusion transcript was still observed. At present, the patient is still undergoing regular chemotherapy and follow-up.