Patients
A retrospective study was conducted on 1,382 patients with hematologic malignancies displaying t(9;22) translocations who were treated at the outpatient department or inpatient ward of the Blood Diseases Hospital (Institute of Hematology), Chinese Academy of Medical Sciences between January 2016 and December 2018. These patients included 834 males and 548 females, giving a male-to-female ratio of 3:2. Five patients (0.36%) had additional rare recurrent genetic abnormalities: 2 patients had t(9;11), 1 patient had t(3;21), 1 patient had t(8;21), and 1 patient had inv(16). The clinical and laboratory data from these 5 patients are described in detail. This study (registration no. NI2020002-EC-1) was approved by the institutional review board of the Institute of Hematology and Blood Diseases Hospital Chinese Academy of Medical Sciences and informed consent was obtained from all subjects.
Cytogenetic analysis
Chromosomal analyses were performed by examining short-term cultures of bone marrow specimens according to standard conventional cytogenetic protocols. Fresh bone marrow was collected from each patient and cultured for 24 h (in RPMI 1640 medium, 20% calf serum) without addition of any growth factors. A methanol–glacial acetic acid fixation method was used for obtaining metaphase cells, and R-banding was performed. Analysis was performed using an Ikaros automated scanning system (Metasystems, Germany). At least 20 cells in metaphase were analyzed in each case. Karyotype descriptions are based on the International System for Human Cytogenomic Nomenclature (ISCN 2016).
Fluorescent in situ hybridization analysis
FISH of interphase cells
Fluorescence in situ hybridization (FISH) was performed on interphase nuclei of samples. FISH analyses were performed to confirm Ph translocation and the presence of t(8;21). Dual color-dual fusion labeled LSI AML1/ETO and LSI BCR/ABL1 probes (Abbott Diagnostics, IL, USA), designed for their respective purposes.The slides were pretreated with 2× sodium saline citrate, pH 7.0, for 30 minutes at 37°C, followed dehydrated in 70%, 85%, and 100% ethanol solutions for 2 minutes. Probes were denatured and hybridized according to the manufacturer’s instructions. Samples were rapidly washed, 10 μl of DAPI/antifade reagent (Abbott Diagnostics, IL, USA) was added to each sample, and slides were covered. Analysis was performed using an Isis system (Metasystems, Germany). Five hundred interphase cells were analyzed per specimen.
FISH of metaphase cells
FISH analyses of metaphase cells were performed to confirm Ph translocation and the presence of t(9;11)、t(3;21)、t(5;7) or inv(16). FISH analysis was performed on a R-banded slide for metaphase mapping to detect BCR/ABL1 fusion gene and MLL/AF9, EVI1, CBFβ, and EGR1/D5S721. After karyotype analysis, a well-dispersed metaphase mitosis figure with clear band and chromosomal abnormalities was identified, photographs were taken, and coordinates were recorded. Metaphase FISH was performed on the same mitotic figure twice. Metaphase FISH was performed using a BCR/ABL1 and MLL/AF9 dual-fusion probe; an EVI1 and CBFβ dual-color separation probe, and an EGR1/D5S721 dual-color deletion probe. BCR/ABL1, CBFβ, and EGR1/D5S721 probes were from Abbott Diagnostics (Vysis, IL); MLL/AF9 and EVI1 probes were from Cytocell (Cambridge, UK). Sample slides were deparaffinized in xylene for 10 min, destained in methanol for 10 min, fixed in fresh fixative solution for 10 min, dehydrated through a graded series of ethanol solutions, and air-dried overnight. Probes were denatured and hybridized according to the manufacturer’s instructions. Since samples had been treated with a hot salt solution, the denaturation temperature was increased to 75-78°C, and the denaturation time was extended to 5 minutes. Samples were rapidly washed, 10 µl of DAPI/antifade reagent was added to each sample, and slides were covered.Analysis was performed using an Isis system (Metasystems, Germany).
Real-time quantitative RT-PCR assay
A real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR) assay was performed for the detection of BCR/ABL1、MLL/AF9、AML/ETO、CBFβ/MYH11 and AML1-MDS/EVI1 fusion gene transcripts. RNA was extracted from bone marrow samples using Trizol reagent (Gibco-BRL, Gaithersburg, MD) according to the manufacturer’s instructions. Reverse transcription was performed on 1 µg of total RNA using random hexamers and superscript II reverse transcriptase (Gibco-BRL) as described previously.The resulting complementary DNA was subjected to PCR to amplify fusion transcripts in an ABI 7500 real-time quantitative PCR instrument. Reaction systems using primers and conditions were as described previously[5,6]. These results were then used to calculate the copy number of each target gene and its internal reference gene ABL1 based on a standard curve. Target gene mRNA level (%) = (copy number of target gene / copy number of ABL1) × 100%.
Flow cytometric immunophenotyping
Eight-color multiparametric flow cytometry (MFC) was performed on peripheral specimens or bone marrow specimens using BD FACSCanto II instruments (BD Biosciences) according to the manufacturer’s instructions. Incubation of cells with monoclonal antibodies at 4°C was followed by RBC lysis with NH4Cl for 10 minutes and washing with phosphate-buffered saline solution. Cells were resuspended and fixed with 1% formaldehyde. A screening panel consisting of 4 tubes was performed: (1)CD64/CD117/CD34/CD33/CD7/HLA-DR/CD38/CD45;(2)CD15/CD13/CD34/CD123/CD56/CD16/CD11b/CD45;(3)CD36/CD10/CD5/CD20/CD4/CD14/CD19/CD45;(4)TdT/MPO/CD9/CD2/cCD79a/mCD3/cCD3/CD45.Data analysis was performed using FCS Express 5 software (De Novo Software). A total of 10,000 events were acquired in each case. Major cell populations were defined by CD45/SSC (side-scatter) characteristics. All populations were further refined by forward and side-scatter gates to exclude nonspecific binding. A positive flow cytometry result was defined as the presence of circulating myeloblasts, lymphoblasts, or monocytes with aberrant antigen expression or lymphoproliferative disorder. Criteria for judging results: For each antibody, appropriate negative levels were determined by comparison with an isotype-matched control sample. leukemia cell membrane surface antigen > 20% was judged to be positive, and intracellular antigen > 10% was judged to be positive[7].