This study was approved by the Institutional Review Board (IRB) of Oklahoma University (IRB Number: 2250). A total of 1,824 AML patient samples were studied cytogenetically from 2000 to 2019 at the Genetics Laboratory of Oklahoma University Health Sciences Center. Bone marrow samples were obtained from three of the 1,824 patients who had t(8;16)(p11.2;p13.3).
Conventional cytogenetic analysis
Short-term cultures of unstimulated bone marrow samples were set up and harvested according to the standard laboratory protocols. Karyotype analysis was performed using giemsa and trypsin technique for G-banding. The cytogenetic abnormalities were described according to the International System for Human Cytogenetic Nomenclature (ISCN 2016).
Fluorescence in situ hybridization analysis
Fluorescence in situ hybridization (FISH) assays were performed according to the manufactures’ instruction in combination with our established laboratory protocols. The PML/RARA dual-color, dual-fusion translocation probe (Abbott Molecular Inc., Des Plaines, IL, USA), the sub-telomere specific probes for chromosome 3 p-arm and q-arm, and the whole chromosome painting (WCP) probes for chromosome 1, 3 and 14 were purchased from Cytocell Ltd, NY, USA. A spectrum green-labeled probe mapping to the 8p11.21 region and a spectrum orange-labeled probe mapping to the 16p13.3 region was created in house, respectively with the following BAC/PAC clones: RP11-642I24[chr8: 41,676,336-41,856,494(hg19)] and RP11-589C21[chr8: 41,873,702-42,036,222(hg19)], RP11-619A23[chr16: 3,720,076-3,914,571(hg19)] and RP11-95J11[chr16: 3,860,374-4,025,510(hg19)] (Children’s Hospital Oakland Research Institute, Oakland, CA, USA). The KAT6A gene located on 8p11.21 and the CREBBP gene located on 16p13.3 was covered by the green-labeled and red-labeled home-brewed probe, respectively. All probes were validated before use. Chromosome spreads were counterstained with 4,6-diamidino-2-phenylindole (DAPI4) in an antifade medium (Vector Laboratories Inc., CA, USA). Digital images carrying specific hybridization signals were captured and processed on CytoVision version 7.0 (Applied Spectral Imaging, Carlsbad, CA, USA).
Genomic DNA was extracted from each three patient’s bone marrow pellet according to the standard operating procedure using the phenol and chloroform method with a commercially-available DNA extraction kit (Puregene blood kit, Qiagen, Valencia, CA) or Nucleic Acid Isolation System (QuickGene-610L, FUJIFILM Corporation, Tokyo, Japan). Two aCGH platforms including NimbleGen and Agilent were used in this study. For the NimbleGen aCGH platform, human reference genomic DNA was purchased from Promega Corporation (Promega Corporation, Madison, WI, USA). The patient’s DNA and the reference DNA were labeled with either Cyanine 3 (Cy-3) or Cyanine 5 (Cy-5) by random priming, and then equal quantity of both labeled products were mixed and loaded onto a 720 K oligonucleotide chip (Roche NimbleGen Inc., Madison, WI, USA) to hybridize at 42°C for 40 h in a MAUI hybridization system (BioMicro Systems, Salt Lake City, UT) according to the manufacturer’s protocols with minor modifications. The slides were washed with washing buffers (Roche NimbleGen Inc.) after hybridization and scanned using Roche Scanner MS200 (Roche NimbleGen Inc.). Images were analyzed using software from NimbleScan software version 2.6 and the SignalMap software version 1.9 (Roche NimbleGen Inc.). The genomic positions were determined using GRCh36/hg18, UCSC Genome Browser. For the Agilent aCGH platform, human reference genomic DNA was purchased from Agilent Corporation (Agilent Corporation, Santa Clara, CA, USA). The patient’s DNA and the purchased reference DNA were labeled with either cyanine 3 (Cy-3) or cyanine 5 (Cy-5) by random priming (Agilent Corporation). Patient DNA (labeled with Cy-3) was combined with a normal control DNA sample (labeled with Cy-5) of the same sex and hybridized to an Agilent 2 × 400 K oligo microarray chip (Agilent Technologies) by incubating in Agilent’s Microarray Hybridization Ovens (Agilent Technologies). After a 40 h of hybridization at 67°C, the slides were washed and scanned using the NimbleGen MS 200 Microarray Scanner (Roche NimbleGen Inc.). Agilent’s CytoGenomics 2.7 software (Agilent Technologies.) was applied for data analysis. The genomic positions were determined using GRCh37/hg19, UCSC Genome Browser.