GoKawiilPatients and samples
This study was reviewed and approved by the regional ethics review board in Stockholm (2008/1330-31/2 and 2017/2085-31/2) and the institutional ethics committees of Kyoto University (G608 and G1110) and participating institutions (Hyogo Prefectural Amagasaki General Medical Center, Chugoku Central Hospital, Dokkyo Medical University Saitama Medical Center, Gifu University Hospital, Gifu Municipal Hospital, Hyogo Medical University, Hokkaido University, Japanese Red Cross Kyoto Daini Hospital, Kobe City Medical Center General Hospital, Kurashiki Central Hospital, Kitano Hospital, Kyoto Medical Center, Kyoto City Hospital, Matsushita Memorial Hospital, Japanese Red Cross Nagano Hospital, National Cancer Center Hospital, NTT Medical Center Tokyo, Osaka International Cancer Institute, Japanese Red Cross Osaka Hospital, University of Osaka, Otsu Red Cross Hospital, Shizuoka City Shizuoka Hospital, Shinko Hospital, Shiga General Hospital, Sumitomo Hospital, Takeda General Hospital, Takatsuki Red Cross Hospital, Uji Tokushukai Medical Center and Japanese Red Cross Society Wakayama Medical Center) (no. G608), and was performed in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants at participating institutions.
A total of 1,563 patients diagnosed with AML and related neoplasms were consecutively enrolled from the participating institutes and hospitals between 1997 and 2022 in Sweden and between 2011 and 2022 in Japan. Although no preselection criteria were applied with respect to clinical characteristics, sample inclusion was contingent on the availability of viable cryopreserved tumour cells suitable for ATAC-seq for consecutively diagnosed patients with AML. Diagnoses were made at each participating institution and hospital in accordance with the WHO classification in use at the time. In Sweden, patients were registered through the national AML registry, which prospectively collects clinical and genomic data on all newly diagnosed cases. In Japan, patients were diagnosed and treated at participating hospitals, with biospecimens and clinical information sent to and managed by the Kyoto University biobank, where clinical annotations were updated annually. Treatment was administered according to institutional standards of care and, for Swedish individuals, according to the national treatment guidelines for AML, with a subset of patients participating in clinical trials. All samples analysed in this study were collected at the time of initial diagnosis before treatment. Detailed clinical annotations, including diagnosis, demographic and laboratory data, treatment regimens and outcomes, were extracted from electronic medical records and the Swedish AML registry. No statistical methods were used to predetermine sample size. Patient characteristics and diagnoses are summarized in Supplementary Tables 1 and 2.
Tumour samples, such as bone marrow or peripheral blood, and matched control buccal samples, were obtained from patients. We also analysed normal bone-marrow samples from 25 individuals without haematological malignancies who underwent hip joint replacement surgery, serving as controls for the ATAC-seq analysis. Bone marrow and peripheral blood cells were isolated, subjected to erythrolysis or mononuclear cell isolation by Ficoll gradient centrifugation, resuspended in CELLBANKER 1 solution (Nippon Zenyaku Kogyo) or in 10% dimethyl sulfoxide (DMSO) (Merck KGaA) with 90% fetal calf serum (FCS; Thermo Fisher Scientific) and cryopreserved in liquid nitrogen. Genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN), the Gentra PureGene Kit (QIAGEN) or the Maxwell RSC Genomic DNA Kit (Promega). RNA was extracted using the RNeasy Mini Kit (QIAGEN) or the Maxwell RSC simplyRNA Tissue Kit (Promega). A summary of this cohort, including diagnosis, ATAC subgroup, AML classifications, driver genes and available multiomics data, is provided in Supplementary Table 3.
Cell lines
K562, KG-1, THP-1, SKM-1, KY821 and NOMO-1 cells were obtained from the RIKEN BioResource Center Cell Bank (Tsukuba, Japan); Kasumi-1, HL-60, MOLM-13 and TF-1 cells from the American Type Culture Collection (ATCC); and OCI-AML3 cells from the German Collection of Microorganisms and Cell Cultures (DSMZ). These cell lines were authenticated by short tandem repeat profiling and tested for mycoplasma by the providing cell banks.
Targeted-capture sequencing
Targeted-capture sequencing was performed using the SureSelect custom kit (Agilent Technologies), with an in-house gene panel including 331 known AML driver genes (Supplementary Table 4) and an additional 1,158–1,317 probes for copy-number detection. Captured targets were sequenced using the NovaSeq 6000 (Illumina) or DNBSEQ-G400 (MGI) with a 150-bp paired-end read protocol.
Sequence alignment and mutation calling were performed using the hg19 reference genome and Genomon pipeline25,51,52,53 (https://github.com/Genomon-Project). Unless otherwise stated, sequencing data were aligned to the hg19 reference genome. The called variants were further filtered by assessing the oncogenicity of variants on the basis of an in-house curation program25,51,52,53 that uses the COSMIC database (v.96), an in-house blacklist of error calls and public SNP databases, including the 1000 Genomes Project (October 2014 release), NCBI dbSNP build 138, National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP) 6500, the Human Genetic Variation Database (HGVD) and our in-house dataset.
For copy number analysis, SNP probes included in the target bait for targeted-capture sequencing were used to allow the detection of copy-number changes and allelic imbalances25,51,52,53. This program (CNACS) is available at https://github.com/papaemmelab/toil_cnacs. A total copy number (TCN) of 2.22 or higher was defined as gain, and a TCN lower than 1.88 was defined as loss. Copy-number-neutral loss of heterozygosity was called with a B-allele frequency lower than 0.90 and a TCN between 1.88 and 2.22. Arm-level changes were called for regions with a total length greater than 1 Mb. Detected copy-number changes were manually curated.
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