Integrated transcriptional analysis reveals gene expression-based AML subtypes with distinct outcomes and drug responses

Subtyping of acute myeloid leukaemia (AML) has made significant progress, exemplified by the recent classification updates by the World Health Organization and International Consensus Classification. AML subclassification is predominantly genetics-based, despite research showing the benefits of transcriptomic profiling on top of known genetic markers. However, a comprehensive survey of subtypes in AML defined by gene expression has yet to be performed. To this end, we integrated mRNAseq data from 1337 patients and five studies, with corresponding biological and clinical data. We defined 19 gene expression-based subtypes, further stratifying AML. We found that KMT2A leukaemias with fusion partners MLLT3, MLLT10 and MLLT1 clustered together, while KMT2A-MLLT4 displayed a distinct gene expression pattern, suggesting differences in their aetiology. We discovered a transcriptional CEBPA subtype, of which only 40% had a CEBPA bZIP indel. Regardless of mutation status, all patients within this CEBPA cluster had the same favourable outcome. We found four NPM1- enriched transcriptomic subtypes, each with distinct co-mutation patterns and associated ex-vivo drug responses. Similarly, we identified nine AML with myelodysplasia-related changes (AML-MRC) subtypes, dividing a subtype making up one-third of the AML patients into novel groups with different outcomes and drug response profiles. In conclusion, we provide an unprecedented overview of the transcriptomic subtypes in AML and illustrate their potential for AML diagnostics. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This project was funded by a strategic investment of the Leiden University Medical Center, embedded within the Leiden Oncology Center, and executed within the Leiden Center for Computational Oncology. EvdA was funded by a personal grant from the Dutch Research Council (NWO; VENI: 09150161810095). The funding bodies had no role in the study design, the collection, analysis, and interpretation of data, the writing of the manuscript, and the decision to submit the manuscript for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All data used in this study was publicly available at the initiation of this study. Transcriptional data: The BEAT, TARGET and TCGA raw gene count data was acquired from the GDC data portal (, project IDs: BEATAML1.0-COHORT, TARGET- AML, TCGA-LAML). The Leucegene transcriptomics FASTQ files were acquired from the following NCBI-GEO IDs: GSE49642, GSE52656, GSE62190, GSE66917, GSE67039. The LUMC data is available under controlled access in the European Genome-phenome Archive under EGAC00001000956 (DAC). Genetic and patient data: We acquired mutation, fusion, karyotyping, and patient characteristics data from doi: 10.1038/s41586-018-0623-z and the Vizome browser () for BEAT. From the TARGET data matrix ([www.ocg.cancer.gov/programs/target/data-matrix][1]) for TARGET. From the cBioPortal ([www.cbioportal.org/study/summary?id=laml\_tcga\_pub][2]) for TCGA. From the Leucegene website () for Leucegene. The LUMC genomics and clinical data was available in-house, but can be acquired together with the transcriptional data as described above. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Differential gene expression results are available from Appendix 1. Other data produced in the present study are available upon reasonable request to e.b.van\_den\_akker@lumc.nl. [https://www.cbioportal.org/study/summary?id=laml\_tcga\_pub][3] [1]: http://www.ocg.cancer.gov/programs/target/data-matrix [2]: http://www.cbioportal.org/study/summary?id=laml_tcga_pub [3]: https://www.cbioportal.org/study/summary?id=laml_tcga_pub