NGS-Based Methylation Profiling Differentiates TCF3-HLF and TCF3-PBX1 Positive B-cell Acute Lymphoblastic Leukemia

Abstract

Aim: To determine whether methylation differences between mostly fatal TCF3-HLF and curable TCF3-PBX1 pediatric acute lymphoblastic leukemia subtypes can be associated with differential gene expression and remission. Materials & methods: Five (extremely rare) TCF3-HLF versus five (very similar) TCF3-PBX1 patients were sampled before and after remission and analyzed using reduced representation bisulfite sequencing and RNA-sequencing. Results: We identified 7000 differentially methylated CpG sites between subtypes, of which 78% had lower methylation levels in TCF3-HLF. Gene expression was negatively correlated with CpG sites in 23 genes. KBTBD11 clearly differed in methylation and expression between subtypes and before and after remission in TCF3-HLF samples. Conclusion:KBTBD11 hypomethylation may be a promising potential target for further experimental validation especially for the TCF3-HLF subtype.

Keywords::

• ALL
• DNA methylation
• epigenetics
• epigenomics
• leukemia
• next-generation sequencing
• NGS
• RNA-Seq
• RRBS
• transcriptomics

A Franke and M Stanulla designed the project. M Stanulla and M Schrappe provided samples or clinical data. F-A Heinsen performed the experiments and sequencing for methylation data. P Kachroo, S Szymczak, G Hemmrich-Stanisak, M Forster, J Bethune and L Baker analyzed the data. P Kachroo and S Szymczak prepared the tables and figures. P Kachroo, S Szymczak and F-A Heinsen drafted and wrote the manuscript. M Forster, M Stanulla and M Schrappe contributed to the review and writing of the manuscript. A Franke supervised the research and approved the final version. All authors critically reviewed the manuscript and its contents.

Acknowledgements

UFO Sequencing Consortium Within the I-BFM Study Group. We thank all participants and personnel involved in the sample collection in Austria, France, Germany, the UK and Switzerland. The authors apologize to the authors whose original work was not cited due to space constraints. We thank the Leukemia and Lymphoma Research (LLR) Childhood Leukemia Cell Bank in the UK for providing primary patient samples. The authors are grateful to the sequencing team of the Department of Vertebrate Genomics of the Max Planck Institute for Molecular Genetics (Berlin). The authors thank N Forgo, Institute for Legal Informatics, Leibniz University Hannover, and HD Tröger, Hannover Medical School, for legal and ethical counseling. The authors thank C Bock and C Mason for advice on methylation data analysis. The authors also thank M Schilhabel, M Vollstedt, CVD Lancken, M Schlapkohl, I Clefsen (ICMB sequencing platform) for sequencing of RRBS libraries. E Zell administrated the childhood acute lymphoblastic leukemia sequencing consortium.

Availability of data & materials

Informed consent for the use of spare specimens for this research work was obtained from study individuals, parents or legal guardians. Sequencing data can be made available from the POPGEN repository (2015-UFO-NG-1; Christian Albrechts University, Kiel). A written request can be made which will proceed upon positive internal review board vote and signing of a material transfer agreement addressing leukemia-related questions.

Financial & competing interests disclosure

This study was carried out as part of the Research Training Group ‘Genes, Environment and Inflammation,’  supported by the Deutsche Forschungsgemeinschaft (GRK 1743). This work was supported by the German Federal Office for Radiation Protection (Grant no. St.Sch. 3611S70014), by the Swiss National Research Foundation SNF 310030–133108, the foundation ‘Kind und Krebs,’ the ‘Krebsliga Zurich,’ the Sassella Foundation, the Fondation Panacée, the clinical research focus program ‘Human Hemato-Lymphatic Diseases’ of the University of Zurich, the ‘Deutsche Forschungsgemeinschaft’ (DFG), Clusters of Excellence ‘Inflammation at Interfaces,’ the German Federal Ministry of Education and Research (BMBF) within the framework of the e:Med research and funding concept (grant number 01ZX1306A, sysINFLAME), the EU Seventh Framework Program [FP7/2007–2013, Grant no. 262055, ESGI; FP7-HEALTH-F2–2011 Grant no. 261474, ENCCA; ERA-Net Transcan, Validation of biomarkers for personalized cancer medicine, TRANSCALL; Health-F2–2010 Grant no. 260791, EUROCANPLATFORM], the ‘Katharina Hardt Stiftung,’ the ‘Deutsche José Carreras Leukämie-Stiftung,’ the ‘Madeleine Schickedanz-Kinderkrebs-Stiftung,’ the ‘Deutsche Krebshilfe – Dr. Mildred Scheel Stiftung (Grant no. 108613),’ the Foundation of Experimental Biomedicine in Zurich, the Max Planck Society and the ‘Elternverein zur Förderung der Behandlung krebskranker Kinder Hannover e.V.’ The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval for all human experimental investigations presented here. The specific research project reported here was approved by the Ethics Committee of the Medical Faculty of the Christian Albrechts University, Kiel, Germany (vote D508/13).

Supplementary data

To view the supplementary data that accompany this paper please visitthe journal website at:www.tandfonline.com/doi/full/10.2217/epi-2017-0080

Additional information

Funding

This study was carried out as part of the Research Training Group ‘Genes, Environment and Inflammation,’  supported by the Deutsche Forschungsgemeinschaft (GRK 1743). This work was supported by the German Federal Office for Radiation Protection (Grant no. St.Sch. 3611S70014), by the Swiss National Research Foundation SNF 310030–133108, the foundation ‘Kind und Krebs,’ the ‘Krebsliga Zurich,’ the Sassella Foundation, the Fondation Panacée, the clinical research focus program ‘Human Hemato-Lymphatic Diseases’ of the University of Zurich, the ‘Deutsche Forschungsgemeinschaft’ (DFG), Clusters of Excellence ‘Inflammation at Interfaces,’ the German Federal Ministry of Education and Research (BMBF) within the framework of the e:Med research and funding concept (grant number 01ZX1306A, sysINFLAME), the EU Seventh Framework Program [FP7/2007–2013, Grant no. 262055, ESGI; FP7-HEALTH-F2–2011 Grant no. 261474, ENCCA; ERA-Net Transcan, Validation of biomarkers for personalized cancer medicine, TRANSCALL; Health-F2–2010 Grant no. 260791, EUROCANPLATFORM], the ‘Katharina Hardt Stiftung,’ the ‘Deutsche José Carreras Leukämie-Stiftung,’ the ‘Madeleine Schickedanz-Kinderkrebs-Stiftung,’ the ‘Deutsche Krebshilfe – Dr. Mildred Scheel Stiftung (Grant no. 108613),’ the Foundation of Experimental Biomedicine in Zurich, the Max Planck Society and the ‘Elternverein zur Förderung der Behandlung krebskranker Kinder Hannover e.V.’ The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.