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Detection of recurrent and of novel fusion transcripts in myeloid malignancies by targeted RNA sequencing

Leukemia volume 32, pages 1229–1238 (2018)Cite this article

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The genomic landscape of hematological malignancies has been resolved mainly based on whole-exome and whole-genome sequencing, primarily targeting gene mutations. Beside mutations also gene fusions—chimeric transcripts where parts of two previously separated genes are expressed in a single transcript—are frequent genetic abnormalities in hematological neoplasms, often contributing to leukemogenesis. The impact of the detection of such fusions is increasing, as they are (1) entity defining, (2) important for risk stratification, (3) actionable targets, and/or (4) can be used as biomarkers for MRD monitoring (minimal residual disease). Classically, the detection of cancer-related gene fusions is performed by cytogenetic analysis for identification of the respective chromosomal rearrangement, followed e.g., by fluorescence in situ hybridization (FISH) or molecular analysis for further characterization of the genes involved in the rearrangement and for potential routine diagnostic applications. More recently, the use of next-generation sequencing (NGS) offers novel possibilities for detection of fusion genes in cancer, e.g., by application of RNA sequencing [1,2,3]. Thus, the aim of this study was to use a combination of cytogenetics and targeted RNA sequencing for the detection of recurrent and novel fusion transcripts in patients with myeloid neoplasms.

One hundred nine cases with myeloid malignancies harboring 114 translocations based on chromosome banding analysis (CBA) were selected and analyzed by targeted RNA sequencing. Bone marrow and/or peripheral blood samples were used, sent for diagnosis to the MLL Munich Leukemia Laboratory 06/2006—11/2016. Patients agreed with the use of laboratory data for research studies. The study followed the rules of the Helsinki Declaration. CBA and classification of karyotypes were performed according to standard methods [4, 5]. The cohort comprised 56 cases with a known rearrangement detected by CBA further confirmed by FISH and/or RT-PCR used for validation of RNA sequencing (Table 1). In 53 cases, novel fusions were suspected as only one partner gene could be identified by CBA/FISH, including rearrangements involving RUNX1 (n = 23), ETV6 (n = 12), PDGFRB (n = 8), KMT2A (n = 6), RARA (n = 2), NPM1 (n = 2), MECOM (n = 1), PDGFRA (n = 1), BCOR (n = 1), TET2 (n = 1), and NUP98 (n = 1) (Table 1). In five cases two rearrangements were present (n = 58 rearrangements). For detection/verification of fusion genes, RNA was extracted according to standard methods. Library preparation for NGS sequencing was performed using TruSight RNA Fusion Panel (Illumina, San Diego, CA) consisting of 7690 targeted exonic regions to cover 507 genes known to be involved in cancer fusions. Samples were sequenced on a NextSeq instrument with a 2 × 76 bp paired-end run on a MID output flowcell with 24 multiplexed samples, according to the manufacturer’s instructions. Analysis with ~8 m reads per sample was performed with the RNA-Seq Alignment App v1.2 (BaseSpace Sequence Hub) using Star for Alignment (to GRCh37/hg19) and Manta for gene fusion calling with default parameters (Illumina).

Table 1 Characteristics of cases with known rearrangements detected by standard methods (cases 1–56) and characteristics of cases with only one known partner gene (cases 1a–53a)
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Acknowledgements

We thank all co-workers at the MLL Munich Leukemia Laboratory for their dedicated work. The authors would like to thank all physicians for providing samples and caring for patients as well as collecting data. We also thank Illumina for providing RNA sequencing kits and discussions during data interpretation.

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  1. MLL Munich Leukemia Laboratory, Max-Lebsche-Platz 31, 81377, Munich, Germany

    Anna Stengel, Niroshan Nadarajah, Torsten Haferlach, Frank Dicker, Wolfgang Kern, Manja Meggendorfer & Claudia Haferlach

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  1. Anna Stengel
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Correspondence to Claudia Haferlach.

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CH, WK, and TH declare part ownership of Munich Leukemia Laboratory (MLL). AS, MM, NN, and FD are employed by the MLL. This study was supported in part by Illumina Inc.

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Stengel, A., Nadarajah, N., Haferlach, T. et al. Detection of recurrent and of novel fusion transcripts in myeloid malignancies by targeted RNA sequencing. Leukemia 32, 1229–1238 (2018). https://doi.org/10.1038/s41375-017-0002-z

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