Novel Non-Coding, Coding and Structural Variants in Hairy Cell Leukemia from Whole Genome Transcriptome Sequencing

Background and Aims Hairy cell leukemia (HCL) is characterized by the BRAF V600E mutation in almost all cases. Other genomic abnormalities include co-mutations in KLF2 and CDKN1B in a proportion of patients (pts) as well as recurrent copy number (CN) abnormalities of 7q and chromosome 5. Targeted genomic approaches to date have not covered the full spectrum of genomic and transcriptomic abnormalities that may be present in HCL, therefore we aimed to comprehensively characterize HCL using whole genome transcriptome sequencing (WGTS) to detect novel genomic abnormalities.

Methods WGTS was performed on 59 pts with HCL (peripheral blood [PB] n=36, bone marrow aspirates [BMA] n=23). WGTS libraries were sequenced on a NovaSeq and variant calling of sequence, CN and structural variants (SV) was performed using multiple tools including Strelka, Manta, STAR-Fusion, Arriba and FusionCatcher.

ResultsBRAF V600E mutations were detected in all 59 pts. Co-mutations were detected in the known genes KLF2 (8%, 5/59), CDKN1B (8%, 5/59) KMT2C (5%, 3/59) and ARID1A (5%, 3/59). In addition we detected novel recurrently co-mutated genes including the JAK/STAT regulator PTPN6 (7%, 4/59), MAPK pathway regulator DUSP6 (5%, 3/59), SGK1 (5%, 3/59) and ACTB (5%, 3/59). CN abnormalities were detected in 32% (19/59) of pts. The most frequent CN change detected was loss of 7q (n=10) with a minimally deleted region (MDR) of approximately 6.2MB involving the telomere maintenance gene POT1. POT1 gene expression was significantly lower in patients harboring 7q deletion compared to those without (median log₂CPM 2.41 vs 2.97, p<0.05). The MDR also involved the putative MAPK pathway inhibitor TMEM229A. Other recurrent CN changes included trisomy 5 (n=3) and monosomy 21 (n=3).

Recurrent SVs were observed involving the TERT locus in 5/59 pts (8%) including two pts with chromosomal inversions and three with inter-chromosomal translocations. TERT SVs juxtaposed the TERT locus to known super-enhancers near genes overexpressed in HCL including MEF2C, PLEKHA2 and MTSS1. TERT expression was markedly elevated in pts with TERT SVs compared to those without (median log₂CPM 2.2 vs -2.7, p<0.05). Non-coding TERT promoter mutations (TERTpmut) were identified in two patients (chr5:g.1295228G>A). Overall, 16/59 (27%) pts had genomic abnormalities affecting genes involved in telomere biology (including TERT promoter mutations, TERT SVs and deletions involving POT1). In order to exclude low variant allele frequency (VAF) TERTpmut we performed UMI-based error corrected sequencing (sensitivity 0.5% VAF) on an extension cohort of 19 pts with HCL and did not detect any low-level TERTpmut in this group. To understand the specificity of these findings in TERT for HCL, a focused evaluation of WGTS was performed on other patients with chronic B-cell neoplasms for both TERTpmut and TERT SVs resulting in locus transposition to regulatory elements. TERT SVs and/or TERTpmut were detected in 38% (24/64), 18% (7/40), 2% (6/349) and 24% (23/94) of patients with SBLPN (HCLv), SMZL, CLL and MCL respectively. TERT was over-expressed in patients with TERT SVs regardless of histological subtype.

Finally, we assessed non-coding variants in pts with HCL by performing an enrichment analysis using bipartite graph models. Using this approach we detected a high frequency of activation-induced cytidine deaminase (AID) induced promoter/enhancer hypermutation of highly expressed genes including RHOH, MTSS1, ZFP36L1, TMSB4X and KLF2.

Conclusion WGTS in this large cohort of pts with HCL has revealed a higher degree of genomic complexity than previously elucidated, with novel diverse coding and non-coding changes involving intracellular pathway regulators (DUSP6, PTPN6, TMEM229A), cytoskeletal proteins (ACTB, MTSS1) and recurrent genomic abnormalities affecting genes involved in telomere maintenance (TERT, POT1). Furthermore, our data reveals HCL has a high incidence of superenhancer juxtaposition and hypermutation associated with increased gene expression. These findings provide multiple future directions to investigate the unique biology of HCL and related neoplasms, in particular the specific consequences of regulatory element hypermutation and the avoidance of oncogene-induced senescence through aberrant telomere regulation.

Blombery:AstraZeneca: Consultancy, Honoraria; Servier: Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria. Walter:MLL Munich Leukemia Laboratory: Current Employment. Hutter:MLL Munich Leukemia Laboratory: Current Employment. Baer:MLL Munich Leukemia Laboratory: Current Employment. Sakuma:MLL Munich Leukemia Laboratory: Current Employment. Mueller:MLL Munich Leukemia Laboratory: Current Employment. Kern:MLL Munich Leukemia Laboratory: Current Employment, Other: Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Current Employment. Haferlach:MLL Munich Leukemia Laboratory: Current Employment, Other: Ownership. Haferlach:Munich Leukemia Laboratory: Current Employment, Other: Part ownership.