Next generation RNA‐sequencing in prognostic subsets of chronic lymphocytic leukemia

Abstract

Advances in next-generation RNA-sequencing have revealed the complexity of transcriptomes by allowing both coding and noncoding (nc)RNAs to be analyzed. However, limited data exist regarding the whole transcriptional landscape of chronic lymphocytic leukemia (CLL). In this pilot-study, we evaluated RNA-sequencing in CLL by comparing two subsets which carry almost identical or ''stereotyped'' B-cell receptors with distinct clinical outcome, that is the poor-prognostic subset #1 (n 5 4) and the more favorable-prognostic subset #4 (n 5 4). Our analysis revealed that 156 genes (e.g. LPL, WNT9A) and 76 ncRNAs, (e.g. SNORD48, SNORD115) were differentially expressed between the subsets. This technology also enabled us to identify numerous subset-specific splice variants (n 5 406), which were predominantly expressed in subset #1, including a splice-isoform of MSI2 with a novel start exon. A further important application of RNA-sequencing was for mutation detection and revealed 16-30 missense mutations per sample; notably many of these changes were found in genes with a strong potential for involvement in CLL pathogenesis, e.g., ATM and NOTCH2. This study not only demonstrates the effectiveness of RNA-sequencing for identifying mutations, quantifying gene expression and detecting splicing events, but also highlights the potential such global approaches have to significantly advance our understanding of the molecular mechanisms behind CLL development.Chronic lymphocytic leukemia (CLL) is a clinically and biologically heterogeneous disease, with many patients following an indolent disease course, whilst others develop a more aggressive disease. The mutation status of the immunoglobulin heavy variable (IGHV) genes divides CLL into two clinically relevant subgroups, where unmutated IGHV genes ( 40% of patients) are associated with a considerably worse prognosis compared to mutated IGHV genes [1,2]. Moreover, CLL is characterized by multiple subsets carrying quasi-identical or ''stereotyped'' B-cell receptors which imply a role for antigen selection in leukemogenesis [3]. Recently, certain subsets have also been shown to share clinico-biological features [3,4]; for instance, subset #1 patients, which uniformly express unmutated IGHV1/5/7 together with IGKV1(D)-39 genes and a stereotyped heavy complementarity determining region 3 (VH CDR3) of 13-14 amino acids, have a significantly inferior outcome compared to patients expressing the same IGHV genes but without stereotypy [3,5]. In contrast, subset #4 patients (IGHV4-34/IGKV2-30 usage and VH CDR3 of 20 amino acids), have a low median age at diagnosis, express surface IgG and have an indolent disease [3]. Furthermore, we have previously shown that stereotyped subsets can carry a different spectrum of genomic alterations as well as diverse gene expression profiles [6,7]. In the present pilot-study, we applied next-generation RNA-sequencing to compare poor-prognostic subset #1 (n 5 4) with the more favorable prognostic subset #4 (n 5 4, Table I), in order to explore the potential...

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