Long Non Coding RNA Signatures with Diagnostic and Prognostic Significance in Aggressive B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) displays significant heterogeneity with regard to clinical, pathological, and genetic features. Using gene expression profiling, we have delineated 3 molecular subgroups within DLBCL: germinal center B cells (GCB-DLBCL), activated B cells (ABC-DLBCL), and primary mediastinal B-cell lymphoma (PMBL),while these defined subgroups show characteristic mutation profiles and oncogenic pathways, a small set of cases of DLBCL still remain "unclassifiable" (UC).

Burkitt lymphoma (BL) is characterized by the t(8;14)(q24;q32) with distinct morphological and immunophenotypic features. The diagnostic distinction between BL and DLBCL is challenging in a subset of cases that have overlapping morphological, immunohistochemical, and even molecular features, but is crucial for effective therapy.

The identification of long non-coding RNA (lncRNA) has added another critical component to cancer biology. LncRNAs are defined as a distinct set of non-protein coding transcripts longer than 200 nucleotides. The functions of a few lncRNAs have been recently elucidated of which some are thought to regulate gene-specific transcription. The goal of the current study was to identify reliable lncRNA signatures for the BL and DLBCL subgroups and evaluate their usefulness as prognostic biomarkers.

We examined the expression of lncRNAs from our earlier studies using Affymetrix-HG-U133 plus 2 arrays to distinguish unique gene expression profiles between BL and DLBCL (PMID: 27292966). In the initial analysis, we compared BL (n=77) with DLBCL (n=474) and identified 275 differentially expressed lncRNAs (p=0.005, fold change>1.5). There was high expression of TCL6 and DDX-AS11 in BL. We confirmed the microarray results for TCL6 and DDX-AS11 by qRT-PCR in a subset of cases. We further tested whether expression of TCL6 and DDX-AS11 was regulated by the MYC oncogene and observed significant downregulation of these transcripts upon CRISPR/cas9 mediated deletion of the MYC promoter in the Raji cell line.

We also sought to identify GCB-DLBCL and ABC-DLBCL associated lncRNAs. We observed 36 lncRNAs highly expressed in ABC-DLBCL and 40 lncRNAs highly expressed in GCB-DLBCL (P=0.005, fold change>1.5). Of the differentially expressed lncRNAs, lnc00487 and DUBR were upregulated in GCB-DLBCL, whereas lnc00944 and FUT8 were upregulated ABC-DLBCL. The observed expression differences were validated in ABC-DLBCL and GCB-DLBCL cell lines. The differentially expressed LncRNAs were also validated in other DLBCL cohorts. LncRNA00487 expression was associated with superior clinical outcome in DLBCL series treated with Rituximab (R)-CHOP (p=0.01), and gene expression and overall survival (OS) were validated in another DLBCL series treated with R-CHOP (PMID:22437443).

In the present study, we found that lncRNAs are differentially expressed in aggressive B cell lymphoma and could be useful as diagnostic or prognostic markers. They may play an important role in lymphoma biology and further studies of their functions are warranted.