Gene Set Enrichment Analysis of Ki-67^high CLL Clones Suggests Complex Interactions of B-Cell Receptor Signaling and Normal Cell Interactions in the Disease

In chronic lymphocytic leukemia (CLL), clonally expanded CD5⁺ B lymphocytes eventually overwhelm healthy immune cells, hindering normal immune function. To determine mechanisms fueling this expansion, gene expression data were gathered by microarray analysis of cells from CLL patients. Samples were grouped based on Ki-67 expression, an indicator of proliferation. To determine mechanisms correlating with B-cell proliferation and impacting on CLL B-cell biology, microarray profiles were compared using Gene Set Enrichment Analysis (GSEA) [Subramanian A, et al. PNAS 2005].

Samples were analyzed for intracellular expression of Ki-67 by flow cytometry and divided into 2 groups based on Ki-67 expression (cutoff at 5%). RNA was then purified from CD5⁺CD19⁺ CLL cells and gene expression microarray assays were performed using Illumina HumanHT12 beadchips. GSEA was carried out using a library of signatures by Dr. Louis Staudt [Shaffer AL, et al. Immunol Rev 2006] containing 305 gene sets encompassing 13, 564 genes biased towards hematopoietic signatures.

Of 61 cases, 14 were Ki-67^high and 47 were Ki-67^low. When time-to-first-treatment (TTFT) was compared between the groups, Ki67^high patients had significantly shorter TTFT (2.76 yrs) compared to Ki-67^low patients (23.46 yrs; P<0.0001). By GSEA, we determined 255/285 gene sets were upregulated in the Ki-67^high group with 50 gene sets significantly enriched at a false discovery rate (FDR) <25%. For the Ki-67^low group, 30/285 gene sets were upregulated with only one significant at FDR <25%. IGHV unmutated CLL (U-CLL) was enriched in only one gene set, termed CLLUNMUT-1, while mutated CLL (M-CLL) was only enriched in CLLMUT-1. CD38^high and CD38^low subsets were similarly enriched in these two gene sets, with 4 additional gene sets in the CD38^high group, including MYD88UP-4 and IFN-2. Of the 50 significantly enriched gene sets in the Ki-67^high group, 17 relate to signaling pathways, 16 to cellular differentiation, 6 to cellular processes, 4 to transcription factor targets, and the remaining 7 relate to cancer. Of these, the percentage of the signaling component is up 13% from its representation in the original Staudt library. The top 5 gene sets enriched in the Ki-67^high group are: upregulated U-CLL compared to M-CLL (CLLUNMUT-1), myeloid tissue compared to other tissues (MYELOID-1), T cell cytokine induced proliferation (TCYTUP-8), BCR crosslinking CLL B cells (CLLBCRUP-1) and BDCA4+ dendritic cells compared to other hematopoietic cells (DC-1). The total number of genes enriched in these 50 sets is 769, with 217 genes shared in two or more gene sets. Twenty genes were enriched in the CLL BCR signature, CLLBCRUP-1 [Herishanu Y, et al. Blood 2011]. Of these, WARS, IRF4, MX1, OAS1, and NAMPT are also enriched in the T cell cytokine induced and T cell activation signatures. Only one gene set was enriched in the Ki-67^low group, CLLMUT-1, upregulated in M-CLL compared to U-CLL. CD274 (PD-L1) was consistently elevated in the Ki-67^low group in all the patients, irrespective of IGHV mutation status.

The observed GSEA profiles in Ki-67^high patients correlated with gene signatures biased towards BCR signaling, signal transduction, and hematopoietic cancer, consistent with the Ki-67^high group containing more (recently) proliferating cells influenced at least in part by BCR signaling. The profiles also suggest that additional cells (T lymphocytes and dendritic cells) may be involved. It is notable these gene sets were not observed for CLL patients subgrouped by IGHV mutation status or by CD38, and that these other subsets did not show as pronounced a distinction by GSEA profiling.

No relevant conflicts of interest to declare.