Differential Expression Genes of CLL Subgroups Defined by Ki67 Expression Level Which Correlated with Clinical Outcome

Chronic Lymphocyte Leukemia (CLL) is characterized by the slow clonal expansion of B-lymphocytes, which eventually overwhelm healthy immune cells, therefore hindering normal function. In order to determine the underlying mechanisms for this uncontrolled proliferation, gene expression data was gathered through cDNA microarray analysis of the B-cells from CLL patients. The data was split into two groups depending on expression levels of Ki67, a strong marker for cellular proliferation. In our study, high Ki67 expression only showed in 60% of unmutated CLL patients and higher in CD38⁺ CLL B cells. The expression of individuals with high levels of Ki67 was compared to the expression profiles of individuals with low levels of Ki67. This allowed for the determination of differences in gene expression correlated to an increase in proliferation of B-cells.

1) The RNA were purified from CLL B cells which were isolated by RosetteSep Human B cell Enrichment Cocktail (StemCell Technologies) during 4 hours after blood drawn from patients. Microarray assay was performed on Illumina HumanWG-6 expression beadchips. 2) The samples were devided to 2 groups according Ki67 surface expression, 9 samples with Ki67^high (>5%) and 26 samples with Ki67^low (<5%). 3) Differential expression of gene were first analyzed using Partek. 98 genes were selected as differentially expressed if the fold change was greater than 1.5-fold and p value is lower than <0.01. 4) This set of genes was further examined by pathway analysis (Ingenuity pathway analyses).

1) Time to first time treatment (TTFT) were compared between Ki67^high and Ki67^low patients. Ki67^high patients had a significantly shorter TTFT (2.76 yr) compared to Ki67^low patients (23.46 years; P<0.0001). 2) 98 genes were selected as differentially expressed between Ki67^high and Ki67^low groups. 78 genes were upregulated and 20 genes downregulated in Ki67^high group. For the location of these genes, Extracellular space: 9 genes; Plasma membrane: 22 genes; Cytoplasma: 21 genes and Nucleus: 18 genes. 3) Several networks, which involved in cell growth and proliferation, cell cycles and cellular development, has revealed in which there were significant changes in gene expression correlated to high levels of Ki-67 activity. 4) As would be expected, distinct changes in expression were those relating to cell proliferation (23 genes), cell movement and migration (14 genes) as well as, cell-signaling and interaction (20 genes) and cell death (23 genes). 5) To compare this gene list to another set microarray analyses in CLL patients. 28 genes were identified in both experiments. 6) One pathway which particular involved with NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) was selected. The genes precipitated in this pathway are CDKN2C, CRY1, DFNA5, FKBP5, HIST2H2AA3, IL15, LPL, MPSK, MYLK, PAK1, ZAP70. NF-κB is a transcription factor involved in the regulation of various genes relating to apoptosis, cell survival, and proliferation. Although unchanged in its own expression, various regulators of NF-κB displayed a significant change in expression.

Ki67 is a nuclear protein which upregulated in G1, S, G2, and M phases of the cell cycle but is absent from resting cells (G0 phase¹). We reported that Ki67⁺ cell is enriched in CD38⁺ cells regardless of the percentage of CD38⁺ cells in a patient's CLL clone². To analyze gene expression profile in Ki67^high and Ki67^low groups will determine the possible interactions responsible for the proliferative nature of B-cells in CLL. Misregulation of the NF-κB complex has been implicated in the development of various immunological diseases as well as cancer, therefore identifying it as a candidate for further research in CLL development and treatment.

1. Scholzen T. et al: J Cell Physiol. 2000; 182:311–322.

2. Damle R. et al: (2007) Blood 110:3352–3359.

No relevant conflicts of interest to declare.