Gene Expression Profiles Document That Recently- and Previously-Divided CLL Fractions Represent a Continuum but Suggest Differing Modes of Activation for These Fractions in U-CLL and M-CLL

Abstract 317

By using reciprocal densities of surface membrane CXCR4 and CD5, chronic lymphocytic leukemia (CLL) B cells can be divided into 3 fractions indicating time since last division (proliferative, intermediate, and resting). It has been suggested that cells in these fractions represent a continuum from resting to intermediate to proliferative. In this study, we made intraclonal gene expression profile (GEP) comparisons of these fractions from 17 CLL patients to try to confirm this notion and interclonal comparisons between U-CLL and M-CLL patients to determine if pathways involved in the actions of these fractions differed between patient subgroups.

PBMCs from 8 U-CLL and 9 M-CLL patients were sorted into 3 fractions (CD19⁺CD3⁻CD5^hiCXCR4^lo, PROLIF), (CD19⁺CD3⁻CD5^intCXCR4^int, INTERM), and (CD19⁺CD3⁻CD5^loCXCR4^hi, REST); RNA was purified from each, and gene expression microarrays using Illumina HumanHT12 beadchips performed. To determine differentially expressed genes in intraclonal comparisons, expression value ratios for fractions from each patient were computed, log-transformed, and Student t-test performed using R (www.r-project.org); for interclonal comparisons, raw GEP data between subpopulations were compared: U-PROLIF and M-PROLIF, and U-REST and M-REST. Sets of significant genes (≥1.5 fold change and P<0.01) were analyzed using Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA).

Upon plotting intraclonal average log ratios of PROLIF/INTERM vs INTERM/REST, it was clear that gene expression levels changed in the same direction, i.e. PROLIF>INTERM>REST, or PROLIF<INTERM<REST, consistent with a continuum between the 3 fractions. Within this pattern, 36 genes were significant for both plotted ratios. Of these, 29 were overexpressed, along with CD5; CD68, ITGAX, CCND2, CRIP1 and LGALS1 were the highest. Functional analysis using IPA showed these genes to be related to NFkB signaling and cell trafficking. Seven genes (ADARB1, BACH2, CNTNAP2, HRK, RHPN2, PRPML, and RXPA) were significantly downregulated, along with CXCR4.

Next we characterized GEP differences between the PROLIF and REST fractions, identifying 390 genes up-regulated in PROLIF and 244 in REST. The top 5 upregulated PROLIF genes were CD68, LY96, ITGAX, CCND2 and CRIP1, and the top 5 REST genes were BACH2, CXCR4, ADARB1, RHPN2 and HRK. Functionally, the upregulated PROLIF genes were related to BCR signaling, cytokines (IFNa, IL12), NFkB, and Akt, whereas the upregulated REST genes related to BCL2, cell death and cell movement. By GSEA, 813/881 gene sets, defined by expression neighborhoods centered on cancer associated genes, were upregulated in the PROLIF with 436 gene sets significant at a false discovery rate (FDR) <10%; 206 sets were significantly enriched with p value <0.01. For the REST, 68/881 gene sets were upregulated, with none significant even at FDR <25%.

Finally, we examined PROLIF and REST fractions from U-CLL vs M-CLL patients. In this interclonal analysis, 93 genes were significantly different between U-PROLIF and M-PROLIF. The top 5 in U-PROLIF were MSI2, TGFBR3, TP53I3, RGCC and IGSF3, and the top 5 in M-PROLIF were MTSS1, BACE2, BRI3BP, AP3B1 and UBE2G2. Similarly, there were 125 genes that were significantly different between U-REST and M-REST. The top 5 in U-REST were DUSP26, CLEC2B, MDK, and EGR2 and in M-REST were NAPSA, RAB24, TARDBP, KCNN4 and ADD3. Interestingly, U-PROLIF and M-PROLIF differed in pathway assignments, with upregulated genes in U-PROLIF contributing to cell signaling and activation, particularly implicating Akt, ERK and P38MAPK.

The intraclonal gene GEP analysis on these 3 fractions confirms that CLL clones contain a spectrum of cells that transition in a sequential manner from PROLIF to INTERM to REST fractions. Functional analyses show that genes upregulated in PROLIF correlate with cell signaling and proliferation, while genes upregulated in REST relate to cell death. Thus the PROLIF fraction is enriched in recently divided cells that likely exit from lymphoid tissue and the REST in older, less vital cells that either traffic to lymphoid tissue or die. The interclonal analysis implies that the stimuli and/or the responses of cells in the PROLIF and REST fractions differ between U-CLL and M-CLL. This last novel finding suggests either distinct cells of origin or distinct activation pathways for the IGHV-defined CLL subsets.