The Correlation of APOBEC Gene Family Member Expression with Worse CLL Patient Outcome Suggests a Role in CLL Mutational Evolution

A mutational signature consistent with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) activity has been identified in somatic mutations found in large-scale surveys of ultra-deep sequencing data from many human cancers including chronic lymphocytic leukemia (CLL). APOBEC is a cytidine deaminase family made up of eleven genes, including AID (activation-induced cytidine deaminase) and APOBEC3B, both of which have been implicated in somatic mutation in various cancers, including CLL. These observations have led to the hypothesis that APOBEC cytidine deaminases may be driving somatic mutations leading to the development of more aggressive cancers. Therefore, we examined APOBEC gene family member RNA expression levels in CLL to test for correlations with expression levels and patient outcome. We further examined if CLL cells generated de novo APOBEC family member mutational patterns in the immunoglobulin variable region gene (IGHV) after implantation in a mouse xenograft model of CLL.

CLL peripheral blood mononuclear cells (PBMCs) and associated clinical data were collected from patients after informed consent as approved by the Institutional Review Board at the North Shore-Long Island Jewish Health System and in accordance with the Helsinki Declaration. CLL samples were chosen based on availability with no pre-established inclusion/exclusion criteria. CLL RNA expression levels were examined by microarray or quantitative real-time PCR (qPCR). For microarray studies, CLL B cells were purified prior to RNA isolation and acquisition of microarray expression data using Illumina Human WG6 and HT12 bead chips, followed by quantile normalization using GenomeStudio software (Illumina). For qPCR, RNA expression from CLL PBMCs was measured relative to glyceraldehyde 3-phosphate dehydrogenase gene expression by Taqman assay with Roche UPL probes and LightCycler 480. To examine de novo mutations in CLL, the IGHV region was ultra-deep sequenced (Roche 454 FLX system) from human CLL cells recovered from the NOD/Shi-scid,γc^null (NSG) xenograft mouse model of CLL as approved by the Institutional Animal Care and Use Committee at the North Shore-Long Island Jewish Health System.

CLL patient (N = 65) RNA expression by microarray showed very low levels of APOBEC1, 2, 3A, 3B, 3D, 4, and AID, modest levels of APOBEC3C and 3H, and high levels of APOBEC3F and 3G. Higher AID expression levels significantly correlated (P <0.05) with shorter time to first treatment (TFT), which was anticipated based on previous reports. Interestingly APOBEC3B and APOBEC3F expression differences showed possible trends correlating with worse patient outcome. Therefore, we tested select APOBEC gene family members by qPCR. For qPCR, we utilized the CLL patient cohort (N= 83) previously found to indicate that AID expression was a risk factor for worse patient outcome in a multivariate analysis (Patten et al. 2012 Blood 120:4802). RNA expression by qPCR followed the same pattern as the microarray data: AID and APOBEC3B had very low levels, APOBEC3H had modest levels, and APOBEC3F and 3G had high levels. Similar to AID, patients could be grouped based on the presence or absence of detectable APOBEC3B, with its presence showing a significant correlation (P <0.05) with worse TFT and overall survival. Higher levels of APOBEC3F and 3H showed a trend towards a correlation with shorter TFT, while differences in APOBEC3G expression had no significant correlation with patient outcome. Thus, not only did we confirm the correlation of AID expression with worse patient outcome, but we also found APOBEC3B and potentially APOBEC3F and 3H correlate with worse patient outcome.

To test if CLL cells can acquire de novo mutations indicative of APOBEC gene family member activity, human CLL cells were transferred into NSG mice. After CLL cells proliferated for 4-14 weeks in this xenograft model, the IGHV region was amplified, ultra-deep sequenced, and analyzed for specific mutational characteristics of various APOBEC gene family members. The results of these ongoing analyses will be presented.

In conclusion, the expression levels of the APOBEC gene family members AID, APOBEC3B, and potentially APOBEC3F and 3H, correlate with worse patient outcome. These data are consistent with the hypothesis that APOBEC gene family member activity may promote new mutations at sites outside the IG gene loci leading to the evolution of aggressive CLL.