CLL Transcript and Serum Profiling Identifies Distinct Oncogenic Pathways in Rai Stage Progression.

Background: Chronic lymphocytic leukemia (CLL) is a common leukemia that is incurable. Survival curves for Rai stage 0/I and III/IV are distinct and a specific gene expression signature may be associated with stage progression (SP). However, distinct oncogenic pathways that differentiate stage progression are not well defined.

Methods: Mononuclear cells from Rai stage 0/I (N=8) and III/IV (N=8) CLL patients were obtained through an IRB approved protocol and analyzed utilizing the HG-U133A 2.0 Affymetrix array after isolating and purifying total RNA (Qiagen, RNAeasy). Control RNA samples are from 4 normal volunteers’ peripheral blood (PB) B-cells (AllCell, CA) and a normal reactive lymph node. Statistical significance of individual genes and pathways was analyzed using ‘BioConductor’. Differential gene expression was analyzed using ‘Limma’. Pathways and cytogenetics regions were analyzed for enrichment using ‘Category’. Real time RT-PCR on specific genes was performed to validate the GEP based on stage. A serum cytokine profile (120 cytokines, RayBiotech) on 8 CLL patients (N=4 low risk; N=4 high risk) and 4 normal volunteers was evaluated to identify a signature for diagnosis and prognosis.

Results: Data are presented as “robust” increases or decreases of relative gene expression common to 8 patients in the low or high risk stages. The Wnt/Ror-1 non-canonical, and TGFb pathways are over-expressed in Rai stage 0/I, implicating a program of cell survival and dysregulated migration. The Hedgehog, Proteasome, TNF/FAS/NF-kB, and MAPK pathways are over-expressed in Rai stage III/IV, suggestive of a tumor microenvironment driven program in SP. Genes located on chromosome 3q27.1 are also dysregulated in Rai stage III/IV. A distinct serum cytokine profile also differentiates low from high stage CLL.

Conclusions: GEP of CLL with RT-PCR validation has identified distinct oncogenic pathways that distinguish Rai stage 0/I from III/IV. A serum cytokine profile also differentiates between low and high risk CLL. These data together provide a ‘molecular signature’ for SP in CLL.