Transcriptosome and Serum Cytokine Profiling of B-CLL Identifies Oncogenic Pathways in Pathogenesis and Survival.

Background: B-cell chronic lymphocytic leukemia (B-CLL) the most common leukemia in the Western world accounts for 25% of all newly diagnosed leukemias. Despite new therapeutic advances, B-CLL is currently not curable. Potential oncogenic signaling pathways in B-CLL require elucidation. We identified ROR-1 receptor tyrosine kinase (RTK) in the WNT/Planar Cell Polarity non-canonical pathway as a possible mechanism of oncogenesis by gene expression profiling (GEP).

Methods: Mononuclear cells from 8 low stage CLL patients were obtained through an IRB approved protocol and analyzed utilizing the HG-U133A 2.0 Affymetrix array (~18,400 transcripts, 22,000 probe sets) after isolating and purifying total RNA (Qiagen, RNAeasy). The control RNA samples were isolated from 8 normal volunteer peripheral blood (PB) B-cells (AllCell, CA) and a normal reactive lymph node. Tumor lineage was confirmed by immunohistochemistry (IHC). Quantitative real time RT-PCR was performed on 20 selected genes to validate the microarray GEP. Twenty one B-CLL patients (includes the 8 patients from the GEP) were evaluated by RT-PCR for several key components of oncogenic signaling pathways based on the GEP with gene specific probes. A serum cytokine profile (120 cytokines) on 12 CLL patients (8 for which GEP is available and 4 additional patients) and 4 normal volunteers were performed for identification of a signature for diagnostic and prognostic value.

Results: Data are represented as “robust” increases or decreases of relative gene expression common to 8 patients. ROR-2, a close member of ROR-1, has been shown to bind Wnt-5A via the cysteine rich domain (CRD) and to activate the JNK signaling pathway. Our findings identify over-expression of members of the non-canonical WNT/PCP- ROR-1- signaling pathway genes that were validated by RT-PCR. A serum cytokine profile of 12 patients provides a signature that may be useful in CLL diagnosis and prognosis.

Conclusions: GEP identified WNT/PCP-ROR-1 as key components of an autocrine pathway that helps B-CLL avoid apoptosis. Several serum cytokines are elevated and require validation as potential diagnostic and prognostic markers. GEP of B-CLL in combination with quantitative real time RT-PCR has identified several novel targets for therapy. The identification of ROR-1 RTK has led to the development of a molecular target for future therapeutic application. Several lead compounds have been identified and are being evaluated as potential therapies in B-CLL.