Levels of Phosphorylated P70 S6 Kinase and Phosphorylated ERK1&2 Predict Overall Survival and Time to First Treatment In Chronic Lymphocytic Leukaemia

Abstract 1363

Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease characterised by an accumulation of malignant B lymphocytes. Central to the survival of CLL-cells is the B-cell Receptor (BCR), a structurally complex moiety which contains an immunoglobulin molecule with antigenic specificity. Stimulation of the BCR by antigen binding may lead to the activation of multiple intracellular kinase cascades with a variety of intracellular functions. Kinases known to be activated include phosphorylated Extracellular Regulated Kinase (pERK) and P70 S6 Kinase (P70S6K).

Increased levels of phosphorylated Extracellular Regulated Kinase (pERK) are found in CLL cells induced to proliferate and inhibition of the ERK pathway promotes chemotherapy induced apoptosis in B-cell lines. P70S6K regulates cell cycle behaviour in B cells and it has been shown that inhibition of P70S6K activation by phosphorylation with rapamycin leads to cell cycle arrest in CLL cells.

We investigated the relationships between pERK isoforms 1&2 (pERK1&2) and phosphorylated-P70S6K concentrations (both baseline and after BCR-crosslinking) and overall survival (OS) as well as time to first treatment (TTFT) in a cohort of patients with CLL.

Lymphocytes were isolated from patients with CLL attending Haematology Clinic at the Hull and East Yorkshire NHS Trust. Collections were made after taking informed consent according to the Declaration of Helsinki with Local Ethics Committee approval (05/Q1104/33). pERK1&2 concentrations and phospho-P70S6K were measured quantitatively using ELISA according to the manufacturer's protocol (#SUV1018 &#DYC896, RnD Systems) in 99 such cases. Kinase concentrations were assayed at baseline in unstimulated cells and also after stimulation by antibody mediated BCR-crosslinking using a goat anti-human IgM antibody (#109-006-129, Jackson ImmunoResearch). The mutational status of the variable region of the immunoglobulin gene (IgV_H status) was determined in all cases. The pERK1&2 and phospho-P70S6K concentrations measured were correlated with clinical stage, IgV_H, ZAP70 and CD38 status. The individual kinase concentration measurements were then ranked and those in the upper quartile were regarded as being elevated. Statistical analysis was performed to assess the relationships between elevated pERK1&2 and phospho-P70S6K concentrations and OS/TTFT in both unstimulated CLL cells as well as after BCR-crosslinking.

An elevated baseline concentration of intracellular pERK1&2 was found to define a subset of IgV_H mutated patients with a reduced mean overall survival (157 vs. 259 months, p=0.011) and, also, to identify patients with a shorter median time to first treatment (8 vs. 31 months, p=0.050). An elevated baseline concentration of P70S6K defined subsets of both IgV_H mutated (168 vs. 254 months, p=0.046) and CD38 positive (57 vs. 234 months, p=0.000) patients with a reduced overall survival. An elevated concentration of pERK after BCR-crosslinking was shown to define a subset of ZAP70 negative patients with reduced overall survival (99 vs. 233 months, p=0.020), as well as identifying patients with a shorter median time to first treatment (8 vs. 37 months, p=0.011).

Our results demonstrate that increased baseline intracellular pERK1&2 and phospho-P70S6K concentrations can be used in conjunction with established prognostic markers to identify patients with a reduced OS and, in the case of pERK1&2, TTFT. An unmutated IgV_H status was associated with higher absolute pERK1&2 and phospho-P70S6K baseline concentrations. This suggests that increased intracellular signalling activity may contribute to the adverse prognosis seen in unmutated IgV_H patients. The findings support the investigation of kinase inhibitors as novel agents in the management of bad risk CLL.