Increased level of phosphorylated akt measured by chemiluminescence-linked immunosorbent assay is a predictor of poor prognosis in primary breast cancer overexpressing ErbB-2

Abstract

Introduction: Akt1, Akt2 and Akt3 kinases are downstream components of phosphoinositol 3-kinase derived signals from receptor tyrosine kinases, which influence cell growth, proliferation and survival. Akt2 overexpression and amplification have been described in breast, ovarian and pancreatic cancers. The present study was designed to investigate the prognostic significance of activated Akt in primary breast cancer and its association with other tumour biomarkers.

Methods: Using a two-site chemiluminescence-linked immunosorbent assay, we measured the quantitative expression levels of total phosphorylated (P-S473) Akt (Akt1/Akt2/Akt3) on cytosol fractions obtained from fresh frozen tissue samples of 156 primary breast cancer patients.

Results: Akt phosphorylation was not associated with nodal status or ErbB-2 protein expression levels. High levels of phosphorylated Akt correlated (P < 0.01) with poor prognosis, and the significance of this correlation increased (P < 0.001) in the subset of patients with ErbB-2 overexpressing tumours. In addition, phosphorylated Akt was found to be associated with mRNA expression levels of several proliferation markers (e.g. thymidylate synthase), measured using quantitative real-time RT-PCR.

Conclusion: Our findings demonstrate that, in breast cancer patients, Akt activation is associated with tumour proliferation and poor prognosis, particularly in the subset of patients with ErbB2-overexpressing tumours.

Figure 1

Chemiluminescence-linked immunoassay (CLISA)-quantified phosphorylated Akt (P-Akt) levels. (a) Histogram showing distribution of chemiluminescence-linked immunoassay (CLISA)-determined phosphorylated Akt (P-Akt) expression levels in 156 primary breast cancer samples. P-Akt levels ranged from 0 to 1.08 U/mg, with a median of 0.17 U/mg. (b) Scatter plot of P-Akt versus ErbB-2 expression levels. No correlation was found between the levels of P-Akt and ErbB-2.

Figure 2

Kaplan–Meier survival curves for patients overall. The curves are stratified by phosphorylated Akt (P-Akt) levels. Patients whose tumors express high levels of P-Akt exhibit a significantly worse outcome in terms of disease-free survival (DFS; P < 0.01).

Figure 3

Kaplan–Meier survival curves for the subset of patients with ErbB-2 overexpressing tumours. The curves stratified by (a) median and (b) last quartile values of phosphorylated Akt (P-Akt). Patients whose tumours express high levels of P-Akt exhibit a significantly worse outcome in terms of disease-free survival (DFS; P ≤ 0.005).

Figure 4

Scatter plot of phosphorylated Akt (P-Akt) versus thymidylate synthase (TS) mRNA expression. There is a good positive correlation (r_s = 0.38; P < 0.001) between the two factors.