The association of PI3 kinase signaling and chemoresistance in advanced ovarian cancer

Abstract

Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3β by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3β. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN.

Figure 1

Association of clinical response and signaling output of the PI3K pathway. The differences in the phosphorylation of p70S6K, AKT, and GSK3β in patients, who had a best response of complete response (CR) or partial response (PR) versus patients who had stable disease (SD) or progressive disease (PD), were measured by ELISA in immunomagnetically separated cancer cells from ascites of patients undergoing palliative paracentesis.

Figure 2

Representative samples of PIK3CA and AKT amplification and PTEN deletion. FISH analysis of PIK3CA, AKT2 and PTEN. Each slide underwent immunofluorescence to positively identify cancer cells (Ber-EP4) and negatively identify noncancer cells, CD45 (CD45; white blood cells), CD68 (macrophages), and podoplanin (mesothelial cells). A, an example of a sample of ascites with cells expressing Ber-EP4 and CD45 are presented at a ×20 magnification. B, in excess of 2 copies of the PIK3CA genes (green) in relation to markers for centromere 3 in a cell (red). C, an excess of 2 copies of the AKT2 gene (green) in relation to 19q telomere in a cell (red). D, less than 1 PTEN gene (red) copy in relation to markers for centromere 10 (green). All FISH images are at ×63 magnification.