PIK3CA alterations in Middle Eastern ovarian cancers

Abstract

Background: PI3K/AKTsignaling pathway plays an important role in cell growth, proliferation, and tumorgenesis of various malignancies. This signaling pathway has been shown to be frequently altered in several human cancers including ovarian cancers. However the role of this oncogenic signaling pathway has not been explored in the Middle Eastern epithelial ovarian cancer (EOC). Therefore, we investigated PI3K/AKT genetic alterations such as PIK3CA amplification, PIK3CA mutation, PTEN protein loss and their relationships with various clinicopathological characteristics in 156 EOCs.

Results: Fluorescence in situ hybridization (FISH) technique and DNA sequencing were used to analyze PIK3CA amplification and mutation respectively. Expression of PIK3CA protein expression (p110 alpha), PTEN, p-AKT and Ki-67 was analyzed by immunohistochemistry. PIK3CA amplification was seen in 54 of 152 (35.5%) EOC cases analyzed; PIK3CA gene mutations in 6/153 EOC (3.9%); KRAS mutations in 3/154 EOC (1.9%), BRAF mutations in 3/156 EOC (1.9%), p53 mutation in 50/154 EOC (32.5%), and loss of PTEN protein expression in 33/144 EOC (22.9%). p110 alpha overexpression was associated with increased phosphorylation of AKT-Ser 473 and with the proliferation marker Ki-67.

Conclusion: Our data showed mutual exclusivity between the molecular event of PIK3CA amplification and mutations in PIK3CA, KRAS, BRAF genes, which suggests that each of these alterations may individually be sufficient to drive ovarian tumor pathogenesis independently. High prevalence of genetic alterations in PI3K/AKT pathway in a Middle Eastern ovarian carcinoma provides genetic evidence supporting the notion that dysregulated PI3K/AKT pathways play an important role in the pathogenesis of ovarian cancers.

Figure 1

Examples of PIK3CA, KRAS, and BRAF mutations in EOC cases. A. Typical sequencing traces of PIK3CA mutations are shown for exon 9 (right) and 20 (left) with cancer mutant sequence (bottom) and normal wild-type sequence (top). B. Sequencing traces of BRAF (right) and KRAS (left) mutations with cancer mutant sequence (bottom) and normal wild-type sequence (top). Arrows denote position of the missense mutations with amino acid changes noted.

Figure 2

Determination of PIK3CA gene copy number by FISH on ovarian tissue (A). B. Sample 1 is normal cell selected according to FISH analysis. Samples 2–4 are PIK3CA amplified ovarian tumor cells selected according to FISH. FISH images show cell nuclei (blue) from selected cases, hybridized with probes directed against PIK3CA gene (green RP11-245 C23) and centromere 3 (red). (1). Normal cell (blue) shows 2 centromeric signals (red) and 2 (green) PIK3CA signals. (2–4). Representative cells show amplification two (red) centromeric signals and green PIK3CA amplified Signals.

Figure 3

Immunohistochemical analysis of p-AKT, PI3K-110 alpha subunit protein expression and PTEN in epithelial ovarian carcinoma (EOC). (i) p-AKT over expression was observed along with (iii) low PTEN expressionand (v) over expression of PI3K-110 alpha in EOC TMA specimen and, (ii) Low expression for p-AKT was seen along with (iv) high PTEN expression and (vi) reduced expression for PI3K-110 alpha in EOC TMA specimen. 20 × magnifications with the inset showing a 100 × magnified view of the same.