Correlation between AKT activation (pAKT) and clinico-pathologic features of patients with OC.

a<p>Patients for which pAKT staining was available (N = 93).</p>b<p>Patients for which both Grade and pAKT staining were available (N = 87).</p>c<p>Patients for which both Figo Stage and pAKT staining were available (N = 93).</p><p><b>NS</b>: not significant.</p

AKT activation in OC.

a<p>Patients for which pAKT staining was available (N°).</p>b<p>Normal vs Tumour Tissue.</p><p><b>S-OC: S</b>erous <b>O</b>varian <b>C</b>arcinoma.</p><p><b>E-OC: E</b>ndometrioid <b>O</b>varian <b>C</b>arcinoma.</p><p><b>CC-OC: C</b>lear <b>C</b>ell <b>O</b>varian <b>C</b>arcinoma.</p><p><b>Mu-OC: M</b>ucinous <b>O</b>varian <b>C</b>arcinoma.</p><p><b>M-OC: M</b>ixed <b>O</b>varian <b>C</b>arcinoma.</p><p><b>NS</b>: not significant.</p

Immunostaining and gene copy number analysis of PTEN in OC.

<p>A. Left: S-OC negative for PTEN expression; right: S-OC positive for PTEN expression. B. Left: E-OC negative for PTEN expression; right: E-OC positive for PTEN expression. Magnification 40X. Magnification of the insets 10X. C. Q-RT PCR of PTEN mRNA expression in normal ovarian tissues and OC. D. Q-PCR analysis of PTEN gene copy number in normal ovarian tissues and OC. DNA from peripheral blood leukocytes (PBL) was used as reference. PTEN copy number in PBL was set arbitrarily as 2.</p

Immunostaining analysis of PIK3CA in OC.

<p>A. Left: S-OC negative for PIK3CA expression; right: S-OC positive for PIK3CA expression. B. Left: E-OC negative for PIK3CA expression; right: E-OC positive for PIK3CA expression. Magnification 40X. Magnification of the insets 10X.</p

Mutation analysis of PIK3CA and KRAS genes in OC.

<p>A. Detection of mutations in PIK3CA by LightCycler (left) and direct sequencing (right). On the left, the negative derivative of the fluorescence (−dF/dT) versus temperature graph shows peaks with different Tm. The wild type sample showed a single Tm at 66°C. The heterozygous mutant sample showed an additional peak at 57°C. On the right, GAG→GCG transition in codon 545 of exon 9 inducing the substitution of a glutammic acid with an alanine (E545A). B. pAKT staining of a mutated S-OC (left) and E-OC (right). C. Point mutations in exon 2 of KRAS gene: GGT→GTT (G12V), GGT→CGT (G12R), GGT→ GAC (G13V). D. pAKT staining of a KRAS mutated sample (MU-6).</p

Immunostaining analysis of AKT2 in OC.

<p>A. Left: S-OC negative for AKT2 expression; right: S-OC positive for AKT2 expression. B. Left: E-OC negative for AKT2 expression; right: E-OC positive for AKT2 expression. Magnification 40X. Magnification of the insets 10X.</p

Analysis of pathways activated downstream PI3K in OC: mTOR and SGK3.

<p>A. Immunostaining analysis of phosphorylated AKT, mTOR, S6K1, S6, and 4EBP1 in two representative formalin-fixed samples: left, p110α low (S17) and right, p110α high (S46). B. Protein from fresh-frozen samples were assayed by Western blot with the indicated antibodies. Samples S8, S17, S47, E2: low p110α expression; samples S63, E13: PIK3CA mutation; samples S63, S46, E3: p110α over-expression. Actin expression was used as a control for protein quality and loading. C. Representative pSGK3 immunostaining of pSGK3-positive (upper left panel) and pSGK3-negative (lower left panel) samples. Middle panels, immunostaining analysis of pAKT; right panels, immunostaining analysis of p110α of the same samples. Magnification 40X. Magnification of the insets 10X.</p

Correlation between AKT activation (pAKT) and expression of the different members of the PI3K pathway patients with OC.

<p><b>NS</b>: not significant.</p

Correlation between AKT activation and the presence of genetic alterations in PIK3CA, AKT1 and AKT2 in OC.

a<p>Low polisomy and negative samples for which pAKT staining was available.</p>b<p>High polisomy and amplified samples for which pAKT staining was available.</p

Immunostaining analysis of AKT1 in OC.

<p>A. Left: S-OC negative for AKT1 expression; right: S-OC positive for AKT1 expression. B. Left: E-OC negative for AKT1 expression; right: E-OC positive for AKT1 expression. Magnification 40X. Magnification of the insets 10X.</p