Integrated Molecular and Clinical Analysis of AKT Activation in

Purpose—Activation of the PI3K-AKT pathway has been implicated in melanoma based primarily on the prevalence of mutations in PTEN and NRAS. To improve our understanding of the regulation and clinical significance of the PI3K-AKT pathway in melanoma, we quantitatively measured the levels of phosphorylated AKT (P-AKT), its substrate GSK3α/β, and its negative regulator PTEN in clinical metastases. Results were compared to mutational status, clinical outcomes, and sites of metastasis. Experimental Design—DNA and protein were isolated from dissected frozen melanoma metastases (n=96). Activating mutations of BRAF, NRAS, AKT, PIK3CA, and KIT were detected by mass-spectroscopy genotyping. P-AKT (Ser473 and Thr308), P-GSK3α/β, and PTEN protein expression were measured by reverse phase protein array (RPPA). A panel of human melanoma cells lines (n=58) was analyzed for comparison. Results—BRAF-mutant tumors had higher levels of P-AKT-Ser473 (P=.01), P-AKT-Thr308 (P=. 002), and P-GSK3α/β (P=.08) than NRAS-mutant tumors. Analysis of individual tumors demonstrated that almost all tumors with elevated P-AKT had low PTEN levels; NRAS-mutan