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Neuroblastoma is a tumor derived from primitive ganglion cells of the sympathetic nervous system and is the most common solid tumor in childhood. Akt plays a critical role in controlling the balance between cell survival and apoptosis. Activation of Akt occurs through its phosphorylation by phosphoinositide 3-kinase (PIK3CA), while it is negatively regulated by PTEN phosphatase. Activating mutations of the PIK3CA gene have been found at a high frequency in many human cancers. As neuronal-derived tumors of the brain have been shown to harbor PIK3CA mutations, and as PTEN inactivation by deletion and mutation is rare in neuroblastoma, we considered the possibility of PIK3CA alterations in this tumor. The most frequently mutated regions of PIK3CA gene are exons 9 and 20, which encode the functional helical and kinase domains, respectively, of the enzyme. Therefore, to analyze for mutations in this region, we have developed a highly sensitive screening technique using denaturing High Performance Liquid Chromatography (dHPLC). Using DNA from the MCF7 breast cancer cell line, which harbors a PIK3CA mutation in exon 9, and the LS174 colon cancer cell line, which harbors a PIK3CA mutation in exon 20, we were able to reproducibly detect mutations in both of these exons using dHPLC. We next screened 23 neuroblastoma cell lines and 105 primary neuroblastoma samples (35 stage I, II and IVS samples and 70 stage III and IV samples) for PIK3CA mutations in exons 9 and 20 using dHPLC. No neuroblastoma cell lines were found to have PIK3CA mutations. However, one stage III primary neuroblastoma sample was found to have a dHPLC profile in exon 20 that was analogous to that of the LS174 cell line. Sequence analysis of this sample confirmed the presence of a heterozygous mutation at bp3236, which results in a Leu → Pro amino acid change at codon 1079. This sample contained N-myc amplification but was otherwise unremarkable versus the rest of the neuroblastoma samples analyzed; survival data on this patient was unavailable. In summary, we have developed a highly sensitive, rapid and cost-effective dHPLC technique to screen for mutations in PIK3CA exons 9 and 20 where greater than 75% of the reported mutations have been localized. With regard to neuroblastoma, PIK3CA mutation is an uncommon event that may only rarely be involved in the pathology of this disease.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]