Search

The PIK3CAgene at chromosome 3q26.32 was found to be amplified in up to 45% of patients with squamous cell carcinoma of the lung. The strong correlation between PIK3CAamplification and increased phosphatidylinositol 3-kinase (PI3K) pathway activities suggested that PIK3CAgene copy number is a potential predictive biomarker for PI3K inhibitors. Currently, all microscopic assessments of PIK3CAand chromosome 3 (CHR3) copy numbers use fluorescence in situhybridization. PIK3CA probes are derived from bacterial artificial chromosomes whereas CHR3 probes are derived mainly from the plasmid pHS05. These manual fluorescence in situhybridization assays mandate 12- to 18-hour hybridization and use of blocking DNA from human sources. Moreover, fluorescence in situhybridization studies provide limited morphologic assessment and suffer from signal decay. We developed an oligonucleotide-based bright-field in situhybridization assay that overcomes these shortcomings. This assay requires only a 1-hour hybridization with no need for blocking DNA followed by indirect chromogenic detection. Oligonucleotide probes produced discrete and uniform CHR3 stains superior to those from the pHS05 plasmid. This assay achieved successful staining in 100% of the 195 lung squamous cell carcinoma resections and in 94% of the 33 fine-needle aspirates. This robust automated bright-field dual in situhybridization assay for the simultaneous detection of PIK3CAand CHR3 centromere provides a potential clinical diagnostic method to assess PIK3CAgene abnormality in lung tumors.