title: Oligonucleotide PIK3CA/Chromosome 3 Dual in Situ Hybridization Automated Assay with Improved Signals, One-Hour Hybridization, and No Use of Blocking DNA.
abstract: The PIK3CA gene 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 PIK3CA amplification and increased phosphatidylinositol 3-kinase (PI3K) pathway activities suggested that PIK3CA gene copy number is a potential predictive biomarker for PI3K inhibitors. Currently, all microscopic assessments of PIK3CA and chromosome 3 (CHR3) copy numbers use fluorescence in situ hybridization. PIK3CA probes are derived from bacterial artificial chromosomes whereas CHR3 probes are derived mainly from the plasmid pHS05. These manual fluorescence in situ hybridization assays mandate 12- to 18-hour hybridization and use of blocking DNA from human sources. Moreover, fluorescence in situ hybridization studies provide limited morphologic assessment and suffer from signal decay. We developed an oligonucleotide-based bright-field in situ hybridization 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 situ hybridization assay for the simultaneous detection of PIK3CA and CHR3 centromere provides a potential clinical diagnostic method to assess PIK3CA gene abnormality in lung tumors.
authors.affiliation: Research & Development, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Research & Development, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: Antony
authors.initials: A
authors.lastname: Hubbard
authors.affiliation: Research & Development, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: Leslie
authors.initials: L
authors.lastname: Baca-Parkinson
authors.affiliation: Research & Development, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: Stacey
authors.initials: S
authors.lastname: Stanislaw
authors.affiliation: Research & Development, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: Frank
authors.initials: F
authors.lastname: Vladich
authors.affiliation: Companion Diagnostics, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Robida
authors.affiliation: Research & Development, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: James G
authors.initials: JG
authors.lastname: Grille
authors.affiliation: Research & Development, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: Daniel
authors.initials: D
authors.lastname: Maxwell
authors.affiliation: Research & Development, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: Tsu-Shuen
authors.initials: TS
authors.lastname: Tsao
authors.affiliation: Medical and Scientific Affairs, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: William
authors.initials: W
authors.lastname: Carroll
authors.affiliation: Medical and Scientific Affairs, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: Tracie
authors.initials: T
authors.lastname: Gardner
authors.affiliation: Medical and Scientific Affairs, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: June
authors.initials: J
authors.lastname: Clements
authors.affiliation: Medical and Scientific Affairs, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona.
authors.forename: Shalini
authors.initials: S
authors.lastname: Singh
authors.affiliation: Research & Development, Pathology Office, Ventana Medical Systems, Inc., Roche Group, Tucson, Arizona. Electronic address: lei.tang@ventana.roche.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Tang
mesh_terms: D057205:Automation, Laboratory; D001483:Base Sequence; D044963:Biopsy, Fine-Needle; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000473:pathology; D002893:Chromosomes, Human, Pair 3 / Q000235:genetics*; D058534:Class I Phosphatidylinositol 3-Kinases; D015342:DNA Probes / Q000737:chemistry; D018628:Gene Dosage; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D012815:Signal Processing, Computer-Assisted; D014407:Tumor Cells, Cultured
chemical_list: D058534:Class I Phosphatidylinositol 3-Kinases; D015342:DNA Probes; D009841:Oligonucleotides; D019869:Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human