title: PTEN mRNA detection by chromogenic, RNA in situ technologies: a reliable alternative to PTEN immunohistochemistry.
abstract: Immunohistochemical staining for phosphatase and tensin homolog (PTEN) does not have either an acceptable standard protocol or concordance of scoring between pathologists. Evaluation of PTEN mRNA with a unique and verified sequence probe may offer a realistic alternative providing a robust and reproducible protocol. In this study, we have evaluated an in situ hybridization (ISH) protocol for PTEN mRNA using RNAScope technology and compared it with a standard protocol for PTEN immunohistochemistry (IHC). PTEN mRNA expression by ISH was consistently more sensitive than PTEN IHC, with 56% of samples on a mixed-tumor tissue microarray (TMA) showing high expression by ISH compared with 42% by IHC. On a prostate TMA, 49% of cases showed high expression by ISH compared with 43% by IHC. Variations in PTEN mRNA expression within malignant epithelium were quantifiable using image analysis on the prostate TMAs. Within tumors, clear overexpression of PTEN mRNA on malignant epithelium compared with benign epithelium was frequently observed and quantified. The use of SpotStudio software in the mixed-tumor TMA allowed for clear demonstration of varying levels of PTEN mRNA between tumor samples by the mRNA methodology. This was evident by the quantifiable differences between distinct oropharyngeal tumors (up to 3-fold increase in average number of spots per cell between 2 cases). mRNA detection of PTEN or other biomarkers, for which optimal or standardized immunohistochemical techniques are not available, represents a means by which heterogeneity of expression within focal regions of tumor can be explored with more confidence.
authors.affiliation: Molecular Pathology Programme, Centre for Cancer Research and Cell Biology, Queen's University, Belfast, UK BT9 7AE.
authors.forename: Victoria
authors.initials: V
authors.lastname: Bingham
authors.affiliation: Prostate Cancer Research Group, Centre for Cancer Research and Cell Biology, Queen's University, Belfast, UK BT9 7AE.
authors.forename: Chee Wee
authors.initials: CW
authors.lastname: Ong
authors.affiliation: Molecular Pathology Programme, Centre for Cancer Research and Cell Biology, Queen's University, Belfast, UK BT9 7AE; Tissue Pathology, Belfast Health and Social Care Trust, Belfast City Hospital, Lisburn Rd BT9 7AB.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: James
authors.affiliation: Prostate Cancer Research Group, Centre for Cancer Research and Cell Biology, Queen's University, Belfast, UK BT9 7AE.
authors.forename: Pamela
authors.initials: P
authors.lastname: Maxwell
authors.affiliation: Prostate Cancer Research Group, Centre for Cancer Research and Cell Biology, Queen's University, Belfast, UK BT9 7AE.
authors.forename: David
authors.initials: D
authors.lastname: Waugh
authors.affiliation: Molecular Pathology Programme, Centre for Cancer Research and Cell Biology, Queen's University, Belfast, UK BT9 7AE; Tissue Pathology, Belfast Health and Social Care Trust, Belfast City Hospital, Lisburn Rd BT9 7AB.
authors.forename: Manuel
authors.initials: M
authors.lastname: Salto-Tellez
authors.affiliation: Molecular Pathology Programme, Centre for Cancer Research and Cell Biology, Queen's University, Belfast, UK BT9 7AE; Tissue Pathology, Belfast Health and Social Care Trust, Belfast City Hospital, Lisburn Rd BT9 7AB. Electronic address: s.mcquaid@qub.ac.uk.
authors.forename: Stephen
authors.initials: S
authors.lastname: McQuaid
mesh_terms: D057205:Automation, Laboratory; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002863:Chromogenic Compounds; D006801:Humans; D007090:Image Interpretation, Computer-Assisted; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D051059:PTEN Phosphohydrolase / Q000235:genetics* / Q000378:metabolism; D011237:Predictive Value of Tests; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012984:Software; D046888:Tissue Array Analysis
chemical_list: D014408:Biomarkers, Tumor; D002863:Chromogenic Compounds; D012333:RNA, Messenger; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human
keywords: Chromogenic ISH; Heterogeneity; Immunohistochemistry; PTEN; RNA