Abstract 39: Clinical assessment of PTEN mutation in FFPE tissue: comparison of Sanger sequencing, immunohistochemistry and chromogenic in situ hybridization methods.
Authors: Sakrison Kellen , Wright Jennifer , Bruening Eric , Wong Stephane , Spittle Cynthia , Sankar Sabita , Galderisi Chad 
Published: August 2014

Abstract
Background: PTEN is a tumor suppressor that negatively regulates the PI3K signaling pathway by dephosphorylating PIP3, converting it to PIP2. PTEN loss of function results in activation of Akt and downstream signaling pathways, and has been associated with numerous cancers, most commonly endometrial cancer, glioblastoma, melanoma and prostate cancer. PTEN loss can be a result of multiple different aberrations which include mutations in the coding region leading to frame-shift or stop codons, genomic deletions as well as promoter methylation leading to loss of PTEN protein. Determination of PTEN status, both at the DNA and protein levels, may be important for clinical decision-making because loss of PTEN is associated with resistance to various targeted therapies, while inactivating mutations may confer sensitivity to therapeutics targeting PI3K pathway members. We have developed Sanger sequencing, immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) assays to assess PTEN mutation status in a set of FFPE patient specimens.
Methods: Our Sanger sequencing assay for PTEN was developed to detect known hotspot mutations in exons 5-8, which occur in the majority of PTEN mutations reported in the COSMIC database. The IHC and CISH assays were developed using commercially available reagents. All assays were validated using a combination of plasmids, cell lines and FFPE tissues for specificity, sensitivity, reproducibility and concordance.
Results: 22 patient FFPE tissue specimens from a variety of tumor types including colon, lung, pancreas, uterus and prostate were evaluated for PTEN mutation and expression status by both Sanger sequencing and IHC methods. Sequencing results were: 9 wild-type, 12 heterozygous mutant, and 1 hemizygous mutant. 7 of the 9 specimens reported as wild-type by sequencing exhibited normal protein expression levels by IHC, while 2 were PTEN negative by IHC, suggesting that mechanisms other than mutations in exons 5-8 contributed to the loss of expression of PTEN protein. These 2 specimens were further analyzed by CISH to determine copy number. Of the 13 specimens reported as mutant by sequencing, IHC protein expression results indicated that 6 were PTEN positive and 7 were PTEN negative, underscoring the observation that exon 5-8 mutation status alone is not sufficient to predict PTEN expression levels. Data on CISH analysis of the specimen that was hemizygous mutant by sequencing will be reported to confirm the copy loss.
Conclusion: The data suggest that additional mutations outside the exon 5-8 region as well as promoter methylation may result in loss of PTEN protein, and that accurate clinical assessment of PTEN mutation and protein expression status in FFPE tissues requires complimentary methods including Sanger sequencing, IHC and CISH methodologies.
Citation Format: Kellen Sakrison, Jennifer Wright, Eric Bruening, Stephane Wong, Cynthia Spittle, Sabita Sankar, Chad Galderisi. Clinical assessment of PTEN mutation in FFPE tissue: comparison of Sanger sequencing, immunohistochemistry and chromogenic in situ hybridization methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 39. doi:10.1158/1538-7445.AM2013-39