A Robust Immunohistochemical Assay for Detecting PTEN Expression in Human Tumors


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Abstract

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of the phosphoinositol-3-kinase (PI3K)/AKT signaling pathway that controls cell cycle progression, growth and inhibition of apoptosis. Loss of PTEN protein expression has been associated with tumorigenesis, cancer progression and drug resistance, but conflicting results exist which may be due in part to difficulties inherent in PTEN immunohistochemistry (IHC). We sought a robust PTEN IHC assay. Human tumor cell lines with PTEN status verified by copy number analysis were formalin fixed and paraffin embedded for use as positive and negative controls. PTEN antibodies were optimized on tumor cell lines. Five optimized antibodies were analyzed on 10 molecularly characterized endometrial carcinoma samples. Four antibodies (CST, Millipore, Abcam, Novus) stained 3/10 positive and 7/10 negative, however, all but CST exhibited nonspecific nucleolar staining of negative controls. One antibody (Dako) stained 5/10 positive and 5/10 negative but with areas (≤10%) of positivity. The 4 samples predicted to be negative by sequencing were negative with the CST antibody, however, one was positive with Dako; as a result we chose the CST antibody for our assay. The assay was validated on an automated platform using 50 formalin fixed and paraffin embedded colon, lung, prostate and breast adenocarcinoma cases. Tumor cell lines served as external controls; endothelial cells and peripheral nerves served as internal positive controls. Dichotomous scoring achieved 100% concordance between three independent pathologists. This reproducible PTEN assay (PREZEON) has been implemented in a CLIA certified laboratory.

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