A Robust Immunohistochemical Assay for Detecting PTEN Expression in Human Tumors

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.

Combined analysis of KRAS and PIK3CA mutations, MET and PTEN expression in primary tumors and corresponding metastases in colorectal cancer

“…However, our results should be considered with caution as a common drawback of all studies evaluating expression of PTEN by immunohistochemistry is the lack of a robust universally-acceptable PTEN immunohistochemistry assay and because the differences observed between primary tumors and related metastases could be the result of previous medical treatments. 31,48 Previous studies have shown that loss of PTEN nuclear expression and BRAF mutations were associated with poor clinical outcome while the prognostic significance of PIK3CA and KRAS mutations in colorectal cancer remains elusive. 24,[49][50][51][52][53][54][55][56][57][58] In our series, loss of PTEN nuclear expression in primary tumors and the PIK3CA mutations in metastases were associated with shorter overall survival (Supplementary Table S2); however, neither PTEN expression nor PIK3CA mutations were emerged as independent prognostic factors in multivariate analysis ( Table 4).…”

Progesterone Receptor and PTEN Expression Predict Survival in Patients With Low- and Intermediate-Grade Pancreatic Neuroendocrine Tumors

“…Nuclear staining for PR in 1% or more of the whole tumor section was considered positive, while those cases with staining in less than 1% of the section were considered negative, a cutoff used in breast cancer 29,30 and one previous study on PanNET. 6 PTEN expression was recorded as follows: positive, both cytoplasmic and nuclear staining intensity in greater than 10% of tumor cells was equal to that of stromal cells (internal positive control); low, at least 90% (ie, 90%-100%) of tumor cells showed weak or faint cytoplasmic and nuclear staining, compared to stromal cells; and negative, at least 90% (ie, 90%-100%) of tumor cells showed complete loss of PTEN immunohistochemical staining, as outlined in a validation study of PTEN immunohistochemistry by Sangale et al 31 …”

Phase II study of dual phosphoinositol‐3‐kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor BEZ235 in patients with locally advanced or metastatic transitional cell carcinoma

“…The staining score was calculated based on the percentage of stained cells (%) with the formula (% weak 92% intermediate 93% strong). There was no loss of PTEN expression if at least 10% of the cells were weakly positive [35].…”