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.
Prostate cancer is the most commonly diagnosed malignancy in men, but many patients have indolent disease that is unlikely to cause significant morbidity or mortality. Biomarkers that accurately predict disease severity would enable physicians to more appropriately manage patient care. Data from previous studies indicated that the molecular status of PTEN (phosphatase and tensin homolog) might be prognostic in prostate cancer patients after radical prostatectomy (RP). PTEN expression was evaluated on surgical specimens by IHC using anti-PTEN antibody 138G6 (Cell Signaling Technology) in 132 men treated with RP. Approximately half the patients also had neoadjuvant hormonal blockade therapy (BAT). PTEN IHC scores were derived from the weighted average of percent positive cells x intensity (0, 1 or 2). PTEN status (positive or negative) was determined using a predefined IHC score of ≤ 20 for negatives. Using this scoring system, 24% of the tumors were PTEN negative. Association with biochemical recurrence was evaluated using Cox PH models and likelihood ratio tests. Since we were testing a prior hypothesis about the prognostic utility of PTEN, the corresponding p-values are one-sided. In a univariate analysis on the overall series, PTEN status was associated with biochemical recurrence (p-value = 0.0046, HR = 2.3; 95% CI: 1.27, 4.33). Most of the clinical parameters typically associated with recurrence were affected by neoadjuvant therapy and rendered uninterpretable. Nevertheless, pathological tumor stage (p-value = 4.3 × 10−6, HR = 6.0; 95% CI: 2.35, 15.22) was still associated with outcome. In multivariate analyses, PTEN status was a significant predictor of outcome after adjusting for pathological stage (p-value = 0.009), neoadjuvant therapy (p-value = 0.014), or both (p-value = 0.04). These data support the hypothesis that PTEN status predicts outcome after RP, and suggest further, that PTEN may be a clinically important molecular marker for defining aggressive prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1186.
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