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.
"Furthermore there is variability over the definition of 'loss of PTEN’ based on IHC scoring. In the largest cohort of mCRC patients, PTEN loss was defined as no staining in any cells at any intensity
, while other groups have used various cut-offs of reduced PTEN expression
[17,28-30]. Others groups investigating the predictive role of PTEN have assessed PTEN allelic loss by fluorescent in situ hydridization (FISH)
, PTEN mutation
[6,19,20], and PTEN promoter methylation
 but concordance with loss of PTEN expression by IHC remains unclear. "
[Show abstract][Hide abstract] ABSTRACT: Loss of phosphatase and tensin homologue (PTEN) function evaluated by loss of PTEN protein expression on immunohistochemistry (IHC) has been reported as both prognostic in metastatic colorectal cancer and predictive of response to anti-EGFR monoclonal antibodies although results remain uncertain. Difficulties in the methodological assessment of PTEN are likely to be a major contributor to recent conflicting results.
We assessed loss of PTEN function in 51 colorectal cancer specimens using Taqman(R) copy number variation (CNV) and IHC. Two blinded pathologists performed independent IHC assessment on each specimen and inter-observer variability of IHC assessment and concordance of IHC versus Taqman(R) CNV was assessed.
Concordance between pathologists (PTEN loss vs no loss) on IHC assessment was 37/51 (73%). In specimens with concordant IHC assessment, concordance between IHC and Taqman(R) copy number in PTEN loss assessment was 25/37 (68%).
Assessment PTEN loss in colorectal cancer is limited by the inter-observer variability of IHC, and discordance of CNV with loss of protein expression. An understanding of the genetic mechanisms of PTEN loss and implementation of improved and standardized methodologies of PTEN assessment are required to clarify the role of PTEN as a biomarker in colorectal cancer.
BMC Cancer 02/2014; 14(1):128. DOI:10.1186/1471-2407-14-128 · 3.36 Impact Factor
"A negative sample was regarded as having no staining in either the cytoplasmic or nuclear cellular compartment; for positive samples, only cytoplasmic staining was scored (Sangale et al, 2011). In both the nucleus and the cytoplasm, PTEN staining was seen, but only cytoplasmic staining was used in the scoring. "
[Show abstract][Hide abstract] ABSTRACT: Background:
The natural history of prostate cancer is highly variable and difficult to predict. We report on the prognostic value of phosphatase and tensin homologue (PTEN) loss in a cohort of 675 men with conservatively managed prostate cancer diagnosed by transurethral resection of the prostate.
The PTEN status was assayed by immunohistochemistry (PTEN IHC) and fluorescent in situ hybridisation (PTEN FISH). The primary end point was death from prostate cancer.
The PTEN IHC loss was observed in 18% cases. This was significantly associated with prostate cancer death in univariate analysis (hazard ratio (HR)=3.51; 95% CI 2.60–4.73; P=3.1 × 10−14). It was highly predictive of prostate cancer death in the 50% of patients with a low risk score based on Gleason score, PSA, Ki-67 and extent of disease (HR=7.4; 95% CI 2.2–24.6; P=0.012) ), but had no prognostic value in the higher risk patients. The PTEN FISH loss was only weakly associated with PTEN IHC loss (κ=0.5). Both PTEN FISH loss and amplification were univariately predictive of death from prostate cancer, but this was not maintained in the multivariate analyses.
In low-risk patients, PTEN IHC loss adds prognostic value to Gleason score, PSA, Ki-67 and extent of disease.
British Journal of Cancer 05/2013; 108(12). DOI:10.1038/bjc.2013.248 · 4.84 Impact Factor
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