Pten encodes a well-characterized protein that is important in several cancers due to its tumor suppressor function. Yet, the detection and evaluation of PTEN by immunohistochemistry (IHC) for clinical practice have not been standardized.
Thus, in this study, we performed a literature review of protocols for PTEN assessment by IHC and the possible differences in evaluation, based on our experience with vulvar carcinomas. Also, we report some of our most recent findings regarding the clinical impact of PTEN in this type of tumor.
In total, 150 FFPE vulvar carcinoma samples in a tissue microarray were examined by IHC with regard to PTEN, PI3K, AKT, and mTOR. All evaluations were performed by slide digitalization and quantification using APERIO ImageScope software. All measurements were converted into HScore values for the statistical analysis.
Sharp and specific PTEN expression was observed in the nuclei and cytoplasmic compartments.
Its HScore values ranged from 3.5 to 226, with a median of 92.5. mTOR expression was robust in all cases (mean HScore=248.1). AKT and PI3K had median HScore values of 200.5 and 156.5, respectively.
In addition, PTEN expression was associated with higher rates of patient survival.
The preanalytical step is the first issue in the immunohistochemical evaluation of PTEN. With regard to the analytical procedure, the antigen retrieval step yielded better stains for protocols with high-pH buffers, and antibody clone 6H2.1 effected the most reliable results.
PTEN is a good prognostic marker for vulvar cancer, correlating with higher rates of patient survival. Our data underscore the importance of technical standardization to ensure more reliable and reproducible evaluation of PTEN in clinical practice.
Expression pattern of class I histone deacetylases in vulvar intraepithelial neoplasia and vulvar cancer: a tissue microarray study.
Epigenetic regulation is an important mechanism leading to cancer initiation and promotion. Histone acetylation by histone deacetylases (HDACs) represents an important part of it. The development of HDAC inhibitors has identified the utility of HDACs as a therapeutic target.
Little is known about the epigenetic regulation of vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell cancer (VSCC).
In this study, the expression of class I HDACs (HDAC 1, 2 and 3) was compared in a series of VIN and VSCC tissues.
A tissue micro array (TMA) with specimens from 106 patients with high-grade VIN and 59 patients with vulvar cancer was constructed. The expression of HDACs 1, 2 and 3 were analyzed with immunohistochemistry (IHC). The nuclear expression pattern was evaluated in terms of intensity and percentage of stained nuclei and was compared between vulvar preinvasive lesions and vulvar cancer.
HDAC 2 expression was significantly higher in VIN than in VSCC (p < 0.001, Fisher’s test). Also, 88.7% (n = 94/106) of VIN samples and only 54.5% (n = 31/57) of VSCC samples were scored at the maximum level. Conversely, HDAC 3 expression was significantly higher in VSCC (93%, 53/57) compared to VIN (73.6%, 78/106, p = 0.003), whereas only a small difference in the expression of HDAC 1 was found between these two entities of vulvar neoplasia.
These results suggest that epigenetic regulation plays a considerable role in the transformation of VIN to invasive vulvar neoplasia.
Hemoglobin level predicts outcome for vulvar cancer patients independent of GLUT-1 and CA-IX expression in tumor tissue.
Intratumoral hypoxia has been associated with poor prognosis in several solid tumors.
The aim of this study was to determine whether the hypoxia-associated markers glucose transporter (GLUT)-1 and carbonic anhydrase (CA)-IX expression and preoperative hemoglobin (Hb) levels correlate with presence of inguinofemoral or distant metastases, and disease-free survival (DSS) in vulvar squamous cell carcinoma (SCC) patients.
Vulvar SCC (n=103) were reviewed for histopathological characteristics by an expert gynecopathologist and stained for GLUT-1 and CA-IX.
Clinical data and preoperative Hb levels were obtained from medical records. No significant correlations were observed between GLUT-1 or CA-IX expression patterns and preoperative Hb levels, presence of inguinofemoral or distant metastases and DSS.
However, anemic patients (Hb<11.2 g/dL) had significantly more inguinofemoral metastases and lower Hb level was an independent prognostic factor for a worse DSS (p<0.001).
The number of comorbidic conditions was inversely correlated with preoperative Hb level. Preoperative Hb levels are associated with poor DSS for vulvar SCC patients, whereas tumor hypoxia reflected by GLUT-1 and CA-IX expression does not have a predictive value.
Because preoperative Hb levels inversely correlated with the number of comorbidic conditions and not with GLUT-1 or CA-IX expression, it is most likely that preoperative Hb levels represent overall physical condition.
Validation of tissue microarray technology in vulvar cancer.
The aim of the study was to validate tissue microarray (TMA) for vulvar cancer by comparing immunohistochemical staining results of triplicate core biopsies on TMA with the results of full section analysis. The study material consisted of slides and selected tissue blocks from 40 patients with vulvar cancer.
A TMA was constructed with 3 cores/case. Both the TMA and the slides were stained with the same antibodies against COX-2, Caspase-3, epidermal growth factor receptor, p16INK4, Cyclin D1, and Ki67. For COX-2, 2 different scoring systems were applied.
Agreement in the readings between TMA and slides was expressed in total agreement and kappa. Expression patterns of antibodies can be reproduced on TMA with good reliability (kappa 0.68 to 0.75) for Ki-67, p16INK4, COX-2, Cyclin D1, and epidermal growth factor receptor in comparison with whole slides.
For Caspase-3 agreement is only slight with a kappa of 0.40. The majority of discordant cases for COX-2 and Ki67 were negative on slide and positive on TMA.
For epidermal growth factor receptor and Caspase-3 an opposite pattern was found. For COX-2, the use of an alternative scoring system resulted in a decrease of kappa from 0.68 to 0.21.
Agreement between results on TMA and slides depends on the distribution of the protein in the cancer tissue and on the scoring system used.