Evaluation of p53 Immunohistochemical Expression Using Open-Source Software for Digital Image Analysis. A Tissue Microarray Study of Penile Squamous Cell Carcinomas.

The addition of molecular biomarkers is needed to increase the accuracy of pathologic factors as prognosticators of outcome in penile squamous cell carcinomas (SCC). Evaluation of these biomarkers is usually carried out by immunohistochemistry. Herein we assess p53 immunohistochemical expression on tissue samples of penile SCC using freely-available, open-source software packages for digital image analysis. We also compared the results of digital analysis with standard visual estimation. Percentages of p53 positive cells were higher by visual estimation than by digital analysis. However, correlation was high between both methods. Our study shows that evaluation of p53 immunohistochemical expression is feasible using open-source software packages for digital image analysis. Although our analysis was limited to penile SCC, the rationale should also hold for other tumor types in which evaluation of p53 immunohistochemical expression is required. This approach would reduce interobserver variability, and would provide a standardized method for reporting the results of immunohistochemical stains. As these diagnostic tools are freely-available online, researchers and practicing pathologists could incorporate them in their daily practice without increasing diagnostic costs.CONACYT - Consejo Nacional de Ciencia y TecnologíaPROCIENCI

Iatrogenic pathology of the urinary bladder

Intravesical immunotherapy, chemotherapy, and neoadjuvant systemic chemotherapy are among the most frequent therapeutic procedures to treat malignancies of the urinary bladder. These treatment modalities produce reactive morphologic changes in the urothelium that can mimic urothelial carcinoma in situ, urothelial dysplasia or true invasive urothelial neoplasia. Mitomycin C used after transurethral resection of bladder tumor to reduce recurrences, BCG intravesical immunotherapy to treat high risk non-muscle invasive bladder cancer and urothelial carcinoma in situ, and platinum-based systemic chemotherapy to improve post-cystectomy disease-specific survival some of the causes of therapy related atypia in urinary bladder. In addition, a number of systemic drugs in use to treat other systemic diseases, such as cyclophosphamide used to treat certain auto-immune disorders or hematologic malignancies, or the anesthetics ketamine increasingly used as illegal recreational drug, may produce similarly relevant atypical changes in the urothelium, and therefore, need to be differentiated from intraepithelial neoplasia. Immunohistochemical approach to reactive urothelium from CIS using CK20, p53, and CD44 may also be of utility in the pos-therapy scenario

Tissue expression of CD10 protein in colorectal carcinoma: correlation with the anatomopathological features of the tumor and with lymph node and liver metastases

BACKGROUND: The reduced expression of CD10 may be related to unfavorable prognosis of patients with colorectal carcinoma. The authors analyzed the tissue immunostaining of CD10 protein in colorectal carcinoma and its relationship to clinicopathologic features. METHOD: In 130 patients submitted to colorectal carcinoma surgery, a tissue microarray block was obtained from the tumor and adjacent non-neoplastic mucosa and submitted to immunohistochemistry with monoclonal antibody CD10. The immunostaining was evaluated by semi-quantitative method, with stained cell count in percentage. The results were related to the location, anatomopathological features, presence of lymph node and hepatic metastases and TNM staging of the colorectal neoplasm. The statistical analysis was performed with the Mann-Whitney, Kruskal-Wallis and Fisher exact tests. RESULTS: The expression of CD10 marker was higher in colorectal tumor tissue than in adjacent non-neoplastic mucosa (p<0.0001) and was higher than in exophytic lesions (p=0.04). The expression of CD10 protein was not associated with other clinical and pathological aspects of colorectal neoplasm. CONCLUSIONS: The expression of CD10 protein was more intense in tumor tissue of colorectal carcinoma than in adjacent non-neoplastic mucosa and was related to the exophytic appearance of the tumor.INTRODUÇÃO: A expressão reduzida de CD10 pode estar relacionada com prognóstico desfavorável de doentes com carcinoma colorretal. Analisou-se a imunoexpressão tecidual da proteína CD10 no carcinoma colorretal e sua relação com os aspectos clinicopatológicos. MÉTODO: Em 130 doentes operados por carcinoma colorretal, um bloco de tissue microarray foi obtido do tecido neoplásico e da mucosa não neoplásica adjacente e submetido ao estudo imuno-histoquímico com anticorpo monoclonal CD10. Avaliou-se a imunoexpressão por método semiquantitativo, com contagem do percentual de células coradas. Os resultados foram relacionados com a localização, aspectos anatomopatológicos, presença de metástases linfonodais e hepáticas e estadiamento TNM da neoplasia. O estudo estatístico foi realizado com os testes de Mann-Whitney, Kruskal-Wallis e exato de Fisher. RESULTADOS: A expressão do marcador CD10 foi maior no tecido do carcinoma colorretal do que na mucosa não neoplásica adjacente (p<0,0001) e foi maior nas lesões exofíticas (p=0,04). A expressão da proteína CD10 não apresentou relação com os demais aspetos clínicos e patológicos da neoplasia colorretal. CONCLUSÕES: A expressão da proteína CD10 foi mais intensa no tecido neoplásico do carcinoma colorretal do que na mucosa não neoplásica adjacente e relacionou-se com o aspecto exofítico do tumor.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Department of SurgeryUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Department of PathologyUNIFESP, EPM, Department of SurgeryUNIFESP, EPM, Department of PathologySciEL

Does Valproic Acid Induce Neuroendocrine Differentiation in Prostate Cancer?

Valproic Acid (VPA) is a histone deacetylase inhibitor that holds promise for cancer therapy. Here, we investigate whether VPA treatment induces neuroendocrine differentiation of Prostate Cancer (PCa). A tissue microarray of VPA-treated and untreated tumor xenografts and cell lines of human PCa (LNCaP, C4-2, DU145, and PC-3) were generated and were analyzed by immunohistochemical analysis (IHC) for NE markers chromogranin A (CgA), synaptophysin, and NCAM (neural cell adhesion molecule). Western blot analysis for CgA was performed to confirm the results of the TMA. IHC analysis did not reveal any induction of CgA, synaptophysin, or NCAM in any xenograft after VPA treatment in vivo. In vitro, VPA treatment induced little synaptophysin expression in C4-2 and PC-3 cells and NCAM expression in LNCaP and PC-3 cells. In the case of CgA, VPA treatment decreased its expression in vitro in a dose-dependent manner, as determined by western blot analysis. Thus our data demonstrates that VPA does not induce NE differentiation of PCa cells in the physiologically relevant in vivo setting

Characterization of glycine-N-acyltransferase like 1 (GLYATL1) in prostate cancer

BackgroundRecent microarray and sequencing studies of prostate cancer showed multiple molecular alterations during cancer progression. It is critical to evaluate these molecular changes to identify new biomarkers and targets. We performed analysis of glycine-N-acyltransferase like 1 (GLYATL1) expression in various stages of prostate cancer in this study and evaluated the regulation of GLYATL1 by androgen.MethodWe performed in silico analysis of cancer gene expression profiling and transcriptome sequencing to evaluate GLYATL1 expression in prostate cancer. Furthermore, we performed immunohistochemistry using specific GLYATL1 antibody using high-density prostate cancer tissue microarray containing primary and metastatic prostate cancer. We also tested the regulation of GLYATL1 expression by androgen and ETS transcription factor ETV1. In addition, we performed RNA-sequencing of GLYATL1 modulated prostate cancer cells to evaluate the gene expression and changes in molecular pathways.ResultsOur in silico analysis of cancer gene expression profiling and transcriptome sequencing we revealed an overexpression of GLYATL1 in primary prostate cancer. Confirming these findings by immunohistochemistry, we show that GLYATL1 is overexpressed in primary prostate cancer compared with metastatic prostate cancer and benign prostatic tissue. Low-grade cancers had higher GLYATL1 expression compared to high-grade prostate tumors. Our studies showed that GLYATL1 is upregulated upon androgen treatment in LNCaP prostate cancer cells which harbors ETV1 gene rearrangement. Furthermore, ETV1 knockdown in LNCaP cells showed downregulation of GLYATL1 suggesting potential regulation of GLYATL1 by ETS transcription factor ETV1. Transcriptome sequencing using the GLYATL1 knockdown prostate cancer cell lines LNCaP showed regulation of multiple metabolic pathways.ConclusionsIn summary, our study characterizes the expression of GLYATL1 in prostate cancer and explores the regulation of its regulation in prostate cancer showing role for androgen and ETS transcription factor ETV1. Future studies are needed to decipher the biological significance of these findings.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151252/1/pros23887.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151252/2/pros23887_am.pd

PTEN protein loss by immunostaining: Analytic validation and prognostic indicator for a high risk surgical cohort of prostate cancer patients

PURPOSE: Analytically validated assays to interrogate biomarker status in clinical samples are crucial for personalized medicine. PTEN is a tumor suppressor commonly inactivated in prostate cancer that has been mechanistically linked to disease aggressiveness. Though deletion of PTEN, as detected by cumbersome fluorescence in situ hybridization (FISH) spot counting assays, is associated with poor prognosis, few studies have validated immunohistochemical (IHC) assays to determine whether loss of PTEN protein is associated with unfavorable disease. EXPERIMENTAL DESIGN: PTEN IHC was validated by employing formalin fixed and paraffin embedded isogenic human cell lines containing or lacking intact PTEN alleles. PTEN IHC was 100% sensitive and 97.8% specific for detecting genomic alterations in 58 additional cell lines. PTEN protein loss was then assessed on 376 prostate tumor samples, and PTEN FISH or high resolution SNP microarray analysis was performed on a subset of these cases. RESULTS: PTEN protein loss, as assessed as a dichotomous IHC variable, was highly reproducible, correlated strongly with adverse pathologic features (e.g. Gleason score and pathological stage), detected between 75% and 86% of cases with PTEN genomic loss, and was found at times in the absence of apparent genomic loss. In a cohort of 217 high risk surgically treated patients, PTEN protein loss was associated with decreased time to metastasis. CONCLUSIONS: These studies validate a simple method to interrogate PTEN status in clinical specimens and support the utility of this test in future multi-center studies, clinical trials and ultimately perhaps for routine clinical care

ATF2 promotes urothelial cancer outgrowth via cooperation with androgen receptor signaling

We investigated the functional role of ATF2, a transcription factor normally activated via its phosphorylation in response to phospho-ERK/MAPK signals, in the outgrowth of urothelial cancer. In both neoplastic and non-neoplastic urothelial cells, the expression levels of androgen receptor (AR) correlated with those of phospho-ATF2. Dihydrotestosterone treatment in AR-positive bladder cancer cells also induced the expression of phospho-ATF2 and phospho-ERK as well as nuclear translocation and transcriptional activity of ATF2. Meanwhile, ATF2 knockdown via shRNA resulted in significant decreases in cell viability, migration and invasion of AR-positive bladder cancer lines, but not AR-negative lines, as well as significant increases and decreases in apoptosis or G0/G1 cell cycle phase and S or G2/M phase, respectively. Additionally, the growth of AR-positive tumors expressing ATF2-shRNA in xenograft-bearing mice was retarded, compared with that of control tumors. ATF2 knockdown also resulted in significant inhibition of neoplastic transformation induced by a chemical carcinogen 3-methylcholanthrene, as well as the expression of Bcl-2/cyclin-A2/cyclin-D1/JUN/MMP-2, in immortalized human normal urothelial SVHUC cells stably expressing AR, but not AR-negative SVHUC cells. Finally, immunohistochemistry in surgical specimens demonstrated significant elevation of ATF2/phospho-ATF2/phospho-ERK expression in bladder tumors, compared with non-neoplastic urothelial tissues. Multivariate analysis further showed that moderate/strong ATF2 expression and phospho-ATF2 positivity were independent predictors for recurrence of low-grade tumors (hazard ratio (HR) = 2.956, P = 0.045) and cancer-specific mortality of muscle-invasive tumors (HR = 5.317, P = 0.012), respectively. Thus, ATF2 appears to be activated in urothelial cells through the AR pathway and promotes the development and progression of urothelial cancer

Espesor del músculo aductor del pulgar en adolescentes brasileños y asociaciones con el estado nutricional, la maduración sexual y la actividad física (Estudio EVA-JF)

Objective: The present study aims to assess the associations of adductor pollicis muscle thickness (APMT) with age, skin color, sexual maturation, anthropometric indicators and physical activity in Brazilian adolescents. Materials and methods: Cross-sectional study of adolescents aged 14-19 years. Weight, height, body mass index (BMI), arm circumference (AC), APMT, body fat, fat-free mass (FFM), fat-free mass index (FFMI), sexual maturation, time of physical activity and skin color were evaluated. APMT was associated with categorical variables using Mann-Whitney or Kruskal-Wallis tests and correlated with anthropometric variables using Spearman’s correlation. Linear regression was used with APMT as a dependent and the other variables as predictors. Data analysis was carried out in SPSS® software (version 17.0) with a 5% significance level. Results: 828 adolescents were evaluated, 57.6% female, with a mean age of 16.13 ± 1.20 years. APMT had an average value of 18.0 mm in females and 21.0 mm in males. The measure was greater in males, in more advanced stages of sexual maturation, overweight and physical activity. It presented a moderate correlation with FFM, FFMI, body fat and AC. In the final model of multiple linear regression for females, the variables AC and body fat explain 20.1% of the APMT variability. For men, the variables AC and FFMI explain 30.5% of the APMT variability. Conclusion: It is recommended that APMT be used in a complementary manner in the nutritional assessment of adolescents.Objetivo: El presente estudio tiene como objetivo evaluar las asociaciones del grosor del músculo aductor del pulgar (EMAP) con la edad, el color de la piel, la maduración sexual, los indicadores antropométricos y la actividad física en adolescentes brasileños. Material y métodos: estudio transversal con adolescentes de 14 a 19 años. Peso, talla, índice de masa corporal (IMC), circunferencia del brazo (CB), EMAP, grasa corporal, masa libre de grasa (MLG), índice de masa libre de grasa (IMLG), maduración sexual, tiempo de actividad física y color de piel fueron juzgado. EMAP se asoció con variables categóricas mediante pruebas de Mann-Whitney o Kruskal-Wallis y se correlacionó con variables antropométricas mediante la correlación de Spearman. Se utilizó regresión lineal con EMAP como variable dependiente y el resto de variables como predictores. El análisis de los datos se realizó mediante el software SPSS® (versión 17.0) con un nivel de significancia del 5%. Resultados: se evaluaron 828 adolescentes, 57,6% mujeres, con una edad media de 16,13 ± 1,20 años. EMAP tuvo un valor promedio de 18.0 mm en mujeres y 21.0 mm en hombres. La medida fue mayor en varones, en estadios más avanzados de maduración sexual, sobrepeso y practicantes de actividad física. Presentó una correlación moderada con MLG, IMLG, grasa corporal y CB. En el modelo final de regresión lineal múltiple para mujeres, las variables CB y grasa corporal explican el 20,1%; para los hombres, las variables CB y MLG explican el 30,5% de la variabilidad EMAP. Conclusión: Se recomienda que EMAP se utilice de forma complementaria en la valoración nutricional de adolescentes