Tissue Microarray-based Immunohistochemistry Research Articles

Immunohistochemical Expression of GLUT1 and its Correlation with Unfavorable Histology and TP53 Codon 72 Polymorphism in Wilms Tumors

Reprogramming of energy metabolism, such as increased glycolysis, is a hallmark of cancer cells. One mechanism by which cancer cells fuel glycolysis is through increased uptake of glucose across cell membranes via the glucose transporter GLUT1. One of the transcriptional repressors of GLUT1 is wild-type TP53, and cancer-associated loss of function mutations within the DNA-binding domain of TP53 impairs the repressive effect of TP53 on transcriptional activity of the GLUT1 gene promoter. Because TP53 mutations are associated with unfavorable histology (diffuse anaplasia) in Wilms tumors, we hypothesized increased expression of GLUT1 in these tumors. To evaluate this hypothesis, we performed tissue microarray-based immunohistochemistry for GLUT1 in a set of 50 Wilms tumors, including 5 with unfavorable histology. In a subset of 16 favorable histology Wilms tumors, we compared the GLUT1 immunoexpression with TP53 codon 72 polymorphism status. We found consistently stronger immunoexpression of GLUT1 in unfavorable histology Wilms tumors compared to favorable histology Wilms tumors (P = 0.04). We noted that the favorable histology Wilms tumors with a proline residue at position 72 of TP53 tended to have higher immunoexpression of GLUT1, although this immunoexpression did not reach statistical significance in this small set of cases. In summary, our finding of strong GLUT1 immunoexpression in unfavorable histology Wilms tumors indicates that these tumors are likely to be 2-deoxy-2-((18)F)fluoro-d-glucose avid and that GLUT1 should be evaluated as a therapeutic target for these tumors that otherwise show resistance to conventional therapy.

Rsf-1/HBXAP overexpression is associated with disease-specific survival of patients with gallbladder carcinoma

Dysregulated chromatin remodeling often leads to abnormal gene expression or silencing in cells, thereby implicating tumor development and progression. As a subunit of remodeling and spacing factor (RSF) complex, Rsf-1, a novel nuclear protein with histone chaperon function, mediates ATPase-dependent chromatin remodeling and confer tumor aggressiveness in common carcinomas. We aimed, for the first time, to evaluate the Rsf-1 expression status and its associations with clinicopathological features and patient survival in a well characterized cohort of gallbladder carcinomas. Using tissue microarray-based immunohistochemistry, we assessed Rsf-1 expression in gallbladder carcinomas, yielding 88 cases undergoing surgical intervention with interpretable results. The Rsf-1 overexpression, present in 61 cases (69.3%), was significantly associated with higher histological grades (p = 0.002) and vascular invasion (p = 0.037) and marginally with non-papillary histotypes (p = 0.058). In univariate log-rank analysis, Rsf-1 overexpression was significantly predictive of disease-specific survival (p = 0.0015), which remained prognostically independent [p = 0.0191, risk ratio (RR) = 2.683], along with American Joint Committee on Cancer stages II-IV (p = 0.0265, RR = 2.102). Our findings indicate that Rsf-1 overexpression is common and associated with adverse prognosticators in gallbladder carcinomas. It may confer tumor aggressiveness through chromatin remodeling and represents a potential prognostic biomarker of gallbladder carcinomas.

Expressions of the gamma2 chain of laminin-5 and secreted protein acidic and rich in cysteine in esophageal squamous cell carcinoma and their relation to prognosis

Previous studies have shown that the expressions of the γ2 chain of laminin-5 and secreted protein acidic and rich in cysteine (SPARC) play important roles in oncogenesis and the development of carcinoma. To assess the expressions of laminin-5 γ2 chain and SPARC in esophageal squamous cell carcinoma (SCC), and to clarify the prognostic significance of the expressions of laminin-5 γ2 chain and SPARC in esophageal SCC, we detected the expressions of laminin-5 γ2 chain and SPARC in cancer tissue and corresponding normal mucosa from 116 patients with advanced (stages II–IV) esophageal SCC using the tissue microarray-based immunohistochemistry and analyzed the correlation of the expressions with clinicopathologic characteristics and survival. We found that in normal esophageal tissues, laminin-5 γ2 chain was expressed in the basement membrane, whereas in esophageal SCC tissues, laminin-5 γ2 chain was expressed in the cytoplasm of carcinoma cells, with a positive rate of 72.4%. SPARC was not detected in normal esophageal mucosa, but was expressed in stromal fibroblasts in 84.6% of esophageal SCC cases and in cancer cells in 7.8% of esophageal SCC cases. There was a significant correlation between laminin-5 γ2 chain and stromal SPARC expression in esophageal SCC (Spearman' s rho = 0.423, P < 0.001). The expressions of both laminin-5 γ2 chain and stromal SPARC were correlated with survival (P = 0.032 and P = 0.034, respectively). In stage-II esophageal SCC, the expression of laminin-5 γ2 chain was significantly correlated with survival (P = 0.023), while the expression of SPARC was not significantly correlated with survival (P = 0.154). Patients with elevated levels of laminin-5 γ2 chain and SPARC expressions had a poorer prognosis than did those lacking elevated levels of laminin-5 γ2 chain expression and/or elevated levels of SPARC expression (P = 0.001). In stage-II esophageal SCC, patients with elevated levels of laminin-5 γ2 chain and SPARC expressions had a poorer prognosis (P < 0.001). These results suggest that laminin-5 γ2 chain and SPARC may play roles in the progression of esophageal SCC and their simultaneous expression is correlated with poorer prognosis, especially in patients with stage-II SCC.

Expression profile reveals novel prognostic biomarkers in hepatocellular carcinoma

The purpose of this study was to identify and validate novel prognostic biomarkers in human hepatocellular carcinoma (HCC). We analyzed gene expression profiles not only between 33 HCCs and their corresponding noncancerous liver tissues, but also between 25 HCCs and pooled normal liver tissues using cDNA microarrays containing 12800 genes. Functional analysis of differentially expressed genes involved in HCC carcinogenesis and tumor progression revealed that up-regulated and down-regulated genes are mainly associated with cell cycle and immune response, respectively. We detected two regions of cytogenetic changes only in poorly-differentiated HCCs using the expression data. We identified a 9-gene expression signature, which was able to predict differentiation degree and survival of HCC samples. Among the 9 most discriminatory genes, minichromosome maintenance protein 2 (MCM2), a significantly up-regulated gene involved in cell cycle pathway, was selected for further analysis. Overexpression of MCM2 protein related to poor-differentiation in HCC was validated using tissue microarray-based immunohistochemistry containing 96 HCCs. Our studies show that the 9-gene expression signature may serve as promising prognostic biomarkers involved in hepatocarcinogenesis and tumor progression.

Tissue Profiling of the Mammalian Central Nervous System Using Human Antibody-based Proteomics

A need exists for mapping the protein profiles in the human brain both during normal and disease conditions. Here we studied 800 antibodies generated toward human proteins as part of a Human Protein Atlas program and investigated their suitability for detailed analysis of various levels of a rat brain using immuno-based methods. In this way, the parallel, rather limited analysis of the human brain, restricted to four brain areas (cerebellum, cerebral cortex, hippocampus, and lateral subventricular zone), could be extended in the rat model to 25 selected areas of the brain. Approximately 100 antibodies (12%) revealed a distinct staining pattern and passed validation of specificity using Western blot analysis. These antibodies were applied to coronal sections of the rat brain at 0.7-mm intervals covering the entire brain. We have now produced detailed protein distribution profiles for these antibodies and acquired over 640 images that form the basis of a publicly available portal of an antibody-based Rodent Brain Protein Atlas database (www.proteinatlas.org/rodentbrain). Because of the systematic selection of target genes, the majority of antibodies included in this database are generated against proteins that have not been studied in the brain before. Furthermore optimized tissue processing and colchicine treatment allow a high quality, more extended annotation and detailed analysis of subcellular distributions and protein dynamics.

Expression profiling of liver receptor homologue 1 (LRH-1) in mouse tissues using tissue microarray

Liver receptor homologue 1 (LRH-1) is a nuclear receptor that plays important roles in lipid homeostasis and embryogenesis. To elucidate systemic physiological functions of LRH-1, we used tissue microarray-based immunohistochemistry to examine the tissue distribution and localization of LRH-1 in adult mouse tissues. LRH-1 immunoreactivity was observed in the nucleus of multiple epithelial lineage cells in the digestive system (including absorptive epithelial cells in the small and large intestines, goblet cells, acinar cells of the exocrine glands, chief cells and mucus neck cells in the stomach, granular and prickle layer cells in the tongue and forestomach, and gall bladder epithelium); respiratory system (alveolar type II cells); and urinary system (transitional epithelium). Nuclear LRH-1 immunoreactivity was also localized in cells involved in fatty acid/glucose metabolism, including hepatocytes, brown adipocytes, and cardiomyocytes, and neurons involved in the regulation of food intake, including the arcuate nucleus in the hypothalamus and paraventricular nucleus of thalamus. Additionally, LRH-1 immunoreactivity was observed in testicular Leydig cells and ovarian follicular cells. These data suggest that LRH-1 functions in multiple organ systems to regulate epithelial cell physiology and differentiation, energy metabolism, and reproduction.

Expression of BIRC7 protein and mRNA in non-Hodgkin's lymphoma

Studies have shown that BIRC7, a new member of inhibitor of the apoptosis protein family, is expressed in fetal tissues and most solid tumors in humans. However, there are no reported data concerning BIRC7 expression in lymphomas. We investigated the expression of BIRC7, survivin, Bcl-2, Bax, p53 and p170 proteins in 167 cases of non-Hodgkin's lymphoma (NHL) and 10 cases of non-specific lymphadenitis by tissue microarray-based immunohistochemistry. BIRC7 mRNA in three cell lines and 16 cases of NHL were detected by reverse transcriptase-polymerase chain reaction. BIRC7 protein was exhibited in the cytoplasm of cells in 25 (31%) of 80 cases of B-NHLs, 32 (37%) of 87 cases of T-NHLs, and none in non-specific lymphadenitis. The positive rate of BIRC7 was lower than that of survivin in almost all types of NHL with no significant differences, and similar to that of Bcl-2, Bax or p53. There was no correlation of protein expression between BIRC7 and any other detected markers, except p170 in T-NHL (P < 0.001). BIRC7 expression did not correlate with clinic pathologic factors such as sex, age, stage and grade, but overexpression of BIRC7 was positively correlated with aggression of NHL cells (P < 0.05). BIRC7 mRNA expressed in six (38%) of 16 cases of NHLs. BIRC7 mRNA expression was approximately consistent with BIRC7 protein in NHL. Our results indicate that the BIRC7 gene might play a role in the development and aggression of NHL and that the inhibition of BIRC7 expression may be important in NHL treatment.