Image analysis algorithms for immunohistochemical assessment of cell death events and fibrosis in tissue sections.

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections.

Bcl-B expression in human epithelial and nonepithelial malignancies.

PURPOSE: Apoptosis plays an important role in neoplastic processes. Bcl-B is an antiapoptotic Bcl-2 family member, which is known to change its phenotype upon binding to Nur77/TR3. The expression pattern of this protein in human malignancies has not been reported. EXPERIMENTAL DESIGN: We investigated Bcl-B expression in normal human tissues and several types of human epithelial and nonepithelial malignancy by immunohistochemistry, correlating results with tumor stage, histologic grade, and patient survival. RESULTS: Bcl-B protein was strongly expressed in all normal plasma cells but found in only 18% of multiple myelomas (n = 133). Bcl-B immunostaining was also present in normal germinal center centroblasts and centrocytes and in approximately half of diffuse large B-cell lymphoma (n = 48) specimens, whereas follicular lymphomas (n = 57) did not contain Bcl-B. In breast (n = 119), prostate (n = 66), gastric (n = 180), and colorectal (n = 106) adenocarcinomas, as well as in non-small cell lung cancers (n = 82), tumor-specific overexpression of Bcl-B was observed. Bcl-B expression was associated with variables of poor prognosis, such as high tumor grade in breast cancer (P = 0.009), microsatellite stability (P = 0.0002), and left-sided anatomic location (P = 0.02) of colorectal cancers, as well as with greater incidence of death from prostate cancer (P = 0.005) and shorter survival of patients with small cell lung cancer (P = 0.009). Conversely, although overexpressed in many gastric cancers, Bcl-B tended to correlate with better outcome (P = 0.01) and more differentiated tumor histology (P < 0.0001). CONCLUSIONS: Tumor-specific alterations in Bcl-B expression may define subsets of nonepithelial and epithelial neoplasms with distinct clinical behaviors.

Claudin-1 immunohistochemistry for distinguishing malignant from benign epithelial lesions of prostate.

BACKGROUND: Claudins are a family of approximately 23 integral membrane tight junction (TJ) proteins that maintain cell polarity and paracellular barrier functions in epithelial and endothelial cells. Although Claudin-1 was demonstrated to be typically downregulated in various cancers, the precise expression patterns of this protein in normal and neoplastic tissues remain poorly characterized. METHODS: Using immunohistochemistry, the expression of Claudin-1 was investigated in prostate tissue samples arranged in a tissue microarray (TMA) format and comprising elements of normal prostatic epithelium (n = 6), benign prostatic hyperplasia (BPH; n = 38), prostatic intraepithelial neoplasia (PIN; n = 11), and prostate adenocarcinoma (n = 48). The Claudin-1 expression pattern was compared with that of the basal cell-specific markers, p63, and HMW cytokeratin (34betaE12), by employing double-labeling techniques in conjunction with image analysis methods utilizing color deconvolution algorithms. RESULTS: In benign prostatic epithelium, pronounced Claudin-1 expression was observed in the basal cell layer with no staining in luminal cells. Prostate adenocarcinoma specimens from 98% (47/48) patients lacked Claudin-1 immunostaining, and no cases contained >5% immunopositive tumor cells. CONCLUSIONS: Claudin-1 immunohistochemistry should be considered for use as a new diagnostic tool for distinguishing malignant from benign lesions of the prostate.