Metallographic in situ hybridization.

Metallographic methods, in which a target is visualized using a probe or antibody that deposits metal selectively at its binding site, offers many advantages for bright-field in situ hybridization (ISH) detection as well as for other labeling and detection methods. Autometallographically enhanced gold labeling procedures have demonstrated higher sensitivity than conventional enzyme chromogens. Enzyme metallography, a novel procedure in which an enzymatic probe is used to deposit metal directly from solution, has been used to develop bright-field ISH methods for HER2 gene determination in breast cancer and other biopsy specimens. It provides the highest level of sensitivity and resolution, both for visualizing endogenous gene copies in nonamplified tissues and for resolving multiple gene copies to allow copy enumeration in amplified tissues without the need for oil immersion or fluorescence optics. An automated enzyme metallography procedure, silver ISH, has been developed for use in slide-staining instruments. Metallographic staining also provides excellent results for immunohistochemistry and may be combined with other staining procedures for the simultaneous detection of more than one gene or combinations of genes and proteins.

A new rabbit monoclonal antibody (4B5) for the immunohistochemical (IHC) determination of the HER2 status in breast cancer: comparison with CB11, fluorescence in situ hybridization (FISH), and interlaboratory reproducibility.

The 2 methodologies in current clinical use to assess HER2 status in breast cancer are: fluorescence in situ hybridization (FISH) (gene amplification) and immunohistochemistry (protein over-expression). A consistent finding has been that 3% to 15% of breast cancers over-express HER2 protein without evidence for gene amplification. Accurate determination of the HER2 status has implications for selecting patients most likely to respond to trastuzumab. We report here our preliminary experience with a new anti-HER2 rabbit monoclonal antibody, 4B5. The evaluation of HER2 status in 2 different cohorts of breast cancer cases (Single Institution (SI) and Multinational (MN)) with a total of 322 breast cancer cases was performed on an automated staining system (Ventana Medical Systems, Inc, Tucson, AZ) and scored by 3 pathologists (0-3+), for comparison with CB11 staining results (PATHWAY) and FISH (PathVysion). Interlaboratory reproducibility of automated staining results and interpretation was determined on a subset of the SI cohort at 3 separate laboratories. Rabbit monoclonal 4B5 demonstrated sharper membrane staining with less cytoplasmic and stromal background staining than CB11. In the SI cohort, the staining results for 4B5 were highly comparable with those obtained for CB11 with an overall concordance of 93.3%. In the multinational cohort, the overall concordance with CB11 was 84.7%. This lower level of concordance was associated with a much higher overall agreement of 4B5 with FISH (89.5%), compared with agreement of CB11 with FISH (81.2%). The difference in the performance of CB11 in the MN cohort versus the SI cohort may be due to differences in tissue fixation and processing in a centralized, high volume laboratory in an academic medical center versus multiple sites in the international community with potentially nonstandardized techniques. The staining results with 4B5 indicate that it has a more robust performance than CB11 because the correlation of 4B5 with FISH was nearly equivalent (88.2% MN; 89.3% SI) in both cohorts. Interlaboratory reproducibility was also excellent (kappa 1.0). RMoAb 4B5 provides excellent sensitivity, specificity, and interlaboratory reproducibility for the detection of HER2 status in breast cancer.

A rat monoclonal antibody that recognizes pro- and active MMP-7 indicates polarized expression in vivo.

Matrix metalloproteinases (MMPs) are a family of enzymes named for their ability to degrade proteins of the extracellular matrix. Here we describe the characterization of a rat monoclonal antibody specifically recognizing one member of this enzyme family, MMP-7. This antibody has been tested for its use in multiple assay types and was shown to be useful for direct enzyme-linked immunosorbent assay (ELISA), Western blotting, immunocytochemistry, and immunohistochemistry of frozen or paraffin-embedded tissues. The antibody has been evaluated for its usefulness with tissues from several different species and, by immunohistochemistry, can detect MMP-7 of human, murine, porcine, and gerbil origin. Immunostaining of MMP-7 in normal tissues or benign tumors of intestinal, breast, and prostatic origin indicates that this protein is normally localized luminally in glandular epithelium. The localization pattern would suggest that in normal or early stage tumors, MMP-7 is most likely not directly involved in extracellular matrix degradation. In contrast, advanced colon tumors show MMP-7 in invading cells at the advancing edge of the tumor.

Fibroblast growth factor 8 isoform B overexpression in prostate epithelium: a new mouse model for prostatic intraepithelial neoplasia.

Fibroblast growth factor 8 isoform b (FGF8b), a mitogenic and transforming polypeptide, was demonstrated to be naturally up-regulated in prostatic premalignant and malignant lesions in men. We generated four independent lines of transgenic mice with targeted overexpression of FGF8b in the prostatic epithelium using an improved rat probasin promoter, ARR(2)PB. Transgene expression in the prostate tissue was readily demonstrated by reverse transcription-PCR and localized to the prostatic epithelium by in situ hybridization. The histopathology of the prostate tissues was followed in different age groups of the various lines but most extensively in one line (line 3), starting from 1 month of age up to 24 months. Prostatic hyperplasia appeared in the lateral and ventral prostates in some animals as early as 2-3 months and in other lobes between 6 and 16 months. Beginning at 5-7 months, dysplasia, akin to what may be considered low-grade prostatic intraepithelial neoplasia (LGPIN) in humans, was detected. During the first 14 months, 100% of animals exhibited multifocal epithelial hyperplasia; 35% also had areas of LGPIN. This profile changed in subsequent months (15-24 months) to a higher incidence of LGPIN (66%) along with high-grade PIN (HGPIN) lesions (51%). Similar to HGPIN, stromal proliferation and appearance of papillary hyperplasia with atypia displayed a delayed pattern. The affected stroma consisted primarily of the smooth muscle cell component. The incidence of chronic inflammation, mostly involving T cells, was higher in the prostate of the transgenic mice relative to controls; however, the presence of a direct correlation between inflammation and hyperplasia or preneoplastic lesions was not identified. These transgenic mice represent a "natural" animal model for investigating the mechanism of development and progression of prostatic diseases, such as prostatic hyperplasia and preneoplastic lesions.

Prostatic intraepithelial neoplasia in mice with conditional disruption of the retinoid X receptor alpha allele in the prostate epithelium.

Retinoids, which are important regulators of cell growth, differentiation, and apoptosis, have been used in treatment or chemoprevention of multiple cancers including prostate cancer. To elucidate the mechanism of action of retinoids in the context of the prostate, we used the Cre-loxP system to disrupt the retinoid X receptor alpha (RXRalpha) gene specifically in the prostatic epithelium of the mouse. Evidence for tissue-specific gene inactivation was obtained at DNA, RNA, and protein levels. Phenotypic changes in the prostate in the homozygous animals of different age groups ranging from 1 to 15 months were investigated. Developmentally, prostatic ductal branching appeared to be increased from the loss of RXRalpha function. There was also a significant change in the profile of secretory proteins in the RXRalpha mutant prostate relative to littermate controls with intact RXRalpha allele. Histopathologically, homozygous RXRalpha-deficient prostates showed multifocal hyperplasia as early as 4 months of age. Lesions, which could be described as low-grade prostatic intraepithelial neoplasias, were detected after 5 months. Subsequently, beginning at approximately 10 months, high-grade prostatic intraepithelial neoplasias developed in some animals. The incidences of low-grade prostatic intraepithelial neoplasias and high-grade prostatic intraepithelial neoplasias among the animals 10-15 months of age were 62 and 17%, respectively. The heterozygous mutant mice also developed similar prostatic phenotypes but in a delayed manner, implying a role of haploinsufficiency. Together, these results indicated for the first time that a major component of retinoid action in the prostate is mediated by a retinoid receptor, RXRalpha, the inactivation of which in the prostatic epithelium leads to the development of preneoplastic lesions.