Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

In the present study, epithelium derived lesions of various pathological manifestations were examined histologically and immunohistochemically for mononuclear cell infiltration. The infiltrate under the transformed epithelium of oral lesions, was examined for differences in the composition of immune mononuclear cells as the epithelium moves from hyperkeratosis through various degrees of dysplasia to squamous cell carcinoma. The study was performed on 53 human tongue tissues diagnosed as hyperkeratosis (11 cases), mild dysplasia (nine cases), moderate and severe dysplasia (14 cases) and squamous cell carcinoma (19 cases). A similar analysis was performed on 30 parotid gland tissues diagnosed as pleomorphic adenoma (14 cases) and carcinoma ex-pleomorphic adenoma (16 cases). Immunohistochemical analysis of various surface markers of the tumour infiltrating immune cells was performed and correlated with the transformation level as defined by morphology and the expression of p53 in the epithelium. The results revealed that, in the tongue lesions, the changes in the epithelium from normal appearance to transformed were accompanied by a corresponding increase in the infiltration of CD4, CD8, CD14, CD19+20, and HLA/DR positive cells. The most significant change was an increase in B lymphocytes in tongue lesions, that was in accordance with the transformation level (P50.001). In the salivary gland, a significant number of cases did not show an infiltrate. In cases where an infiltrate was present, a similar pattern was observed and the more malignant tissues exhibited a higher degree of immune cell infiltration.

In an effort to improve affinity biomarker validation in fixed patient tissue specimens, we have developed a novel quantum dotbased bioimaging system that utilizes chicken IgY antibody for high sensitivity and specificity relative quantitation of cancer proteins. Monospecific, polyclonal IgYs were generated against human HER2 and telomerase, and analytically validated for specificity by western blot and immunohistochemistry on tumor and normal cells and for relative affinity by layered peptide array (LPA). IgYs bound desired targets in cell lines and fixed tissues and showed greater affinity than commercial mammalian antibodies for both HER2 and telomerase proteins. In tissue microarray experiments, HER2 quantitation with IgY antibody and quantum dot imaging correlated well with chromogenic in situ hybridization (CISH), whereas telomerase quantitation suggested a trend toward correlation with prostate cancer Gleason Grade and differentiation. Although patient numbers were small, these findings demonstrate the feasibility of relative quantitation of cancer biomarkers with IgY and quantum dot fluorophores, and show promise for rigorous clinical validation in large patient cohorts. ' 2008 Wiley-Liss, Inc.Key words: IgY antibody; cancer biomarker; HER2; telomerase; quantum dots Few new early cancer biomarkers have surfaced in recent years. 1 Among novel proteomic biomarkers for early cancer detection, some have proven controversial. 2,3 Analytical and clinical validation of cancer biomarkers has suffered from bias in the design, conduct and interpretation of such research, 4 incompletely validated imaging, 5 and lack of affinity standards 6 and antibodies that did not, in fact, detect correct targets. 7 Here, we describe a novel approach to cell-based bioimaging with relative quantitation for biomarker validation. We report characterization of two new IgY antibodies for quantitation of model cancer biomarker systems, HER2 and telomerase, 8 and explore analytical improvements, including low cross-reactivity IgY-isotype chicken polyclonal antibodies raised against recombinant polypeptides; digital quantification of antibody signals with streptavidin-conjugated semiconductor nanocrystals to obviate photobleaching of organic fluorescent dyes; complete z-plane fluorescence image capture using 3D-deconvolution microscopy; high-throughput, automated, robotic slide processing; and quantitative, massively parallel, high-throughput analysis of peptide antigen-antibody interactions by layered peptide array (LPA) technology. 9

Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.

Layered peptide array is a new methodology for multiplex molecular measurements from two-dimensional life science platforms. The technology can be used in several different configurations depending on the needs of the investigator. Described here is an indirect layered peptide array (iLPA) that is capable of measuring proteins on a solid surface, such as target antigens on a tissue section. A prototype iLPA system was developed and subsequently examined for reproducibility and specificity and then compared with standard immunohistochemistry. Semiquantitative, multiplex proteomic analysis of histological sections was achieved with up to 20 membranes. The experimental variability was 18%. Overall, the data suggest that iLPA technology will be a relatively simple and inexpensive method for molecular measurements from tissue sections.

No abstract

No abstract

Proteomics, the global study of protein expression and characteristics, has recently emerged as a key component in the field of molecular analysis. The dynamic nature of proteins, from ion channels to chaperones, presents a challenge, yet the understanding of these molecules provides a rich source of information. When applying proteomic analysis directly to human tissue samples, additional difficulties arise. The following article presents an overview of the current proteomic tools used in the analysis of tissues, beginning with conventional methods such as western blot analysis and 2D polyacrylamide gel electrophoresis. The most current high-throughput techniques being used today are also reviewed. These include protein arrays, reverse-phase protein lysate arrays, matrix-assisted laser desorption/ionization, surface-enhanced laser desorption/ionization and layered expression scanning. In addition, bioinformatics as well as issues regarding tissue preservation and microdissection to obtain pure cell populations are included. Finally, future directions of the tissue proteomics field are discussed.