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Recently the suspected precursor lesions of ductal adenocarcinoma of the pancreas have been called Pancreatic intraepithelial neoplasia (PanIN) and graded according to the degree of dysplasia. To correlate each grade of PanIN with molecular genetic alterations, we determined the frequency of allelic losses at chromosomal arms 9p (the location of the p16 gene), 17p (p53 gene) and 18q (DPC4/SMAD4 gene) in 81 microdissected PanINs, using a combination of whole genome amplification and microsatellite analysis. In addition, p53 and Dpc4 protein expression was determined by immunohistochemistry. Essentially no allelic losses were identified in the non-dysplastic PanIN-1 lesion. In PanIN-2 with low grade dysplasia the frequency of allelic losses at chromosomal region 9p, 17p and 18q was 20%, 33% and 17%, respectively, which increased to 46%, 77% and 58%, respectively, in PanIN-2 with moderate dysplasia, to 87%, 60% and 88% in PanIN-3 with high grade dysplasia, and to 100%, 91%, and 82% in the invasive carcinomas. The progressive occurrence of allelic losses at all three loci strongly supports the PanIN progression model for pancreatic carcinoma. Nuclear p53 and loss of Dpc4 protein expression was associated only with PanIN-3 and invasive carcinomas, consistent with the model that inactivation of p53 and DPC4 are late events in pancreatic carcinogenesis. Since the aberrant protein expression patterns, were preceded, however by a sharp increase in allelic losses from PanIN-2 with low grade dysplasia to PanIN-2 with moderate dysplasia it is suggested that the increasing grade of dysplasia in the PanIN lesions identify biologically relevant steps towards invasive carcinoma. The discrepancy between alleic loss frequencies and p53 and DPC4 expression also raises the possibility that additional tumor suppressor genes on chromosomes 17p and 18q promote early pancreatic carcinogenesis.

Surface-enhanced Raman scattering (SERS) microscopy is a novel method of optical imaging for the localization and quantification of target molecules in cells and tissue specimens. The major advantages of SERS over fluorescence are quantification and spectral multiplexing due to the small line width of vibrational Raman bands. The position of the plasmon band of both hollow gold/silver nanoshells and silica-encapsulated gold nanoclusters can be tuned for maximum SERS enhancement upon red laser excitation, which is optimal for minimizing the disturbing autofluorescence of tissue. In this work, silica-encapsulated and non-encapsulated SERS particles were used for the localization of target proteins in prostate tissue specimens. Two different biofunctionalization methods were established for each type of SERS particles. The cross-linking method based on s-NHS/EDC chemistry was modified for covalently conjugating proteins to hollow gold/silver nanoshells and gold nanostars in order to minimize the aggregation of SERS nanoparticles during and after cross-linking. As an alternative to covalent conjugation chemistry, the noncovalent binding of antibodies to the SERS particles via an adapter protein (protein A/G) was established. The influence of several factors that determine the quality of results obtained by SERS imaging, such as the number of immuno-SERS conjugates, incubation time, antigen retrieval and blocking buffer, were investigated. Rapid SERS microscopy with 30 msec acquisition time per pixel was enabled by using silica-encapsulated gold nanoclusters for the localization of p63 proteins on prostate tissue specimens from healthy donors. Two-color SERS experiments for the parallel localization of PSA and p63 were performed with silica-encapsulated and non-encapsulated nanoshells. The quality of the results depends less on the nature of the surface chemistry of the nanoparticles (with or without silica encapsulation), but more on the blocking buffer and the antigen retrieval method. Silica-encapsulated gold nanoclusters were also used for the simultaneous quantification of three cytokines (IL1, IL8 and TNF- α) in a SERS-based sandwich immunoassay with a detection limit of ca. 0.3 pM. Keywords: Raman, SERS microscopy, biocompatibility of nanoparticles, cross-linking, antigen unmasking methods, antigen detection, immunohistochemistry, immunoassay.

No description available.