Molecular Analysis of Microdissected Tumors and Preneoplastic Intraductal Lesions in Pancreatic Carcinoma

Little or no data exist concerning the inactivation of tumor suppressor genes in intraductal lesions surrounding invasive ductal pancreatic carcinomas. Using a novel improved primer extension and preamplification polymerase chain reaction, we analyzed microdissected paraffin-embedded specimens of pancreatic carcinoma (n = 29) and their corresponding pancreatic intraductal lesions (PIL, n = 331) for loss of heterozygosity (LOH) of p16(INK4), DPC4, and p53 by microsatellite analysis and for p53 protein by immunohistochemistry. LOH at the p16(INK4) locus (9p21) was found in nine of 22 informative tumors (41%), in 15 of 25 tumors (60%) at the DPC4 locus (18q21.1), and in 22 of 27 tumors (81%) at the p53 locus (17p13). Homozygous deletions of p16(INK4) and DPC4 were found in eight of 22 (36%) and four of 25 tumors (16%), respectively. Furthermore, 24 of 29 tumors (83%) revealed considerable intratumoral genetic heterogeneity. In 165 of 277 PILs (60%) having suitable DNA for microsatellite analysis, alterations in at least one tumor suppressor gene were found. In individual PILs, up to three alterations were detected, and p53 LOH occurred even in morphologically normal-appearing ductal epithelium near the tumor. Although deletions of all three tumor suppressor genes were found in PILs without nuclear atypia, there was a tendency toward earlier LOH of p16(INK4) compared to DPC4 and p53 in these lesions. LOH in tumors accompanied positive p53 immunohistochemistry in 81% but only in 38% in PILs

Single-Cell Immunohistochemical Mutation Load Assay (SCIMLA) Using Human Paraffin-Embedded Tissues

It would be advantageous to measure mutation load in situ in order to determine the relationship between a high mutation load and increased risk for cancer or other diseases and to evaluate sources of possible mutagen exposure. Previously, in situ mutation detection assays have been plagued with multiple rounds of amplification and high rates of false-positives and false-negatives. The single cell immunohistochemical mutation load assay (SCIMLA) was developed to measure somatic mutation frequency, pattern, and spectrum in normal tissues with a single round of amplification. The P53 gene was utilized as a mutation reporter because of the unusual property that missense mutations often cause P53 protein to accumulate in the cell, allowing the mutant proteins to be detected by immunohistochemical staining. Alternative reporter genes with stabilized mutant proteins may be envisioned. Single cells that stain positively for P53 protein overabundance (red cells) were microdissected from ethanol-fixed and paraffin-embedded tissues. A novel stimulated-PCR (S-PCR) protocol permitted successful amplification of a 1.8-kb segment of the P53 gene (i.e., exons 5-9) in 87% of single mammary cells. Subsequent sequence analysis demonstrated that 35% of the amplified red-stained epithelial cells from normal breast tissue have missense mutations at evolutionarily conserved amino acids. Jackpot mutations, presumably due to clonal expansion, were common. False-positive missense mutations at conserved residues were observed in 3% of the clear cells (i.e., without red stain), presumably due to DNA polymerase error in early PCR cycles. The allele dropout rate was measured at 40% of the amplified cells. SCIMLA is applicable to a variety of tissues, utilizes a single amplification of an endogenous gene, displays mutant cells in situ, and may be adapted to other species. © 2003 Wiley-Liss, Inc

Microdissection and molecular analysis of single cells or small cell clusters in pathology and diagnosis -Significance and challenges

Colour figures can be viewed on http://www.esacp.org/acp/2002/24‐4_5/heinmoeller.ht

Toward efficient analysis of mutations in single cells from ethanol-fixed, paraffin-embedded, and immunohistochemically stained tissues

Only a few studies have demonstrated successful molecular analysis after whole genome amplification using single cells dissected from paraffin-embedded tissues. The results in these studies were limited by low-amplification efficiency and high rates of allele dropout. In the present study, the amplification rate using a thoroughly modified primer extension and preamplification-PCR protocol was improved significantly for single cells microdissected from paraffin-embedded and immunohistochemically stained tissues. Tissue fixation with ethanol (85%) and the addition of 0.2 mmol/L EDTA helped to achieve an amplification rate between 67% (segments 200 to 400 bp) and 72% (segments \u3c200 bp). Normal tissue sections were immunohistochemically double stained for overabundance of p53 protein and proliferating cell nuclear antigen. Microdissection of single cells was performed with a manual micromanipulator equipped with a Tungsten needle. Sequence analysis of the TP53 gene was performed after improved primer extension preamplification-PCR and multiplex PCR from single microdissected cells. The rate of allele dropout was at least 68%. These technical advances facilitate routine mutation analysis using a single cell or a few cells microdissected from routinely processed paraffin-embedded normal and tumor tissues. Allele dropout still represents a serious problem in single-cell mutation analysis, especially in samples with limited template DNA and prone to DNA damage. © 2002 by The United States and Canadian Academy of Pathology, Inc