Laser Microbeams for Genomics and Proteomics

Laser microtools can be of significant value for genomics and proteomics in molecular profiling of cancer and other genetically based diseases. As discussed above, laser microbeam microdissection (LMM) coupled with laser pressure catapulting (LPC) or laser capture microdissection (LCM) allows isolation of a single cell, as well as a small number of specific cells from an archival tissue in a noncontact mode.Thus laser microdissection can be used to extract specific cell populations such as normal cells, precancerous cells, and invasive cancer cells. The purity of these specific cells then can permit one to compare and identify tumor suppressor genes as well as novel transcriptions and proteins that change in neoplastic cells (Best and Emmert-Buck, 2001; Maitra et al., 2002).

Genetic changes manifested in multistep progression of cancer can involve gain of mutation in dominant oncogenes, or loss of a function by delection, mutation, or methylation in repressive tumor suppressor genes. This loss of supressor gene function in a tumor is called loss of heterozygosity (LOH) (Gillespie et al., 2000). Laser microdissection has made a significant contribution to applications of LOH analysis to cancer studies because virtually pure populations of tumor cells or preneoplastic foci necessary for LOH analysis can be isolated without contamination even by a few unwanted cells. The LOH analysis has proved valuable in the mapping of tumor suppressor genes, localization of putative chromosomal "hot spots," and the study of sequential genetic changes in preneoplastic lesions. LCM in conjunction with fluorescence in situ hybridization (FISH, discussed in Chapter 8) demonstrated LOH on chromosome sp21 in prostate cancer. Loss of the dematin gene was observed, leading to dysregulation of cell shape (Lutchman et al., 2000). Study of preneoplastic lesions has revealed that genetic alterations in cancers actually starts in histologically "benign" tissue.

LCM used in conjunction with polymerase chain reaction (PCR) such as reverse transcriptase-PCR (RTPCR) provides an opportunity to study only a few hundred cells. The advantage is that even microscopic preneoplastic lesions can be studied. In addition to LOH analysis, other studies have been performed using laser microdissection. They include X-chromosome inactiva-tion analysis to access clonality, single-strand conformation polymorphism (SSCP) analysis for mutations in critical genes, comparative genomic hybridization (CGH), and the analysis of promoter hypermethylation.

Microdissected cells have been used to obtain differential gene expression which is a useful parameter to differentiate tumors from their normal cells. Methods to study gene expression include expressed sequence tag (EST) sequencing, differential display, subtractive hybridization, serial analysis of gene expression (SAGE), and cDNA microarray technique. The microarray technology has been discussed in Chapter 10. LCM has been used to generate cDNA libraries for a number of cancers. These data can be accessed at the NIH Cancer Genomic Anatomy Project (CGAP) website ( The cDNA libraries can be used for identification of novel genes that are either overexpressed or underexpressed in the multistage pathogenesis of cancer, which can eventually lead to genetic profiling of individual patient samples to customize treatment on an individual basis.

Laser microdissection for molecular profiling of global protein patterns will play an important role because it is crucial for protein analysis and differentiation to obtain pure populations of tumor cells and their preneo-plastic lesions. Identification of proteins dysregulated during cancer progression will be valuable in formulating treatment and developing intervention strategies.

LCM has been used in conjunction with high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), a technique used to analyze populations of proteins in different cell types to resolve more than 600 proteins or their isoforms and identify dysregulated products in cancer cells. Sequencing of the altered peptide products unique to the tumor population can be used to identify novel tumor-specific alterations. For example, pro-teomic analysis of microdissected prostate cancers and benign prostatic epithelium revealed six differentially expressed proteins (Ornstein et al., 2000). Microdissected specimen of colon cancer has shown increased levels of gelatinase and cathepsin B, both implicated in cancer invasion and metastasis (Emmert-Buck et al., 1994).

To conclude, it can be envisioned that the use of a rapid microdissection technique, together with biomolecule amplification protocols, can provide more sensitive detection and database integration which can become a standard practice for cancer diagnostics. The molecular profile information on DNA, RNA, and protein alterations can lead to diseases management as well as to design of optimal, low-risk, and patient-tailored treatment.

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