NEWS

Laser Capture Microdissection Comes Into Mainstream Use

Tracy Webb

Pathologists recognize subtle changes in tissue and cellular organization. But their qualitative impressions can sometimes yield different assessments. "One pathologist’s moderately differentiated cell is somebody else’s mild and somebody else’s severe," said James Resau, Ph.D., from the Van Andel Research Institute, Grand Rapids, Mich. "So as we get into a global diagnosis, and a global grouping of cases, what we are lacking are objective, measurable, reproducible standards that are not qualitative."

But one relatively new technology—laser capture microdissection—has come on the scene and has narrowed the window of interpretation. LCM’s ability to readily obtain and identify normal or cancerous cells has made it an attractive technology to the field of genomics. "The real advantage of current technologies such as LCM and microarrays is that they are more objective, measurable, and reproducible," said Resau. "Data can be grouped across hospitals and across disciplines so that standardization and objective measurements of patients are available and we can treat more scientifically and appropriately."



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The images above are of prostate tissue before (a) and after (b) laser capture microdissection, and of a captured cell (c).

 
Birth of LCM

Nearly 4 years ago, Lance Liotta, M.D., Ph.D., chief of the Laboratory of Pathology at the National Cancer Institute, and his laboratory published an article in Science that described the process of LCM. This invention, driven by the need to isolate pure premalignant cells from their native tissue to facilitate the study of molecular events leading to invasive cancer, has helped to promote scientific advancement in many ways.

"Tissues are very complicated structures that contain many different types of cells," explained Liotta. To study the genetic changes that occur in transition from a normal to an invasive cell, tiny cell subpopulations, sometimes less than 5% of the total tissue, need to be isolated to reduce contamination from other cell types. Before LCM, the process of isolating specific cell populations from tissue was very tedious and inefficient.

To perform LCM, a slide containing tissue of interest is placed under a microscope and the image is transferred to a computer screen. Cells of interest are selected with a joystick and at the push of a button, an infrared laser melts a special film above the targeted cells. The film then sets, pulling the chosen cells away from the tissue slide. These selected cells are ready for molecular analysis. The remaining tissue on the slide is intact and can be subjected to further dissection.

Microdissected cells display very different genetic profiles when compared to cells in culture, Liotta said. Many new genes have been identified in microdissected cells; in contrast to cells in culture, cells obtained through LCM are in their natural environment and express their true set of genes, and are therefore better suited for molecular analysis.

This will make the molecular pathologist even more important in the future, Liotta said. "Pathologists will be diagnosing the molecular reason rather than just trying to correlate patterns," he said, and this will help give a "scientific basis for diagnosis."

Emanuel Petricoin, Ph.D., of the Tissue Proteomic Program in the Division of Therapeutic Products at the U.S. Food and Drug Administration, said pathologists will be "seamlessly linked throughout the whole process." In the past, they were linked either at the very beginning with a diagnosis or at the end after the patient had died. Now, he believes, pathologists will become molecular profiling specialists and will work with oncologists from diagnosis through individually tailored treatment.

Putting LCM to Use

Two projects are using LCM to make the concept of individually tailored treatment a reality. The Cancer Genome Anatomy Project, launched by the NCI in 1998, is using LCM to create a complementary DNA library. The cDNA library, a collection of all genes being expressed in the cell, contains powerful information regarding cancer gene expression when normal and cancer cDNA libraries are compared. This project will help identify most of the genes involved in cancer development. To date, cDNA libraries from both normal and premalignant prostate, breast, ovary, lung, and colon have been collected and examined.

Another project, Tissue Proteomics, uses LCM as a launching point for proteomics—the study of proteins expressed by cells or tissue with the goal of identifying and understanding how proteins change in the disease process. The project, which began 3 years ago, is a joint effort between FDA and NCI.

"Looking at proteins in microdissected tissue will help aid in early cancer detection, cancer prevention and therapy, and the development of clinical trials," Liotta said. Scientists are moving beyond the genome to understand the events taking place at the protein level. This program is using existing technologies, such as two-dimensional gel electrophoresis and surface enhanced laser deabsorption and ionization (SELDI), and developing new technologies along the way to generate protein "fingerprints."

"Greater than 100 proteins have been identified to date, and we are currently working on validating all of these proteins," Petricoin said.

In the long run, the Tissue Proteomics initiative hopes to find its ultimate impact in the drug development process. Petricoin pointed out that the FDA usually rejects pharmaceutical drugs for two reasons, unforeseen toxicity or inaccurate drug targets.

The joint initiative was designed to address both of these issues to prevent loss of both resources and time. "Some drugs are in the pipeline for 6 to 10 years, and then go down the drain," Petricoin said. "The FDA would like to see technologies help [get] effective and safe drugs to market."

With the help of LCM, the joint initiative is generating protein fingerprints at each stage of cancer development. This information will identify the critical proteins that are involved in cancer development and progression, will provide new therapeutic targets, and will potentially match tumor stage with therapeutic strategies to improve treatment regimens. In addition, the FDA can test new drugs on the market for efficacy by examining changes in protein profiles before and after drug treatment.

The initiative will also generate early toxicity fingerprints, such as those occurring during vascular damage, and identify specific toxicity markers. With this collection of information, they can develop algorithms and test new drugs for early toxicity based on their defined protein fingerprints.

According to Liotta, this initiative will reduce the worry of biological relevancy and early toxicity. "We would like to go from the bench to the clinic with less toxicity and greater success."



             
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