6th ESACP Congress, Heidelberg, April 7-11, 1999

A023
IDENTIFICATION OF EXPRESSED GENES IN ARCHIVAL TISSUE BY LASER-MEDIATED MANIPULATION OF SINGLE CELLS: CLUES TO TUMOR PROGRESSION OR TUMOR HETEROGENEITY
Lahr G, Schütze K

Laserlabor und Molekularbiologie 1.Med.Abteilung Städt.Krankenhaus Müchen-Harlaching, München, Germany

Fundamental questions concerning the early events preceding cancer formation as well as questions dealing with clonal progression and timing of different mutations, deletions, etc. remain to be resolved. The melange of cell types in a typical tumor has long been a problem for researchers analyzing gene expression in cancer cells. Analysis of RNAs extracted from a whole tumor does not result in an accurate picture because it originated from a mix of molecules from all of the different cell types included in the tumor. The challenge has been to separate distinct cells from the others to analyze the mRNAs of the selected cells. The ROBOT-MICROBEAM SYSTEM (P.A.L.M. GmbH, Bernried) was used to procure single cells from a colon adenocarcinoma tissue slice by Laser Microbeam Microdissection (LMM). With a few laser shots (Laser Pressure Catapulting; LPC) the laser- isolated specimens were catapulted away from the object slides directly into the cap of a reaction tube. Reverse transcription followed by nested PCR amplification of the Ki-ras2 mRNA allowed to detect point mutations within codon 12 in isolated colon carcinoma cells by artificial restriction length polymorphism, even from a single paraffin-embedded archival tumor cell. Point mutations in the Ki-ras2 gene are common genetic alterations associated with development and progression of human colon cancer. Our method allows to analyze retrospectively expressed genes from routine histopathological tissue slices to gain more information about the molecular defects underlying specific diseases.