New Method for Measuring Breast Cancer Gene Expression

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Daniel Pinkel, Ph.D. -
Award Cycle: 1995 (Cycle I) Grant #: 1IB-0003 Award: $85,706
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1995)
This IDEA grant provides the opportunity to perform proof of principle studies on a new concept for analysis of genetic function in breast cancer. If successful, this technique will provide new information on fundamental biological characteristics of breast tumors and their associated risk factors, and help contribute to the identification of new genes involved in breast cancer development. Such information is critical to the development of early detection and prevention strategies.

Malignant breast cancer cells contain abnormalities in the genetic code of their DNA which alter critical aspects of normal function. The first step in translation of the genetic code into function is the production of messenger RNA (mRNA) molecules, a different type for each gene. A mistake in the genetic code in a gene will result in an abnormal type of mRNA. In many cases this leads to an increase or decrease in the amount of that type of mRNA in the cell. Thus detection of abnormal mRNA levels provides one important view of the genetic events involved in the development of malignancy. Current techniques are only able to analyze mRNA produced by a few genes at a time, and these genes must have already been discovered. Since a large number of breast cancer genes are already known, and many remain to be discovered, more comprehensive analytical approaches are needed. We propose to develop a method that will: a) measure the expression of multiple genes in a single test; and b) contribute to discovery of new breast cancer genes. The proposed method involves extraction of the mRNA produced by all of the genes in a breast tumor and labeling that mixture with a green fluorescent dye. Similarly all the mRNA is extracted from normal cells and labeled with a red dye. These are then combined and reacted with an array. Each element of the array contains many copies of the same DNA molecule, but the molecules differ among the elements. The mRNA produced from a gene binds specifically to the element that contains DNA with the genetic code from which it was originally made. Thus each element becomes stained with a mixture of green and red dyes, and the ratio of the two colors is proportional to the ratio of that mRNA in the tumor and normal cells. For example if a type of mRNA in a tumor becomes elevated its green to red ratio would be higher than the ratio for an mRNA that remains the same in tumor and normal cells, while if it became reduced in abundance its ratio would be lower. We will test this method by analyzing the expression of the gene cERBB2, which is known to be at elevated levels in some breast tumors and is related to a poorer prognosis.

Final Report (1997)
Breast cancer cells, like cells of other tumor types, contain genes that are functioning abnormally. It is now clear in each breast tumor several such genes are involved, but different sets of genes may be involved in different tumors. The discovery of these genes and the understanding of their functions and interactions in health and disease forms the basis on which the development of breast cancer can be better understood and rational prevention and therapeutic strategies developed. Although simply stated, gene discovery and evaluation are very complex and form one of the major areas of current research. This goal of this project was to develop a method to permit the coordinated, simultaneous analysis of many genes.

Genetic information is carried in the DNA of each cell. In most cases the information corresponding to a gene in the DNA is transcribed into a number of RNA copies, which contain the coded information from the gene and transport it to where it can be translated into functional products such as proteins. We proposed to develop a technique to simultaneously measure RNA levels from numerous genes. This involved extracting the RNA from the cells, labeling it (or copies of it) with fluorescent molecules, and chemically reacting it with target spots of DNA that contain the genetic code of the genes to be analyzed. The chemical reaction is such that the cellular RNA produced by a particular gene would stick to the target spot containing the corresponding gene sequence. Microarrays with thousands of spots, each containing the genetic sequence of a different gene, can be made. The use of RNA from two cell populations, for example normal and tumor, each labeled with a different color fluorescent molecule, permits determination of relative amount of many specific gene sequences in the two by measuring the ratio of the intensities of the two colors on each target spot. Thus expression changes in individual genes, and coordinated changes involving multiple genes can be detected. A critical aspect of this approach is minimizing the number of cells required for the analysis since breast tumor material is highly limited. The techniques we developed for attaching the targets and performing the hybridizations appear to require a much lower number of starting cells than other microarray techniques developed by others.