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Gene-expression Profiling in Cancer | A Brief Review July 24, 2007

It seems I’m getting more and more interested in gene-expression profiling (GEP) oncotests. In the Internet the information about this topic is quite messy and dispersed, so here I will try to draw some focus on this emerging technology (what will be added to a Genetic testing section later).

So, if we want to make gene-expression profiling, or in the other words, to answer an important question – what genes are expressed in a particular cell type of an organism, at a particular time, under particular conditions? [ref.]- first we need to have a sample of a tumor tissue to analyze its RNA pattern (or more precisely, messenger RNA (mRNA)). Here, unlike in mutations search, we’re not interested in DNA. RNA is quite instable molecule, so you need be quite cute and smart to manage preserve and analyse it.

A breast cancer is currently a holy grail for gene-expression profiling research to identify genes associated with breast cancer and to measure their activity in tumor cells, i.e. obtain its gene expression profiles.

Several techniques for measuring gene expression are available: a) a microarray based analysis and b) reverse transcriptase-polymerase chain reaction (RT-PCR). To measure gene expressions with a microarray you need to have a “fresh” (or “unpreserved”) tumor tissue samples. Tumor samples are frequently preserved in this way in Europe. Whereas in the United States tumor samples are more commonly preserved in formalin and embedded in paraffin blocks. Microarray analysis cannot be done on tissue that has been preserved in this way [ref.]. Since our pathologists are accredited by College of American Pathologists, they’re using paraffin blocks as well. Instead, for formalin-fixed and paraffin embedded (FFPE) tumor tissue blocks RT-PCR can be used to measure gene expression.

Therefore its important to know, that because of this difference in common methods of tissue processing has led to the development of tests in Europe and the United States that are quite different from one another, and that are based on measurements of the activity of different sets of genes.

In Europe, using microarray analysis, researchers are studying the usefulness of at least two different groups, or “panels”, of genes in studies on early stage tumors from node negative patients. One is a 70-gene panel, and another is a 76-gene panel. ^1,2 Only three genes are common to both panels, while all the others are unique to one test or the other^B. In spite of this difference, both panels have yielded results that appear to be promising,³ and a commercial test based on the 70-gene panel is already available in the United States under the name MammaPrint^®. (via)

Based on 76-gene panel Johnson&Jonson’s companies Veridex research, a two genes expression The GeneSearch™ Breast Lymph Node (BLN) Assay test was recently approved dy FDA, and is based on RT-PCR (i.e. for analysis in FFPE).

Common use of FFPE and a need for gene expression profiling using RT-PCR has led researchers in the United States to investigate several other panels of genes that produce too much or too little protein in early stage, node-negative, and estrogen receptor-positive breast cancer cells. A 21-gene panel commercial Oncotype DX test is one of example, which recently was approved by TEC to inform decision making about adjuvant chemotherapy only for a women with estrogen receptor-positive, node-negative, tamoxifen-treated breast cancer.

Let’s have a closer look at these genes [ref.]:

Measurements of five of those 21 genes (Beta-actin, GAPDH, RPLPO, GUS, and TFRC ) are used as “controls” – that is, to verify that the test has not been contaminated or run improperly.

The other 16 genes, which produce their proteins at varying levels in different tumors, include:

• genes associated with cell proliferation (Ki-67, STK15, Survivin, Cyclin B1, and MYBL2);
• genes associated with cellular invasion (Stromolysin 3, and CathepsinL2);
• genes associated with HER2 activity (GRB7 and HER2);
• genes associated with estrogen activity (ER, PR, Bc12, and SCUBE2); and
• three other genes with distinctly different activity in cancer cells (GSTM1, BAG1, and CD68).

This Oncotype DX panel has only a single gene overlap with the 70-gene MammaPrint^® panel. The reason for such diversity between gene panels under investigation are because of differences in tissue preparation, differences in laboratory methodologies, and differences in measurement techniques. At present, these gene-expression profile tests have only been validated on stored sample tissue.

Whether one of the existing panels will prove to be significantly superior to others, or whether newer panels will emerge that have better predictive power remains to be seen. It is clear that additional validation studies and more clinical experience are needed to establish the reliability of gene expression profiling for predicting tumor recurrence and response to specific treatments (ref.)

TBC