Prior research on the RhoC gene had shown its potential as a biomarker for breast cancer. The current research was conducted with 182 tissue samples. The test detected invasive cancer that had the potential to metastasize with 88 percent specificity and had 92 percent specificity for tiny tumors that had already metastasized. Samples of normal breast, benign breast cysts, or noninvasive breast cancer had little RhoC.

"This is a very promising marker for small but invasive breast cancers that may metastasize, which right now are hard to identify," says presenter Celina Kleer, M.D. "While more research is needed before clinical testing can begin, we hope it will help identify early-stage cancer that could be vulnerable to aggressive treatment, perhaps with drugs that target Rho protein."

Kleer and her colleagues designed the current research to learn how much RhoC was produced in different kinds of breast cancer cells compared with normal breast cells.
Previously, they had documented overexpression of RhoC in inflammatory breast cancer. Overexpression of the gene, they believed, might also occur in other kinds of aggressive breast cancer -- leading to larger quantities of the RhoC protein in cells of those cancers.

RhoC, whose full name is RhoC-GTPase, is an enzyme involved in changing the internal skeleton of a cell -- changes that allow a cell to polarize or move. That ability is important in muscle cells, which produce a lot of RhoC. However, in cancerous non-muscle cells, RhoC is vital to the structural changes that give a cell the ability to separate from a primary tumor and metastasize.

In finding the inflammatory breast cancer correlation, the team was the first to show that RhoC, already implicated in liver, pancreas, and skin cancer, was also involved in breast cancer. Investigators then showed that transplanting the RhoC gene into normal breast cells in mice resulted in malignant transformation with the resultant cells showing high metastatic potential.

The new research started with the development of a RhoC test. The team created an antibody to RhoC protein. A stain specific to the antibody then allowed researchers to see how concentrated the protein was in different tissue samples from breast tumors and surrounding areas.

The 182 tissue samples used in the study came from 164 patients whose breasts had been biopsied; information on diagnosis as well as presence of conditions such as fibrocystic change was available. Samples of breast cancer tissue were accompanied by information describing exactly what variety of cancer it was, how large the tumor was, whether it was invasive (spreading beyond the layer of cells where it started and into healthy tissue), what stage it was in, and whether it had metastasized through the body.

The cancerous samples included everything from ductal carcinoma in situ to stage IV invasive ductal carcinoma. The wide spectrum allowed the team to look for differences between cancers of different sizes, stages, types, and levels of invasiveness.

Antibody staining revealed the expected high levels of RhoC in muscle and blood vessel cells surrounding the breast tissue, and none in normal breast tissue. However, the concentrations of the reddish-brown stain in the cancerous tissue varied greatly. When the researchers correlated the samples that stained darkest with their clinical characteristics, they found the connection.

"We found RhoC only in invasive cancers, and it almost always correlated with the presence of metastases. Very few non-metastatic cancers contained high levels of RhoC," Kleer says. "The level of RhoC expression also increased as the stage of the breast cancer increased, which is another confirmation that it's a marker of more aggressive cancer. We had enough samples from invasive metastatic cancers of less than one centimeter in size to show that RhoC is highly specific for those tumors, but we'd like to look at more samples to be sure."

Kleer and colleagues are preparing to examine even more breast samples for the presence of RhoC to see if their initial results hold. The team is also in the process of planning clinical studies on the predictive power of RhoC.