A New and Advanced Imaging Technique Allows Researchers to Monitor Protein Changes in Mouse Tumors

A new imaging technique can monitor, in living mice, the HER2 protein found in above-normal amounts in many cases of breast cancer as well as some ovarian, prostate and lung cancers. This new approach, once validated in mice and pending further experiments, could provide a real-time noninvasive method for identifying tumors in women who express HER2 and who would be candidates for targeted therapy directed against this protein.

The new technique may also provide real-time information that will help clinicians optimize treatment for individual patients. The study by Kramer-Marek and colleagues, published in the July 2009 issue of The Journal of Nuclear Medicine, was conducted at the National Cancer Institute (NCI) and the National Institute of Biomedical Imaging and Bioengineering, both parts of the National Institutes of Health.

Overexpression of HER2
The HER2 protein is overexpressed in approximately 20 percent to 25 percent of breast cancers. Numerous studies have shown that HER2 is associated with shorter disease-free and overall survival. In breast cancer, HER2 gene amplification was significantly associated with pathologic stage at diagnosis, axillary node involvement, and histologic subtype. In ovarian cancer HER2 has been associated with decreased overall survival and with an increase in relative risk of death.

Tumors that overexpress HER2 are more aggressive and more likely to recur than tumors that do not overexpress the protein. Targeted therapies directed against HER2 can slow or stop the growth of tumors that overexpress it.

Determining HER2
HER2 expression can be determined by any of several methods. The most commonly used methods include Fluorescence in situ hybridization or FISH, which detects gene amplification by measuring the number of copies of the HER2 gene in the nuclei of tumor cells and Immunohistochemistry or IHC, which measures the number of HER2 receptors on the cell surface and therefore detects receptor overexpression.

Chromogenic in situ hybridization or CISH, which measures gene amplification using a light microscope rather than a fluorescent microscope required for FISH and reverse-transcriptase polymerase chain reaction or RT-PCR, which detects HER2 gene amplification are among some other methods of HER2 testing whicj have been used increasingly in clinical studies and may eventually be incorporated into routine practice.

Not Perfect
In 2007, Wolff et al showed that Immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH) test results can be affected by testing conditions including the use of suboptimally fixed tissue, failure to use specified reagents, deviation from specific instructions, and failure to include appropriate controls. Therefore, in order to increase the accuracy of HER2 testing results, testing should be performed by laboratories with demonstrated aptitude in the specific test requested. Even if tested in these high-end laboratories, expression of HER2 in test samples may still not accurately represent HER2 expression in the tumor as a whole. Moreover, follow-up biopsies are not always routinely performed after the initial diagnosis, and there are no means to evaluate how long a targeted therapy takes to reach its target, how effective it is, and how long its effects last.

In this study, the researchers used an imaging compound that consists of a radioactive atom (fluorine-18) attached to an Affibody molecule, a small protein that binds strongly and specifically to HER2. Affibody molecules are developed by Affibody AB, (Bromma, Sweden), a Swedish biotech company focused on developing next generation products for therapy, diagnostic imaging, and other applications based on its unique proprietary Affibody® molecules and albumin binding technology platforms. The Affibody molecules are much smaller than antibodies and can reach the surface of tumors more easily. The radioactive atom allows the distribution of the Affibody molecules in the body to be analyzed by positron emission tomography (PET) imaging.

The researchers first used the radiolabeled Affibody molecule to visualize tumors that expressed HER2 in mice. The mice were injected under the skin with human breast cancer cells that varied in their levels of HER2 expression, from no expression to very high expression. After three to five weeks, when tumors had formed, the mice were injected with the Affibody molecule and PET images were recorded. The levels of HER2 expression as determined by PET were consistent with the levels measured in surgically removed samples of the same tumors using established laboratory techniques.

To determine whether their method could be used to monitor possible changes in HER2 expression in response to treatment, the team next injected the Affibody molecule into mice with tumors that expressed very high or high levels of HER2 and then treated them with the drug 17-DMAG, which is known to decrease HER2 expression. PET scans were performed before and after 17-DMAG treatment. The researchers found that HER2 levels were reduced by 71 percent in mice with tumors that expressed very high levels of HER2 and by 33 percent in mice with tumors that expressed high levels of HER2 in comparison with mice that did not receive 17-DMAG. The researchers confirmed these reductions by using established laboratory techniques to determine the concentrations of HER2 in the tumors after they were removed from the mice.

"Our work shows that PET imaging using Affibody molecules was sufficiently sensitive to detect a twofold to threefold decrease in HER2 expression," said senior author Jacek Capala, Ph.D., of NCI’s Center for Cancer Research. "Therefore, PET imaging may provide a considerable advantage over current methods. Our technique would allow a better selection of patients for HER2-targeted therapies and also early detection of tumors that either do not respond to or acquire resistance to these therapies."

"This approach might easily be extended to forms of cancer other than breast cancer," Capala continued. "Because Affibody molecules may be selected to target specific cell proteins, similar compounds can be developed to target proteins that are unique to other types of tumors."

For more information:

• Kramer-Marek G, Kiesewetter DO, Capala J. Changes in HER2 Expression in Breast Cancer Xenografts After Therapy Can Be Quantified Using PET and 18F-Labeled Affibody Molecules. J. Nucl Med. 2009 Jun 12. [Epub ahead of print].
• Wållberg H, Ahlgren S, Widström C, Orlova A. Evaluation of the Radiocobalt-Labeled [MMA-DOTA-Cys(61)]-Z (HER2:2395)-Cys Affibody Molecule for Targeting of HER2-Expressing Tumors. Mol Imaging Biol. 2009 Jun 26. [Epub ahead of print]

Also read PubMed Abstracts:

• Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989 May 12;244(4905):707-12
• Ménard S, Tagliabue E, Campiglio M, Pupa SM. Role of HER2 gene overexpression in breast carcinoma. J Cell Physiol. 2000 Feb;182(2):150-62. Review
• Sergina NV, Rausch M, Wang D, Blair J, Hann B, et al. Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3. Nature. 2007 Jan 25;445(7126):437-41. Epub 2007 Jan 7
• Konecny GE, Meng YG, Untch M, Wang HJ. et al. Association between HER-2/neu and vascular endothelial growth factor expression predicts clinical outcome in primary breast cancer patients. Clin Cancer Res. 2004 Mar 1;10(5):1706-16
• Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131(1):18-43.

More information:

• View an animation of how Affibody® molecules will enable cancer specialists to locate and treat tumors and metastases!

Images courtesy of Affibody AB.