U.S. Army Medical Research and Materiel Command

June 9, 2005

FOCUSING ON NEW TARGETS TO ENHANCE BREAST CANCER TREATMENT

Points of Contact:
Lynn Rudinsky phone 212/886-2200, email lrudinsky@cooneywaters.com
In Philadelphia 6/8 - 6/11
Pennsylvania Convention Center, 215-418-2155
Gail Whitehead, Public Affairs Coordinator, phone (301) 619-7783, email gail.whitehead@amedd.army.mil

Research Results from the "Era of Hope "Department of Defense Breast Cancer Research Program Meeting

PHILADELPHIA, June 9, 2005 - Studies presented at the "Era of Hope" Department of Defense Breast Cancer Research Program meeting offer a glimpse into the array of research exploring new breast cancer treatment options to improve outcomes. Examples include: using an immunotoxin to suppress breast cancer growth in mice by stimulating the immune system; correlating genetic changes in normal-looking tissues near breast tumors with risk of recurrence; constructing a new radiation technique to target cancerous breast tissue and spare healthy breast tissue; and finding the right gene combination to create a simple test to predict treatment success with tamoxifen.

Immunotoxin Suppresses Breast Cancer Growth in Mice by Targeting Tumor Defense Mechanism
Recent studies finger regulatory T cells (T-regs) as one mechanism that allows breast cancer to thwart the body's natural immune response. Using a hybrid molecule to slash the population of T-regs, researchers overcame this defense tactic and significantly reduced the growth of breast tumors in mice.

The anti-T-reg molecule is a commercially available fusion protein called denileukin diftitox (Ontak ®), which joins a diphtheria toxin to interleukein-2 (IL-2). The resulting IL-2 immunotoxin is both specific and lethal to the target T-regs. It is currently approved for treatment of cutaneous T-cell lymphoma.

"Developing breast cancers can recruit T-regs into the tumor. The IL-2 immunotoxin kills these T-regs by blocking protein synthesis, a function that's essential to cell survival," explained Keith Knutson, Ph.D., immunology researcher at Mayo Clinic in Rochester, MN.

The researchers studied the effects of the IL-2 immunotoxin in 10 to 12 mice with minimal disease and an equal number with established disease. Both groups received the same dose (1 or 5 µg) and treatment schedule (every two to three days for a total of six doses), and each had its own control arm. In the minimal-disease group, treatment began the day after the mice were injected with human invasive breast cancer. The other group started treatment when tumors were palpable.

At the end of the treatment cycles, the IL-2 immunotoxin significantly delayed the growth of tumors in the animals with minimal disease and slowed the growth of tumors in both treatment groups. The mean tumor sizes were about 90% smaller in the minimal-disease mice and 79% smaller in the established-disease mice compared with their respective control groups, and the anti-tumor response lasted for more than a month after treatment ended. This sustained response may be a result of immunologic memory, suggested Dr. Knutson. In addition, the researchers found that the anti-tumor and T-reg effects were dose-dependent, whereby animals that received 5 µg fared better than those that had 1 µg, but all treated animals had reduced tumor burden.

"Evidence is emerging that some of the effects of chemotherapy are due to depleting T regulatory cells. This may be a way to target the T-regs directly instead of using the indirect route of chemotherapy. Depleting T regulatory cells may boost natural immunity against breast cancer," Dr. Knutson noted.

When the researchers treated the removed tumors with the immunotoxin, they saw no impact, which supports the idea that it acts on the immune system and not as a direct-acting drug. Further, they cancelled out the effect of the drug completely by stopping therapy and reinfusing T-regs. Dr. Knutson hopes to pursue this treatment in women with advanced breast cancer.

Disease Recurrence Correlated with Genetic Changes in Normal-looking Tissue Near Breast Tumors
By identifying telltale genetic changes in breast tissues that appear normal, a new test may help predict the return of breast cancer after treatment, report researchers from the University of New Mexico School of Medicine. The assay measures the DNA content of telomeres (TC), specialized nucleoprotein complexes that cap the ends of chromosomes, which are altered in tumors.

"We wanted to confirm that shortening of telomeres leads to genomic instability, which is a critical step in the progression to recurrent cancer," said Christopher Heaphy, a Ph.D. student in the Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine in Albuquerque.

In previous studies, the group defined normal-length telomeres as having a TC value of 70% to 136% of a standard control. Telomeres with a TC value of less than 70% are considered tumor-like telomeres. Using mastectomy specimens from 11 women, the investigators examined tissue from the tumor at 1 cm and 5 cm distances from its visible edges. As expected, all 11 tumors had TC values of less than 70%. The assay the group developed also found tumor-like TC values in tissues that looked normal on standard laboratory analysis in nine of 11 tissues at the 1 cm point, and in zero at 5 cm. There was also an increase in allelic imbalance (AI), a reflection of genomic instability.

The researchers then examined samples from 25 women diagnosed with breast cancer around 1990 who were monitored on health status information after their mastectomies. The majority of tumors were large and had signs of spread to underarm lymph nodes. In addition to tumor specimens, they analyzed nearby tissue taken an unknown distance from the tumor margin.

With the aid of long-term follow-up data on these women, Mr. Heaphy and his colleagues determined that tumor-like TC in normal-appearing tissues was associated with the return of breast cancer within 84 months of surgery. Specifically, TC values predicted relapse in seven of the 12 women whose breast cancer recurred. In 12 of the13 women who remained free of cancer, TC values of 70% or higher predicted this outcome. Earlier this year, the same research team published a paper showing that TC predicts outcome in prostate cancer.

"Our current TC assay is great for research labs, but it takes two days. We're now trying to develop a lower-cost, faster, and easier assay that will be practical in a clinical setting, to help identify women at high risk for disease progression," Mr. Heaphy said. "In roughly 30% of women with breast cancer, metastatic disease will develop even after treatment. We believe that our TC assay can help identify these patients and allow the physician to make better informed decisions about further treatment."

The researchers also expect that their work showing genetic alterations in histologically normal breast tissue adjacent to the tumor will lead to a better definition of relevant surgical margins and a better assessment of risk of local recurrence after breast-conserving therapy.

In a much larger prospective project, the researchers are in the process of measuring TC and AI from tumors of 569 breast cancer survivors enrolled in the New Mexico Women's Health Study. This will allow the researchers to draw conclusions about the role of ethnicity in treatment, particularly in Hispanics, and whether TC can predict clinical outcome in a wider range of breast tumor types.

Advanced Electron Beam Targets Cancerous Breast Tissue, Spares Normal Tissue
A new, high-precision version of electron beams used in radiation therapy may be able to target only cancerous breast tissue, leaving healthy breast tissue unharmed, according to research conducted at the University of Maryland, Baltimore.

"Women prefer to avoid radiation therapy as part of breast cancer treatment because it can cause skin reactions and other side effects," said Lijun Ma, Ph.D., associate professor of radiation oncology physics at the University of Maryland School of Medicine. "This new technique could potentially minimize some of these important quality-of-life concerns for women."

Traditional electron beams are used in conjunction with radiation therapy, but are prone to excessive scattering, causing irradiation of normal breast tissue. This can cause complications such as long-term skin reactions and fibrosis or tissue hardening. Dr. Ma and his team constructed a cylindrical magnetic collimater as an alternative to conventional electron beams. A collimater is a device used to control a beam's size and shape. The advanced beam device was awarded a U.S. patent in April.

"The intrinsic physical properties of conventional electron beams cause wobbling and scattering," Dr. Ma said. "The technique we developed allows the electrons to travel more accurately to reach only the areas of the breast that carry the cancer cells."

In the research, the beam characteristics of the new, high-precision electron beam were compared to those of conventional electron beams. The measurements were carried out using radiographic films inside water-equivalent material shaped like a human breast.

The researchers found that magnetically collimated electron beams significantly lower the surface dose compared to conventional electron beams with the same entrance energies. The magnetically collimated arc beam also allows for a reduced surface dose - to less than 25% of the traditional procedure. Electron scattering is significantly reduced, and the focused electron beam can pinpoint the cancerous breast tissue.

The technique will be further refined over the next few years, according to Dr. Ma. Then, the research team plans to initiate clinical trials and, eventually, the University of Maryland School of Medicine may collaborate with a medical device company to manufacture the advanced electron beam for commercial use.

Two-Gene Pattern Predicts Tamoxifen Treatment Outcomes
A simple measurement of the activity of two specific genes in breast cancer tissue appears to predict the outcome of tamoxifen treatment in patients with early-stage breast cancer, according to research presented here today.

Currently, tumor cells are tested for the presence of estrogen, which is likely to feed the tumor. Estrogen-receptor positive tumors are treated with an estrogen blocker, such as tamoxifen. Because only two-thirds of patients currently being treated with tamoxifen have a prolonged response to the drug, this novel two-gene pattern may help identify which women should receive earlier treatment with an alternative therapy.

Researchers from Massachusetts General Hospital analyzed frozen tumor samples from patients who received tamoxifen treatment for early-stage, estrogen-receptor-positive breast cancer. They started with 22,000 genes and, through their analysis, identified a subset of two - HOXB13 and IL17BR - that demonstrate the strongest predictor of whether a tumor would recur.

"The higher the expression level of HOXB13 and the lower the expression level of IL17BR, the greater the chance of cancer recurrence," explained Dennis Sgroi, M.D. associate professor of pathology at Harvard Medical School and director of breast pathology at Massachusetts General Hospital.

The predictive power of the two-gene signature in the initial test set of 20 patients was 80%. In subsequent work looking at a larger cohort of patients with lymph node negative breast cancer, the accuracy was ~78%. Further research continues to test the pattern, adding other genes that may improve the predictive power of the pattern.

"Another exciting discovery from our research is that the HOXB13 gene proved to be more than a biomarker," said Dr. Sgroi. "It may have importance in the development and progression of breast cancer and may give us a target for breast cancer treatment in the future."

Many clinical laboratories currently have the capability to utilize the test and the research team, in collaboration with Arcturus Bioscience, Inc., hopes to commercialize the user-friendly test in the future.

"Era of Hope" is a forum for the presentation of research supported by the U.S. Department of Defense's Breast Cancer Research Program (BCRP), an unprecedented partnership between the military, scientists, clinicians, and breast cancer survivors. Since 1992, the BCRP has been working to prevent and cure breast cancer by fostering new directions in research, addressing underserved populations and issues, encouraging the work of new and young scientists and inviting the voice of breast cancer survivors to be heard in all aspects of the program. One of many congressional research programs managed by the U.S. Army Medical Research and Materiel Command, the BCRP has received more than $1.8 billion to date from Congress for innovative breast cancer research.

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"Inhibition of Breast Tumor Growth in the Neu-Transgenic Mouse Following Depletion of Regulatory T Cells with an Interleukin-2 Immunotoxin"
K Knutson, Y Dang, H Lu, E Makary, M Disis
Concurrent Symposia Session IV, Symposia 22: Friday, June 10, 6:45 p.m. - 8:45 p.m., Room 204A

"Telomere DNA Content and Allelic Imbalance in Histologically Normal Tissue Adjacent to Breast Tumors"
C Heaphy, M Bisoffi, C Fordyce, A Mangalik, J Griffith Poster Session: Saturday, June 11, 7:00 p.m. - 9:00 p.m., Exhibit Hall A

"Characterization of Helical Electron Beams for Breast Radiation Therapy"
L Ma
Poster Session: Thursday, June 9, 6:30 p.m. - 8:30 p.m., Exhibit Hall A

"Discovery of a Novel Two-Gene Expression Ratio That Predicts Clinical Outcome in Breast Cancer Patients Treated with Tamoxifen"
D Sgroi, X Ma, D Haber, P Ryan, Z Wang, J Younger, S Isakoff, B Smith, J Bruggs, F Couch, M Goetz, V Suman, W Lingle, J Ingle, M Erlander Concurrent Symposia Session IV, Symposia 20: Friday, June 10, 6:45 p.m. - 8:45 p.m., Room 201B