Breast Cancer SPORE Research Projects

Designed to meet the fundamental requirements of the National Cancer Institute (NCI) for translational research, Mayo Clinic Breast Cancer SPORE research projects capitalize on pre-existing research to find the most innovative ways to generate advances in the understanding of breast cancer biology. The Breast Cancer SPORE research projects reflect Mayo Clinic's strengths in basic, clinical, translational and clinical trials research in breast cancer. The investigative teams are interdisciplinary and include both breast cancer researchers and clinicians who are experienced in patient-oriented cancer research.

There are four research projects in the Mayo Clinic Breast Cancer SPORE:

Project 1. Risk assessment for carriers of variants of uncertain significance (VUS) in the BRCA1 and BRCA2 breast cancer predisposition genes

Co-leaders: Fergus J. Couch, Ph.D.; Noralane M. Lindor, M.D.
Co-investigators: David Goldgar, Ph.D. (Huntsman Cancer Institute, University of Utah); Susan Vadaparampil, Ph.D. (H. Lee Moffitt Cancer Center & Research Institute)

Women who carry inactivating mutations in the BRCA1 and BRCA2 genes in their germline are at significantly elevated risk of breast and ovarian cancers. In addition, many other women are found to carry variants of uncertain significance (VUS), which are predominantly mutations. Few of these variants have been classified as neutral or as a predisposing factor for breast cancer. As a result, many women carrying VUS that may be neutral unnecessarily undergo prophylactic surgery. Surgeries dramatically reduce the risk of developing these cancers but are associated with significant long-term side effects.

With this research, we propose to determine the cancer relevance of VUS found throughout the BRCA1 and BRCA2 genes by establishing genetic and laboratory assay-based methods of VUS analysis.

This project has three aims:

• Aim 1. Classify VUS using a series of studies focusing on family history of cancer in individuals with VUS and on breast tumor pathology in individuals with VUS. To facilitate this approach, we will use data and analysis from the recently established Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA), a consortium working to classify VUS through pooling available family information from many research centers. Only through the data sharing proposed in ENIGMA will it be possible to classify a substantial number of these genetic mutations.
• Aim 2. Establish the sensitivity and specificity of BRCA2 functional assays for classification of BRCA2 VUS. We will not study BRCA1 because many of the variants in that gene have already been characterized by functional studies. By establishing the sensitivity and specificity of the assays relative to the genetic data from aim one, it may be possible to classify many additional VUS with insufficient family data for direct classification by genetic methods.
• Aim 3. Develop methods for providing these results to providers and patients. This will involve evaluating the current use of reclassification results, providing the results of the project's reclassification efforts and offering educational material to clinicians. Our final goal is to improve this process and evaluate improvements in the use of these results.

To learn about a new model developed by Drs. Couch and Lindor for interpreting the meaning of some VUS in the BRCA genes, see "Progress in Clinical Interpretation of DNA Variants of Uncertain Significance in BRCA1 and BRCA2." (PDF file requiring Adobe Reader)

Results from this project were recently published in Human Mutation: "A Review of a Multifactorial Probability-Based Model for Classification of BRCA1 and BRCA2 Variants of Uncertain Significance (VUS)."

Project 2. Endoxifen as a novel hormonal therapy for breast cancer

Co-leaders: Matthew P. Goetz, M.D.; Matthew M. Ames, Ph.D.
Co-investigators: John R. Hawse IV, Ph.D.; Michael A. Mahr, M.D.; Joel M. Reid, Ph.D.

Tamoxifen (TAM) continues to be an important drug for the treatment of estrogen receptor positive (ER positive) breast cancer. We have demonstrated that endoxifen, a potent metabolite resulting in part from cytochrome P450 2D6 (CYP2D6) metabolism, is crucial for TAM's anti-proliferative effects.

Our observation that reductions in CYP2D6 activity were associated with a higher risk of recurrence in TAM-treated breast cancer led us to focus our studies on endoxifen, providing the preliminary data for this study.

In tumor-bearing animals, endoxifen is superior to TAM. Our in vitro data also indicate that endoxifen can overcome TAM resistance associated with human epidermal growth factor receptor 2 (HER2) expression because endoxifen does not stimulate ER/HER2 cross talk as TAM does.

We presented these data to the National Cancer Institute, which proceeded with endoxifen drug development, including production of clinical-grade endoxifen hydrochloride and preclinical toxicology and pharmacology for investigational new drug (IND) submission. Our preliminary data indicate that several questions need to be addressed:

• What are the metabolic pathways responsible for elimination of endoxifen, and are endoxifen-related toxicities similar to TAM, such as uterine stimulation?
• Does endoxifen have in vivo anti-tumor activity similar to or greater than aromatase inhibitors (AIs), and does endoxifen exhibit anti-tumor activity in cells resistant to TAM or AIs?
• In humans, can we identify a tolerable endoxifen dose and its toxicity profile?
• Is this tolerable dose of endoxifen biologically relevant, as indicated by reductions in proliferation (Ki-67) and growth factor signaling in vivo, as well as in clinical responses?

To address these questions, we have proposed four aims:

• Aim 1. To further characterize the pharmacokinetics, metabolism and toxicology of endoxifen
• Aim 2. To study endoxifen's anti-tumor activity and its effects on cell signaling in a murine xenograft model in comparison to TAM and letrozole
• Aim 3. To describe the anti-tumor activity of endoxifen in TAM- and letrozole-resistant tumors
• Aim 4. To conduct a phase I study of endoxifen in humans to determine the maximum tolerated dose (MTD), and describe its toxicity profile

After these aims are addressed, we will enroll additional clinical trial participants to explore two different doses of endoxifen: the maximum tolerated dose (MTD) and the endoxifen dose associated with steady state concentrations of 1 micromolar. At these doses, we will examine the impact of endoxifen on uterine thickness and measure the frequency and severity of hot flashes. We will also perform paired tumor biopsies to determine endoxifen's effect on proteins important in growth factor signaling and proliferation.

Project 3. Regulation of hormone-resistant breast cancer by IGF and insulin system signaling

Co-leaders: Paul Haluska Jr.; M.D., Ph.D.; Douglas Yee, M.D. (Masonic Cancer Center, University of Minnesota)

Hormonal agents such as tamoxifen and aromatase inhibitors are both effective and tolerable. They have had a profound impact on the treatment of estrogen receptor positive (ER positive) breast cancer in the adjuvant and metastatic setting. ER positive breast cancers represent the majority of all breast cancers. However, both primary and acquired resistance to these agents is common, and developing new methods to overcome or prevent resistance would have a major impact on treating breast cancer.

Insulin-like growth factor (IGF) signaling has been implicated as a major resistance pathway to therapies directed at the ER through the activation of two membrane receptor tyrosine kinases:

1. IGF-I receptor (IGF-IR) through binding in IGF-I and IGF-II
2. InsR isoform A (InsR-A) through binding of IGF-II

We now have evidence that complete blockade of both IGF signaling receptors with a tyrosine kinase inhibitor (BMS-754807) is sufficient to reverse resistance to hormonal therapy in vivo. In contrast, IGF-IR inhibition alone did not induce regression and led to upregulation of InsR-A isoforms. Upregulation of InsR-A is important, as we have shown that this isoform is upregulated in breast cancer. It is also expressed at an even higher level than IGF-1R in hormone-resistant breast cancer. This provides evidence in clinical samples that both IGF-1R and InsR-A may be driving IGF signaling and that both need to be blocked.

It is unclear if the metabolic isoform of the InsR (InsR-B), which binds Ins, but not IGF-I or IGF-II, is important to proliferation and survival signaling in hormonal therapy resistance. This question is important because BMS-754807 inhibits both InsR isoforms and IGF-IR and may have potential metabolic liabilities of blocking the action of Ins on InsR-B. However, it is also plausible that Ins signaling blockade is important to reversal of hormone therapy resistance.

Our overarching goal is to determine which IGF and insulin signaling components are necessary for this effect. We hypothesize that IGF blockade is sufficient to overcome hormonal therapy resistance in vivo in multiple models, has less potential to upregulate alternative signaling mechanisms and will be tolerable at effective doses in breast cancer patients.

To test these hypotheses, we propose to:

• Optimize BMS-754807 combinations in hormonal therapy resistant models
• Determine if Ins and InsR-A signaling, or both, are sufficient for IGF-IR-independent resistance to hormonal therapy
• Evaluate the efficacy of BMS-754807 and letrozole
• Perform correlative studies in patients with breast cancer

Project 4: Improving breast cancer risk prediction for women with benign breast disease

Co-leaders: Lynn C. Hartmann, M.D.; Derek C. Radisky, Ph.D.
Co-investigators: William Dupont, Ph.D. (Vanderbilt-Ingram Cancer Center); Marlene Frost, Ph.D.; Melody L. Stallings-Mann, Ph.D.; Celine M. Vachon, Ph.D.

Most of the 288,000 women diagnosed annually in the United States with in situ or invasive breast cancer were not recognized as being at increased risk of the disease, according to the American Cancer Society. Assessing a woman's breast cancer risk accurately is key to improving early detection and prevention.

The most widely used risk-prediction model for breast cancer is the Gail model, which generally performs well in predicting breast cancer risk across groups of women but is limited when predicting individualized risk. In general, it's easiest to predict whether someone is likely to be susceptible to cancer when the at-risk tissue can be examined.

In the breast, benign tissue is available for risk assessment from women who have had a benign biopsy. Women with so-called benign breast disease are a common and clinically important group with a known increased risk of breast cancer. About 25 percent of women with breast cancer report having had a prior benign biopsy.

We hypothesize that features in benign breast tissue, especially from subgroups of women known to be at increased risk of breast cancer, can help identify women who are at high risk of breast cancer progression. Our purpose with this project is to develop a risk-prediction model for women with benign breast disease based on examination of benign breast tissue.

Two recognized high-risk groups within benign breast disease are women with atypia and women in whom normal, age-related regression or involution of breast lobules has not occurred. Utilizing a cohort of more than 11,000 women with benign breast disease at Mayo Clinic, we will identify novel breast cancer-predictive biomarkers obtained from transcriptional profiling of women with atypia who either did or did not progress to breast cancer. We will also investigate the mechanisms underlying lobular involution, a physiological process that we recently found to be associated with decreased cancer risk.

Using a newly developed technique, we will also measure the specific extent of lobular involution that has occurred in these women to generate a continuous risk feature. Then, in a nested case control series within our benign breast disease cohort, we will build a risk-prediction model that incorporates the top predicting elements from:

• Histology
• Clinical-epidemiologic features
• Mammographic density
• Quantitation of involution and molecular biomarkers

Finally, building on a strong collaboration fostered by Breast Cancer SPORE developmental funds, we will assess our breast cancer risk model using the Nashville benign disease cohort supported by the Vanderbilt-Ingram Cancer Center SPORE.

Together, these studies will test the ability to use tissue-based features to enhance risk prediction of breast cancer and potentially provide insights about important early events in breast cancer development.