Trial Information

Epigenetics and Breast Cancer Risk in African American Women

Increasing evidence supports the importance of the role of pregnancy, lactation and
post-weaning breast remodeling in the etiology of certain types of early onset aggressive
breast cancers, including basal breast cancers, which are difficult to detect and treat and
disproportionately affect African American women. Thus, improving methods for detecting or
preventing early onset tumors is important from a clinical, public health and racial
disparities perspective. Recent evidence indicates that analysis of breast milk during the
postpartum period may advance the discovery of mechanisms and biomarkers related to risk of
early onset, aggressive tumors. However, developing methods for collecting, processing and
testing milk for biomarkers poses challenges.

A research team co-led by Drs. Mark Sherman (NCI/DCEG/HREB) and Kathleen Arcaro (University
of Massachusetts) has received an NIH Bench-to-Bedside award for a project entitled,
Molecular Epidemiology of Postpartum Involution of the Breast: Development and Demonstration
of Tools for Understanding the Postpartum Period in Relation to Risk for Early Onset Breast
Cancer. The specific bench objectives of this project include:

1. To develop improved methods for fractionating breast milk into epithelial cell rich and
liquid components

2. To optimize assays for DNA methylation, proliferation, apoptosis, p16 expression and
telomere lengths using epithelial enriched breast milk fractions, and

3. To develop assays for TGF-beta ligands, prolactin and sex -steroid hormone using liquid
milk fractions.

To achieve these objectives, NCI will initially work with fresh specimens that are being
prospectively collected under the University of Massachusetts IRB-approved open protocol
entitled Epigenetics and Breast Cancer Risk in African American Women , funded by the Avon
Foundation for Women. Dr. Arcaro's lab is studying breast cancer risk and promoter
hypermethylation in breast cells obtained from the milk of nursing women. They have analyzed
breast cells from nearly 400 women and are continuing with long-term follow-up. However, the
majority of the data has been from White women. Since disease risk factors differ between
ethnic groups, it is important to test risk assessment methods on a wide population. The
main purpose of this specific UMass study is to extend their findings of breast cancer risk
to African American women. They plan to recruit 200 lactating African American women to
participate in the breast milk study. This involves collecting questionnaire data,
completing methylation analyses for eight genes, archiving milk and the remaining DNA for
future studies, and annual follow-up.

The key aims that we seek to address through the current protocol are related to objective
(1) listed above: To develop improved methods for fractionating breast milk into epithelial
cell rich and liquid components. Given that Dr. Arcaro has an IRB approved open protocol to
collect fresh milk at University of Massachusetts, NCI can only pursue this collaborative
aim (effectively within the timeframe of the bench-to-bedside award) in the context of this
ongoing study. Specifically, the collaborative project that is the subject of this IRB
application will assess the following aims through in vitro manipulation of fresh liquid
milk collections: 1) the effects of modifying the initial rinse of epithelial pellets (rinse
solution: saline vs. media; centrifugation speeds, temperature); 2) yields of cells and
nucleic acids achievable via fractionation of milk using different types of immunomagnetic
beads (coated with antibodies to remove leukocytes ( negative selection ) vs. coated with
antibodies to remove epithelial cells ( positive selection ); 3) the relative preservation
at room temperature at 24, 48 and 72 hours of milk suspended in cellular fixatives (e.g.
formalin, Proclin) as compared to fresh milk without fixation; 4) the possibility that
strategies 1 and 2 be combined. This project brings together specific experience and
expertise at University of Massachusetts with regard to milk processing with technical
knowledge from NCI, provided by Drs. Kopp, Heckman, Yang and Sherman. In particular, NCI
Frederick and Dr. Heckman have successfully used immunomagnetic bead technology to purify
epithelial cells from blood products and lactating mouse glands by removing leukocytes.