Morphometry of Breast Cells in Ductal Lavage
WHY THE STUDY IS BEING DONE
Breast nipple aspirate fluids (NAF) can be obtained non-invasively from women, making
analyses of this fluid relevant to studies of breast cancer risk. The inter-individual
variation for lipid oxidation products (a type of damage to fats) in NAF is more than
100-fold, and these oxidation products are likely to have a toxic effect on the breast
epithelial cells. During breast ductal lavage, NAF is first aspirated, and the ducts
producing fluid are then subjected to lavage to obtain epithelial cells. The nuclear
features (morphology) of these cells can then be examined. Nuclear morphology is a
quantitative method to observe subtle changes in nuclear features, and these measures in
benign breast biopsies have been shown to have prognostic value for breast cancer risk.
This exploratory study will help establish the feasibility of using these measures as
endpoints in dietary intervention studies. Since the differences in lipid oxidation levels
in NAF are markedly large, it is reasonable to believe that there must be some influence on
nuclear morphology. These measures also should be applicable to investigations that seek to
examine the impact of certain genetic polymorphisms and environmental exposures on
biomarkers of oxidative stress and breast cancer risk.
We will study women who have been diagnosed previously with proliferative breast disease.
These women may be good candidates for dietary prevention studies since they are at somewhat
increased risk of breast cancer, receive little beyond surgery for treatment, and have been
shown to have higher levels of lipid oxidation in the breast than women with no such
WHAT WE HOPE TO ACCOMPLISH
We hope to determine the feasibility of obtaining and analyzing both NAF and breast
epithelial cells from women with benign proliferative breast disease. These women are at
increased breast cancer risk, and the composition of the NAF, which bathes the breast ducts
may affect the morphological features of the nuclei in the breast epithelial cells. If the
levels of lipid oxidation products are higher in women with large nuclei (a risk factor for
breast cancer), then interventions which reduce oxidative stress and cancer risk may be
planned in the future.
The aims are:
1. To determine if levels of fats, damaged or oxidized fats, oxidized or damaged vitamins
and vitamins (8-isoprostane, cholesterol, fatty acids, tocopherols, carotenoids and
2,6-cyclolycopene-4,5-diol) can be quantified in breast NAF obtained before breast
lavage. Levels in NAF will be compared to that in blood. These measures were chosen
based on their potential relationships to dietary intakes and to oxidative stress
levels, which is relevant to intervention studies. Tocopherols and carotenoids are
dietary antioxidants, and certain fatty acids are oxidized more readily than others.
Both 8-isoprostane and 2,6-cyclolycopene-4,5-diol appear to be markers of oxidative
2. To obtain breast lavage fluids from women with proliferative breast disease and to
characterize the nuclear morphology of breast epithelial cells in the breast lavage
fluid. We will quantify various measures commonly used in biopsies including nuclear
diameter, circumference, area, roundness and nuclear:cytoplasmic area ratio. These
measures have been shown to be related to breast cancer risk. If there are sufficient
cells, analysis of oxidative DNA damage will be made using the comet assay.
3. To examine whether nuclear morphology is associated with levels of oxidative stress in
the NAF. Correlations will be evaluated with levels of 8-isoprostane, cholesterol,
specific fatty acids, 2,6-cyclolycopene-1,5-diol and fat-soluble micronutrients. The
potential influence of diet, demographic factors and nature of previous biopsy
diagnosis on nuclear morphology will also be examined as a sub-aim in an effort to
better understand differences among women in the nuclear morphology of epithelial cells
from ductal lavage.
The materials gathered for research purposes will be:
Consent Form Health Status Questionnaire Food Frequency Questionnaire Height and body weight
Breast nipple aspirate fluid Breast lavage fluid 10-ml Blood sample biopsy date, diagnosis
and history of medical problems from the medical chart
It was desired to equip our study so that the variables of interest can be determined within
a coefficient of variation (CV) of 25%, with 90% confidence. Sixty subjects would yield
estimates of the mean level of 8 of the 9 study variables to within a CV of 25%, with 90%
confidence. The one exception is the highly variable 8-isoprostane measure, whose mean
level could be estimated to within a CV of 37%, with 90% confidence.
HOW THE STUDY WILL BE CONDUCTED
A total of 75 women will be recruited for this study over roughly 13 months (ten months of
year 1 and 3 months of year 2) by a clinician. She will confirm from the patient chart the
previous diagnosis of proliferative breast disease, obtain the type of breast disease
diagnosed, how long ago it was diagnosed to the nearest month, and any breast or health
problems that could affect the lavage results. The study subjects will fill out
questionnaires, be weighed and measured for height, donate a 10-ml blood sample and undergo
a breast lavage procedure. During the lavage procedure, breast nipple fluid and the lavage
fluid will be collected.
Breast ductal lavage is an important new procedure that can be used to help determine a
woman's risk for breast cancer. It is an attractive diagnostic method since it is
associated with very little discomfort. At present, it is only used in certain women who
are thought to be at very high-risk for breast cancer. During the ductal lavage procedure,
the fluid-producing ducts must first be identified. This is done by first freeing the ducts
at the nipple surface of keratin plugs, and a woman then uses massage to produce fluid.
This fluid, called nipple aspirate fluid (NAF), is what bathes the breast ducts and will be
collected for analysis in this proposed project. Once the ducts that produce NAF are
identified, the physician then lavages the duct. The lavage fluid washes out breast cells
that can then be evaluated by a pathologist to determine whether or not they are normal.
The entire procedure is done using anesthetic at the nipple surface initially and in the
lavage fluid to minimize any discomfort, and levels of discomfort is generally less than
that with a mammogram.
An early indicator of breast cancer risk may be the characteristics of the epithelial cells
from the lavage fluid. These breast cells are dislodged upon lavage, they are placed onto a
slide and a pathologist examines them for abnormalities. Beyond the pathological diagnosis,
there may be subtle changes in the appearance of the cells. Changes in the size and shape
of the cell nucleus can be measured using a computer-driven microscope that has quantitative
image analysis software. Increases in nuclear area, diameter and perimeter and less uniform
roundness of nuclei are known to be associated with breast cancers of higher grade. These
kinds of measures have also been measured in breast biopsies that were either normal or
hyperplastic, and those women with larger nuclei in completely benign breast biopsies were
more likely to develop breast cancer 1-33 years later.
One factor that may affect the shape of nuclei in breast cells is the level of oxidative
stress in the breast. The breast fluid is very rich in fats, or lipids, and certain kinds
of lipids are easily oxidized. NAF also contains vitamins obtained from fruits and
vegetables that function as antioxidants to stop lipid oxidation. For example, carotenoids
can react with harmful oxidants to form oxidized carotenoids. We will therefore measure
levels of one lipid peroxidation marker and one oxidized carotenoid in NAF as markers of
oxidative stress. We also will measure fatty acid levels and levels of un-oxidized
carotenoids. We then will determine if differences in the composition of the NAF are
related to the measures of nuclear shape made on breast cells.
The breast NAF and blood will be analyzed for cholesterol using spectrophotometric kits.
Fatty acids will be analyzed using gas chromatography. Total 8-isoprostanes will be
analyzed by immunoassay kits. Micronutrients and vitamins will be analyzed by high pressure
liquid chromatography. Nuclear morphology will be analyzed by a computer-driven microscope.
DNA damage in breast cells will be analyzed using gel electrophoresis of lysed cells (comet
assay). Statistical analyses will allow us to determine if the NAF and blood measures are
related to each other and to the nuclear measures.
Observational Model: Cohort, Time Perspective: Prospective
Zora Djuric, PhD
University of Michigan
United States: Institutional Review Board
|Karmanos Cancer Institute||Detroit, Michigan 48201|