Coming soon to your lab: NAF for breast-cancer risk assessment

Oct. 1, 2008

Aside from non-melanoma skin cancer, breast cancer is the most common form of cancer in women. It is the second most common cause of cancer death in women.1 Yet, there is currently no definitive way to predict who is most likely to develop the disease. Of the nearly 200,000 women diagnosed each year, 70% have no identifiable risk factors, other than age. In order to see a decline in breast-cancer mortality, there is a need to develop tools to identify women who are at an elevated risk, allowing women and their physicians to take a more proactive approach to reducing breast disease.

Multiple long-term studies show that the presence of atypical cells in nipple aspirate fluid (NAF) has been associated with an increased risk of developing breast cancer. It has been suggested that cytologic examination of NAF collected from asymptomatic women might be a useful tool for breast-cancer risk assessment.2,3

To date, the lack of a reliable, standardized and practical method to acquire NAF — as well as some misconceptions among pathologists and other clinicians — has limited the adoption of NAF assessment for routine screening in the clinic. With the recent marketing of automated acquisition of NAF in the primary-care setting, however, labs can expect to see more requests for NAF cytology analysis. Despite misgivings about the clinical utility early on, the practice appears to be growing. What are the salient points for pathologists and lab personnel to keep in mind regarding NAF?

Understanding NAF

It is important to emphasize the difference between the cytologic evaluation of nipple secretions or discharges, and NAF. Nipple secretions are spontaneously produced specimens that could be symptoms of a variety of conditions such as mastitis, hormonal imbalances, or tumors. Cytologic evaluation of these samples for detection of breast lesions has been less than fruitful, with known false-positive and false-negative results. Nipple aspirate fluid, on the other hand, is fluid that can be extracted from the breasts of asymptomatic women for risk assessment, rather than for pathologic diagnosis.

NAF cytology has been demonstrated in scientific literature to be a reliable predictor of breast-cancer risk. Wrensch and Petrakis, et al, of the University of California-San Francisco conducted the first and most significant prospective study of breast-cancer risk assessment using NAF cytologic examination in asymptomatic women. The results of the study published in the American Journal of Epidemiology in 1992 showed that women with atypia in NAF were 4.9 times more likely to develop breast cancer.4 A subsequent study published in the Journal of the National Cancer Institute in 2001 included more than 7,600 women over 21 years of follow up, confirmed the researchers’ previous finding.5 More recent work reported by Buehring, et al, and Baltzell, et al, provide continuing evidence on the predictive value of NAF as an indicator of breast cancer risk.6,7

A second key point is that breast-cancer risk assessment with NAF represents a significant shift in emphasis from the traditional paradigm for pathologists of pathologic diagnosis, prognosis, and assessment of treatment, toward screening for risk and preventing disease. Until now, the cytologic method has been used primarily for screening for disease (e.g., Pap smears), to answer the question “Is there a chance I have cancer today?” To fully realize the value of NAF evaluation, we need to shift our thinking further back in the disease-management continuum, to risk assessment and prevention, to answer the question “Am I at risk of developing cancer?”

NAF acquisition

There are several methods to acquire breast ductal epithelium for analysis. These acquisition methods are ductal lavage (DL), random peri-areaolar fine-needle aspiration (FNA) and manual breast compression and nipple aspiration. While effective at obtaining specimens, these methods have limitations.

Ductal lavagehas not become a widely accepted practice for many reasons. The method was time consuming, minimally invasive, and not well tolerated by women. Physicians and laboratories had to undergo training and certification prior to acquisition and analysis of these specimens. Its use was limited to women already identified as high risk. Therefore, the 70% of women who display no risk factors — the very group risk assessment seeks to help — were excluded from this procedure. Ductal lavage was particularly challenging for the pathologist. Pathologists were required to differentiate between subtle degrees of mild, moderate, or marked atypia. In contrast, the latest NAF cytology is a simple binary evaluation for the presence or absence of atypia. Since the results from NAF are used only for risk stratification rather than pathologic diagnosis, the subtle differences in degree of atypia are much less important.

Manual nipple aspiration of breast fluid requires the successive application of heat and massage, followed by the extraction of NAF, typically with a suction cup and syringe. Like the previous methods, it entails a certain level of clinical expertise, often requiring physicians to perform the procedure. Though more tolerable than DL or random FNA, it is often time consuming and is not reproducible for practical applications.

Automated NAF collection. There is a non-invasive system which automates the process of NAF collection via bilateral, simultaneous collection using heat, massage, and suction. The cycle can be completed in five minutes. In addition, this procedure can be performed by a technician or medical assistant, making it more useful in clinical settings, such as an Ob-Gyn office.

Educating pathologists

Laboratories and pathologists need to be prepared to accurately evaluate and report on NAF specimens, preferably following a minimal amount of training. Currently, the optimal specimen collection, transport, and processing of NAF is liquid-based cytology (LBC), as is used with non-Gyn cytology specimens. This method is convenient to pathologists interpreting these aspirates since it yields optimal cellularity for evaluation. NAF is a protein-rich material and is usually hypocellular in asymptomatic patients. Today, LBC thin-layer slide preparation from NAF specimens rinsed in cytofixative solution (e.g., CytoLyt or PreservCyt) has gained wide acceptance.

Laboratory evaluation of NAF

The primary objective of NAF cytology is to identify if the specimen contains ductal epithelial cells, and, if so, whether the cells are atypical. Unlike cervical cytology specimens, it is not necessary to make a statement regarding an NAF specimen’s adequacy in the physician report. As these specimens are not obtained for diagnostic purposes and are normally hypocellular, it is not necessary to classify these samples as “unsatisfactory” or “non-diagnostic.” Many women do not produce fluid and are labeled “non-yielders.” Those who do produce NAF commonly have an acellular specimen. This is a normal result, indicating the woman’s risk is only slightly higher than someone who does not produce fluid. Neither of these situations should be considered “inadequate.”

Classification and reporting of NAF

The following categories (see table directly below) are proposed to make reporting and subsequent management recommendations as clear and concise as possible.

No or


Using these general categories (0 through IV) to indicate benign, atypical, or suspicious can quickly and unambiguously communicate results. Similar to the Pap report and the Bethesda System, this reporting format allows physicians to have administrative staff separate pathology reports requiring their immediate attention from others that do not. It is also common for laboratories to include educational notes or comments with these NAF reports.


Ultimately, the success of NAF as a risk-assessment tool depends not only on proper reporting procedures in the laboratory but also on adequate patient follow up and identification of the appropriate care path. A small number of asymptomatic women will have an abnormal NAF cytology in the range of 0.7% to 2.7% in most studies. These women should be referred to a breast surgeon or comprehensive breast center. There are established algorithms to guide the management of high-risk patients,8 to include a conservative management plan of increased surveillance, lifestyle counseling, and enhanced imaging modalities with the option of chemoprevention (Tamoxifen). As a result of NAF screening and other studies, an individual woman and her physician can make more informed decisions about minimizing her personalized risk of breast cancer.

Joel Bentz, MD, is clinical professor of Pathology at the University of Utah in Salt Lake City. He is a member of the Scientific Advisory Board at Neomatrix LLC, in Irvine, CA.


1. United States Cancer Statistics: 2004 Incidence and Mortality. Atlanta (GA): Department of Health and Human Services, Centers for Disease Control and Prevention, and National Cancer Institute. 2007.

2. Fabian CJ, Kimler BF. Breast cancer risk prediction: should nipple aspiration fluid cytology be incorporated into clinical practice? J Natl Cancer Inst. 2001;93(23):1762-1763.

3. West JG, Hollingsworth AB, Screening for breast cancer risk in the obstetric/gynecological setting: a breast surgeon’s perspective. Expert Rev Obstet Gynecol. 2008. 3(1):59-63.

4. Wrensch MR, et al., Breast cancer incidence in women with abnormal cytology in nipple aspirates of breast fluid. Am J Epidemiol. 1992. 135(2):130-141.

5. Wrensch MR, et al. Breast cancer risk in women with abnormal cytology in nipple aspirates of breast fluid. J Natl Cancer Inst. 2001. 93(23):1791-1798.

6. Buehring GC, et al., Presence of epithelial cells in nipple aspirate fluid is associated with subsequent breast cancer: a 25-year prospective study. Breast Cancer Res Treat. 2006. 98(1):63-70.

7. Baltzell KA, et al., Epithelial cells in nipple aspirate fluid and subsequent breast cancer risk: a historic prospective study. BMC Cancer, 2008. 8:75.

8. Hollingsworth AB, et al. Current comprehensive assessment and management of women at increased risk for breast cancer. Am J Surg. 2004. 187(3):349-362.

Category Interpretation Characteristics
Category 0 Negative for atypical or malignant cells.
Category I Benign – normal ductal epithelial cells identified Ductal cells. Foam cells. Apocrine cells
Category II Benign – hyperplastic ductal epithelial cells identified Cell distribution predominately in cohesive groups with >10-50 cells. Minimal nuclear changes. Fine chromatin
Category III Atypical Distinct nuclear enlargement, increasing N/C ratio, irregular nuclear borders and nuclear variation. Course chromatin. Prominent chromocenters.
Category IV Suspicious for malignancy Single cells and groups of cells with nuclear features suspicious for cancer.