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  • National Reference Laboratory for Breast Health

Role of nipple aspirate fluid cytopathology in risk stratification for breast cancer.

Presence of epithelial cells in nipple aspirate fluid is associated with subsequent breast cancer: a 25-year prospective study.

Gertrude Case Buehring, Amy Letscher, Kathleen M. McGirr, Shruti Khandhar, Lisa H. Che, Christine T. Nguyen, and Adeline J. Hackett, Division of Infectious Diseases, School of Public Health, University of California, Berkeley, CA.

Screening for breast cancer risk in the obstetric/ gynecological setting: a breast surgeon’s perspective.
John G. West, Alan Hollingsworth, Breast Care Center,
Cordelia Knott Center for Wellness, Orange, CA.

Epithelial cells in nipple aspirate fluid and subsequent breast cancer risk: A historic prospective study.

Baltzell KA, Moghadassi M, Rice T, Sison JD and Wrensch M., University of California San Francisco, Department of Physiological Nursing, San Francisco, CA.

Breast Cancer Risk in Women with Abnormal Cytology in Nipple Aspirates of Breast Fluid.

Wrensch MR, Petrakis NL, Miike R, et al. Dept. of Epidemiology and Biostatistics, School of Medicine,
University of California San Fransisco, CA.

Nipple Aspirate Fluid Cytology and the Gail Model for Breast Cancer Risk Assessment in a Screening Population.

Tice JA, Miike R, Adduci K, et al. Division of General Internal Medicine, Department of Medicine, University of California, San Francisco, San Francisco, CA.

Breast Cancer risk in women with abnormal cytology in nipple aspirates of breast fluid.

Margaret R. Wrensch, Nicolas L. Petrakis, Eileen B. King Rei Miike, Lynn Mason, Karen L. Chew, Marlon M. Lee, Virginia L. Ernster, Joan F. Hilton, Robert Schweitzer, William H. Goodson Ill, and Thomas K. Hunt. Samuel Merritt Hospital, Oakland CA, Dept of Surgery, Dept of Epidemiology and Biostatistics, School of Medicine, University of California, San Francisco, CA.

Best Practices in Diagnostic Immunohistochemistry: Myoepithelial Markers in Breast Pathology.

Rajan Dewar, MD, PhD, Oluwole Fadare, MD, Hannah Gilmore, MD, Allen M. Gown, MD, Beth Israel Deaconess Medical Center of Pathology, Vanderbilt University Medical Center, Nashville, TN; Department of Pathology and Laboratory Medicine, Univ of British Columbia, Canada; PhenoPath Laboratories, Seattle, WA.

Nipple aspirate fluid: a promising non-invasive method to identify cellular markers of breast cancer risk.
Sauter ER, Ross E, Daly M, Kelin-Szanto A, Engstrom PF, Sorling A, Malick Jm Ehya H, Division of Population Science, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

Approaches to applying breast cancer risk prediction models in clinical practice. Cecelia Bellcross, PhD, MS, CGC, Dean Health system, Madison, WI

The presence of epithelial cells in NAF is associated with increased breast cancer risk in a 25-year prospective study.

A well-recognized care path exists for management and treatment of high risk women identified by NAF cytology.

Presence of epithelial cells in nipple aspirate fluid is associated with subsequent breast cancer: a 25-year prospective study. Gertrude Case Buehring, Amy Letscher, Kathleen M. McGirr, Shruti Khandhar, Lisa H. Che, Christine T. Nguyen, and Adeline J. Hackett, Division of Infectious Diseases, School of Public Health, University of California, Berkeley, CA.

OBJECTIVE:
Fluid and epithelial cells obtained from the breast of non-pregnant, non-lactating women by nipple aspiration, can be used for early diagnosis of breast neoplasms. However, since nipple aspirate fluid (NAF) with cells is obtainable from less than half of women sampled, the question arises: Is this method capable of targeting the women most likely to develop breast cancer?

BACKGROUND & METHODOLOGY:
We approached this question with a 25-year prospective study to determine if subjects yielding NAF with or without epithelial cells were more likely to develop breast cancer during the follow-up period than subjects from whom no NAF or epithelial cells were obtained. The follow-up cohort of 972 was representative of the eligible cohort of 1605 for factors related to breast cancer risk and nipple aspiration outcome, and representative of the general population for breast cancer risk.

CONCLUSIONS:
• “Our data suggest the potential usefulness of NAF epithelial
cells as a biomarker for breast cancer risk, particularly in
women <55 years of age.

• “A possible clinical application of this is routine nipple aspiration and NAF cytology during general physicals and gynecologic examinations of premenopausal women.”

• “Breast cancer is curable when detected at an early stage. The fact that it remains the second most frequent cause of cancer death in women indicates that detection is not occurring early enough. NAF cytology may be an important adjunct to the battery of early detection methods currently available.”

Screening for breast cancer risk in the obstetric/ gynecological setting: a breast surgeon’s perspective. John G West, Alan Hollingsworth, Breast Care Center, Cordelia Knott Center for Wellness, Orange, CA.

OBJECTIVE:
Despite recent advances in screening and detection of breast cancer in post-menopausal women there are significant challenges in risk assessment in premenopausal women. We review the use of nipple aspirate fluid (NAF) cytology in the obstetric/ gynecological practice setting for risk stratifying of women with respect to future breast cancer.

RESULTS:
A finding of atypia in NAF cytology confers a four- to five-fold increase risk of breast cancer. The major advantage of screening with NAF is that atypia can be detected before cancer is detected by imaging technologies.

Women who are found to have atypia would be candidates for more aggressive follow up and would be candidates for tamoxifen, which has been demonstrated to provide high-risk women with atypia an 86% reduction in the risk of future breast cancer.

CONCLUSIONS:
• “It has been shown that 70% of breast cancers have no identifiable risk factors.”

• “Premenopausal women generally have dense breasts that render screening mammography less effective.”

• “The obstetrician-gynecologist is uniquely positioned to perform a breast health screening test for asymptomatic women during their well women visit.”

• “A finding of atypia in NAF is well recognized as an indicator of increased risk for breast cancer, with a four- to five-fold increase in relative risk.”

• “A well-recognized care path exists within the comprehensive breast center for management and treatment of high risk women, such as identified by NAF with atypia.”

Women with epithelial cells in NAF have a 1.9-fold increase in breast cancer over an 18-year follow up.

Epithelial cells in nipple aspirate fluid and subsequent breast cancer risk: A historic prospective study: Baltzell KA, Moghadassi M, Rice T, Sison JD and Wrensch M. University of California San Francisco, Department of Physiological Nursing , San Francisco, CA.

OBJECTIVE:
Does the presence of epithelial cells in NAF increase the likelihood of future breast cancer?

BACKGROUND & METHODOLOGY:
This study analyzed NAF results from a group of women seen in a breast clinic from 1970 – 1991 (N = 2480). The analysis presented here is an aggregate of two sub-groups: women with questionnaire data (n = 712) and those with NAF visits beginning in 1988 and ending in 2006.

RESULTS:
Overall, 10% ( 93 ) of the 946 women developed breast cancer during the follow-up period. Age-adjusted ORs and 95% confidence intervals ( C.I.) compared to non-yielders were 1.4 (0.3 to 6.4), 1.7 (0.9 to 3.5), and 2.0 (1.1 to 3.6) for women with fluid without epithelial cells, normal epithelial cells and hyperplasia/atypia, respectively. Comparing the presence or absence of epithelial cells in NAF, women with epithelial cells present in NAF were more likely to develop breast cancer than non-yielders or women with fluid without epithelial cells (RR = 1.9, 1.2 to 3.1).

CONCLUSIONS:
These results support previous findings that:

• women with abnormal epithelial cells in NAF have an increased risk of breast cancer when compared to non yielders or women with normal epithelial cells in NAF and;

• women with epithelial cells present in NAF have an increased risk of breast cancer when compared to non-yielders or women who had NAF without epithelial cells present.

Atypia/hyperplasia in NAF cytology predicts breast cancer over 2 years with a sensitivity of 84% and specificity of 73%.

Breast cancer risk in women with abnormal cytology in nipple aspirates of breast fluid.
Margaret R. Wrensch, Nicholas L. Petrakis, Eileen B, King, Rei Miike, Lynn Mason, Karen L. Chew, Marion M. Lee, Virginia L. Ernster, Joan F. Hilton, Robert Schweitzer, William H. Goodson III, and Thomas K. Hunt. Samuel Merritt Hospital, Oakland, CA, Dept of Surgery, Dept of Epidemiology and Biostatistics, School of Medicine, University of California, San Fransisco, CA.

OBJECTIVE:
A previous study demonstrated that women with abnormal cytology in breast fluid obtained by nipple aspiration had an increased relative risk (RR) of breast cancer compared with women from whom was not obtained and with women whose fluid had normal cytology. The study extends the follow up in the original study group of women (n = 4046) to 21-years and presents the 8-year follow up for a second group of women (n = 3627).

BACKGROUND AND METHODOLOGY:
Nipple aspirate fluid (NAF) was collected, women were classified according to the most severe epithelial cytology observed in fluid specimens, and determined breast cancer incidence. RRs were estimated for breast cancer using Cox regressions, adjusting for age and year of study entry.


Adding NAF cytology results to the Gail breast cancer risk model significantly improves the model fit (P <0.0001).

Nipple aspirate fluid cytology and the Gail Model for breast cancer risk assessment in a screening population Tice JA, Miike R, Adduci K, et al. Division of General Internal Medicine, Department of Medicine, University of California, San Francisco, CA.

OBJECTIVE:
The Gail model is a validated breast cancer risk assessment tool that is primarily based on non-modifiable breast cancer risk factors. Conversely, nipple aspirate fluid (NAF) is strongly correlated with breast cancer risk and responds to risk-modifying interventions. To determine whether nipple aspirate fluid (NAF) cytology combined with the Gail model provides breast cancer risk assessment that is superior to either method alone.

BACKGROUND & METHODOLOGY:
Recent guidelines suggest that chemoprevention with tamoxifen may be appropriate for women who have a 5-year risk of breast cancer greater than 1.66% calculated using the Gail model. A prospective study of 6,904 women with NAF cytology and 14.6 year follow up was performed.

RESULTS:
Adding NAF cytopathology to Gail model significantly improved the model fit (p <0.0001).

CONCLUSIONS:
• Clinically, NAF cytology may be useful for women at highest absolute risk by the Gail model because modest differences in relative risk are amplified.

• In this cohort, the incidence of breast cancer by NAF cytology ranged from 5.3 to 10.3 per 1,000 women years (non yielder to hyperplasia/ atypia) for women in the highest tertile of Gail risk.


Women aged 18-54 with atypical cytology on NAF have a 16-fold increase incidence of breast cancer over a 14-year interval.

Breast Cancer Risk in Women with Abnormal Cytology in Nipple Aspirates of Breast Fluid.
Wrensch MR, Petrakis NL, Miike R, et al. Dept. of Epidemiology and Biostatistics, School of Medicine, University of California San Francisco, San Francisco, CA.

OBJECTIVE:
To determine the relationship of the incidence of breast cancer among 2701 women age 18 to >65 and the cytology of nipple aspirate fluid (NAF) in a long term, prospective study.

RESULTS:
The incidence of breast cancer over a 14-year internal in 2343 women age 18 – 54, based on initial NAF cytology, is shown below:

The incidence of breast cancer over a 14-year internal in 2701 women age 18 to >65 with a first degree relative with breast cancer and NAF cytology findings is shown below:

CONCLUSIONS:
• NAF cytology provides a strong predictor of future breast cancer and failure to obtain NAF is a physiological condition denoting low risk of future cancer.

Best practices for distinguishing low risk “usual” ductal hyperplasia from higher risk “atypical” ductal hyperplasia in breast samples involves multiplex IHC staining for nuclear p63 and cytokeratins 5, 7, 14, and 18 and can reduce unnecessary surgeries from incorrect ADH diagnoses.

Best Practices in Diagnostic Immunohistochemistry: Myoepithelial Markers in Breast Pathology
Rajan Dewar, MD, PhD, Oluwole Fadare, MD, Hannah Gilmore, MD, Allen M. Gown, MD, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA; Department of Pathology, Vanderbilt University Medical Center, Nashville, TN; Department of Pathology and Laboratory Medicine, Univ of British Columbia, Canada; PhenoPath Laboratories, Seattle, WA.

CONTEXT:
Numerous immunohistochemical stains have been shown to exhibit exclusive or preferential positivity in breast Myoepithelial cells relative to their luminal/ epithelial counterparts. These myoepithelial markets provide invaluable assistance in accurately classifying breast proliferations. Although numerous myoepithelial markers are available, they differ in their sensitivity, specificity and ease of interpretation, which may be attributed, to a large extent, to the variable immunoreactivity of these markers in stromal cells.

OBJECTIVE:
To review commonly used myoepithelial markers in breast pathology.

DATA SOURCES:
The information outlined in this review article is based on our experiences with routine cases and a review of English-language articles published between 1987 and 2008.

CONCLUSIONS:
To demonstrate the presence or absence of myoepithelial cells, a panel-based approach of 2 more markers is recommended.

Markers that most effectively combine sensitivity, specificity, and ease of interpretation include p63 and basal cytokeratins, particularly CK5 and CK14. Luminal/epithelial cells can be distinguished with CK7 and CK18. Myoepithelial markers highlighting nuclei (p63) have a distinctive advantage in certain settings.

Atypical ductal hyperplasia: interobserver and intraobserver variability Rohit K Jain*, Rutika Mehta*, Rosen Dimitrov, Lisbeth G Larsson, Paul M Musto, Kurt B Hodges, Thomas M Ulbright, Eyas M Hattab, Narasimhan Agaram, Muhammad T Idrees and Sunil Badve, Department of Pathology and Laboratory Science, I U School of Medicine, Indianapolis, IN, USA

OBJECTIVE:
Interobserver reproducibility in the diagnosis of benign intraductal proliferative lesions has been poor. The aims of the study were to investigate the inter- and intraobserver variability and the impact of the addition of an immunostain for high- and low-molecular weight keratins on the variability.

METHODS:
Nine pathologists reviewed 81 cases of breast proliferative lesions in three stages and assigned each of the lesions to one of the following three diagnoses: usual ductal hyperplasia, atypical ductal hyperplasia and ductal carcinoma in situ. Hematoxylin and eosin slides and corresponding slides stained with cytokeratins CK 5, 14. 7, 18 and p63 by Immunohistochemistry (IHC) were evaluated. Concordance was evaluated at each stage of the study.

RESULTS:
The interobserver agreement among the nine pathologists for diagnosing the 81 proliferative breast lesions was fair (κ- value=0.34). The intraobserver κ-value ranged from 0.56 to 0.88 (moderate to strong). Complete agreement among nine pathologists was achieved in only nine (11%) cases, at least eight agreed in 20 (25%) cases and seven or more agreed in 38 (47%) cases. Following IHC stain, a significant improvement in the interobserver concordance (overall κ-value=0.50) was observed (P=0.015). There was a significant reduction in the total number of atypical ductal hyperplasia diagnosis made by nine pathologists after the use of the multiples IHC.

Nipple aspiration fluid cytology in young women, for whom mammography is often non-revealing due to breast density, was effective in 96%.
Nipple aspirate fluid: a promising non-invasive method to identify cellular markers of breast cancer risk. Sauter ER, Rose E, Daly M, Kelin-Szanto A, Engstrom PF, Sorling A, Malick Jm Ehya H, Division of Population Science, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

OBJECTIVE:
To evaluate the feasibility of nipple aspiration and to identify intermediate markers of breast cancer risk, nipple aspirate fluid (NAF) was collected from 177 subjects using a modified breast pump.

BACKGROUND AND METHODOLOGY:
Nipple fluid was aspirated using a modified breast pump.

Cytological criteria for nipple aspirate specimens
Scant mammary epithelial cells: This includes acellular specimens, those with only foam cells or with fewer than ten mammary epithelial cells.
Benign mammary epithelial cells: This consists of specimens containing more than ten mammary epithelial cells without cytological atypia. This category encompasses normal mammary cells, apocrine metaplasia, and duct hyperplasia without atypia. We subdivide this category into: (a) Normal/ non-papillary – single cells or small, loosely cohesive aggregates of cells. (b) Hyperplastic – large three-dimensional clusters of cells (indicative of duct hyperplasia or papillomatosis).
Atypical mammary epithelial cells: This category of specimens with ten or more mammary epithelial cells that exhibit atypical features. Atypia is defined as nuclear to cytoplasmic ratio or a chromatin distribution abnormality short of obvious malignant criteria. It is noted whether the atypical cells are single or in papillary clusters.
Malignant cells present: This category consists of specimens that contain cells with unequivocal criteria of malignancy.

RESULTS:
NAF was obtained in 167 out of 177 (94%) subjects (age 30-65) overall and in 99% of the 144 most recent subjects. Sufficient NAF was obtained to evaluate cytology in 160 out of 167 (96%). NAF cytology correlated with increased breast cancer risk (P = 0.002)

CONCLUSIONS:
Our data indicates that NAF can be obtained in essentially all eligible subjects and that breast epithelial cells are evaluable in >95% of NAF samples for cytology.


In comparison to others, including the Gail model, the IBIS model is the most consistently accurate for prediction of breast cancer.

Approaches to applying breast cancer risk prediction models in clinical practice. Cecelia Bellcross, PhD, MS, CGC, Dean Health system, Madison, WI

Overview:
Breast cancer risk stratification is becoming increasingly important as additional screening and prevention options are now available for women at different levels of risk. Use of screening breast MRI, tamoxifen chemoprevention, and genetic counseling and testing for hereditary breast cancer are appropriate for some women at increased susceptibility.

BREAST CANCER RISK PREDICTION MODELS:

· Three models dominate the medical literature,
the Gail model, the Claus model, and the
International Breast Intervention Study (IBIS)
model, also known as the Tyler-Cuzick model.
· The factors of the IBIS model are shown
below.

· The IBIS model incorporates genetic,
nongenetic, and cytophathology findings
· Adjusts for number/ age of unaffected first –
and second-degree relatives
· Provides age-adjusted risks compared to
population in graphical form
· Model assumes nongenetic factors are
multiplicative on risk
· Important to communicate to patient that
risk can change, by for example, tamoxifen
chemoprevention

Case Study: A 36 y female with family history of breast and ovarian
cancer. What is her future risk of breast cancer?

· Clinical breast examination and mammogram were normal

- Mammogram conducted per clinical
trial protocol; not standard of care at
age 36

· The woman’s family and genetic data are entered into the IBIS Breast Cancer personalized Risk Assessment Tool before obtaining nipple aspirate fluid

· Based on this information the patient has a lifetime risk of breast cancer of 14.79%

This places her at NORMAL risk of breast cancer
· Nipple aspirate fluid collection and cytology is performed

- Usual ductal hyperplasia is noted in the
right breast NAF specimen

· This new data is added to the IBIS Assessment Tool and the algorithm is re-run

· By including this new information the patient’s lifetime risk of breast cancer increases to 27.4%

· This places her HIGH risk of breast cancer and changes her breast health management