BANU ARUN. The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. Key Words. Breast cancer Ductal lavage Chemoprevention Tamoxifen

The Oncologist Breast Cancer Ductal Lavage and Risk Assessment of Breast Cancer BANU ARUN The University of Texas MD Anderson Cancer Center, Houston, Texas, USA Key Words. Breast cancer Ductal lavage Chemoprevention Tamoxifen LEARNING OBJECTIVES After completing this course, the reader will be able to: 1. List the risk factors for breast cancer. 2. Explain the rationale using ductal lavage in the risk assessment of breast cancer. 3. Describe breast cancer risk reduction options. CME ABSTRACT Access and take the CME test online and receive 1 hour of AMA PRA category 1 credit at CME.TheOncologist.com INTRODUCTION Following recent advances in breast cancer chemoprevention, much emphasis has been placed on risk assessment to evaluate whether women at increased risk for developing breast cancer should proceed with breast cancer risk reduction strategies. The currently available risk-reduction approaches include screening, chemoprevention, and preventive surgeries. Breast cancer arises from the epithelial linings of the ductal system, and it is believed that hyperplasia and atypical hyperplasia represent early changes in the breast carcinogenesis Following recent advances in breast cancer chemoprevention, much emphasis has been placed on risk assessment to evaluate whether women at increased risk for developing breast cancer should proceed with breast cancer risk reduction strategies. The currently available risk-reduction approaches include screening, chemoprevention, and preventive surgeries. Breast cancer arises from the epithelial linings of the ductal system, and it is believed that hyperplasia and atypical hyperplasia represent early changes in the breast carcinogenesis process. The ductal lavage procedure offers a minimally invasive method to obtain breast epithelial cells from the ductal system for cytopathologic analysis to provide individualized risk assessment. This paper reviews breast cancer risk factors, with an emphasis on cytological atypia and the role of ductal lavage in breast cancer risk assessment. The Oncologist 2004;9:599-605 process. The ductal lavage procedure offers a minimally invasive method to obtain breast epithelial cells from the ductal system for cytopathologic analysis to provide individualized risk assessment. This paper reviews breast cancer risk factors, with an emphasis on cytological atypia and the role of ductal lavage in breast cancer risk assessment. RISK FACTORS FOR BREAST CANCER Several factors are associated with an increased risk for the development of breast cancer. Age is one of the most Correspondence: Banu Arun, M.D., The University of Texas MD Anderson Cancer Center, Breast Medical Oncology, 1515 Holcombe Boulevard 424, Houston, Texas 77030, USA. Telephone: 713-792-2817; Fax: 713-794-4385; e-mail: barun@mdanderson.org Received September 10, 2003; accepted for publication May 6, 2004. AlphaMed Press 1083-7159/2004/$12.00/0 The Oncologist 2004;9:599-605 www.theoncologist.com

600 Ductal Lavage in Breast Cancer Risk Assessment important factors; a woman s breast cancer risk increases throughout her life. The incidence of breast cancer is 27.9 cases per 100,000 women per year at 30-35 years of age; whereas the incidence increases to 412 cases per 100,000 in women 80 years of age. One of the other risk factors for breast cancer is family history. It is known that 5%-10% of breast cancers are due to inherited genetic mutations [1, 2]. Two of these genes, BRCA1 and BRCA2, have been recently cloned and are estimated, together, to be involved in 60%-70% of all hereditary breast cancers [3]. Women with mutations of these genes have an approximately 50%-80% lifetime risk of developing breast cancer [4, 5]. Germline mutations in the tumor suppressor gene p53 are also associated with increased breast cancer risk and account for 1% of breast cancers in young women [6]. However, it is necessary to point out that most women with a family history of breast cancer do not have genetically inherited disease; it is important to distinguish this group from the genetically inherited group, since the former carries a lower lifetime risk for developing breast cancer. In fact, a 30-year-old woman with a mother and sister diagnosed with unilateral breast cancer has up to an 18% lifetime risk of having breast cancer. This risk increases to 25% if her mother and sister had bilateral breast cancer [7]. Epidemiologic data also strongly suggest an association between ovarian hormones and the risk of breast cancer. This is supported by the observation that prolonged estrogen exposure, such as with early menarche [8], late menopause [9], nulliparity, and late age at first pregnancy [8], is associated with a higher risk for breast cancer. For example, menarche at age 16 is associated with a 10%-30% reduction in breast cancer risk. Pregnancy at a young age, especially before the age of 20, also reduces the risk for subsequent breast cancer. Studies reporting on the relationship between breast cancer risk and abortion are controversial [10, 11], as are studies on lactation and breast cancer risk [12]; recent data suggest that prolonged lactation can reduce breast cancer risk [13]. Many studies have reported on the exogenous use of hormones and breast cancer risk. One recent meta-analysis revealed that the use of hormonal replacement therapy (HRT) was associated with a slightly increased risk [14]. Another meta-analysis revealed essentially the same results; in that report, the increased risk was noted in patients who used estrogen for at least 5 years [15]. A metaanalysis of 51 case-controlled and cohort studies, which included more than 52,000 women with breast cancer, showed that breast cancer risk was 35% higher in women who used HRT for more than 5 years [16]. Finally, the recent Women s Health Initiative Study showed that HRT was associated with an increased risk of developing breast cancer (hazard ratio 1.26, 95% confidence interval = 1.00-1.59) [17]. Studies investigating the relationship between the use of oral contraceptives and breast cancer have revealed that the use of oral contraceptives slightly increases the risk for breast cancer, especially if they are used before first birth [18, 19]. Other studies have found no such correlation [20]. ATYPICAL BREAST EPITHELIUM AS A RISK FACTOR Several studies with long-term follow-up have shown that women with atypical hyperplasia have an elevated risk of developing breast cancer. Dupont et al. evaluated outcomes of women who had pathological diagnoses of atypical hyperplasia. With 10-17 years of follow-up, they reported that atypical ductal hyperplasia conferred a 4.3-5.3-fold higher relative risk of developing breast cancer [21, 22]. A significantly greater risk for developing breast cancer in women with the diagnosis of atypical hyperplasia diagnosed by biopsy was also shown by other investigators [23]. Wrensch et al. reported on the outcome of 2,343 women with atypical hyperplasia, diagnosed using nipple aspiration fluid (NAF). At a follow-up period of 12.7 years [24], the presence of cellular atypia in NAF was associated with a 4.9- fold higher relative risk of developing breast cancer. Furthermore, the addition of family history was also evaluated; women who had cellular atypia in NAF and a family history of breast carcinoma had an 18-fold higher relative risk of developing breast cancer than those with atypia but without a family history [24]. Fabian et al. evaluated 480 high-risk women for the presence of atypia using the periareolar fine-needle aspiration (FNA) method [25]. Atypia was detected in 21% of the highrisk women. After a median of 45 months follow-up, 4% of the women with an elevated 10-year Gail risk developed breast cancer, but the breast cancer rate was 15% in women who had an elevated 10-year Gail risk and the presence of atypical cytology. Cellular atypia was found to be predictive of an increased risk of developing breast carcinoma in each of those studies. The higher relative risks of developing breast carcinoma were found to be similar in these studies regardless of whether the atypical cells were identified by cytology of NAF, and in random FNAs, or whether the atypical hyperplasia was identified histologically on a breast biopsy. Other diseases in the breast epithelium associated with breast cancer risk include lobular carcinoma in situ (LCIS). Specifically, the relative risk of LCIS patients developing breast cancer has been estimated to be seven to nine times that of the normal population, with an absolute lifetime risk of 20% [26].

Arun 601 DUCTAL LAVAGE AS A TOOL TO DETECT ATYPIA As discussed previously, histologic and cytologic features of breast epithelial cells and the subsequent risk of breast cancer have been investigated for over 30 years. The finding of atypia can be demonstrated by core biopsy, FNA, or nipple aspiration. Although at this point, there is no direct evidence linking a cytologic diagnosis of atypia to histologically diagnosed atypical hyperplasia, the presence of atypical cells in the ductal fluid is associated with an increased risk of developing breast carcinoma [21-24, 27]. Even though it is more convenient to obtain and analyze NAF, the limitation of this approach is that it yields an inadequate number of breast epithelial cells for cytopathologic analysis. The ductal lavage procedure provides a noninvasive way to evaluate atypia in the breast epithelium. During this procedure, a local anesthetic cream is applied to the nipple for 20-30 minutes. A suction cup is then attached to the nipple to produce NAF, which enables the orifice of the natural milk duct opening to be located. A small double-lumen microcatheter is inserted into the duct, the duct is rinsed with normal saline through the first lumen, and the effluent fluid is collected through the second lumen. The collected fluid is then cytologically analyzed (described below). Recently, in a landmark study, Dooley et al. [28] compared breast epithelial cellular yield, cytology, and feasibility of ductal lavage versus nipple aspiration in 507 high-risk women. High risk was defined as a 5-year Gail risk >1.7%, a previous history of breast cancer, ductal carcinoma in situ (DCIS), LCIS, or a mutation in the BRCA1 or BRCA2 gene. All participants had to have a normal mammogram and normal breast examination within the previous 12 months. Participants could not be on tamoxifen (Nolvadex ; AstraZeneca; Wayne, PA) or raloxifen (Evista ; Eli Lilly and Company; Indianapolis, IN). All participants underwent nipple aspiration; if NAF was obtained, participants also underwent ductal lavage on the ducts that yielded fluid. Cytologic analyses were performed and the NAF specimens (n = 417 women) and ductal lavage specimens (n = 383 women) were compared. Cytology was interpreted as insufficient cellular material for diagnosis (ICMD), benign, atypical (mild or marked), or malignant. The median epithelial cell yield from NAF was 120 cells per breast. NAF samples from 73% of the study participants were described as ICMD (<10 epithelial cells or a poorly prepared slide). Epithelial cell yield was 13,500 per duct in specimens obtained via ductal lavage. Only 22% of the ductal lavage samples were interpreted as ICMD. The rate of identifying abnormal cells (atypical and malignant) in ductal lavage samples was higher than that for NAF samples. Abnormal cells were found in 24% of the high-risk women via ductal lavage, whereas detection of abnormal cells was only 10% in NAF from the same women. When ductal lavage and NAF samples from the same patient were compared, ductal lavage had an overall relative sensitivity that was 3.2 times greater than that of NAF in detecting abnormal intraductal cells (p = 0.001). That study also revealed that tolerance of the procedure was acceptable; on a 0-10 pain scale, the median intensity of pain was reported to be 2.4. It appears that ductal lavage is a more convenient way to evaluate the presence of atypia in breast epithelium of high-risk women. Even though the procedure is convenient, under some circumstances NAF cannot be produced and, therefore, the duct cannot be cannulated. Several factors are associated with the ability to obtain fluid, such as age, age at menarche, non-asian versus Asian ethnicity, and history of parity and/or lactation. Furthermore, our experience has been that individuals on tamoxifen or who have received recent chemotherapy also might not produce NAF. ROLE OF DUCTAL LAVAGE IN THE RISK ASSESSMENT OF BREAST CANCER The evaluation of risk factors for breast cancer, mainly by epidemiologic studies, has helped to develop models to identify women at increased risk. The most commonly used model is the Gail model, which used data from 4,496 matched pairs of cases in the Breast Cancer and Diagnosis and Demonstration Project, a mammography screening program [29]. This model evaluates a woman s 5-year and lifetime risks for developing breast cancer. The risk factors for this model included age at menarche, age at first live birth, number of previous breast biopsies, and number of first-degree relatives with breast cancer. This model has been modified to include ethnicity, estimate risk for invasive cancer only, and account for the presence of atypical hyperplasia in prior breast biopsies. Unfortunately, the model does not take into account the risk for individuals who have second-degree relatives diagnosed with breast cancer, or first- and second-degree relatives with ovarian cancer. It also underestimates the risk in individuals with a history of LCIS, DCIS, or invasive breast cancer [27] and may overestimate the risk in women with nonproliferative disease at biopsy, and in those who are age 50 and greater. The Gail model was validated by two subsequent studies [30, 31] and was shown to be accurate in predicting breast carcinoma incidence in the Nurses Health Study, but was found not to be accurate at the individual level [30, 32]. Furthermore, the Gail risk model also does not account for the use of HRT. As mentioned above, it was shown in the Women s Health Initiative Study that HRT use is associated with an increased risk of developing breast cancer [17].

602 Ductal Lavage in Breast Cancer Risk Assessment Nevertheless, this risk model is currently being used to determine eligibility for chemoprevention trials [33] and for the use of tamoxifen, a drug now approved for risk reduction in breast cancer [34]. The Breast Cancer Prevention Trial P1 (BCPT-P1) randomized more than 13,000 high-risk women to receive either tamoxifen or placebo for 5 years. Eligibility criteria, in addition to the Gail risk model, included age >60 years or a diagnosis of LCIS. At an almost 5-year follow-up, the results of the study showed that tamoxifen use reduced the risk of developing breast cancer by 49%. However, that study also demonstrated significant side effects associated with tamoxifen use. These included an increased risk of endometrial cancer the relative risk in the tamoxifen group was 2.5 and it was 4.01 in women age 50 or older. Deep vein thrombosis and pulmonary emboli were also seen more often in the tamoxifen group, with women aged 50 or older again at higher risk (relative risk 1.71 for deep vein thrombosis, 3.00 for pulmonary emboli) [34]. An apparently greater risk for stroke among women taking tamoxifen did not reach statistical significance. Marginally significant higher occurrences of cataract formation and the need for requiring cataract surgery were noted for the tamoxifen group. Other side effects included increased hot flushes and vaginal discharge. Given these side effects, a careful risk and benefit assessment needs to be performed before recommending tamoxifen to high-risk, but still healthy, individuals [35]. Identification of a subgroup of women who will derive benefit from tamoxifen despite its side effects is the main aspect of risk counseling, which depends on the optimal use of risk-assessment models and methods. Ductal lavage, by detecting atypical breast epithelial cells, can be used to improve risk stratification for women who already are at an increased risk for developing breast carcinoma. Indeed, Fabian et al. have shown that cytologic atypia detected on random periareolar FNA specimens is predictive of additional risk, independent of Gail risk assessment [27]. In that study, 15% of women with a Gail risk >4% and atypia developed breast cancer within the following 3 years [27]. These data suggest that the additional information of atypia adds to the known Gail risk and further increases the knowledge on the short-term risk of developing breast cancer. The importance of demonstrating atypia is further substantiated by the fact that women who have a history of previous atypical hyperplasia are the ones who derive the most benefit from tamoxifen. The BCPT-P1 showed that tamoxifen reduced the risk of breast cancer by 86% in women who had atypical hyperplasia [34]. As discussed previously, the higher relative risks of developing breast cancer were found to be similar between studies regardless of whether the atypical cells were identified by cytology using NAF, and in random FNAs, or whether the atypical hyperplasia was identified histologically on a breast biopsy. Ductal lavage seems to be a convenient method to assess the presence of atypia in high-risk women, which can help to refine each individual s risk level. However, there are several issues that need to be taken into consideration. First, it is assumed that atypical cells identified from ductal lavage fluid confer the same risk of breast cancer as atypia identified by FNA or core biopsy; currently, there are no long-term studies evaluating the outcomes of women who were found to have atypia in ductal lavage fluid. Second, since the negative predictive value of normal cytology with ductal lavage is not known, demonstrating normal cytology in ductal lavage fluid in a highrisk woman does not reduce her already greater risk for developing breast cancer. DUCTAL LAVAGE IN CLINICAL PRACTICE Currently, ductal lavage and cytologic evaluation are considered in women who are at increased risk for developing breast cancer. High-risk women who are considering risk-reduction options, such as screening, prevention with tamoxifen, or prophylactic surgeries, are candidates for this procedure, which could help them weigh the risks and benefits of using a risk-reduction intervention. Under some circumstances, the knowledge of cytologic atypia might not add further information when contemplating a management decision, for example, in women with an already known histologic diagnosis of atypical hyperplasia or LCIS. Women who have a history of DCIS or invasive breast cancer and who are taking tamoxifen might also not benefit further from the knowledge of cytologic atypia, since the management options would not change. The role of ductal lavage in women who have strong family histories of breast cancer or who are carriers of BRCA1 or BRCA2 mutations has also not yet been studied. In summary, ductal lavage is indicated if the additional information of cellular atypia in a high-risk woman will help her decide on an appropriate risk-reduction strategy. Furthermore, even though it has not yet been studied, the presence of cellular atypia might influence a woman s decision to take HRT. All these aspects of risk assessment are being discussed in high-risk assessment programs. CYTOLOGIC EVALUATION OF DUCTAL LAVAGE FLUID AND MANAGEMENT OF ABNORMAL CYTOLOGY The cytologic preparation and evaluation of ductal lavage specimens are similar to those used for breast FNA and nipple discharge samples [27, 36-39]. Usually, most ductal lavage samples contain an adequate number of cells for cytopathologic evaluation in a recent study, the number of epithelial cells recovered per duct was 13,500 [28].

Arun 603 Usually, ductal lavage specimens are divided into several cytologic categories benign cells, atypical cells, malignant cells, and ICMD. Benign cells include nonproliferative breast disease, apocrine metaplasia, fibroadenoma, and pregnancy-associated changes. Even though, there are currently no prospective follow-up data, it is recommended that, if a ductal lavage specimen in a high-risk woman shows benign cells, ductal lavage should be repeated after 1-3 years [40]. Further prospective studies to evaluate the most appropriate frequency of ductal lavage in these individuals are needed. As discussed previously, since the negative predictive value of benign cytology for breast cancer development is not known, a high-risk woman with benign cytology should still be considered for risk-reduction interventions. Atypical cells can range from mild atypia to marked atypia (Fig. 1). Mild atypia may represent histologic lesions of hyperplasia, atypical ductal hyperplasia, or low-grade DCIS, all of which are considered to be lesions of the progressive breast carcinogenesis process [41]. The presence of markedly atypical cells indicates the possibility of a neoplastic process, but these cells do not have all the features of malignant cells yet. Markedly atypical findings could be associated with atypical hyperplasia or low- to intermediate-grade DCIS. Even though no prospective studies have yet been done, in cases where atypical hyperplasia is found, it is recommended that the ductal lavage is repeated within 6-12 months to confirm the diagnosis. It is advisable to record the duct location on a nipple grid and with photography, which could help to recannulate that specific ductal system at subsequent visits. However, the reproducibility of ductal lavage findings over time has not yet been established [40]. In cases where marked atypia in ductal lavage samples of high-risk women is found, a repeat lavage should be performed to confirm the findings and to exclude malignancy. Additional breast imaging studies, including mammogram, ultrasound, and, if necessary, magnetic resonance imaging (MRI) should be performed. If necessary, ductoscopy or ductography to define the size and location of the ductal system and to exclude filling defects, should be performed. If suspicious lesions are found, a biopsy should be done. If no suspicious lesions are found, high-risk women can be followed closely, and risk-reduction options, including tamoxifen, should be discussed. At that point, since the predictive value of atypical findings on ductal lavage is not known and the atypical cells cannot be mapped to a specific duct system, blind duct excision and further surgical management is controversial. Malignant cells are defined as cells that demonstrate morphologic features of carcinoma. The finding of malignant cells on ductal lavage is rather rare (<1%) [28], but once found, a repeat ductal lavage should be performed to confirm the diagnosis. As in the case of markedly atypical findings, additional breast imaging studies should be performed, including mammography, ultrasound, ductography, ductoscopy, and MRI. Suspicious lesions should be biopsied. If the work-up is negative and imaging studies fail to demonstrate any lesions, a terminal duct excision could be considered. Since there are no data regarding the correlation between malignant lavage cytology findings and pathologic diagnosis on tissue biopsy, the outcome of further surgical excision has not yet been established. If no surgical intervention is performed, it is advisable to closely monitor the patient with regular breast examinations, imaging studies, and repeat ductal lavage. Risk-reduction strategies, including tamoxifen, should be considered. Finally, if a ductal lavage specimen from any fluid-yielding duct is interpreted as ICMD, lavage of that duct should be repeated. If the second ductal lavage reveals ICMD, that individual might be followed in the same way as with a finding of benign cells [40]. In summary, at this point, there are no evidence-based standardized guidelines on how to incorporate ductal lavage findings into clinical practice, but algorithms established by physicians experienced in breast cancer risk assessment have been developed (Table 1) and should guide physicians who are incorporating ductal lavage into their risk-assessment practice. Figure 1. Ductal lavage specimen showing atypical hyperplasia. DUCTAL LAVAGE AS A DIAGNOSTIC TOOL Since the specificity and sensitivity of ductal lavage for the early detection of breast cancer are not known, it should not be used for this purpose. At this point, it is also not recommended to use ductal lavage as an adjunct to other standard imaging methods, such as mammograms. One large study of 507 high-risk women with normal mammograms evaluated the finding of abnormal cytology in ductal lavage specimens [28]. Eleven of 92 women whose samples showed

604 Ductal Lavage in Breast Cancer Risk Assessment Table 1. Incorporating ductal lavage into clinical practice Cytologic evaluation Inadequate cellular material for diagnosis Benign cells Atypical cells Atypical cells marked atypia Malignant cells atypia underwent biopsies, and four were found to have DCIS. This is an intriguing finding, but it is not sufficient to consider ductal lavage as a diagnostic tool. Further large, randomized trials are needed to evaluate the role of ductal lavage in this setting. Until sufficient data become available, ductal lavage should remain as a risk-assessment tool. CONCLUSIONS AND FUTURE DIRECTIONS Ductal lavage is a promising and noninvasive method to obtain breast epithelial cells from high-risk women for cytologic analysis. The finding of atypia on ductal lavage can refine the risk level of a woman with an already increased Gail risk. This additional information may assist in the decision-making process for risk-reduction options, including the use of tamoxifen and preventive surgeries. The incorporation of ductal lavage into a risk-assessment program requires careful counseling. First, this procedure should not be regarded as a diagnostic tool; second, it does not replace standard screening methods, such as mammograms; and third, a negative result (no atypia) does not reduce the already increased risk of developing breast cancer in high-risk women. Ideally, the Recommendation Repeat ductal lavage; if normal, follow-up in 1-3 years Follow-up in 1-3 years Discuss tamoxifen or chemoprevention trials Repeat ductal lavage in 6-12 months Repeat ductal lavage to confirm diagnosis Perform additional imaging: mammogram, ultrasound, ductography, ductoscopy, MRI Biopsy if abnormal lesion found Discuss tamoxifen Repeat ductal lavage to confirm diagnosis Perform additional imaging: mammogram, ultrasound, ductography, ductoscopy, MRI Biopsy if abnormal lesion found Surgical management? Discuss tamoxifen Repeat ductal lavage in 3 months additional finding of cytologic atypia should help the highrisk woman to decide whether a specific risk-reduction intervention is appropriate for her and whether the benefits of that intervention outweigh the risks. If marked atypia or malignant cells are identified, further appropriate imaging studies and procedures should be performed. The positive and negative predictive values of ductal lavage for breast cancer, its role in the early detection of breast cancer, and its clinical utility for following the progression and regression of intraepithelial neoplasia with and without the use of antiestrogen therapy should be studied in prospective clinical trials. A prospective study is currently planned to evaluate the long-term outcome of women who have cytologic atypia found in ductal lavage fluid. Furthermore, the sampling of breast epithelial cells via ductal lavage can expand the capacity to perform tissue-acquisition-based translational research. There are several ongoing studies evaluating intermediate end point biomarkers in ductal lavage fluid, with the aim of identifying factors that are associated with the initiation and progression of breast cancer [42]. REFERENCES 1 Newman B, Austin MA, Lee M et al. Inheritance of human breast cancer: evidence for autosomal dominant transmission in high-risk families. Proc Natl Acad Sci USA 1988;85:3044-3048. 2 Yang X, Lippman ME. BRCA1 and BRCA2 in breast cancer. Breast Cancer Res Treat 1999;54:1-10. 3 Ford D, Easton DF, Stratton M et al. Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium. Am J Hum Genet 1998;62:676-689. 4 Easton DF, Bishop DT, Ford D et al. Genetic linkage analysis in familial breast and ovarian cancer: results from 214 families. The Breast Cancer Linkage Consortium. Am J Hum Genet 1993;52:678-701. 5 Struewing JP, Hartge P, Wacholder S et al. The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews. N Engl J Med 1997;336:1401-1408. 6 Malkin D, Li FP, Strong LC et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 1990;250:1233-1238. 7 Anderson DE, Badzioch MD. Bilaterality in familial breast cancer patients. Cancer 1985;56:2092-2098.

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