Breast-tissue sampling for risk assessment and prevention

http://www.100md.com 《肿瘤与内分泌》

     1 Departments of Internal Medicine

    2 Radiation Oncology

    3 Preventive Medicine and Public Health, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KA 66160, USA

    4 Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Abstract

    Breast tissue and duct fluid provide a rich source of biomarkers to both aid in the assessment of short-term risk of developing breast cancer and predict and assess responses to prevention interventions. There are three methods currently being utilized to sample breast tissue in asymptomatic women for risk assessment: nipple-aspirate fluid (NAF), random periareolar fine-needle aspiration (RPFNA) and ductal lavage. Prospective single-institution trials have shown that the presence of atypical cells in NAF fluid or RPFNA specimens is associated with an increased risk of breast cancer. Furthermore, RPFNA-detected atypia has been observed to further stratify risk based on the commonly used Gail risk-assessment model. A prospective trial evaluating risk prediction on the basis of atypical cells in ductal-lavage fluid is ongoing. The ability of other established non-genetic biomarkers (mammographic breast density; serum levels of bioavailable estradiol, testosterone, insulin-like growth factor-1 and its insulin like growth factor binding protein-3) to stratify risk based on the Gail model is as yet incompletely defined. Modulation of breast intra-epithelial neoplasia (i.e. hyperplasia with or without atypia) with or without associated breast-tissue molecular markers, such as proliferation, is currently being used to evaluate response in Phase II chemoprevention trials. RPFNA has been the method most frequently used for Phase II studies of 6–12 months duration. However, ductal lavage, RPFNA and random and directed core needle biopsies are all being utilized in ongoing multi-institutional Phase II studies. The strengths and weaknesses of each method are reviewed.

    Use of breast-tissue biomarkers in risk assessment and prevention

    Need for risk biomarkers

    Over 211 000 women are estimated to develop invasive breast cancer in the USA in 2005 (Jemal et al. 2005). These women will generally undergo some combination of surgery, radiation, antihormone and/or chemotherapy which for many will result in appreciable long-term morbidity (Kuehn et al. 2000, Stanton et al. 2001, Ganz et al. 2002). Despite advances in early detection and treatment, 40 000 women previously diagnosed with invasive breast cancer were predicted to die in 2004 (Jemal et al. 2005). Prevention would be a preferable alternative to treatment of established disease, if those women most likely to benefit from the prevention intervention could be readily identified.

    Tamoxifen has been identified as a cost-effective intervention for primary risk reduction for asymptomatic women of 35–70 years without prior invasive cancer if they have previously had a biopsy exhibiting atypical ductal hyperplasia (ADH), ductal or lobular carcinoma in situ (DCIS, LCIS), or currently have an estimated 5-year Gail model risk of >1.67% (Fisher et al. 1998, Cuzick et al. 2002, Hershman et al. 2002). Tamoxifen has been recommended by the US Preventive Services Task Force (Kinsinger et al. 2002), the American Society of Clinical Oncology (Chlebowski et al. 1999) and the Canadian Task Force on Preventive Health (Levine et al. 2001) under these circumstances. Yet, despite a relative reduction in cancer incidence of 32–49%, only a minority of high-risk women without a prior diagnosis of DCIS or invasive cancer agree to take it following a recommendation by their health-care provider (Port et al. 2001, Vogel et al. 2002, Bober et al. 2004, Tchou et al. 2004). A woman’s reluctance to take 5 years of tamoxifen as preventive therapy appears to be based on the fear of side effects coupled with uncertainty of the benefits, particularly if the 5-year Gail model risk of >1.67% is the primary tool used to determine suitability for prevention therapy (Port et al. 2001). The Gail risk model is based on five variables captured as part of the Breast Cancer Detection Project (BCDP): current age, age at menarche, first live birth, number of breast biopsies and number of affected first-degree relatives, as well as a correction factor for atypical hyperplasia if it has been observed in a diagnostic biopsy (Gail et al. 1989; Table 1). The Gail model is simple to use and has been validated for populations undergoing regular screening and an updated version with 5-, 10-, 20- and 30-year risk calculated by race is available on the National Cancer Institute (NCI) website (http://bcra.nci.nih.gov/brc/). Unfortunately, it has only modest discriminatory value for the individual woman, and thus may not be helpful in decision-making with regards to whether to take tamoxifen for prevention (Rockhill et al. 2001). Indeed, Freedman et al. (2003) have suggested that benefit from tamoxifen prevention therapy is likely to accrue to less than 25% of Caucasian women of ages 35–70 identified as high risk on the basis of a 5-year Gail risk of >1.67%. A large number of risk factors are not considered by the Gail model and this may provide partial explanation for its modest individual discriminatory value.

    In an attempt to improve the individual discriminatory value of the Gail model, other models such as the Tyrer–Cuzick model have recently been developed which incorporate additional common risk factors including height, weight, presence of affected relatives with ovarian cancer, second-degree relatives with breast cancer, age of affected relatives and whether the relative had unilateral or bilateral breast cancer (Tyrer et al. 2004). The Tyrer–Cuzick model was developed from data obtained from the International Breast Cancer Intervention Study I (IBIS-1) prevention study and may be more relevant to women seeking risk assessment in anticipation of a prevention intervention than the Gail model, which is based on a screening cohort. The discriminatory value of the Tyrer–Cuzick model may be superior to the Gail model for women with a single affected relative (Tyrer et al. 2004).

    Although risk models based on historical personal and family history are useful, increasing attention is being given to risk biomarkers that may improve short-term predictive accuracy for the individual woman. Biomarkers may be particularly useful in helping women who are identified as being at increased risk from epidemiologic models make decisions about medical or surgical prevention options. To the extent they can be modulated, biomarkers may also be used to monitor response to prevention interventions and/or predict response to a particular type of intervention. Of particular interest are breast-tissue changes which are highly associated with later cancer development. These changes are currently being utilized to select cohorts and assess response in Phase I and II trials of potential new prevention agents (Boone & Kelloff 1993).

    We will review the concept of risk biomarkers with emphasis on those derived from breast tissue and the methods to acquire specimens for the purpose of both risk assessment and prevention.

    Characteristics of ideal risk biomarkers

    Characteristics of ideal risk biomarkers include: biologic plausibility, differential expression in low- versus high-risk populations, presence in a reasonable proportion of the high-risk population, association with cancer in prospective studies, expression minimally influenced by normal physiologic processes, the ability to obtain the marker by minimally invasive techniques and an assessment method that provides reproducible results (Kelloff et al. 1996, Boone et al. 1997).

    Established risk biomarkers

    Deleterious germline mutations in highly penetrant genes such as BRCA1/BRCA2 are strong predictors of breast cancer development but occur in less than 5–10% of women with breast cancer and in only 1% of the general population (Peto et al. 1999, Nathanson et al. 2001, Rebbeck 2002). Common single nucleotide polymorphisms of genes whose protein products are involved in carcinogen and hormone metabolism and/or DNA repair are associated with relative risks of 1.4–2.0; but two and three gene polymorphism combinations may be associated with much higher relative risks (Coughlin & Piper 1999, Feigelson et al. 2001, Pharoah et al. 2002, Comings et al. 2003, Aston et al. 2005). The established risk biomarkers serum-bioavailable estradiol and testosterone in postmenopausal women (Missmer et al. 2004, Tworoger et al. 2005), serum insulin-like growth factor-I (IGF-I) and its binding protein-3 (IGFBP-3) in premenopausal women (Hankinson et al. 1998), mammographic breast density (Boyd et al. 1998) and breast intra-epithelial neoplasia (Page & Dupont 1990; Table 2) have much broader applicability than germline mutations in tumor-suppressor genes. Further, since they are subject to modulation, these risk biomarkers might be used to monitor change in breast cancer susceptibility from a prevention intervention. Mammographic breast density and intra-epithelial neoplasia are the most attractive risk biomarkers of the potentially modulatable markers as they are useful in both pre- and postmenopausal women. However, Tice et al. (2004b) has reported recently that mammographic density adds only modestly to the Gail model in improving discriminatory accuracy.

    Breast intra-epithelial neoplasia: the risk biomarker with the closest biologic association with cancer

    The established risk biomarker with the closest direct biologic association with invasive breast cancer, and least likely to be affected by normal physiologic processes, is intra-epithelial neoplasia. This includes proliferative breast disease without atypia, atypical ductal and lobular hyperplasia and in situ cancer (Wellings et al. 1975, Boone et al. 1997, Fitzgibbons et al. 1998). Within the spectrum of intra-epithelial neoplasia, an increase in morphologic abnormality is associated with a progressive increase in relative risk and decrease in latency (Page et al. 1985, Page & Dupont 1990, Page et al. 1991, Tavassoli & Norris 1990, Ottesen et al. 1993, Modan et al. 1997).

    Proliferative breast disease without atypia (moderate to florid hyperplasia, sclerosing adenosis, papillomas, etc.) is found in approximately 25–30% of diagnostic biopsies and is associated with a 1.4–2.0-fold increase in the relative risk for breast cancer (Dupont & Page 1985, Carter et al. 1988, London et al. 1992, Fitzgibbons et al. 1998, Wang et al. 2004). Higher relative risks associated with proliferative disease without atypia (e.g. 2.0 versus 1.4) may be associated with older age (>50 years) or a positive family history (London et al. 1992, Wang et al. 2004).

    Atypical hyperplasia in diagnostic biopsies, whether ductal or lobular, is associated with an approximate 5-fold increase in relative risk without regard to other risk factors (Dupont & Page 1985, Tavassoli & Norris 1990, Page et al. 1991, Dupont et al. 1993). Women with atypia without a positive family history have an approximately 4-fold increase whereas women with a positive family history have an approximately 10-fold increase in their relative risk of breast cancer (Dupont & Page 1985, Dupont et al. 1993).

    Atypical ductal and lobular hyperplasia are observed in 3–10% of unselected diagnostic surgical and stereotactic core biopsies (Hutchinson et al. 1980, Dupont & Page 1985, Lieberman et al. 1995, Brown et al. 1998). Those women who ultimately develop cancer have a higher proportion of prior benign biopsies exhibiting atypical hyperplasia than those who do not (London et al. 1992, McDivitt et al. 1992, Dupont et al. 1993).

    Several investigators, including Wellings & Jensen (1973) and more recently Allred et al. (1998, 2001) and Reis-Filho & Lakhani (2003), have suggested that atypical hyperplasia may arise more commonly from an intermediate lesion called an unfolded lobule (A for ductal, B for lobular) than hyperplasia of the usual type (HUT; Fig. 1). In fact, both atypical hyperplasia and HUT may both arise from unfolded lobules (Wellings & Jensen 1973, Allred et al. 1998, 2001). These unfolded lobules are characterized by increased cellularity and proliferation with distension of the terminal lobule duct unit.

    ADH often shares molecular and genetic changes with DCIS as assessed by immunostaining (Boecker et al. 2002) or mRNA gene profiles (Ma et al. 2003). The frequency of loss of heterozygosity at at least one locus occurs in approximately 50% of ADH lesions and somewhat less frequently for HUT lesions (O’Connell et al. 1998). The most prevalent chromosomal losses are at 16q and 17p for both HUT and atypical hyperplasia, similar to what is observed for DCIS (Lakhani et al. 1995, Amari et al. 1999, Gong et al. 2001). Comparative genomic hybridization also indicates patterns of similar chromosomal gains and losses for non-invasive and invasive lobular cancer. Of particular importance is the loss of 16q, which contains E-cadherin, a tumor-suppressor gene involved in cell adhesion and cell-cycle regulation. E-cadherin is expressed in normal cells, but is lost in LCIS and invasive lobular cancer (reviewed in Reis-Filho & Lakhani 2003).

    Although ADH is reported in 5% or less of diagnostic biopsies, it has been reported in 9% of autopsy specimens from average-risk women (Nielsen et al. 1987) and 39% of prophylactic mastectomy specimens from high-risk women (Hoogerbrugge et al. 2003). In the series by Hoogerbrugge et al. (2003), 57% of women with a family history consistent with that of a mutation in BRCA1 and/or BRCA2 had atypical ductal or lobular lesions and/or in situ cancer and these lesions were often multifocal or multicentric. Most women at increased risk for breast cancer by virtue of family history or other factors have never had a diagnostic biopsy. The question then becomes, how might we best detect intra-epithelial neoplasia, particularly atypical hyperplasia, via non-diagnostic tissue sampling Further, do morphologic changes suggestive of intra-epithelial neoplasia detected as part of non-diagnostic tissue sampling carry similar predictive weight as those found in diagnostic biopsies performed following an abnormal exam or breast-imaging procedure

    Methods of detecting breast intra-epithelial neoplasia for risk assessment

    Core biopsy, nipple aspiration for collection of nipple-aspirate fluid (NAF), ductal lavage and random periareolar fine-needle aspiration (RPFNA) are all being utilized to collect breast epithelial tissue for risk assessment in asymptomatic women without suspicious lesions on physical exam or mammography. Morphology has been prospectively correlated with later breast cancer development for only two of these methods: NAF and RPFNA.

    NAF

    NAF is generally collected following 5–10 min of manual massage, with or without the use of a Sartorius-type breast pump. Warming of the breast with a heating pad and scrubbing the nipple to dislodge keratin plugs are also often advocated (Sartorius et al. 1977). Ability to produce NAF is influenced by cohort selection and the number of attempts (Sauter et al. 1996, Klein et al. 2001, Wrensch et al. 2001, King et al. 2004). Approximately 80% of women are reported to produce NAF after five or more attempts (Sauter et al. 1996, King et al. 2004). NAF production has been reported in 39–66% of women without regard to risk (Wrensch et al. 1992, 2001), and 50–95% of high-risk women (Sauter et al. 1997, Dooley et al. 2001, Antill et al. 2004, Kurian et al. 2004, Sharma et al. 2004). Young age (30–50 years), prior lactation and non-Asian ethnicity are positively associated with the ability to produce NAF (Wrensch et al. 1990). Use of oxytocin nasal spray (50 units) has been reported to increase the volume of NAF which is generally in the range of a few microliters (Zhang et al. 2003). Women with a contralateral breast cancer or spontaneous nipple discharge have higher rates of NAF production (Khan et al. 2002, Cazzaniga et al. 2003). Series reporting a very high proportion of NAF producers (83–95%) often obtain participants from surgical practices where one would expect a larger percentage of women to have initially presented with a nipple discharge or contra-lateral breast cancer than series in which participants were drawn primarily from screening or high-risk clinics (Sauter et al. 1997, Dooley et al. 2001, Sharma et al. 2004). The ability to obtain at least the 10 epithelial cells required for a cytomorphologic interpretation has been reported in 53–83% of cases (Dooley et al. 2001, Wrensch et al. 2001). The median number of epithelial cells in NAF specimens in the series reported by Dooley et al. (2001) was modest at 120. Multiple sampling attempts improve not only the ability to harvest NAF but also the frequency with which atypia is discovered. In a recent series by King et al. (2004) where NAF attempts were performed every 6 months for 2 years, atypical cells were discovered in initial NAF in 6.7% of women, but in a total of 18.2% by the fifth visit. These investigators recommend three or four NAF attempts rather than a single attempt (King et al. 2004). Use of a MilliporeTM filter rather than a cytospin is reported to maximize cell collection (King et al. 1983). NAF production as well as epithelial cell morphology may be useful in risk assessment. Wrensch et al. (1992) originally reported a stepwise increase in the relative risk of breast cancer, from women who did not produce NAF, to NAF producers without proliferative epithelium, with proliferative epithelium and with proliferative epithelium with atypia (Fig. 2). The relative risk for women producing NAF with atypia was five times that of women who did not produce NAF (Wrensch et al. 1992). In an update of their original series, women producing NAF exhibiting proliferative epithelium with or without atypia had a 2.4–2.8-fold risk of breast cancer compared with those who did not produce NAF with a median follow-up time of 21 years (Wrensch et al. 2001). Tice et al. (2004a) recently reported that adding NAF cytomor-phology to the Gail risk model improved model fit in a cohort of 6904 women with 100 000 patient years of follow-up. The relative incidence for the highest quintile compared with the lowest was 3.2 for the Gail model and 5.3 for the model including NAF cytology. There was no significant interaction with age.

    In summary, both NAF production and NAF cyto-morphology have been associated with elevated risk in prospective trials and NAF is easy and inexpensive to collect. There is preliminary evidence that NAF cytomorphology may also stratify risk based on the Gail model. Unfortunately, up to 50% of high-risk women fail to produce NAF, and up to 73% of NAF samples have insufficient cells for morphologic assessment (Dooley et al. 2001, Sharma et al. 2004, Francescatti et al. 2004). Given these limitations, several investigators have turned to molecular analysis of NAF fluid including hormone levels (Elia et al. 2002, Chatterton et al. 2004), proteomic patterns (Sauter et al. 2004, Alexander et al. 2004) and gene methylation (Evron et al. 2001, Krassenstein et al. 2004). Others have sought more reliable methods of obtaining epithelial cells from breast tissue.

    RPFNA

    A second method of non-lesion directed tissue sampling is RPFNA. This technique is based on the premise that if there are widespread proliferative changes within the breast, then there is an appreciable chance that these changes might be detected by random tissue sampling. The rationale is supported by the multifocal, multicentric proliferative changes observed in autopsy series (Bhathal et al. 1985, Nielsen et al. 1987) as well in prophylactic mastectomy series from high-risk women (Hoogerbrugge et al. 2003). Rather than assessing specific ducts that produce NAF, RPFNA attempts to detect a field change. Presumably those individuals who have atypia which can be detected by random tissue sampling would have the highest density of precancerous lesions within the breast tissue and a higher short-term risk of breast cancer than those women in whom atypia was not detected by this technique.

    Skolnick et al. (1990) performed four-quadrant FNA on first-degree relatives of cancer patients and compared these aspirates to age-matched controls without affected family members. Cytologic evidence of proliferative breast disease with or without atypia was observed in 35% of high-risk women compared with 13% of controls. Fabian et al. (1994, 2000) used a modification of this technique. Instead of four-quadrant aspirates, two sites per breast were aspirated approximately 1 cm from the nipple areolar complex in both the upper-outer and upper-inner quadrants. Buffered lidocaine was used to anesthetize the skin and deeper subcutaneous tissue. Utilizing a 1.5 inch 21-gauge needle and a 12 cc syringe prewetted with RPMI, four or five aspirations were performed through each of the anesthetized areas. To reduce risk of bleeding and hematoma formation, women are asked to discontinue non-steroidal anti-inflammatory drugs, vitamin E or fish oil products 3 weeks prior to the procedure. Currently the majority of women are also offered vitamin K (10 mg) for 3 days prior to the procedure. Cold packs are applied to the breasts for approximately 10 min after the aspirations and then the breasts and chest wall are bound firmly with a soft gauze for several hours. Women are then instructed to wear a tight-fitting sports bra for several days. Severe hematoma formation requiring surgical evacuation and/or infection requiring oral antibiotics occurred in fewer than 1% of aspiration visits (Fabian et al. 2000). RPFNA produces minimal discomfort with a median reported pain score of 1 on a 0–10 scale (Chamberlain et al. 2003).

    Although the procedure is called random as it is not directed towards a palpable mass or lesion detected by breast imaging, areas in which some resistance is encountered with the tip of the needle are sampled preferentially. Material from all aspiration sites is pooled in a single 15 cc tube and processed for cyto-morphology and biomarkers. In our original series (Fabian et al. 2000), material was expressed into RPMI and processed via a MilliporeTM filter on to slides (Barrett & King 1976). Since 1999, RPFNA specimen processing has been modified such that material is expressed directly into 10 cc of a modified CytolytTM fixative (9 cc of CytolytTM plus 1 cc of 10% neutral buffered formalin). Cells remain in the modified CytolytTM for 24–48 h on a test-tube rocker prior to transfer to PreservecytTM. ThinPrepTM slides are then made according to standard instructions provided by Cytyc. Generally four slides are made: one for cytomorphology, with the remainder reserved for other biomarkers. The addition of formalin is useful in preserving estrogen receptor (ER) and preventing cellular degeneration if cells are exposed to extreme temperatures during shipment as part of multicenter collaborations. The number of epithelial cells obtained is related directly to the cytomorphology pattern observed. For the RPFNA procedure, we categorize cell number for each slide as <10, 10–99, 100–499, 500–999, 1000–5000 and >5000. In general, non-proliferative specimens have 100–499 cells per slide and it is possible to make only one or two slides. Specimens with hyperplasia have a median of 1000–5000 cells/slide and it is generally possible to make three or four slides per aspiration setting. Women with atypia have a median of >5000 cells/slide and it is almost always possible to make four or more slides per aspiration (C J Fabian et al., unpublished observations).

    A cohort of 480 women with a median age of 44 years and a median 10-year Gail risk of 4% underwent an initial RPFNA and were asked to return for a follow-up RPFNA 6–12 months later. 82% returned for the follow-up RPFNA. Results from the first and second aspiration were combined for a baseline data set and subjects were followed for cancer development. 94% of subjects had adequate cytology for morphologic assessment from the initial aspiration. Utilizing the combined baseline dataset, 30% exhibited non-proliferative cytology, 49% hyperplasia and 21% hyperplasia with atypia. Considering only the initial aspiration, 12% were considered to have hyperplasia with atypia (Zalles et al. 1995, Fabian et al. 2000). 60% of the women were premenopausal. Premenopausal and postmenopausal women on hormone-replacement therapy (HRT) had a higher prevalence of RPFNA atypia than postmenopausal women not on HRT (P=0.001; Fabian et al. 2000). At a median follow-up time of 45 months, women with baseline hyperplasia with atypia were more likely to have developed DCIS and/or invasive cancer than women without atypia (Fig. 3). Further, women with 10-year Gail risks above the median of 4% (corresponding roughly to a 5-year Gail risk of 1.7%) could be stratified into very high and moderately high risk on the basis of RPFNA atypia (Fig. 4). Women with both RPFNA atypia and 10-year Gail risks of > 4% had a 15% incidence of DCIS and/or invasive cancer at 3 years, whereas women with a 10-year Gail estimate of <4% had a 4% incidence of DCIS or invasive cancer within 3 years. For the entire cohort, both 10-year Gail risk and RPFNA atypia were predictive of cancer development. For women premenopausal at the time of study entry, RPFNA atypia and prior precancerous diagnostic biopsy (atypical hyperplasia, LCIS) were predictive of subsequent breast cancer development of DICS or invasive cancer (P=0.044). Although this subcohort analysis must be viewed with caution, it is possible that RPFNA atypia may be a more sensitive risk predictor in premenopausal than postmenopausal women.

    Proliferative breast disease is a continuum with overlapping morphologic features. Thus, the substantial intra- and inter-observer variance described previously in the interpretation of both cytologically and histologically prepared specimens is not surprising (Rosai 1991, Schnitt et al. 1992, Sidawy et al. 1998). Using a single experienced cytopathologist and pre-defined criteria for non-proliferative specimens, hyperplasia or hyperplasia with atypia (Zalles et al. 1995), intra-observer variance was approximately 25% in our RPFNA series (Fabian et al. 2000, 2002). Masood et al. (1990) has developed a semiquantitative scoring index in which six cytologic characteristics are assigned 1-4 points depending on the degree of abnormality observed. Although there is overlap, non-proliferative samples generally score in the 6–10 range, hyperplasia 11–14 and hyperplasia with atypia 15–18 (Masood et al. 1990). Use of this index allows identification of samples that are borderline between hyperplasia and atypia (e.g. a score of 14) and may also reduce interpretive variance. Intra-observer variance was reduced from 25% with traditional categorical descriptors to 16% with the Masood index system when significant variance was considered to be a change in the index score of three or more points (Fabian et al. 2002).

    In 1996, at a National Cancer Institute Conference, a Uniform Approach for diagnostic fine-needle aspiration biopsies (FNABs) was adopted (Uniform Approach 1997). Five categories were recognized: (1) unsatisfactory/insufficient cellularity; (2) benign; (3) atypical/indeterminate; (4) suspicious, probably malignant and (5) malignant. It was suggested that diagnostic FNABs falling into the atypical/indeterminate category be followed by surgical biopsy (Uniform Approach 1997). In a series reported by Boerner et al. (1999), 5% of diagnostic FNABs were atypical/ indeterminate and cancer was found in approximately half of these specimens at follow-up excisional biopsy. At the present time it is unknown whether either the Masood scoring index or the Uniform Approach criteria, when applied to RPFNA specimens, would result in less inter- and intra-observer variance; nor whether it would provide superior or inferior predictive ability for development of breast cancer.

    In summary, RPFNA utilizing the technique developed by Fabian et al. in which four or five aspirates are taken from each of two anesthetized sites per breast is associated with 94% cytomorphologic evaluability in a high-risk cohort where age is predominately 30–60 years. A random single aspiration from the upper-outer quadrant is not likely to produce the same results (only 60% morphologic evaluability reported; Khan et al. 1998). Further, cytologic evidence of atypia confers a 5-fold increase in risk compared with the absence of evidence of atypia and allows stratification of women with elevated Gail risk into high and very high categories. Although more invasive than NAF, the procedure may be performed comfortably and supply costs are modest. The primary drawback to this procedure is that the location of marked atypia, if observed, is unknown.

    Ductal lavage

    Ductal lavage is an extension of the NAF technique. In this procedure, NAF-producing ducts are cannulated with a microcatheter, saline or other physiologic solution is infused, the breast is massaged and the ductal lavage effluent is collected and expressed into a tube of fixative. In the multi-institution study published by Dooley et al. (2001), the ductal-lavage effluent was expressed into tubes of CytolytTM and mailed to a central processing location. The liquid fixative/cell mixture was then poured through a MilliporeTM filter system, and cells captured on a filter paper that must be transferred subsequently to a glass side and dissolved with chloroform or other suitable solvent. This is a very efficient system for maximum cell capture but nuclear morphology can be suboptimal if the filter is not completely dissolved.

    The multicenter study indicated that NAF production was possible in 83% of 500 eligible women from a high-risk cohort (57% of whom had a contra-lateral breast cancer and 39% with a 5-year Gail risk of >1.7%). 92% of women with NAF production underwent successful duct cannulation. Adequate samples for cytomorphologic assessment (>10 cells) were obtained from 78% of women who underwent successful duct cannulation. Thus, 60% of women presenting for breast-tissue-based risk assessment produced NAF, underwent successful cannulation and had evaluable epithelial cells in their lavage specimen (Dooley et al. 2001).

    The number of epithelial cells was estimated by counting the number of cell clusters and multiplying the number of clusters by the average number of cells in a cluster. Adequate epithelial cells for a morphologic designation (>10 cells) were obtained in NAF from 27% (111/417) of women versus 78% (299/383) of women undergoing successful duct cannulation. The median number of epithelial cells from evaluable NAF specimens was 120 (range 10–74 300) versus 4000 (range 24–143 000) or 13 500 (range 43–492 000) depending upon which microcatheter was used for ductal lavage.

    Morphologic assessment was also performed centrally by two expert cytopathologists using modified Uniform Approach criteria (Uniform Approach 1997). Morphology was reported as insufficient, benign, mild atypia, marked atypia or malignant. 8% of the 500 eligible subjects had atypia by NAF compared with 18% by ductal lavage. Most cases of atypia were mild and inter-observer variance between two cytopatho-logists was reported as 11% utilizing the modified Uniform Approach criteria. Concordance between NAF and ductal-lavage cytomorphology was poor. Half the women with atypia in their NAF specimens had ductal-lavage specimens interpreted as benign. Three-quarters of atypical lavage specimens were associated with benign or acellular NAF specimens (Dooley et al. 2001).

    Both NAF and ductal-lavage procedures were reported as well tolerated with a median pain score of 8 mm for NAF and 24 mm for ductal lavage on a 0–100 mm visual analogue scale. However, 28% of subjects underwent the procedure in the operating room under general anesthesia. In the multi-institutional series, sterile technique was used for ductal lavage. Subsequently, Francescatti et al. (2004) have reported on a series of 114 subjects undergoing lavage using aseptic but not sterile technique: no infections were noted. Similar to the Dooley study, the mean age was 52 years, mean 5-year Gail risk was 3.1%, and 39% had contralateral breast cancer. This group found that 56% of subjects presenting for risk assessment via ductal lavage had cytologically evaluable results. Reasons for non-evaluability included lack of NAF production (23%), inability to cannulate a NAF-producing duct (5%) or insuffi-cient cells in the effluent (16%). The 57% cytologic evaluability rate for all women presenting for study is similar to the 60% rate reported by Dooley et al. (2001).

    There are two mechanical challenges during ductal lavage which are responsible for a relatively modest portion of cases of cytologic inevaluability. These are passage of the catheter through the nipple sphincter and successful navigation through the lactiferous sinus into a duct without piercing the wall of the duct. The group at Northwestern has suggested several modifications aimed at sphincter relaxation and/or increasing patient comfort, which, in their experience, increase the rate of successful duct cannulation. These include use of nitroglycerin paste to relax the sphincter and subcutaneous nipple block with lido-caine (Golewale et al. 2003). Far more frequent causes for cytologic inevaluability are the inability to produce NAF to guide catheter placement and lack of epithelial cells. Several investigators highly skilled in performing ductal lavage have been able to cannulate non-NAF-producing ducts. They report that NAF-non-producing ducts are often cellular, particularly if there are other NAF-producing ducts within the breast (Cazzaniga et al. 2003, Love & King 2004). Whether this is an approach which can be transferred to less-experienced clinicians remains to be seen.

    Other investigators have not been able to reproduce the yields of high epithelial cells reported in the original multi-institutional study by Dooley et al. (2001), even when cannulating only NAF-producing ducts. Currently, the ThinPrepTM technique described for RPFNA is also used to process specimens obtained by ductal lavage. This processing technique is associated with improved nuclear morphology compared with the previous MilliporeTM system even though cellularity may be reduced. Experienced investigators report an average cell yield of 5000 cells per duct successfully lavaged (Khan et al. 2002).

    An advantage of lavage over RPFNA is that investigation of ducts producing atypical or frankly malignant cells can be investigated via ductoscopy. In a series by Noga et al. (2002) mild to marked ductal lavage atypia was found in 42 ducts from 68 patients with pathologic nipple discharge. The majority (71%) of ducts with atypia were found to have intraductal papilloma and only 5.7% were found to have cancer. Pleomorphic spindle-shaped cells which may be confused with atypical proliferative lesions may be a result of uneven or incomplete fixation. These fixation artifacts are probably secondary to the saline lavage solution. Use of a more isotonic fluid such as lactated Ringers or PlasmolyteTM for lavage and not allowing the CytolytTM lavage fluid ratio to exceed 3 may reduce fixation artifact.

    The potential for early detection of breast cancer sets ductal lavage apart from other minimally invasive techniques for risk assessment. Khan et al. (2004) studied 44 breasts from 39 women, 38 of which had histologic evidence of cancer (although one had only lobular carcinoma in situ). Mean age was 50 years. 87% of breasts with cancer produced NAF. In only 5/38 (13%) of cancerous breasts were markedly atypical or malignant cells observed and in only 16/38 breasts were mildly or markedly atypical cells observed (Khan et al. 2004). Thus, in the study reported by Khan et al., the sensitivity for cancer detection was 13–42% depending on whether mild or marked atypia is used as a threshold. In a second study of women with suspicious microcalcifications undergoing core needle biopsy, NAF was obtained in six of 10 breasts with DCIS, but the DCIS-containing ducts yielded fluid in only one woman (Khan S A et al. 2005). The disappointingly low sensitivity for detection of cancer with ductal lavage may in part be due to ductal anatomy and distribution of cancer. Going & Moffat (2004) have demonstrated that a minority of ducts drain the majority of breast-tissue volume. Further, only approximately one-third of ducts would be readily accessible by ductal lavage or ductoscopy: the rest taper to a minute orifice and some do not communicate with the skin surface. Badve et al. (2003) reviewed 801 mastectomies performed for DCIS or invasive cancer and found nipple and central duct involvement in only 22% of cases.

    The sensitivity of ductal lavage for cancer detection is lower than that of mammography (61–81%) in a young screening population (Humphrey et al. 2002, Carney et al. 2003) or breast magnetic resonance imaging (79%) in a high-risk population (Kriege et al. 2004). However, the sensitivity of ductal lavage may be similar to that of mammography (33%) in a young high-risk population (Kriege et al. 2004).

    Women with ductal-lavage atypia are presumed to be at increased risk of breast cancer based on the elevated risk observed for women with atypical cells in NAF or RPNFA specimens (Vogel 2004). However, the impact of ductal-lavage-detected atypia on the short-term risk for breast cancer is presently unknown since there was no follow-up of participants in the multicenter study reported by Dooley et al. (2001). A prospective trial is currently underway at multiple centers in the US in which women at increased risk for breast cancer will undergo ductal lavage at 6 month intervals over a 3-year period and will be followed for clinical breast cancer development. In this study, cytomorphology will be assessed at the individual participating institutions rather than through a central review board. Several published reviews suggest that women with ductal-lavage-detected atypia should be offered standard risk-reduction options such as tamo-xifen (Morrow et al. 2002, O’Shaughnessy et al. 2002). Women with moderate to marked atypia may undergo ductoscopy; however, reimbursement for either ductal lavage or ductoscopy by third-party carriers is variable. Given the low specificity for cancer, removal of breast tissue on the basis of ductal-lavage atypia alone in the absence of a suspicious lesion on ducto-scopy or breast-imaging modalities is discouraged (Morrow et al. 2002).

    In summary, ductal-lavage cytomorphology (atypia) is currently being utilized for clinical risk stratification although the magnitude of risk conferred by ductal-lavage atypia has yet to be defined. Ductal lavage produces evaluable material for cytomorphology in 56–60% of women presenting for lavage when production of NAF guides attempts at duct cannulation, and 78% of women with a successful duct cannulation. Lavage is reported as well tolerated. Costs for procedure-related materials are substantially higher than NAF and RPFNA. Ductal lavage has low sensitivity for cancer detection and should not be used for that purpose. However, ductal-lavage atypia can be further investigated by ductoscopy.

    Core needle biopsy

    Core needle biopsy holds the promise of better architectural definition and large numbers of epithelial cells for study; but non-lesion-directed core biopsies as a method of harvesting tissue for risk assessment and prevention trials have had mixed results. Mansoor et al. (2000) reported predominately atrophic terminal lobular duct units in 11-gauge core needle biopsies of normal breast tissue adjacent to benign lesions requiring stereotactic biopsy. In this series, the median number of normal cores per patients was two (range 1–7). Non-atrophic terminal lobule duct units were present in only 47% of patients. Postmenopausal women on HRT and women with dense heterogenous parenchyma were most likely to have non-atrophic terminal lobule duct units (Mansoor et al. 2000). To date, prevention trials using core needle biopsy as the sampling technique have not accrued subjects at a rapid rate although the procedure is described as well-tolerated (Harper-Wynne et al. 2002, Mohsin et al. 2003, Palomares et al. 2004). A 60–90% success rate has been described for obtaining adequate tissue at both the baseline and follow-up core biopsy (Harper-Wynne et al. 2002, Mohsin et al. 2003, Palomares et al. 2004, Stearns et al. 2004). The relative risks for non-directed core biopsy findings of hyperplasia with or without atypia have yet to be defined.

    Which procedure is superior for obtaining epithelial cells for risk assessment

    This is a complicated question without a simple answer and will to a great extent depend on the skill set of the health-care professional performing the tissue sampling and the available resources. For this comparison, we have not included random core biopsy as there is minimal experience with this technique used in this fashion at the present time.

    Are risk-eligible women with evidence of atypia more likely to undertake a prevention intervention

    Although this question has yet to be directly addressed, Bober et al. (2004) reported that risk-eligible post-menopausal women who had a prior abnormal biopsy including atypical hyperplasia were significantly more likely to accept prevention treatment with tamoxifen or participation in the STAR trial of tamoxifen versus raloxifene than women without a history of abnormal biopsy. Having a first-degree relative with breast cancer and/or a biopsy per se did not predict acceptance of prevention drug treatment. However, women with a history of an abnormal biopsy were much more likely to perceive that their physician was advising them to undergo a prevention intervention than women with a family history alone (Bober et al. 2004). On multivariable analysis, only perceived recommendation by a physician, not atypia, was significant in predicting uptake of prevention drug therapy. A second study of factors affecting tamoxifen acceptance among high-risk women found that a history of ADH or LCIS is the strongest determinant of willingness to take tamoxifen (Tchou et al. 2004). Conversely, Didwania et al. (2003) reported that only 2/11 subjects with mild ductal-lavage atypia were influenced by the results to take tamoxifen.

    Potential breast-tissue molecular risk biomarkers

    Considering the inter- and intra-observer variance observed with cytomorphology for both ductal lavage and diagnostic and/or RPFNA, there is a great deal of interest in supplementing morphologic interpretations with molecular markers (Fabian et al. 2002, Ljung et al. 2004, Gornstein et al. 2004, Sneige 2004). Simple assessment of ploidy has been accomplished for both RPFNA and ductal-lavage samples (Fabian et al. 2000, Sauter et al. 2004). Sauter et al. (2004) noted higher frequencies of aneuploidy and hypertetroploidy in ductoscopy lavage specimens from women known to have breast cancer.

    Genetic markers of allelic imbalance such as loss of heterozygosity and comparative genomic hybridization suggest a close relationship between atypical duct hyperplasia, DCIS and invasive cancer (O’Connell et al. 1998, Amari et al. 1999, Allred et al. 2001, Gong et al. 2001). Comparative genomic hybridization studies further indicate that well-differentiated DCIS and poorly differentiated DCIS are distinct genetic entities separately evolving into low- and high-grade invasive cancer (Buerger et al. 1999, 2001). These types of study also suggest that ductal and lobular cancers appear to evolve from different precursor lesions (Reis-Filho & Lakhani 2003).

    Gene-expression profiling provides an estimate of the relative abundance of a particular gene compared with a reference sample. RNA is isolated, reverse transcribed to cDNA, labeled with a fluorescent dye and hybridized to a microarray. In the past, mRNA expression profiling has required appreciable amounts of fresh or frozen tissue which made the study of precancerous lesions difficult (Das & Singal 2002). However, laser-assisted microdissection, RNA linear amplification techniques (Van Gelder et al. 1990, Zhao et al. 2002) and specialized processing (Baunoch et al. 2003, Ma et al. 2003) allow mRNA expression profiling or quantitative real-time PCR to be performed on discrete lesions from formalin-fixed paraffin-embedded tissue obtained from core needle biopsies or fine-needle aspirations (Ellis et al. 2002, Fabian et al. 2003). Using mRNA expression profiling, Ma et al. (2003) provided additional evidence that ADH is a genetically advanced precancerous lesion and that ADH and DCIS are direct precursors of invasive ductal cancer. Relatively few genetic differences were found between ADH, DCIS and invasive cancer in the same breast, although appreciable differences were found between low- and high-grade in situ and invasive cancers from different individuals. Genes whose expression increased between DCIS and invasive cancer were related to proliferation and cell-cycle regulation (Ma et al. 2003). Based on studies such as these, some investigators have hypothesized that ADH is a committed precursor lesion whose molecular phenotype may predict the type of later in situ or invasive cancer (Jeffrey & Pollack 2003). It follows that markers of allelic imbalance or gene expression profiling might be utilized to supplement morphologic interpretations in the identification of high-risk lesions such as atypical hyperplasia.

    Many early changes in carcinogenesis may be at the translational rather than at the transcriptional level, which could be theoretically useful in identifying women with non-proliferative or proliferative changes at high risk of developing more-advanced precancer-ous lesions and eventually cancer. Further, gene-specific levels of mRNA and their protein products do not necessarily correlate, indicating the importance of post-transcriptional influences (Gygi et al. 1999, Celis et al. 2000). Several proteomic technologies are available including two-dimensional gel analysis, surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) and sandwich antigen capture or direct immunoassays. Investigators have used both antibody arrays and two-dimensional gel profiling to study differences between normal and malignant ductal and lobular units. A large number of proteins differentially expressed in normal and malignant tissue were identified (Czerwenka et al. 2001, Hudelist et al. 2004). These included proteins involved in cellular trafficking and cytoskeletal and extracellular matrix regulation (including e1F), cell signaling and apoptosis (including Rho, 14-3-3 proteins and proteins involved in epidermal growth factor receptor (EGFR) pro-sphorylation; Fish et al. 1995, Wulfkuhle et al. 2002, Hudelist et al. 2004). A disadvantage of current proteomics techniques is they must be performed on fresh or frozen tissue/fluid that has not been fixed in formalin. This limits proteomic analysis on diagnostic biopsies, especially for multi-institutional trials.

    Proteomics pattern assessment is readily performed on NAF using the SELDI technique. Paweletz et al. (2001) observed different protein profiles in NAF fluid in women with and without breast cancer. Interestingly, Pawlik et al. (2004) have reported differences in protein patterns in NAF samples in healthy women versus those with early-stage breast cancer, but no significant differences between the involved and uninvolved breasts. Sauter et al. (2002) found five differentially expressed protein profiles present in over 75% of women with invasive cancer but <10% of NAF samples from normal women.

    Another method of assessing gene/protein function is with methylation-specific PCR. DNA methylation is an important process in epigenetic cellular memory that restricts or permits differential gene expression in descendent cells (Widschwendter & Jones 2002a, 2002b). This may be particularly important for evaluating the function of tumor-suppressor genes whose expression is attenuated or lost during the initiation or promotional phases of breast carcinogenesis (Das & Singal 2004). The tumor-suppressor genes HIC-1, RASSFIA and 14-3-3 are methylated in a substantial proportion of cases of hyperplasia with and without atypia (Fujii et al. 1998, Ferguson et al. 2000, Umbricht et al. 2001, Lehmann et al. 2002). RASSFIA is involved with the regulation of cell-cycle progression via inhibition of cyclin D1 accumulation and promotion of apoptosis. Low-frequency promoter methylation of GSTP, CDH, BRCA1, p16 and RAR2 have all been observed in benign breast tissue (Troch et al. 2003, Bae et al. 2004, Bean et al. 2005). These abnormalities allow escape from normal senescence and apoptosis and an extended period of susceptibility to proliferation and carcinogenic influences (Widschwendter et al. 1997, Huschtscha et al. 1998, Nuovo et al. 1999, Tlsty et al. 2001, Neumeister et al. 2002, Holst et al. 2003). Methylation abnormalities such as these may be detected from fixed microdissected or whole-slide scrapings from RPFNA (Troch et al. 2003, Sukumar et al. 2004) or ductal-lavage samples (Fackler et al. 2004).

    The increased proportion of cells expressing ER and/or the proliferation marker Ki-67 may signal the transition from non-proliferative to proliferative epithelium. Proliferation in terminal lobular duct units varies with age, menopausal status and phase of menstrual cycle; and is highest for premenopausal women in the luteal phase of the cycle (Soderqvist et al. 1997, Potten et al. 1998). In the normal breast epithelium, proliferation, as measured by Ki-67, is positively correlated with serum progesterone levels but not serum estradiol, prolactin, bioavailable testosterone, androstenedione or IGF-I (Soderqvist et al. 1997). For premenopausal women, the proportion of normal breast epithelial cells expressing Ki-67 expression has been reported as approximately 1% in the follicular phase and 2–3% in the luteal phase (Soderqvist et al. 1997, Shoker et al. 1999). For post-menopausal women, the proportion of breast epithelial cells expressing Ki-67 is less than 1% (Shoker et al. 1999). The proportion of epithelial cells expressing Ki-67 increases in hyperplasia (>1%) and hyperplasia with atypia (2–5%) in both histologic and RPFNA specimens (Shoker et al. 1999, Allred et al. 2001, Khan Q J et al. 2005).

    The proportion of breast epithelial cells expressing ER also varies with age and menopausal status as well as cell-cycle phase. The proportion of cells expressing ER is lowest in the luteal phase of the menstrual cycle and highest in postmenopausal women. ER has been reported to average 20% in the follicular portion of the cycle and 0–5% in the luteal portion of the cycle in normal lobules from premenopausal women and 18–40% in normal lobules from postmenopausal women (Soderqvist et al. 1993, Markopoulos et al. 1998, Khan et al. 1999, Shoker et al. 1999). The proportion of cells expressing PR in non-proliferative breast tissue is generally greater than those expressing ER and progesterone receptor (PR) expression does not vary significantly over the cycle (Soderqvist et al. 1993, Khan et al. 1999). In hyperplasia the proportion of ER-positive cells increases to 45% or greater and 90% for ADH (Shoker et al. 1999, Allred et al. 2001). In a cross-sectional study of women with known outcome, Khan et al. (1999) found that an increased level of ER relative to PR in benign biopsies was associated with increased risk of breast cancer relative to those in whom the proportion of cells staining positive for PR was greater than or equal to those staining positive for ER (Khan et al. 1999).

    In normal, non-proliferative breast tissue, ER-positive cells rarely express proliferation antigens; rather, proliferation is observed in adjacent ER-negative cells, which respond to paracrine influences from their ER-positive neighbors (Clarke et al. 1997). Studies in normal premenopausal breast tissue showed that only 0.01% of cells are dual-labeled for both ER and Ki-67 (Clarke et al. 1997, Shoker et al. 1999). Despite a lower overall percentage of epithelial cells expressing Ki-67, Shoker et al. (1999) reported that the proportion of dual-labeled cells was 4–20-fold higher in postmenopausal than premenopausal women. The proportion of epithelial cells expressing Ki-67 or dual-labeled for both Ki-67 and ER shows a progressive increase between hyperplasia ADH and carcinoma in situ (Shoker et al. 1999). A negative association between Ki-67 and ER expression is maintained in hyperplasia of the usual type but this is lost in ADH. This would indicate a lack of suppression of ER expression as cells enter the cell cycle in atypical hyperplasia (Shoker et al. 1999). An increase in the proportion of cells staining individually as well as dually for Ki-67 and ER with progression to atypia may indicate a shift from paracrine to autocrine control of proliferation.

    Combining morphologic and molecular markers for risk stratification

    Molecular markers have the potential to further stratify risk prediction based on epidemiologic models and breast-tissue morphology although few prospective studies have been performed. Immunocytochem-ical expression of ER, p53, EGFR and HER-2 was assessed in cytospin preparations along with morphology from MilliporeTM preparations in high-risk women in our prospective study (Fabian et al. 2000). Cytoplasmic and/or membrane staining of 10% or more of ductal cells (>2+ intensity) was considered as evidence of expression for both EGFR and HER-2. Given the use of cytospin preparations, lack of immediate fixation in a formalin fixative and/or antigen retrieval of ER expression was likely underestimated and thus >1+ intensity staining for ER in 10% or more of ductal cells was considered evidence of expression.

    All single markers were strongly predictive of cyto-logic atypia (P < 0.001 in univariate analysis) and multiple marker expression of the three-biomarker set EGFR, ER and p53 was strongly predictive of atypia in multivariable analysis (P < 0.001). ER was the only single molecular marker predictive of cancer development (P=0.048) in a univariate analysis, although multiple markers in the three-set test (EGFR, ER and p53) were strongly predictive in univariate analysis for cancer development and for time to cancer development (P=0.021 and 0.003, respectively). Neither single nor multiple biomarkers (p53, EGFR, ER) were predictive for DCIS or invasive cancer in a multivariable analysis when RPFNA cytomorphology, 10-year Gail risk and prior diagnosis of LCIS or atypical hyperplasia in a diagnostic biopsy were included in the equation (Fabian et al. 2000). RPFNA hyperplasia with atypia, 10-year Gail risk and prior LCIS or atypical hyperplasia in a diagnostic biopsy were all predictive for cancer development (Fabian et al. 2000).

    Quantitative PCR may be readily performed for 6–12 biomarkers on cellular material available from one of the four slides generally made from a RPFNA (Petroff et al. 2004). Quantitative PCR may provide for more accurate and reproducible assessments than immunochemistry especially for biomarkers expressed primarily in the membrane or cytoplasm. Quantitative PCR may also allow for measurements of more biomarkers in small samples than can be performed with immunochemistry. There is, however, poor correlation with focally expressed markers such as Ki-67, or those in which protein stabilization not elevated mRNA levels give rise to enhanced expression (Ginestier et al. 2002).

    Methylation-specific PCR to determine loss of expression of tumor-suppressor genes is being utilized to help identify cancer in markedly atypical cytology specimens, and currently is being studied to determine whether it might identify proliferative disease likely to progress to cancer. Methylation-specific PCR can easily be performed on cells available from RPFNA or ductal-lavage samples (Evron et al. 2001, Fackler et al. 2004, Krassenstein et al. 2004, Moore et al. 2004). A new technique called quantitative multiplex methylation-specific PCR (QM-MSP) allows quantitative assessment of the extent of methylation of several genes (Fackler et al. 2004).

    Chromosomal alterations in ductal lavage specimens matching those in corresponding cancers measured by comparative genomic hybridization or fluorescent in situ hybridization matching those in corresponding tumors have been observed in women who have breast cancer regardless of whether atypical or malignant cytomorphology is present (Adduci et al. 2004).

    Whether one or more of these molecular techniques improves prediction based on epidemiologic models combined with cytomorphology with or without mammographic breast density needs to be addressed in a prospective trial.

    Use of breast-tissue biomarkers in prevention

    Biomarkers highly associated with short-term risk and subject to modulation may be used to select cohorts and measure a response to a prevention intervention. Biomarkers used to measure a response to an intervention are called surrogate endpoint biomarkers or SEBs (Kelloff et al. 1994, 1996). Important properties of an SEB are similar to those of risk biomarkers discussed above. In addition to risk-marker properties, favorable modulation of the SEB by established prevention interventions should be associated with reduced cancer risk. Risk biomarkers which are currently being used to assess response to a prevention intervention in Phase I and II trials are (1) the ratio of serum IGF-I to its binding protein IGFBP-3, (2) serum-bioavailable estradiol and sex hormone-binding globulin, (3) mammographic breast density and (4) breast intra-epithelial neoplasia (hyperplasia with and without atypia) and associated changes within breast intra-epithelial neoplasia, such as proliferation (Table 4).

    Tamoxifen, an established prevention drug, has been shown to favorably modulate all of the above biomarkers except serum hormones (Brisson et al. 2000, Chang et al. 2000, Bonanni et al. 2001, Tan-Chiu et al. 2003, Cuzick et al. 2004). Individuals who did not exhibit favorable biomarker modulation in response to tamoxifen do not necessarily go on to develop breast cancer (Tan-Chiu et al. 2003, Cuzick et al. 2004). In fact, the observed benefit from drug treatment is greater than would be expected from the extent of modulation of many biomarkers, particularly mammographic density (Cuzick et al. 2004).

    Sampling breast tissue for SEB also provides the potential to assess biomarkers predictive of the response to a particular class of agents (e.g. ER for selective estrogen receptor modulators (SERMs)). This would allow appropriate matching of an individual with an intervention to which she is most likely to respond presuming the mechanism of action is known and predictive biomarkers have been identified (Paik et al. 2004). Repeated sampling also allows for assessment of markers which may provide evidence that the individual is in fact benefiting from drug treatment, i.e. reduction in proliferation or improvement in abnormal morphology.

    Clinical models for Phase I trials

    The toxicity profile is generally well known for most drugs being considered as potential prevention agents. As even minimal side effects are often unacceptable when a drug is to be given to a healthy women over a prolonged period, Phase I prevention trials focus on establishing the lowest dose at which a drug modulates a risk and/or a mechanism-of-action biomarker (Kelloff et al. 1994, Fabian et al. 1998, 2004a, 2005). Phase IA trials explore the effects of dose on several biomarkers. Phase IB trials are usually placebo-controlled and confirm that a given drug dose modulates a biomarker reliably (Fig. 5).

    At present, the most popular Phase IA model is the so-called presurgical model in which women with a DCIS or a small invasive cancer who have undergone a diagnostic core biopsy are randomized between one of several drug doses in the interval (generally 2–4 weeks) between biopsy and re-excision, lumpect-omy or mastectomy. A no-treatment control (either randomized or non-randomized) may be used as well to determine the effect of biopsy and other influences (such as stopping HRT) on the biomarkers being assessed. Generally, a proliferation biomarker such as Ki-67 is used as the primary endpoint and 8–12 subjects are entered at each dose level (Fabian et al. 2004a, 2005). In Phase IB, the dose(s) from Phase IA which have shown favorable modulation in 80% of subjects is chosen and subjects are randomized to study drug or placebo (Decensi et al. 2003, Fabian et al. 2004a). Restriction of entry to a relatively homogenous population (e.g. similar menopause status and grade) may reduce the wide variation seen in Ki-67 and thus the number of subjects needed. For example, for a postmenopausal cohort not previously on HRT with non-high-grade tumors and a median Ki-67 of 10% and a standard deviation of 9%, a 50% reduction in Ki-67 could be detected with 40 evaluable subjects in the treatment group and 20 subjects in the placebo group (Fabian et al. 2004a).

    Significant problems with the presurgical model include (1) difficulties with accrual (Singletary et al. 2000, Fabian et al. 2004a), (2) significant variation in Ki-67 between different parts of the tumor, especially when proliferation is low (<5%), (3) confounding effects of stopping HRT between diagnostic biopsy and re-excision in postmenopausal women (Conner et al. 2003, Fabian et al. 2004a), (4) confounding effects of initial and follow-up biopsy in different phases of the menstrual cycle in premenopausal women (Soderqvist et al. 1997), (5) effects of tissue reaction to injury (Urban et al. 1999) and (6) minimal tumor at re-excision and fixation and processing differences between core biopsy and re-excision (Grizzle et al. 1995, 1998). Despite these problems, adequate accrual to presurgical model trials has been successfully accomplished utilizing multi-institutional consortia (Decensi et al. 2003, Fabian et al. 2004a). A modest reduction in Ki-67 compared with placebo or no treatment control has been demonstrated for tamoxifen and other SERMs (Dowsett et al. 2001, Decensi et al. 2003).

    Clinical models for Phase II trials

    Phase II trials are generally randomized, double-blind, placebo-controlled studies in which high-risk subjects are enrolled for 6–12 months. However, in cases in which the effect size of the agent on the primary endpoint biomarker is uncertain, a single-arm pilot study may be very useful. Primary response endpoints most often employed include modulation of morphology in high-risk women with baseline intra-epithelial neoplasia, modulation of proliferation or modulation of mammographic breast density (Boyd et al. 1997, Fabian et al. 2002, Harper-Wynne et al. 2002). Serum IGF-I/IGFBP-3 may be employed as well (Bonnani et al. 2001, Decensi et al. 2004). These SEBs are measured at baseline and study completion (Fig. 6).

    In the US, Phase II trials using breast-tissue biomarkers as the primary endpoint have to date only been completed successfully in a reasonable time frame using RPFNA as the tissue-sampling method, although trials with ductal lavage and serial random and ultrasound-guided biopsy are ongoing.

    Fabian et al. (2002) reported accrual of 119 subjects in 23 months to a single-institution trial of 6 months of -difluromethylornithine versus placebo. Approximately four women underwent aspiration for every woman placed on study as the study required demonstration of hyperplasia or hyperplasia with atypia at baseline, sufficient cells for three other biomarker studies, as well as a number of other medical parameters including a reasonably normal auditory evaluation at baseline. Median age of entrants was 46 years. Considering only eligibility based on cytomorphology, approximately one in two women aspirated would have been eligible. 96% of subjects completed the study including a follow-up RPFNA 6 months after randomization.

    The main endpoint of the study was improvement in cytologic morphology which was similar for both the -difluromethylornithine- and placebo-treated groups. Samples from 28% of subjects randomized to the placebo were interpreted as showing improvement using the traditional categories of non-proliferative, hyperplasia and hyperplasia with atypia. 18% of samples from placebo-treated subjects were interpreted as showing improvement when improvement was defined as reduction of 3 or more Masood score points. Average baseline Masood score was 13.5 with a mean decrease of 0.46±2.5 points in the placebo group. Although this amount of variation in the placebo group is within the range of discordance reported for benign breast specimens (Sidawy et al. 1998) with the modest sample size (119 subjects) utilized for this trial, a 60% improvement in cytologic category and/or a mean reduction of 2 Masood score points (from an average baseline of 13.5) would be needed to detect a significant difference relative to placebo with an 80% power and a type I error rate of 5%. Alternatively, the sample size could be increased to detect smaller differences. Two models have been proposed in this regard which would facilitate sample-size estimation and statistical analysis. These have been termed the prevention-of-progression and reversal-of-atypia models (O’Shaughnessy et al. 2002, Fabian et al. 2005). In the prevention-of-progression model, women without atypia but with hyperplasia (traditional criteria) are randomized to placebo or drug for 6–12 months. The primary endpoint is hyperplasia with atypia in the follow-up RPFNA sample (Fig. 7). Even if up to 25% of subjects randomized to placebo showed atypia in the follow-up sample, a 50% reduction in the incidence of hyperpalsia with atypia could be detected in 335 subjects, with 80% power and a 5% type I error rate (Fabian et al. 2005). Approximately 670 high-risk subjects would have to be screened by RPNA for this type of trial design.

    A reversal of the hyperplasia-with-atypia model in which all subjects had atypia at entry would require two or three times as many screening aspirations but fewer subjects entered on trial (Fig. 7). As few as 130 subjects would need to be entered into the treatment portion to detect a 50% reduction in fine-needle aspiration atypia after 6–12 months and 280 to detect a 33% reduction. Corresponding numbers of women who would need to be screened with an aspiration are 650 and 1400. It is unknown at present whether hyper-plasia with atypia can be reversed after 6–12 months, and what effect failure to detect atypia on follow-up RPFNA might have on subsequent cancer incidence. The Phase II reversal of atypia model at present is probably best employed for drugs/regimens predicted to have a strong apoptotic effects (such as withdrawal of estrogen).

    Because of the large number of subjects required for modulation of morphology for either the prevention-of-progression or reversal-of-atypia design, pilots and smaller Phase II studies often utilize modulation of proliferation as the primary response indicator. Ki-67/MIB-1 is an attractive proliferation marker because of its reproducibility (Keshgegian & Cnaan 1995, Biesterfeld et al. 1998). The proportion of cells expressing MIB-1 varies with age, menopause status and phase of the menstrual cycle as well as morphologic abnormality. Utilizing RPFNA cytology specimens (obtained in the follicular phase for premenopausal women), we have observed that median Ki-67 in premenopausal women with hyperplasia is 2% and 1% for postmenopausal women. Further, women with hyperplasia without atypia had a median Ki-67 of 1.1% versus 2.8% for hyperplasia with atypia (Khan Q J et al. 2005). Similar values for Ki-67 have been reported for ductal lavage specimens from high-risk women (Cazzaniga et al. 2003). Given the small proportion of cells expressing Ki-67 it is necessary to count at least 500 ductal cells and 1000 would be optimum. Several small chemoprevention studies with modulation of Ki-67 in RPFNA cytology specimens as the main endpoint are ongoing (Fig. 8), and one using letrozole on postmenopausal women on HRT for control of menopausal symptoms shows evidence of modulation of Ki-67 despite continuing HRT during letrozole treatment (Fabian et al. 2004b).

    Detection of a 50% reduction in the proportion of cells expressing Ki-67 would require approximately 50 subjects in each arm, assuming a median Ki-67 of 3.5% at baseline and a standard deviation of a similar value, with 80% power and a 5% type I error rate. Detecting a 33% reduction in proliferation would require approximately 200 subjects (Fabian et al. 2005). These are theoretical estimates as studies assessing the variability of Ki-67 in postmenopausal women or premenopausal women in the same phase of the menstrual cycle over 6–12 months have not yet been performed.

    Ductal lavage is an attractive alternative tissue-sampling method for Phase II prevention trials if reproducibility of cytomorphology or biomarkers is superior to that of RPFNA. In a preliminary analysis of an ongoing Phase II study of women undergoing repeat ductal lavage after 6 months of either tamoxifen or no intervention, 230/266 ducts (86%) could be recannulated 6 months later. Of the women who had at least one duct recannulated at a second lavage procedure, 75% had sufficient cells for cytologic diagnosis at both time points. There was no fall-off in cell yield or overall success of the procedure after 6 months of tamoxifen, although there was a trend towards declining nipple-fluid yield with longer durations of tamoxifen use (Bhandare et al. 2004). Reduction in incidence of cytologic atypia was seen in both the tamoxifen and no-intervention groups, with no sig-nificant difference between groups. Further, since NAF production and a successful ductal lavage can be expected in only approximately 60% of high-risk subjects, a larger number of subjects may have to be screened in order to meet the accrual goals, and supplies for ductal lavage are considerably more expensive than for RPFNA. Finally, it is possible that a successful drug intervention may reduce NAF production, making it less likely to obtain an adequate follow-up ductal-lavage specimen. The pros and cons of using of RPFNA, ductal lavage and NAF in prevention studies are detailed in Table 3.

    As it is not clear at present which technique is most likely to give the most satisfactory results, both before and after a chemoprevention intervention, the NCI is sponsoring a multi-institutional trial comparing ductal lavage and RPFNA in high-risk premenopausal women before and after 12 months of celecoxib (400 mg) twice daily for 12 months. Women eligible for the screening phase undergo a NAF attempt and, if successful, ductal lavage during the follicular phase of the menstrual cycle. Women then undergo an RPFNA the same day. Women who do not produce NAF and/or whose lavage is unsuccessful also have an RPFNA the same day. Four slides are made for both the ductal lavage and RPFNA procedure for cytomorphology, Ki-67, ER and cyclo-oxygenase 2 (COX-2). In order to be eligible for the treatment portion women must have >1000 ductal cells on the cytomorphology slide, >500 ductal cells on the Ki-67 slide with >1% of cells showing evidence of proliferation and >100 ductal cells for ER and COX-2. Although the primary endpoint is modulation of proliferation by RPFNA, significant secondary endpoints include comparison of screening eligibility, adequacy of follow-up specimens, differences in cytomorphology and immunochemistry assessment, and procedure tolerance.

    A number of Phase II clinical prevention trials are currently ongoing utilizing RPFNA or ductal lavage or core biopsy as the tissue-sampling technique and these are listed in Table 5.

    Summary

    RPFNA, ductal lavage and NAF are all commonly used methods to obtain breast tissue for risk stratification as well as for response monitoring in pilot and Phase II prevention trials. Both NAF and RPFNA atypia are associated with increased risk of subsequent breast cancer in prospective trials of average and high-risk women, respectively. Currently, the magnitude of increase in relative risk for detected atypia is known for only RPFNA (5-fold) and NAF (approximately 2.8-fold) but will probably be at least as high for ductal-lavage-detected atypia as for NAF-detected atypia. The most efficient method of obtaining epithelial cells for evaluation at a reasonable cost is RPFNA. Considerable issues remain regarding the reproducibility of morphologic assessment across a wide range of settings. Research is ongoing for methods to improve morphologic assessment reproducibility, including the addition of molecular tools. Therefore, breast-tissue-based risk stratification is still best performed within the context of a clinical trial.

    RPFNA, ductal lavage and core biopsy are being utilized for tissue sampling in pilot and Phase II prevention studies in which modulation of morphology and/or molecular markers are used as the primary response endpoints following 1–12 months of study drug or placebo. It is not clear at present which method of repeated tissue sampling is the most cost-effective for prevention studies, but an NCI-sponsored study comparing RPFNA and ductal lavage has nearly completed accrual and will address this question directly.

    Acknowledgements

    The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

    References

    Adduci K, Devries-Troxel S, Chew K, Annis C, Boutin J, Magrane G, Ljung B-M, Waldman F & Esserman LJ 2004 Fluorescence in situ hybridization (FISH) of ductal lavage samples identifies malignant phenotypes from cytologically normal cells in women with breast cancer. Breast Cancer Research and Treatment 88 S223 (abstract 6010).

    Alexander H, Stegner AL, Wagner-Mann C, Du Bois GC, Alexander S & Sauter ER 2004 Proteomic analysis to identify breast cancer biomarkers in nipple aspirate fluid. Clinical Cancer Research 10 7500–7510.

    Allred DC, Harvey JM, Berardo M & Clark GM 1998 Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Modern Pathology 11 155–168.

    Allred DC, Mohsin SK & Fuqua SA 2001 Histological and biological evolution of human premalignant breast disease. Endocrine-related Cancer 8 47–61.

    Amari M, Suzuki A, Moriya T, Yoshinaga K, Amano G, Sasano H, Ohuchi N, Satomi S & Horii A 1999 LOH analyses of premalignant and malignant lesions of human breast: frequent LOH in 8p, 16q, and 17q in atypical ductal hyperplasia. Oncology Report 6 1277–1280.

    Antill Y, Murray W, Lindeman G, House C, Phillips G & Mitchell G 2004 Ductal lavage in BRCA1/2 mutationcarriers: initial experience. Breast Cancer Research and Treatment Supplement 1 88 S153 (abstract 4013).

    Arun B, Lammy BG, Hortobagyi G & Sneige N 2003 Phase II chemoprevention trial of celecoxib using ductal lavage. Breast Cancer Research and Treatment Supplement 1 82 S26 (abstract 126).

    Aston CE, Ralph DA, Lalo DP, Manjeshwar S, Gramling BA, Defreese DC, West AD, Branam DE, Thompson LF, Craft MA et al. 2005 Oligogenic combinations associated with breast cancer risk in women under 53 years of age. Human Genetics 116 208–221.

    Badve S, Wiley E & Rodriguez N 2003 Assessment of utility of ductal lavage and ductoscopy in breast cancer-a retrospective analysis of mastectomy specimens. Modern Pathology 16 206–209.

    Bae YK, Brown A, Garrett E, Bornman D, Fackler MJ, Sukumar S, Herman JG & Gabrielson E 2004 Hypermethylation in histologically distinct classes of breast cancer. Clinical Cancer Research 10 5998–6005.

    Barrett DL & King EB 1976 Comparison of cellular recovery rates and morphologic detail obtained using membrane filter and cytocentrifuge techniques. Acta Cytologica 20 174–180.

    Baunoch D, Moore M, Reyes M, Cotter P, Bloom K, Erlander M, Ma X-J & Sgroi D 2003 Microarray analysis of formalin fixed paraffin-embedded tissue: the development of a gene expression staging system for breast carcinoma. Breast Cancer Research and Treatment Supplement 1 82 S116 (abstract 474).

    Bean GR, Scott V, Yee L, Ratliff-Daniel B, Troche MM, Seo P, Bowie ML, Marcom PK, Slade J, Kimler BF et al. 2005 Retinoic acid receptor-beta2 promoter methylation in random periareolar fine needle aspiration. Cancer Epidemiology Biomarkers and Prevention 14 790–798.

    Bhandare D, Bryk M, Nayar R, Hou N, Rademaker A, Chatterton R & Khan SA 2004 Effect of tamoxifen (TAM) on estrogen-related biomarkers in ductal lavage (DL) samples. a study on follow-up lavages. Breast Cancer Research and Treatment Supplement 1 88 S158 (abstract 4031).

    Bhathal PS, Brown RW, Lesueur GC & Russell IS 1985 Frequency of benign and malignant breast lesions in 207 consecutive autopsies in Australian women. British Journal of Cancer 51 271–278.

    Biesterfeld S, Kluppel D, Koch R, Schneider S, Steinhagen G, Mihalcea AM & Schroder W 1998 Rapid and prognostically valid quantification of immunohistochemical reactions by immunohistometry of the most positive tumour focus. A prospective follow-up study on breast cancer using antibodies against MIB-1, PCNA, ER, and PR. Journal of Pathology 185 25–31.

    Bober SL, Hoke LA, Duda RB, Regan MM & Tung NM 2004 Decision-making about tamoxifen in women at high risk for breast cancer: clinical and psychological factors. Journal of Clinical Oncology 22 4951–4957.

    Boecker W, Moll R, Dervan P, Buerger H, Poremba C, Diallo RI, Herbst H, Schmidt A, Lerch MM & Buchwalow IB 2002 Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ. Journal of Pathology 198 458–467.

    Boerner S, Fornage BD, Singletary E & Sneige N 1999 Ultrasound-guided fine-needle aspiration (FNA) of nonpalpable breast lesions: a review of 1885 FNA cases using the National Cancer Institute-supported recommendations on the uniform approach to breast FNA. Cancer 87 19–24.

    Bonanni B, Johansson H, Gandini S, Guerrieri-Gonzaga A, Torrisi R, Sandri M T, Cazzaniga M, Mora S, Robertson C, Lien EA & Decensi A 2001 Effect of low dose tamoxifen on the insulin-like growth factor system in healthy women. Breast Cancer Research and Treatment 69 21–27.

    Boone CW & Kelloff GJ 1993 Intraepithelial neoplasia, surrogate endpoint biomarkers, and cancer chemoprevention. Journal of Cellular Biochemistry Supplement 17F 37–48.

    Boone CW, Bacus JW, Bacus JV, Steele VE & Kelloff GJ 1997 Properties of intraepithelial neoplasia relevant to cancer chemoprevention and to the development of surrogate end points for clinical trials. Proceedings of the Society for Experimental Biology and Medicine 216 151–165.

    Boyd NF, Greenberg C, Lockwood G, Little L, Martin L, Byng J, Yaffe M & Tritchler D 1997 Effects at two years of a low-fat, high-carbohydrate diet on radiologic features of the breast: results from a randomized trial. Journal of the National Cancer Institute 89 488–496.

    Boyd NF, Lockwood GA, Bying JW, Tritchler DL & Yaffe MJ 1998 Mammographic densities and breast cancer risk. Cancer Epidemiology Biomarkers and Prevention 7 1133–1144.

    Brown TA, Wall JW, Christensen ED, Smith DV, Holt CA, Carter PL, Patience TH, Babu SS & Williard WC 1998 Atypical hyperplasia in the era of stereotactic core needle biopsy. Journal of Surgical Oncology 67 168–173.

    Buerger H, Otterbach F, Simon R, Poremba C, Diallo R, Decker T, Riethdorf L, Brinkschmidt C, Dockhorn-Dworniczak B & Boecker W 1999 Comparative genomic hybridization of ductal carcinoma in situ of the breast-evidence of multiple genetic pathways. Journal of Pathology 187 396–402.

    Buerger H, Mommers EC, Littmann R, Simon R, Diallo R, Poremba C, Dockhorn-Dworniczak B, van Diest PJ & Boecker W 2001 Ductal invasive G2 and G3 carcinomas of the breast are the end stages of at least two different lines of genetic evolution. Journal of Pathology 194 165–170.

    Bundred NJ, Anderson E, Nicholson RI, Dowsett M, Dixon M& Robertson JF 2002 Fulvestrant, an estrogen receptor downregulator, reduces cell turnover index more effectively than tamoxifen. Anticancer Research 22 2317–2319.

    Carney PA, Miglioretti DL, Yankaskas BC, Kerlikowske K, Rosenberg R, Rutter CM, Geller BM, Abraham LA, Taplin SH, Dignam M, Cutter G & Ballard-Barbash R 2003 Individual and combined effects of age, breast density, and hormone replacement therapy use on the accuracy of screening mammography. Annals of Internal Medicine 138 168–175.

    Carter CL, Corle DK, Micozzi MS, Schatzkin A & Taylor PR 1988 A prospective study of the development of breast cancer in 16 692 women with benign breast disease. American Journal of Epidemiology 128 467–477.

    Cazzaniga M, Casadio C, Severi G, Chiapparini L, Mora S, Gall A, Veronesi U & Decensi A 2003 Relationships between breast cancer risk, nipple discharge, atypia and Ki67 expression in ductal lavage. Cancer Epidemiology Biomarkers and Prevention 11 1287S (abstract 127).

    Celis JE, Kruhoffer M, Gromova I, Frederiksen C, Ostergaard M, Thykjaer T, Gromov P, Yu J, Palsdottir H, Magnusson N & Orntoft TF 2000 Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics. FEBS Letters 480 2–16.

    Chamberlain C, Simonson M, Kennedy T, Hall M & Fabian CJ 2003 High risk women report minimal pain rating score with random periareolar fine-needle aspirations. Proceedings of the American Society of Clinical Oncology 22 95.

    Chang J, Powles TJ, Allred DC, Ashley SE, Makris A, Gregory RK, Osborne CK & Dowsett M 2000 Prediction of clinical outcome from primary tamoxifen by expression of biologic markers in breast cancer patients. Clinical Cancer Research 6 616–621.

    Chatterton RT Jr, Geiger AS, Khan SA, Helenowski IB, Jovanovic BD & Gann PH 2004 Variation in estradiol, estradiol precursors, and estrogen-related products in nipple aspirate fiuid from normal premenopausal women. Cancer Epidemiology Biomarkers and Prevention 13 928–935.

    Chlebowski RT, Collyar DE, Somerfield MR & P.ster DG 1999 American Society of Clinical technology assessment of breast cancer risk reduction strategies: tamoxifen and raloxifene. Journal of Clinical Oncology 17 1939–1955.

    Clarke RB, Howell A, Potten CS & Anderson E 1997 Dissociation between steroid receptor expression and cell proliferation in the human breast. Cancer Research 57 4987–4991.

    Comings DE, Gade-Andavolu R, Cone LA, Muhleman D & MacMurray JP 2003 A multigene test for the risk of sporadic breast carcinoma. Cancer 97 2160–2170.

    Conley B, O’Shaughnessy J, Prindiville S, Lawrence J, Chow C, Jones E, Merino MJ, Kaiser-Kupfer MI, Caruso RC, Podgor M et al. 2000 Pilot trial of the safety, tolerability, and retinoid levels of N-(4-hydroxyphenyl) retinamide in combination with tamoxifen in patients at high risk for developing invasive breast cancer. Journal of Clinical Oncology 18 275–283.

    Conner P, Soderqvist G, Skoog L, Graser T, Walter F, Tani E, Carlstrom K & von Schoultz B 2003 Breast cell proliferation in postmenopausal women during HRT evaluated through fine needle aspiration cytology. Breast Cancer Research and Treatment 78 159–165.

    Coughlin SS & Piper M 1999 Genetic polymorphisms and risk of breast cancer. Cancer Epidemiology Biomarkers and Prevention 8 1023–1032.

    Cummings SR, Eckert S, Krueger KA, Grady D, Powles TJ, Cauley JA, Norton L, Nickelsen T, Bjarnason NH, Morrow M et al. 1999 The effect of raloxifene on risk of breast cancer in postmenopausal women: Results from the MORE Randomized Trial. Multiple outcomes of raloxifene evaluation. Journal of the American Medical Association 281 2189–2197.

    Cuzick J, Forbes J, Edwards R, Baum M, Cawthorn S, Coates A, Hamed A, Howell A & Powles T 2002 First results from the International Breast Cancer Intervention Study (IBIS-I): a randomised prevention trial. Lancet 360 817–824.

    Cuzick J, Warwick J, Pinney E, Warren RM & Duffy SW 2004 Tamoxifen and breast density in women at increased risk of breast cancer. Journal of the National Cancer Institute 96 621–628.

    Czerwenka KF, Manavi M, Hosmann J, Jelincic D, Pischinger KI, Battistutti WB, Behnam M & Kubista E 2001 Comparative analysis of two-dimensional protein patterns in malignant and normal human breast tissue. Cancer Detection and Prevention 25 268–279.

    Das PM & Singal R 2004 DNA methylation and cancer. Journal of Clinical Oncology 22 4632–4642.

    Decensi A, Robertson C, Viale G, Pigatto F, Johansson H, Kisanga ER, Veronesi P, Torrisi R, Cazzaniga M, Mora S et al. 2003 A randomized trial of low-dose tamoxifen on breast cancer proliferation and blood estrogenic biomarkers. Journal of the National Cancer Institute 95 779–790.

    Decensi A, Bonanni B, Guerrieri-Gonzaga A, Robertson C, Cazzaniga M, Mariette F, Gulisano M, Latronico M, Franchi D & Johnson K 2004 A randomized 2x2 biomarker trial of low-dose tamoxifen and fenretinide in premenopausal women at-high risk for breast cancer. Proceedings of the American Society of Clinical Oncology 23 97 (abstract 1001).

    DeFriend DJ, Howell A, Nicholson RI, Anderson E, Dowsett M, Mansel RE, Blamey RW, Bundred NJ, Robertson JF & Saunders C 1994 Investigation of a new pure antiestrogen (ICI 182780) in women with primary breast cancer. Cancer Research 54 408–414.

    Didwania A, Golewale NH, Khan SA, Priyanath A, Gann P & Hou N 2003 Influence of ductal lavage(DL) findings on tamoxifen decision for high risk women. Breast Cancer Research and Treatment 82 S179 (abstract 1037).

    Dixon JM, Jackson J, Hills M, Renshaw L, Cameron DA, Anderson TJ, Miller WR & Dowsett M 2004 Anastrozole demonstrates clinical and biological effectiveness in oestrogen receptor-positive breast cancers, irrespective of the erbB2 status. European Journal of Cancer 40 2742–2747.

    Dooley WC, Ljung BM, Veronesi U, Cazzaniga M, Elledge RM, O’Shaughnessy JA, Kuerer HM, Hung DT, Khan SA, Phillips RF et al. 2001 Ductal lavage for detection of cellular atypia in women at high risk for breast cancer. Journal of the National Cancer Institute 93 1624–1632.

    Dowsett M, Dixon JM, Horgan K, Salter J, Hills M & Harvey E 2000 Antiproliferative effects of idoxifene in a placebo-controlled trial in primary human breast cancer. Clinical Cancer Research 6 2260–2267.

    Dowsett M, Bundred NJ, Decensi A, Sainsbury RC, Lu Y, Hills MJ, Cohen FJ, Veronesi P, O’Brien ME, Scott T & Muchmore DB 2001 Effect of raloxifene on breast cancer cell Ki67 and apoptosis: a double-blind, placebocontrolled, randomized clinical trial in postmenopausal patients. Cancer Epidemiology Biomarkers and Prevention 10 961–966.

    Dupont WD & Page DL 1985 Risk factors for breast cancer in women with proliferative breast disease. New England Journal of Medicine 312 146–151.

    Dupont WD, Parl FF, Hartmann WH, Brinton LA, Winfleld AC, Worrell JA, Schuyler PA & Plummer WD 1993 Breast cancer risk associated with proliferative breast disease and atypical hyperplasia. Cancer 71 1258–1265.

    Elia M, Handpour S, Terranova P, Anderson J, Klemp JR & Fabian CJ 2002 Marked variation in nipple aspirate fluid (NAF) estrogen concentration and NAF/serum ratios between ducts in high risk women. Proceedings of the Ameerican Association for Cancer Research 43 820 (abstract 4072).

    Ellis M, Davis N, Coop A, Liu M, Schumaker L, Lee RY, Srikanchana R, Russell CG, Singh B, Miller WR et al. 2002 Development and validation of a method for using breast core needle biopsies for gene expression microarray analyses. Clinical Cancer Research 8 1155–1166.

    Evron E, Dooley WC, Umbricht CB, Rosenthal D, Sacchi N, Gabrielson E, Soito AB, Hung DT, Ljung B, Davidson NE & Sukumar S 2001 Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR. Lancet 357 1335–1336.

    Fabian CJ, Zalles C, Kamel S, Kimler BF, McKittrick R, Tranin AS, Zeiger S, Moore WP, Hassanein RS, Simon C et al. 1994 Prevalence of aneuploidy, overexpressed ER, and overexpressed EGFR in random breast aspirates of women at high and low risk for breast cancer. Breast Cancer Research and Treatment 30 263–274.

    Fabian CJ, Kimler BF, Elledge RM, Grizzle WE, Beenken SW & Ward JH 1998 Models for early chemoprevention trials in breast cancer. Hematology/Oncology Clinics of North America 12 993–1017.

    Fabian CJ, Kimler BF, Zalles CM, Klemp JR, Kamel S, Zeiger S & Mayo MS 2000 Short-term breast cancer prediction by random periareolar fine-needle aspiration cytology and the Gail risk model. Journal of the National Cancer Institute 92 1217–1227.

    Fabian CJ, Kimler BF, Brady DA, Mayo MS, Chang CHJ, Ferraro JA, Zalles CM, Stanton AL, Masood S, Grizzle WE et al. 2002 A phase II breast cancer chemoprevention trial of oral alphadi fluoromethylornithine: breast tissue, imaging, and serum and urine biomarkers. Clinical Cancer Research 8 3105–3117.

    Fabian CJ, Petroff BK, Kimler BF, Clark J, Metheny T, Stecker K, Salunga R & Erlander MG 2003 Quantitative RT-PCR to assess change in molecular expression in methanol-formalin fixed cytology specimens obtained by random periareolar FNA from women on phase II breast cancer chemoprvention trials. Cancer Epidemiology Biomarkers & Prevention 12 1301S (abstract A194).

    Fabian CJ, Kimler BF, Anderson J, Tawfik OW, Mayo MS, Burak Jr WE, O’Shaughnessy JA, Albain KS, Hyams DM, Budd GT et al. 2004a Breast cancer chemoprevention phase I evaluation of biomarker modulation by arzoxifene, a third generation selective estrogen receptor modulator. Clinical Cancer Research 10 5403–5417.

    Fabian CJ, Kimler BF, Simonsen S, Metheny T, Zalles CM & Hall M 2004b Reduction in epithelial proliferation after six months of letrozole in high risk women on HRT with evidence of RPFNA atypia. Breast Cancer Research Treatment Supplement 1 88 S8 (abstract 5).

    Fabian CJ, Kimler BF, Mayo MS, Grizzle WE, Masood S & Ursin G 2005 Clinical approaches to the discovery and testing of new breast cancer prevention drugs. In Cancer Chemoprevention, vol 2, Strategies for Cancer Chemoprevention, pp 213–238. Eds GJ Kelloff, ET Hawk, CC Sigman. Totowa, NJ: Humana Press.

    Fackler MJ, McVeigh M, Mehrotra J, Blum MA, Lange J, Lapides A, Garrett E, Argani P & Sukumar S 2004 Quantitative multiplex methylation-specific PCR assay for the detection of promoter hypermethylation in multiple genes in breast cancer. Cancer Research 64 4442–4452.

    Feigelson HS, McKean-Cowdin R, Coetzee GA, Stram DO, Kolonel LN & Henderson BE 2001 Building a multigenic model of breast cancer susceptibility: CYP17 and HSD17B1 are two important candidates. Cancer Research 61 785–789.

    Ferguson AT, Evron E, Umbricht CB, Pandita TK, Chan TA, Hermeking H, Marks JR, Lambers AR, Futreal PA, Stampfer MR & Sukumar S 2000 High frequency of hypermethylation at the 14-13-3 sigma locus leads to gene silencing in breast cancer. PNAS 97 6049–6054.

    Fish KJ, Cegielska A, Getman ME, Landes GM & Virshup DM 1995 Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family. Journal of Biological Chemistry 270 14875–14883.

    Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M, Cronin WM, Vogel V, Robidoux A, Dimitrov N, Atkins J et al. 1998 Tamoxifen for prevention of breast cancer: Report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. Journal of the National Cancer Institute 90 1371–1388.

    Fitzgibbons PL, Henson DE & Hutter RV 1998 Benign breast changes and the risk for subsequent breast cancer: an update of the 1985 consensus statement. Cancer Committee of the College of American Pathologists. Archives of Pathology and Laboratory Medicine 122 1053–1055.

    Francescatti DS, Kluskens L & Shah L 2004 Ductal lavage using medically aseptic technique in women at high risk for breast cancer. Clinical Breast Cancer 5 299–302.

    Freedman AN, Graubard BI, Rao SR, McCaskill-Stevens W, Ballard-Barbash R & Gail MH 2003 Estimates of the number of US women who could benefit from tamoxifen for breast cancer chemoprevention. Journal of the National Cancer Institute 95 526–532.

    Fujii H, Biel MA, Zhou W, Weitzman SA, Baylin SB & Gabrielson E 1998 Methylation of the HIC-1 candidate tumor suppressor gene in human breast cancer. Oncogene 16 2159–2164.

    Gail MH, Briton LA, Byar DP, Corle DK, Green SB, Schair C & Mulvihill JJ 1989 Projecting individualized probabilities of developing breast cancer for white females who are being examined annually. Journal of the National Cancer Institute 81 1879–1886.

    Ganz PA, Desmond KA, Leedham B, Rowland JH, Meyerowitz BE & Belin TR 2002 Quality of life in long-term, disease-free survivors of breast cancer: a follow-up study. Journal of the National Cancer Institute 94 39–49.

    Geisler J, Detre S, Berntsen H, Ottestad L, Lindtjorn B, Dowsett M & Einstein Lonning P 2001 Influence of neoadjuvant anastrozole (Arimidex) on intratumoral estrogen levels and proliferation markers in patients with locally advanced breast cancer. Clinical Cancer Research 7 1230–1236.

    Ginestier C, Charafe-Jauffret E, Bertucci F, Eisinger F, Geneix J, Bechlian D, Conte N, Adelaide J, Toiron Y, Nguyen C et al. 2002 Distinct and complementary information provided by use of tissue and DNA microarrays in the study of breast tumor markers. American Journal of Pathology 161 1223–1233.

    Going JJ & Moffat DF 2004 Escaping from Flatland: clinical and biological aspects of human mammary duct anatomy in three dimensions. Journal of Pathology 203 538–544.

    Golewale NH, Bryk M, Nayar R, Didwania A, Hou N & Khan SA 2003 Technical modifications of ductal lavage to improve cell yield. Breast Cancer Research and Treatment Supplement 1 82 S175 (abstract 1024).

    Gong G, DeVries S, Chew KL, Cha I, Ljung BM & Waldman FM 2001 Genetic changes in paired atypical and usual ductal hyperplasia of the breast by comparative genomic hybridization. Clinical Cancer Research 7 2410–2414.

    Gornstein B, Jacobs T, Bedard Y, Biscotti C, Ducatman B, Layfleld L, McKee G, Sneige N & Wang H 2004 Interobserver agreement of a probabilistic approach to reporting breast fine-needle aspirations on ThinPrep. Diagnostic Cytopathology 30 389–395.

    Grizzle WE, Meyers RB & Oelschlager DK 1995 Prognostic biomarkers in breast cancer: factors affecting immunohistochemical evaluation. The Breast Journal 1 243–250.

    Grizzle WE, Myers RB, Manne U & Srivastava S 1998 Immunohistochemical Evaluation of Biomarkers in Prostatic and Colorectal Neoplasia. In John Walker’s Methods in Molecular Medicine-Tumor Marker Protocols, pp 143–160. Eds M Hanausek & Z Walaszek. Totowa, NJ: Humana Press.

    Gygi SP, Rochon Y, Franza BR & Aebersold R 1999 Correlation between protein and mRNA abundance in yeast. Molecular and Cellular Biology 19 1720–1730.

    Hankinson SE, Willett WC, Colditz GA, Hunter DJ, Michaud DS, Deroo B, Rosner B, Speizer FE & Pollak M 1998 Circulating concentrations of insulin-like growth factor-I and risk of breast cancer. Lancet 351 1393–1396.

    Harper-Wynne C, Ross G, Sacks N, Salter J, Nasiri N, Iqbal J, A’Hern R & Dowsett M 2002 Effects of the aromatase inhibitor letrozole on normal breast epithelial cell proliferation and metabolic indices in postmenopausal women: a pilot study for breast cancer prevention. Cancer Epidemiology Biomarkers and Prevention 11 614–621.

    Hershman D, Sundararajan V, Jacobson JS, Heitjan DF, Neugut AI & Grann VR 2002 Outcomes of tamoxifen chemoprevention for breast cancer in very high-risk women: a cost-effectiveness analysis. Journal of Clinical Oncology 20 9–16.

    Holst CR, Nuovo GJ, Esteller M, Chew K, Baylin SB, Herman JG & Tlsty TD 2003 Methylation of p16(INK4a) promoters occurs in vivo in histologically normal human mammary epithelia. Cancer Research 63 1596–1601.

    Hoogerbrugge N, Bult P, deWidt-Levert LM, Beex LV, Kiemeney LA, Ligtenberg MJ, Massuger LF, Boetes C, Manders P & Brunner HG 2003 High prevalence of premalignant lesions in prophylactically removed breasts from women at hereditary risk for breast cancer. Journal of Clinical Oncology 21 41–45.

    Hudelist G, Pacher-Zavisin M, Singer CF, Holper T, Kubista E, Schreiber M, Manavi M, Bilban M & Czerwenka K 2004 Use of high-throughput protein array for profiling of differentially expressed proteins in normal and malignant breast tissue. Breast Cancer Research and Treatment 86 281–291.

    Humphrey LL, Helfand M, Chan BK & Woolf SH 2002 Breast cancer screening: a summary of the evidence for the U.S. Preventive Services Task Force. Annals of Internal Medicine 137 347–360.

    Huschtscha LI, Noble JR, Neumann AA, Moy EL, Barry P, Melki JR, Clark SJ & Reddel RR 1998 Loss of p16INK4 expression of methylation is associated with lifespan extension of human mammary epithelial cells. Cancer Research 58 3508–3512.

    Hutchinson WB, Thomas DB, Hamlin WB, Roth GJ, Peterson AV & Williams B 1980 Risk of breast cancer in women with benign breast disease. Journal of the National Cancer Institute 65 13–20.

    Jeffrey SS & Pollack JR 2003 The diagnosis and management of pre-invasive breast disease: promise of new technologies in understanding pre-invasive breast lesions. Breast Cancer Research 5 320–328.

    Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ & Thun MJ 2005 Cancer statistics 2005. CA A Cancer Journal for Clinicians 55 10–30.

    Kelloff GJ, Boone CW, Steele VE, Crowell JA, Lubet R & Sigman CC 1994 Progress in cancer chemoprevention: perspectives on agent selection and short-term clinical intervention trials. Cancer Research Supplement 54 2015S–2024S.

    Kelloff GJ, Boone CW, Crowell JA, Nayfield SG, Hawk E, Malone WF, Steele VE, Lubet RA & Sigman CC 1996 Risk biomarkers and current strategies for cancer chemoprevention. Journal of Cellular Biochemistry 25 1–14.

    Keshgegian AA & Cnaan A 1995 Proliferation markers in breast carcinoma. Mitotic figure count, S-phase fraction, proliferating cell nuclear antigen, Ki-67 and MIB-1. American Journal of Clinical Pathology 104 42–49.

    Khan QJ, Kimler BF, Clark J, Metheny T, Zalles CM & Fabian CJ 2005 Ki-67 Expression in benign breast ductal cells obtained by random periareolar fine needle aspiration. Cancer Epidemiology Biomarkers and Prevention 14 786–789.

    Khan SA, Masood S, Miller L & Numann PJ 1998 Random fine needle aspiration of the breast of women at increased breast cancer risk, and standard risk controls. The Breast Journal 4 409–419.

    Khan SA, Sachdeva A, Naim S, Meguid MM, Marx W, Simon H, Halverson JD & Numann PJ 1999 The normal breast epithelium of women with breast cancer displays an aberrant response to estradiol. Cancer Epidemiology Biomarkers and Prevention 25 867–872.

    Khan SA, Baird C, Staradub VL & Morrow M 2002 Ductal lavage and ductoscopy: the opportunities and the limitations. Clinical Breast Cancer 3 185–191.

    Khan SA, Wiley EL, Rodriguez N, Baird C, Ramakrishan R, Nayar R, Bryk M, Bethke KB, Staradub VL, Wolfman J, Rademaker A, Ljung B-M & Morrow M 2004 Ductal lavage findings in women with known breast cancer undergoing mastectomy. Journal of the National Cancer Institute 96 1510–1517.

    Khan SA, Wolfman JA, Segal L, Benjamin S, Nayar R, Wiley EL, Bryk M & Morrow M 2005 Ductal lavage findings in women with mammographic microcalcifications undergoing biopsy. Annals of Surgical Oncology (in press).

    King EB, Chew KL, Hom JD, Miike R, Wrensch MR & Petrakis NL 2004 Multiple sampling for increasing the diagnostic sensitivity of nipple aspirate fluid for atypical cytology. Acta Cytologica 48 813–817.

    Kinsinger LS, Harris R, Woolf SH, Sox HC & Lohr KN 2002 Chemoprevention of breast cancer: a summary of the evidence for the US Preventive Services Task Force. Annals of Internal Medicine 137 59–69.

    Kisanga ER, Gjerde J, Guerrieri-Gonzaga A, Pigatto F, Pesci-Feltri A, Robertson C, Serrano D, Pelosi G, Decensi A & Lien EA 2004 Tamoxifen and metabolite concentrations in serum and breast cancer tissue during three dose regimens in a randomized preoperative trial. Clinical Cancer Research 10 2336–2343.

    Klein P, Glaser E, Grogan L, Keane M, Lipkowitz S, Soballe P, Brooks L, Jenkins J, Steinberg SM, DeMarini DM & Kirsch I 2001 Biomarker assays in nipple aspirate fluid. The Breast Journal 7 378–387.

    Krassenstein R, Sauter E, Dulaimi E, Battagli C, Ehya H, Klein-Szanto A & Cairns P 2004 Detection of breast cancer in nipple aspirate fluid by CpG island hypermethylation. Clinical Cancer Research 10 28–32.

    Kriege M, Brekelmans CT, Boetes C, Besnard PE, Zonderland HM, Obdeijn IM, Besnard PE, Zonderland HM, Obdeijn IM, Manoliu RA et al. 2004 Magnetic Resonance Imaging Screening Study Group. Efficacy of MRI and mammography for breast-cancer screening in women with a familial or genetic predisposition. New England Journal of Medicine 351 427–437.

    Kuehn T, Klauss W, Darsow M, Regele S, Flock F, Maiterth C, Dahlbender R, Wendt I & Kreienberg R 2000 Long-term morbidity following axillary dissection in breast cancer patients–clinical assessment, significance for life quality and the impact of demographic, oncologic and therapeutic factors. Breast Cancer Research and Treatment 64 275–286.

    Kurian AW, Daniel BL, Mills MA, Nowels KW, Ford JM, Plevritis SK, Kingham KE, Chun NM, Herfkens RJ, Dirbas FM et al. 2004 A pilot breast cancer screening trial for women at high inherited risk using clinical breast exam, mammography, breast magnetic resonance imaging, and ductal lavage: updated results after median follow-up of fourteen months. Breast Cancer Research and Treatment Supplement 1 88 S187 (abstract 5013).

    Lakhani SR, Collins N, Stratton MR & Sloane JP 1995 Atypical ductal hyperplasia of the breast: clonal proliferation with loss of heterozygosity on chromosomes 16q and 17p. Journal of Clinical Pathology 48 611–615.

    Lehmann U, Langer F, Feist H, Glockner S, Hasemeier B & Kreipe H 2002 Quantitative assessment of promoter hypermethylation during breast cancer development. American Journal of Pathology 160 605–612.

    Levine M, Moutquin JM, Walton R, Feightner J & Canadian Task Force on Preventive Health Care and the Canadian Breast Cancer Initiative’s Steering Committee on Clinical Practice Guidelines for the Care and Treatment of Breast Cancer 2001 Chemoprevention of breast cancer. A joint guideline from the Canadian Task Force on Preventive Health Care and the Canadian Breast Cancer Initiative’s Steering Committee on Clinical Practice Guidelines for the Care and Treatment of Breast Cancer. Canadian Medical Association Journal 164 1681–1690.

    Lieberman L, Cohen MA, Dershaw DD, Abramson AF, Hann LE & Rosen PP 1995 Atypical ductal hyperplasia diagnosed at stereotaxic core biopsy of breast lesions: an indication for surgical biopsy. AJR American Journal of Roentgenology 164 1111–1113.

    Ljung BM, Chew KL, Moore DH 2nd & King EB 2004 Cytology of ductal lavage fluid of the breast. Diagnostic Cytopathology 30 143–150.

    London SJ, Connolly JL, Schnitt SJ & Colditz GA 1992 A prospective study of benign breast disease and the risk of breast cancer. Journal of the American Medical Association 267 941–944.

    Love SM & King BL 2004 Ductal lavage in nipple aspirate fluid producing and non-producing ducts in healthy volunteers. Breast Cancer Research and Treatment Supplement 1 88 S189–S190 (abstract 5019).

    Ma XJ, Salunga R, Tuggle JT, Gaudet J, Enright E, McQuary P, Payette T, Pistone M, Stecker K, Zhang BM et al. 2003 Gene expression profiles of human breast cancer progression. PNAS 100 5974–5979.

    Makris A, Powles TJ, Allred DC, Ashley S, Ormerod MG, Titley JC & Dowsett M 1998 Changes in hormone receptors and proliferation markers in tamoxifen treated breast cancer patients and the relationship with response. Breast Cancer Research and Treatment 48 11–20.

    Mansoor S, Ip C & Stomper PC 2000 Yield of terminal ductal lobule units in normal breast stereotactic core biopsy specimens: implications of biomarker studies. The Breast Journal 6 220–224.

    Masood S, Frykberg ER, McLellan GL, Scalapino MC, Mitchum DG & Bullard JB 1990 Prospective evaluation of radiologically directed fine-needle aspiration biopsy of nonpalpable breast lesions. Cancer 66 1480–1487.

    McDivitt RW, Stevens JA, Lee NC, Wingo PA, Rubin GL & Gersell D 1992 Histologic types of benign breast disease and the risk for breast cancer. The Cancer and Steroid Hormone Study Group. Cancer 69 1408–1414.

    Missmer SA, Eliassen AH, Barbieri RL & Hankinson SE 2004 Endogenous estrogen, androgen, and progesterone concentrations and breast cancer risk among postmenopausal women. Journal of the National Cancer Institute 96 1856–1865.

    Modan B, Lubin F, Alfandary E, Cheritrit A & Papa M 1997 Breast cancer following benign breast disease-a nationwide study. Breast Cancer Research and Treatment 46 45.

    Mohsin SK, Elledge RM, Arun B, Miller A, Wu K, Johnson K, Lamph WW & Brown PH 2003 Breast cancer prevention using RXR selective retinoid (Targretin) in high risk women – initial report of a phase II randomized clinical trial. Breast Cancer Research and Treatment Supplement 1 82 S176 (abstract 1027).

    Moore C, Bean G, Ratliff B, Kimler B, Fabian C, Zalles C & Seewaldt V 2004 Can RARbeta P2 promoter methylation in breast random periareolar fine-needle aspiration serve to risk stratify women at high risk for breast cancer Breast Cancer Research and Treatment 88 S162 (abstract 4040).

    Morrow M, Vogel V, Ljung BM & O’Shaughnessy JA 2002 Evaluation and management of the woman with an abnormal ductal lavage. Journal of the American College of Surgeons 194 648–656.

    Nathanson KL, Wooster R, Weber BL & Nathanson KN 2001 Breast cancer genetics: what we know and what we need. Nature Medicine 7 552–556.

    Neumeister P, Albanese C, Balent B, Greally J & Pestell RG 2002 Senescence and epigenetic dysregulation in cancer. International Journal of Biochemistry and Cell Biology 34 1475–1490.

    Nielsen M, Thomsen JL, Primdahl S, Dyreborg U & Andersen JA 1987 Breast cancer and atypia among young and middle-aged women: a study of 110 medicolegal autopsies. British Journal of Cancer 56 814–819.

    Noga CM, Brainard JA, Dietz JR 2002 Ductoscopy in patients with pathologic nipple discharge: correlation of ductal lavage and duct excision findings. Laboratory Investigations 82 83 (abstract).

    Nuovo GJ, Plaia TW, Belinsky SA, Baylin SB & Herman JG 1999 In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis. PNAS 96 12754–12759.

    O’Connell P, Pekkel V, Fuqua SA, Osborne CK, Clark GM & Allred DC 1998 Analysis of loss of heterozygosity in 399 premalignant breast lesions at 15 genetic loci. Journal of the National Cancer Institute 90 697–703.

    O’Shaughnessy JA, Ljung BM, Dooley WC, Chang J, Kuerer HM, Hung DT, Grant MD, Khan SA, Phillips RF, Duvall K et al. 2002 Ductal lavage and the clinical management of women at high risk for breast carcinoma: a commentary. Cancer 94 292–298.

    Ottesen GL, Graversen HP, Blichert-Toft M, Zedeler K & Andersen JA 1993 Lobular carcinoma in situ of the female breast. Short-term results of a prospective nationwide study. The Danish Breast Cancer Cooperative Group. American Journal of Surgical Pathology 17 14–21.

    Ozanne EM & Esserman LJ 2004 Evaluation of breast cancer risk assessment techniques: a cost-effectiveness analysis. Cancer Epidemiology Biomarkers and Prevention 13 2043–2052.

    Page DL & Dupont WD 1990 Anatomic markers of human premalignancy and risk of breast cancer. Cancer 66 1326–1335.

    Page DL, Dupont WD, Rogers LW & Rados MS 1985 Atypical hyperplastic lesions of the female breast. A long-term follow-up study. Cancer 55 2698–2708.

    Page DL, Kidd Jr TE, Dupont WD, Simpson JF & Rogers LW 1991 Lobular neoplasia of the breast: higher risk for subsequent invasive cancer by more extensive disease. Human Pathology 22 1232–1239.

    Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, Baehner FL, Walker MG, Watson D, Park T et al. 2004 A multigene assay to predict recurrence of tamoxifentreated, node-negative breast cancer. New England Journal of Medicine 351 2817–2826.

    Palomares MR, Hopper L, Goldstein L, Lehman CD, Lampe JW, Storer BE & Gralow JR 2004 Effect of soy isoflavones on breast proliferation in postmenopausal breast cancer survivors. Breast Cancer Research and Treatment 88 S149 (abstract 4002).

    Paweletz CP, Trock B, Pennanen M, Tsangaris T, Magnant C, Liotta LA & Petricoin 3rd EF 2001 Proteomic patterns of nipple aspirate fluids obtained by SELDI-TOF: potential for new biomarkers to aid in the diagnosis of breast cancer. Disease Markers 17 301–307.

    Pawlik TM, Fritsche H, Coombes KR, Xiao L, Krishnamurthy S, Hunt KK, Pusztai L, Chen J-N, Clarke CH, Arun B, Hung M-C & Kuerer HM 2004 Significant differences in ductal fluid protein expression in healthy women versus those with breast cancer identified by timeof-fiight mass spectrometry. Breast Cancer Research and Treatment 88 S189 (abstract 5018).

    Peto J, Collins N, Barfoot R, Seal S, Warren W, Rahman N, Easton DF, Evans C, Deacon J & Stratton MR 1999 Prevalence of BRCA1 and BRCA2 gene mutations in patients with early-onset breast cancer. Journal of the National Cancer Institute 91 943–949.

    Petroff BK, Gum SL, Phillips TA, Clark JS, Kimler BF & Fabian CJ 2004 Assessment of risk biomarker gene expression by real time reverse transcription-polymerase chain reaction using amplified ribonucleic acids from formalin-.xed fine needle aspirates of human breast tissue: A preliminary report. Cancer Epidemiology Biomarkers and Prevention 13 1921S (abstract C61).

    Pharoah PD, Antoniou A, Bobrow M, Zimmern RL, Easton DF & Ponder BA 2002 Polygenic susceptibility to breast cancer and implications for prevention. Nature Genetics 31 33–36.

    Port ER, Montgomery LL, Heerdt AS & Borgen PI 2001 Patient reluctance toward tamoxifen use for breast cancer primary prevention. Annals of Surgical Oncology 8 580–585.

    Potten CS, Watson RJ, Williams GT, Tickle S, Roberts SA, Harris M & Howell A 1998 The effect of age and menstrual cycle upon proliferative activity of the normal human breast. British Journal of Cancer 58 163–170.

    Rebbeck TR 2002 The contribution of inherited genotype to breast cancer. Breast Cancer Research 4 85–89.

    Reis-Filho JS & Lakhani SR 2003 The diagnosis and management of pre-invasive breast disease: genetic alterations in pre-invasive lesions. Breast Cancer Research 5 313–319.

    Robertson JF, Nicholson RI, Bundred NJ, Anderson E, Rayter Z, Dowsett M, Fox JN, Gee JM, Webster A, Wakeling AE, Morris C & Dixon M 2001 Comparison of the short-term biological effects of 7alpha-[9-(4,4,5,5,5- pentafluoropentylsulfinyl)-nonyl]estra-1,3,5,(10)-triene- 3,17beta-diol (Faslodex) versus tamoxifen in postmenopausal women with primary breast cancer. Cancer Research 61 6739–6746.

    Rockhill B, Spiegelman D, Byrne C, Hunter DJ & Colditz GA 2001 Validation of the Gail et al. Model of breast cancer risk prediction and implications for chemoprevention. Journal of the National Cancer Institute 93 358–366.

    Rosai J 1991 Borderline epithelial lesions of the breast. American Journal of Pathology 15 209–221.

    Sartippour MR, Rao JY, Apple S, Wu D, Henning S, Wang H, Elashoff R, Rubio R, Heber D & Brooks MN 2004 A pilot clinical study of short-term isoflavone supplements in breast cancer patients. Nutrition and Cancer 49 59–65.

    Sartorius OW, Smith HS, Morris P, Benedict D & Friesen L 1977 Cytologic evaluation of breast fluid in the detection of breast disease. Journal of the National Cancer Institute 59 1073–1080.

    Sauter ER, Daly M, Linahan K, Ehya H, Engstrom PF, Bonney G, Ross EA, Yu H & Diamandis E 1996 Prostatespecific antigen levels in nipple aspirate fluid correlate with breast cancer risk. Cancer Epidemiology Biomarkers and Prevention 5 967–970.

    Sauter ER, Ross E, Daly M, Klein-Szanto A, Engstrom PF, Sorling A, Malick J & Ehya H 1997 Nipple aspirate fluid: a promising non-invasive method to identify cellular markers of breast cancer risk. British Journal of Cancer 76 494–501.

    Sauter ER, Zhu W, Fan XJ, Wassell RP, Chervoneva I & Du Bois GC 2002 Proteomic analysis of nipple aspirate fluid to detect biologic markers of breast cancer. British Journal of Cancer 86 1440–1443.

    Sauter ER, Shan S, Hewett JE, Speckman P & Du Bois GC 2005 Proteomic analysis of nipple aspirate fluid using SELDI-TOF-MS. International Journal of Cancer 114 791–796.

    Schnitt SJ, Connolly JL, Tavassoli FA, Fechner RE, Kempson RL, Gelman R & Page DL 1992 Interobserver reproducibility in the diagnosis of ductal proliferative breast lesions using standardized criteria. American Journal of Surgical Pathology 16 1133–1143.

    Sharma P, Klemp JR, Simensen M, Welsko CM, Zalles CM, Kimler BF & Fabian CJ 2004 Failure of high risk women to produce nipple aspirate fluid does not exclude detection of cytologic atypia in random periareolar fine needle aspiration specimens. Breast Cancer Research and Treatment 87 59–64.

    Shoker BS, Jarvis C, Clarke RB, Anderson E, Hewlett J, Davies MP, Sibson DR & Sloane JP 1999 Estrogen receptor-positive proliferating cells in the normal and precancerous breast. American Journal of Pathology 155 1811–1815.

    Sidawy MK, Stoler MH, Frable WJ, Frost AR, Masood S, Miller TR, Silverberg SG, Sneige N & Wang HH 1998 Interobserver variability in the classification of proliferative breast lesions by fine-needle aspiration: results of the Papanicolaou Society of Cytopathology Study. Diagnostic Cytopathology 18 150–165.

    Singletary E, Lieberman R, Atkinson N, Sneige N, Sahin A, Tolley S, Colchin M, Bevers T, Stelling C, Fornage B et al. 2000 Novel translational model for breast cancer chemoprevention study: accrual to a presurgical intervention with tamoxifen and N-[4-hydroxyphenyl] retinamide. Cancer Epidemiology Biomarkers and Prevention 9 1087–1090.

    Skolnick MH, Cannon-Albright LA, Goldgar DE, Ward JH, Marshall CJ, Schumann GB, Hogle H, McWhorter WP, Wright EC & Tran TD 1990 Inheritance of proliferative breast disease in breast cancer kindreds. Science 250 1715–1720.

    Sneige N 2004 Utility of cytologic specimens in the evaluation of prognostic and predictive factors of breast cancer: current issues and future directions. Diagnostic Cytopathology 30 158–165.

    Soderqvist G, von Schoultz B, Tani E & Skoog L 1993 Estrogen and progesterone receptor content in breast epithelial cells from healthy women during the menstrual cycle. American Journal of Obstetrics and Gynecology 168 874–879.

    Soderqvist G, Isaksson E, von Schoultz B, Carlstrom K, Tani E & Skoog L 1997 Proliferation of breast epithelial cells in healthy women during the menstrual cycle. American Journal of Obstetrics and Gynecology 176 123–128.

    Stanton AL, Krishnan L & Collins CA 2001 Form or function Part 1. Subjective cosmetic and functional correlates of quality of life in women treated with breast-conserving surgical procedures and radiotherapy. Cancer 91 2273–2281.

    Stearns V, Gallagher A, Kleer CG, Singh B, Freedman M, Haddad BR, Isaacs C, Warren R, Brown M, Cullen J, Trock B & Hayes DF 2004 A pilot study to establish a clinical model to perform phase II studies of breast cancer chemopreventive agents in women at high risk with biomarkers as surrogate endpoints for activity. Clinical Cancer Research 10 8332–8340.

    Sukumar S, Fackler MJ, Argani P, Garrett-Mayer E & Lange J 2004 Methylated genes as risk assessment markers for breast cancer. Breast Cancer Research and Treatment Supplement 1 88 S2–S3 (abstract MS 1–1).

    Tan-Chiu E, Wang J, Costantino JP, Paik S, Butch C, Wickerham DL, Fisher B & Wolmark N 2003 Effects of tamoxifen on benign breast disease in women at high risk for breast cancer. Journal of the National Cancer Institute 95 302–307.

    Tavassoli FA & Norris HJ 1990 A comparison of the results of long-term follow-up for atypical intraductal hyperplasia and intraductal hyperplasia of the breast. Cancer 65 518–529.

    Tchou J, Hou N, Rademaker A, Jordan VC & Morrow M 2004 Acceptance of tamoxifen chemoprevention by physicians and women at risk. Cancer 100 1800–1806.

    Tice JA, Miike R, Adduci K, Petrakis NL & Wrensch MR 2004a Improving risk prediction for breast cancer: Does nipple aspiration fluid cytology enhance the Gail model Proceedings of the American Association for Cancer Research 45 1084 (abstract).

    Tice JA, Ziv E & Kerlikowske KM 2004b Mammographic breast density combined with the Gail model for breast cancer risk prediction. Breast Cancer Research and Treatment Supplement 1 88 S11 (abstract 13).

    Tlsty TD, Romanov SR, Kozakiewicz BK, Holst CR, Haupt LM & Crawford YG 2001 Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence. Journal of Mammary Gland Biology and Neoplasia 6 235–243.

    Troch M, Ratliff B, Kimler B, Fabian C, Yee L & Seewaldt V 2003 RAR P2 promoter methylation in breast random fine-needle aspiration: a biologically relevant marker for breast cancer chemoprevention. Cancer Epidemiology Biomarkers & Prevention 12 1334S (abstract C122).

    Tworoger SS, Missmer SA, Barbieri RL, Willett WC, Colditz GA & Hankinson SE 2005 Plasma sex hormone concentrations and subsequent risk of breast cancer among women using postmenopausal hormones. Journal of the National Cancer Institute 97 595–602.

    Tyrer J, Duffy SW & Cuzick J 2004 A breast cancer prediction model incorporating familial and personal risk factors. Statistics in Medicine 23 1111–1130.

    Uniform Approach 1997 The uniform approach to breast fine-needle aspiration biopsy. National Cancer Institute Fine-Needle Aspiration of Breast Workshop Subcommittees. Diagnostic Cytopathology 16 295–311.

    Umbricht CB, Evron E, Gabrielson E, Ferguson A, Marks J & Sukumar S 2001 Hypermethylation of 14-13-3 sigma (stratifin) is an early event in breast cancer. Oncogene 20 3348–3353.

    Urban D, Myers R, Manne U, Weiss H, Mohler J, Perkins D, Markiewicz M, Lieberman R, Kelloff G, Marshall M & Grizzle W 1999 Evaluation of biomarker modulation by fenretinide in prostate cancer patients. European Urology 35 429–438.

    Van Gelder RN, von Zastrow ME, Yool A, Dement WC, Barchas JD & Eberwine JH 1990 Amplified RNA synthesized from limited quantities of heterogeneous cDNA. PNAS 87 1663–1667.

    Vogel VG 2004 Atypia in the assessment of breast cancer risk: implications for management. Diagnostic Cytopathology 30 151–157.

    Vogel VG, Costantino JP, Wickerham DL & Cronin WM 2002 Re: tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. Journal of the National Cancer Institute 94 1504.

    Wang J, Costantino JP, Tan-Chiu E, Wickerham DL, Paik S & Wolmark N 2004 Lower-category benign breast disease and the risk of invasive breast cancer. Journal of the National Cancer Institute 96 616–620.

    Wellings SR & Jensen HM 1973 On the origin and progression of ductal carcinoma in the human breast. Journal of the National Cancer Institute 50 1111–1118.

    Wellings SR, Jensen HM & Marcum RG 1975 An atlas of subgross pathology of the human breast with special reference to possible precancerous lesions. Journal of the National Cancer Institute 55 231–273.

    Widschwendter M & Jones PA 2002a DNA methylation and breast carcinogenesis. Oncogene 21 5462–5482.

    Widschwendter M & Jones PA 2002b The potential prognostic, predictive, and therapeutic values of DNA methylation in cancer. Commentary re: J. Kwong et al., Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin. Cancer Res., 8: 131–137 2002, and H-Z. Zou et al. Detection of aberrant p16 methylation in the serum of colorectal cancer patients. Clin. Cancer Res., 8: 188–191 2002. Clinical Cancer Research 8 17–21.

    Widschwendter M, Berger J, Daxewnbichler G, Muller- Holzner E, Widschwendter A, Mayr A, Marth C & Zeimet AG 1997 Loss of retinoic acid receptor B expression in breast cancer and normal adjacent tissue but not in normal tissue distinct from the cancer. Cancer Research 57 4158–4161.

    Wrensch MR, Petrakis NL, Gruenke LD, Ernster VL, Miike R, King EB & Hauck WW 1990 Factors associated with obtaining nipple aspirate fluid: analysis of 1428 women and literature review. Breast Cancer Research and Treatment 15 39–51.

    Wrensch MR, Petrakis NL, King EB, Miike R, Mason L, Chew KL, Lee MM, Ernster VL, Hilton JF & Schweitzer R 1992 Breast cancer incidence in women with abnormal cytology in nipple aspirates of breast fluid. American Journal of Epidemiology 135 130–141.

    Wrensch MR, Petrakis NL, Miike R, King EB, Chew K, Neuhaus J, Lee MM & Rhys M 2001 Breast cancer risk in women with abnormal cytology in nipple aspirates of beast fluid. Journal of the National Cancer Institute 93 1791–1798.

    Wulfkuhle JD, Sgroi DC, Krutzsch H, McLean K, McGarvey K, Knowlton M, Chen S, Shu H, Sahin A, Kurek R et al. 2002 Proteomics of human breast ductal carcinoma in situ. Cancer Research 62 6740–6749.

    Zalles C, Kimler BF, Kamel S,0 McKittrick R & Fabian CJ 1995 Cytologic patterns in random aspirates from women at high and low risk for breast cancer. The Breast Journal 1 343–349.

    Zhang L, Shao ZM, Beatty P, Sartippour M, Wang HJ, Elashoff R, Chang H & Brooks MN 2003 The use of oxytocin in nipple fluid aspiration. The Breast Journal 9 266–268.

    Zhao H, Hastie T, Whitfield ML, Borresen-Dale AL & Jeffrey SS 2002 Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis. BMC Genomics 3 31.(C J Fabian, B F Kimler, M)

    http://www.100md.com/html/DirDu/2007/01/01/33/00/33.htm