Intraductal Approach Research Articles

Anatomical association of fluid yielding ducts with location of the breast cancer affected segment in screen detected and symptomatic breast cancer

The concept of the intraductal approach to evaluating the breast microenvironment assumes direct access to the breast cancer containing duct. Previous studies on duct anatomy infusing a dye or contrast found fluid-yielding ducts to be associated with the cancer containing duct in approximately 50% of fluid-yielding breasts with cancer. A concordant anatomical relationship between accessible ductal sytems and the cancer-affected lobe is essential if cytology or other cell markers are to be successfully identified as indicators of cancer or of early cancerous change. The concordant relationship is less important if field change effects are considered to predictors of malignant change. The aim of this study was to determine how often duct lavage effluent drains the breast cancer affected segment.

Biomarkers for Risk Assessment and Prevention of Breast Cancer

Breast carcinogenesis is a multistep and multipath disease process which occurs in the epithelium lining of the ductal system in the vast majority of cases. Several studies have shown that the relative risk of breast cancer is increased in every step of this progression and many tumour associated antigens or biomarkers appear during each phase of carcinogenesis. However, their ability to predict for a substantial likelihood of developing breast cancer remains unclear. The acquisition of breast tissue samples, representative of an individual's cellular stability and subcellular biochemical and molecular state could lead to definition of surrogates for risk, early detection, pharmacodynamic determination and finally chemopreventive intervention. The intraductal approach includes nipple aspiration fluid (NAF), ductal lavage (DL) and mammary ductoscopy (MD). These techniques together with random periareolar fine needle aspiration (RPFNA) represent the available techniques for the sampling of breast fluid and exfoliated epithelial cells. At the moment, these procedures are not considered a screening procedure for early breast cancer detection but might provide a powerful research tool for studying breast carcinogenesis in vivo. We summarize the current knowledge regarding the vast array of molecules involved at all stages of carcinogenesis, which can be studied by intraductal approach, and the possibility to utilize them as candidate biomarkers to refine risk assessment, and their possible use in prevention strategies.

Protein profile ana lysis of the breast microenvironment to differentiate healthy women from breast cancer patients

Protein and proteomic high-throughput technologies provide the polypeptide signatures of nipple aspirate fluid (NAF), a breast secretion collected noninvasively from healthy individuals and cancer patients. As breast cancer develops from ductal–lobular epithelium, the analysis of NAF (mirroring the ductal–lobular microenvironment) is a useful tool for the analysis of metabolic pathways within the mammary gland, deepening our knowledge of the biomolecular mechanisms of breast cancer initiation and progression. The different protein expression of major NAF proteins, separated using 1D polyacrylamide gels, has proven valuable for the early detection of women with increased risk of cancer. The failure to recognize a single marker with sufficient clinical sensitivity and/or specificity has driven the identification of breast cancer multiple proteins by 2D electrophoresis. Mass spectrometry-based proteomic approaches (SELDI- and MALDI-TOF technologies) have allowed the characterization of differential NAF proteomic fingerprints between healthy individuals and breast cancer patients. The intraductal approach of protein and proteomic analyses may provide a panel of biomarkers to strengthen the armory against breast cancer.

Internalization and retention of nanodiamonds by MCF-7 cells in vitro: a potential drug delivery platform.

Abstract Abstract #2134 Background: Selective uptake and prolonged retention of drugs will facilitate pharmacotherapy to the breast. This may be accomplished by complexing of drugs to carbon-based nanodiamond (ND) particles, 2-8nm size. ND particles are attractive for this purpose because they are inert and non-toxic.We tested the uptake and retention of ND particles by the estrogen receptor positive breast cancer cell line MCF-7.
 Methods: Polylysine coated ND were labeled with fluorescein isothiocyanate (FITC-ND), and examined by UV-visible spectra and found to be stable. MCF-7 cells were cultured on Lab-Tek II chamber slides using DMEM with 10% FCS, insulin, sodium pyruvate, L-glutamine, and Pen/Strep. To assess the efficiency of ND internalization, we added 10ug/ml ND-FITC in complete media to the chamber slides and incubated for 30 min, 1 hr, 3 hr, 4 hr, 17 hr, and 24 hr. FITC-ND was withdrawn from the medium after an initial exposure of 17 hr by washing of cells, and retention of fluorescence was measured at intervals, up to 3 days after withdrawal. To assess the ability of MCF-7 cells to take up NDs under low metabolic conditions MCF-7 cells were incubated in serum-free medium. As a control soluble FITC alone was incubated at 10 ug/ml with MCF-7 cells. Cells were imaged on the laser confocal microscope and FITC detection at 488nm was analyzed by three dimensional Z stack imaging.
 Results: Fluorescence was initially associated with the membrane surface, and later was visualized in the cytoplasm and in the perinuclear zone. Cellular uptake of FITC and ND-FITC occurred at 30 minutes and reached a maximum at 17 hr of incubation. After washing of cells, the retention studies showed little decrease in the intensity of the ND-FITC signal within the cells up to 96 hours. The FITC molecule was readily released from the cells by passive diffusion within 24 hours while NDs appeared to be compartmentalized in cytoplasmic vesicles.
 
 Conclusions: Internalization of ND particles within the cell cytoplasm occurs within a relatively short time, and the ND particles are retained for at least 96 hours following removal from the culture medium. This prolonged retention is likely to be an advantage when considering novel routes of drug delivery to the breast, such as transdermal or intraductal approaches.
 Funded by Avon. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2134.

Abstract CN13-03: Intraductal route of breast cancer prevention and therapy: from preclinical studies to Phase I trials

Abstract CN13-03 We hypothesized that it is possible to prevent and treat breast cancer by accessing the mammary ductal network through the teat. This intraductal approach, which delivers a high dose of drug locally to the tumors but a low dose systemically, could be an alternative approach to therapy and prevention of breast cancer. Our previous work has demonstrated the effectiveness of intraductal administration of pegylated liposomal doxorubicin, (PLD) and 4-hydroxytamoxifen (4-OHT) using the rat N-methyl N’-nitrosourea (MNU)-induced and the spontaneous, HER2/neu transgenic mouse (neu-N) models of breast cancer. 4-OHT had a preventive effect in the rat MNU-induced models, and PLD had a preventive and therapeutic effect in both the rat and Her2/neu transgenic mouse models (Murata et al, Cancer Res. 2006). Recently we evaluated the effectiveness of other drugs such as carboplatin, methotrexate, Abraxane, 5-Fluorouracil and PLD by whole mount analysis of the treated mammary glands, histopathology following H&E staining, and immunohistochemistry for the proliferation marker, Ki67. We found that carboplatin, 5FU and PLD-treated groups showed significant protection in the prevention setting. PLD had long term effects on the mammary gland structure and function in treated rats and mice. In response to pregnancy, significantly reduced proliferation and milk production was observed in PLD treated Her2/neu mice compared to vehicle treated mice. The litter size and weight at birth were normal in the PLD treated group. Notably, unlike the control group, the PLD-treated Her2/neu transgenic mice remained protected from spontaneous tumor development despite exposure to vast hormonal modulations inherent to pregnancy. Based on the preclinical findings, we initiated a clinical trial to determine the feasibility, safety, and maximum tolerated dose of PLD administered into one duct of women with breast cancer awaiting a mastectomy. PLD was administered using a dose escalation schema based on our preclinical pharmacokinetic and safety data. Serial doxorubicin and doxorubicinol concentrations were determined in plasma and tissue by LC/MS/MS. At the time of mastectomy, blue dye was injected into the treated duct and tissue was obtained for pharmacokinetic and biomarker analysis. To date 14 women entered the study, and doses of 0 to 10 mg PLD per were tested without serious adverse events. Intraductal PLD was associated with a dose-dependent increase in both systemic and local exposure to doxorubicin and doxorubicinol. Neither doxorubicin nor the metabolite was detected in contralateral breast tissue. Doxorubicin and doxorubicinol were detectable in the breast tissue and plasma of women receiving the highest dose (10 mg/duct). Systemic exposure (in plasma) was lower than with conventionally administered PLD. No inflammatory or morphological changes were seen in the breast tissue. The study established safety and feasibility of administering PLD in an outpatient setting. Higher doses of PLD will be tested to achieve the maximum tolerated dose. Future studies will evaluate other agents administered to one or more ducts. In summary, these approaches may find particular use in treatment of DCIS as a safer alternative to surgery, and prevention of breast cancer in women at high risk of developing breast cancer due to inherited mutations or other factors. High local, but low circulating levels of drug, and thereby lower systemic toxicity are the major advantages of the ductal access to breast cancer treatment. This study demonstrates that intraductal injection provides more direct access to breast lesions, and has potential in the prevention and neo-adjuvant therapy of breast cancer. Citation Information: Cancer Prev Res 2008;1(7 Suppl):CN13-03.

Analysis of the intraductal microenvironment for the early diagnosis of breast cancer: identification of biomarkers in nipple-aspirate fluids

Breast cancer, a complex and multifactorial disease, is the most commonly diagnosed malignancy that affects women. Methods of breast cancer detection that are available at present have well-described limitations; the intraductal approaches directly assess the microenvironment of the breast. The aim of this overview is to highlight the application of nipple-aspirate fluid studies in the field of biomarker discovery, useful for early detection and prevention of breast cancer risk. Nipple-aspirate fluids can be obtained non-invasively from the breast in most women and represent a promising biological tool to assess metabolic and molecular changes of cells lining the ducts from which breast cancer arises.

Intraductal approach to the detection of intraductal lesions of the breast

Bloody nipple discharge is a clue in the detection of ductal carcinoma of the breast that do not display a mass. Since sensitivity of discharge cytology is not sufficiently high and mammary ductendoscopy (MS) contributes to the diagnosis of intraductal lesions. We set out to determine whether the intraductal approach is effective for detection of ductal carcinoma. We performed 445 MS procedure in 323 patients who had nipple discharge but no overt mass. The diagnostic accuracy rates of discharge cytology and intraductal breast biopsy (IDBB) were studied in detecting malignancy. The therapeutic value of IDBB for intraductal papillomas was studied in 73 patients. Out of 323 patients, 80 had breast cancer and 155 had intraductal papilloma. MS detected intraductal tumors in 47 cases (58.8%). IDBB was performed in 35 of these 47 cases. The sensitivity was 37.1% by touch cytology, 68.6% by IDBB, and 82.8% by directed ductal lavage cytology. Of the 73 intraductal papilloma patients who were followed for more than 3 years, the therapeutic effectiveness of IDBB was recognized in 57 (78.1%). Directed ductal lavage cytology was the most sensitive method in detecting malignancy. MS and IDBB were benefit in the treatment of intraductal papilloma.

The Role of Mammary Ductoscopy in Breast Cancer: a Review of the Literature

Breast cancer is the most frequently diagnosed malignancy among American women. It is the second most common cause of cancer death. Genetic analysis using comparative genetic hybridization (CGH) has shown evidence that the majority of breast cancers, approximately 85%, begin in the ductal epithelium with normal cells progressing to atypia and finally to carcinoma. Mammary ductoscopy, also referred to as the intraductal approach, is a new tool that allows direct visualization of the breast ductal system. It enables one to sample the ductal epithelium and may allow identification of early changes cytologically as well as potentially play an important role in aiding surgical excision. This may aid in detection of breast masses long before they are palpable or visible via mammography. Mammary ductoscopy may have a role in the evaluation of women with nipple discharge, high-risk women, or limiting the amount of tissue removed in breast conservation surgery for cancer.

The Intraductal Approach to Breast Cancer

Breast cancer is a disease that has been common in the United States and Western Europe for many years. As countries around the world develop more prosperous economies, we are seeing rapid rises in the rates of breast cancer—especially in the younger generations who have ‘‘benefited’’ from the new prosperity during their pubertal growth spurts and young adult years. 1 We can no longer see breast cancer as a disease of just aging Western populations. Our primary successful tool to increase diagnosis at earlier stages in the West has been mammography. In a postmenopausal woman in a relatively wealthy Western country, annual screening with mammography, although it may only give a lead time bias of 12–18 months over clinical exam, saves lives by finding tumors smaller, when our therapies are more effective. Unfortunately, for all our Western advances in breast cancer we still arguably do not have effective means of screening young premenopausal women for the earliest stages of this disease. Our best prevention strategies for premenopausal women are either bilateral amputation (alias prophylactic mastectomy) or a solitary SERM (tamoxifen), which may reduce incidence but hardly has an effect on death rates. At a time when the world needs effective screening for the earliest stages of breast cancer in the youngest women, we have little promising to offer. Kapenhas-Valdes et al. 2 have provided a compre

Magnetic Resonance Galactography: a Feasibility Study in Women with Prior Atypical Breast Duct Cytology

To the Editor: For women who inherit a high risk of developing breast cancer, early detection is a key strategy to reduce morbidity and mortality (1). Multiple studies have found that standard mammographic screening often fails to detect breast cancer early in women at high inherited risk of breast cancer, prompting inclusion of emerging methods such as magnetic resonance imaging (MRI) into high-risk screening protocols (2– 13). The evaluation of breast duct cells is also under investigation as a potential cancer risk assessment technique. Approximately 80 percent of breast cancers originate in the ductal system, and early changes of the ductal epithelium, which might predate malignant transformation, have been reported (14–16); for these reasons, analysis of duct cells is a rational approach to risk stratification and biomarker development. At this time, understanding of the normal breast ductal anatomy, including an appreciation of variation in the number of ducts per breast, continues to evolve (17). A prior anatomic study on mastectomy specimens has also reported wide variation in the proportion of breast volume drained by individual ducts (18). With increasing study of intraductal approaches, a better comprehension of ductal structure and the appearance of the ductal system on MRI has practical relevance. We undertook a pilot study of a novel technique, magnetic resonance (MR) galactography, which combines MRI and intraductal evaluation. Our goals were to map the breast ductal system, quantify the breast volume drained by individual ducts, and evaluate sources of prior cytologic atypia in high-risk women.

Breast Ductal Secretions: Clinical Features, Potential Uses, and Possible Applications

Nipple discharge accounts for approximately 5% of visits to a breast specialist surgical practice and may be encountered as the chief complaint by many other types of physicians. The vast majority of breast cancers originate in the ductal system, which prompted interest in the evaluation of the intraductal approach to breast cancer. Ductoscopy, nipple aspiration, and ductal lavage have emerged as innovative fields of study that may have clinical applications. We performed a literature search of published manuscripts using the keywords nipple discharge, breast ductal secretions, and intraductal approach. We also report our single-institution experience in managing nipple discharge. We present our institutional algorithm for the management of nipple discharge. The possible etiologies of nipple discharge and the appropriate workup are reviewed. Three evolving minimally invasive techniques for the evaluation of high-risk patients include ductoscopy, nipple aspiration, and ductal lavage. Nipple aspiration and ductal lavage fluid may be assayed for cytology, genomic, gene expression, and proteomic studies. Several different translational approaches are being undertaken to investigate the local microenvironment associated with the development and progression of breast carcinoma. Nipple aspiration fluid and ductal lavage offer the opportunity to study the local microenvironment of the ductal system, which is where most breast cancers originate. These powerful approaches to biomarker analysis could be applied to the prevention and treatment of breast cancer.

Predicting breast cancer risk in BRCA1 and BRCA2 carriers: Methylation studies using intraductal fluid

10537 Background: Genomic alterations are likely to precede the phenotypic changes of breast cancer (BC) and include hyper- methylation of tumour suppressor genes. An intraductal approach, such as ductal lavage (DL), is an attractive means of accessing ductal epithelial and other cells in a prospective fashion. Methods: Women with at least one breast unaffected by BC, with a germline BRCA1 or BRCA2 mutation, attended for 6-monthly DL collection for up to 3 years. Standard methods were used for DNA extraction and bisulfite conversion. Hyper-methylation of p16, RASSF1A, twist and RARβ genes was investigated using a qualitative, real-time, nested PCR assay. Associations between methylation status and categorical variables were tested using Fisher's exact test including cytological findings and BC incidence. Logistic regression was used to examine the relationship between methylation status and patient age, and to examine the effects of multiple variables. Analyses were performed at three levels; samples from a single breast over time and from a single duct over time and each sample in isolation. Results: A total of 173 DL samples from 98 ducts in 56 breasts were analysed in 34 women (16 BRCA1 and 18 BRCA2 mutation carriers) with a median age of 43 years (range 27- 60). DL fluid was collected from all women on at least one occasion (median 2.5 visits, range 1–5). Five women developed BC on study. Methylation of p16 was strongly associated with a known BRCA1 mutation (p=0.0003, p<0.0001 and p<0.0001 at the breast, duct and sample levels respectively) and was also more likely in women with a previous history of contralateral BC (p=0.0008 and p<0.0001 at the duct and sample level respectively). Independent associations were seen for women who developed BC on study and methylation of p16 and RASSF1A (p16: p=0.0023 at the sample level only) and (RASSF1A: p=0.0760, p=0.0178 and p<0.0001- at the breast, duct and sample level respectively). Conclusions: The methylation pattern in a panel of genes could be used as a biomarker for risk of future breast cancer. In addition p16 methylation may be a predictor of BRCA1 mutation status. Further research is required to corroborate these findings as the numbers recruited for this study are small with a short duration of clinical follow up. No significant financial relationships to disclose.

Human breast duct anatomy, the ‘sick lobe’ hypothesis and intraductal approaches to breast cancer

Information about central and peripheral duct anatomy is a requirement for developing intraductal approaches to human breast cancer, but remains sparse. This study looks at the acquisition and digital modelling of data describing breast duct branching from thick ('subgross') sections using data structures from the neurosciences, and at high-throughput imaging of duct anatomy in the nipple. The branching of a large breast duct system was modelled using data extracted from cleared and stained 2 mm 'subgross' sections of an autopsy breast using a public-domain neuron modelling program (CVAPP), and episcopic fluorescence image capture (EFIC) was used to collect a stack of 1100 autofluorescence images of ducts in a mastectomy nipple. The duct skeleton was captured in 440 line segments with some pruning of terminal ducts. Extracting this data manually in a usable form was, however, laborious and error prone, emphasising the need for improved morphological informatics. EFIC captured anatomical detail and subsequent 3D reconstruction was consistent with the distinction between 'type A' and 'type B' nipple ducts proposed by Going and Moffat (J Pathol 203: 538-544, 2004). Whole-lobe duct modelling and EFIC reveal central and peripheral duct anatomy in human breast. Such knowledge is required for understanding normal breast development, the growth of cancer precursors, and for developing the intraductal approach to breast cancer.

The Intraductal Approach to Breast Cancer Biomarker Discovery

Established methods of breast cancer detection have well-described limitations, and new diagnostic techniques are evolving continually to improve diagnostic accuracy. The intraductal approach encompasses the modalities of nipple aspiration, ductal lavage, and duct endoscopy, and is a means of directly accessing the microenvironment of the breast and either sampling or visualizing this intraductal milieu. The aim of sampling this mammary microenvironment is to obtain samples from the physical surroundings of cells that are undergoing malignant transformation, thereby providing a new method of detection before the development of a clinically or radiologically discernible mass. A literature review was conducted to investigate the evolution of the intraductal approach and its particular application in the field of biomarker discovery, primarily using the intraductal technique of nipple aspiration, in combination with emerging protein profiling techniques.

Combined ductal lavage and ductoscopy: What is the future for the intraductal approach?

Combined ductal lavage and ductoscopy: What is the future for the intraductal approach?

Patterns of reduced nipple aspirate fluid production and ductal lavage cellularity in women at high risk for breast cancer

IntroductionNipple aspiration is a noninvasive technique for obtaining breast fluids from the duct openings of the nipple for the evaluation of abnormalities associated with breast cancer. Nipple aspirate fluid (NAF) can be elicited from 48 to 94% of healthy women, and its production has been linked to an increased relative risk for breast cancer development. NAF production has been used in studies to guide the selection of ducts for ductal lavage, a procedure in which ducts are cannulated and flushed with saline to collect cells. In a previous multicenter trial to evaluate intraductal approaches in women at high-risk for breast cancer, NAF production was observed in 84% of the subjects. However, we observed a significantly lower proportion of fluid-yielding subjects in a similar series of high-risk women. The purpose of the present study was to identify variables associated with this reduction.MethodNipple aspiration was performed on 33 high-risk women (defined as having a 5-year Gail model index of more than 1.7, a personal or family history of breast cancer, and/or a BRCA1 or BRCA2 germline mutation) to identify ductal orifices for lavage procedures. Lavage was performed on all fluid-yielding ducts and on nine non-fluid-yielding ducts.ResultsFluid-yielding ducts were identified in 12 of 33 (36%) of the subjects in the present series, compared with 16 of 19 (84%) of the subjects undergoing identical procedures at our facility during a multicenter trial (P = 0.001). Reduced NAF yields were associated with postmenopausal status (P = 0.02), BRCA germline mutations (P = 0.004), and risk reduction therapies, including bilateral salpingo-oophorectomy (BSO) and/or selective estrogen receptor modulators (SERMs; P = 0.009). All nine (100%) of the ductal lavage specimens collected from non-fluidyielding ducts were acellular, in comparison with 3 of 13 specimens from fluid-yielding ducts (P < .001).ConclusionAnalysis of high-risk women in the present series revealed patterns of reduced NAF production and ductal lavage cellularity compared with a previous multicenter trial. The present series included more BRCA-positive women, many of whom had undergone BSO and/or were using SERMs. Our data suggest that endocrine mechanisms associated with these risk-reducing therapies may be related to patterns of diminished breast fluid production.

The Fourth International Symposium on the Intraductal Approach to Breast Cancer, Santa Barbara, California, 10–13 March 2005

Intraductal approaches encompass procedures and technologies that are designed to access and interrogate the ductal–alveolar systems of the human breast, and include nipple aspiration, ductal lavage, random periareolar fine needle aspiration, and ductoscopy. These approaches are being used to collect and analyze fluids and cells to develop methods for breast cancer detection and risk assessment; to introduce imaging technologies to explore the mammary tree for abnormalities; to administer therapeutic and/or preventive agents directly to the breast tissue; and to explore the biology of the normal mammary gland. The latest research findings in these areas, presented at The 4th International Symposium on the Intraductal Approach to Breast Cancer in 2005, are summarized in this report.

Anatomy of the nipple and breast ducts revisited.

Increasing interest in the intraductal approach to the breast has necessitated revisiting the anatomy of the breast. Using six different complementary in vivo and in vitro approaches, the authors determined the number, distribution, and anatomic properties of the ductal systems of the breast, which extend from the nipple orifices to the terminal duct lobular units. More than 90% of all nipples examined contained 5-9 ductal orifices, generally arranged as a central group and a peripheral group. Each nipple orifice communicated with a separate, nonanastomosing ductal system, which extended to the terminal duct lobular unit. Increased knowledge of the ductal anatomy of the breast and the ability to access the nipple ductal orifices will provide a foundation for the intraductal approach to the breast.

Transoesophageal ultrasound applicator for sector-based thermal ablation: first in vivo experiments

New curative and palliative treatments must be proposed to respond to the bad long-term prognosis of oesophageal cancers. It has been demonstrated that high intensity ultrasound (US) can induce rapid, complete and well-defined coagulation necrosis. For the treatment of this cancer, we designed an applicator that uses an intraductal approach. The active part is an air-backed plane transducer. It has an external water-cooling system and operates at 10 MHz. Ex vivo experiments conducted on pig liver demonstrated the ability of this applicator to generate, by rotating the transducer, circular or sector-based coagulation necroses at predetermined depths up to 13 mm, with an excellent angular precision. The treatment of sector-based oesophageal tumours may be critical, where both malignant and healthy tissues are covered by the US beam. Thus, in vivo trials were conducted on five healthy pig oesophaguses to determine the maximal thermal dose that will not induce a perforation of the oesophagus or surrounding tissues. From the results of previous studies, this dose is high enough to treat pathological tissues. These promising results indicate that this US system represents a safe and effective tool for the clinical treatment of oesophageal tumours. (E-mail: melodemia@lyon.inserm.fr)

Ductal lavage, nipple aspiration, and ductoscopy for breast cancer diagnosis.

The intraductal approach to breast cancer has been invigorated this year by a series of papers exploring ductal-based screening through nipple aspiration and lavage and ductal exploration through endoscopy. The merging of these efforts to define the earliest biologic changes in the progression toward breast cancer is opening new fields for both bench-translational and clinical research. These techniques have already begun to show value in defining the presence and extent of proliferative disease in high-risk patients, allowing for more informed therapeutic decision making.