Intraductal Approach Research Articles

Ductal Architecture: Corrosion Casting of Canine Mammary Glands Using an Intraductal Approach.

Detailed knowledge related to the morphology, anatomy, physiology, and pathology of the canine mammary gland is scarce. Mammary tissue undergoes massive changes instructed by hormones multiple times within the lifespan of every bitch, affecting its appearance. To address the ductal system's appearance and to present how different our findings regarding the canine mammary gland are in comparison with the available literature, we obtained cadaveric specimens after euthanasia and mastectomy. All bitches were euthanised due to poor prognosis for their recovery from maladies unrelated to mammae. Using intraductal cannulation ex vivo, milk- or fluid-yielding ducts were perfused using VasQtec (polyurethane resin), which revealed casts, i.e., imprints of ducts and glandular structures in situ. We observed primary, vertically positioned ducts that ascended throughout the teat and continued branching to secondary, tertiary, etc., horizontally positioned ducts, which drained mammary gland lobes under the skin located close to the abdominal wall. The ascendant teat canal could be split into two and could be connected to gland alveoli or end blind. Alveolar formations were located along ducts and ductules in bigger and/or smaller clusters. This study is the first to generate a 3D image of canine ducts and glandular tissue using an intraductal approach.

Using Endoscopy in the Diagnosis of Pancreato-Biliary Cancers

Pancreatic cancer and cholangiocarcinoma are life threatening oncological conditions with poor prognosis and outcome. Pancreatic cystic lesions are considered precursors of pancreatic cancer as some of them have the potential to progress to malignancy. Therefore, accurate identification and classification of these lesions is important to prevent the development of invasive cancer. In the biliary tract, the accurate characterization of biliary strictures is essential for providing appropriate management and avoiding unnecessary surgery. Techniques have been developed to improve the diagnosis, risk stratification, and management of pancreato-biliary lesions. Endoscopic ultrasound (EUS) and associated techniques, such as elastography, contrasted-enhanced EUS, and EUS-guided needle confocal laser endomicroscopy, may improve diagnostic accuracy. In addition, intraductal techniques applied during endoscopic retrograde cholangiopancreatography (ERCP), such as new generation cholangioscopy and in vivo cellular evaluation through probe-based confocal laser endomicroscopy, can increase the diagnostic yield in characterizing indeterminate biliary strictures. Both EUS-guided and intraductal approaches can provide the possibility for tissue sampling with new tools, such as needles, biopsies forceps, and brushes. At the molecular level, novel biomarkers have been explored that provide new insights into diagnosis, risk stratification, and management of these lesions.

Abstract PR005: Intraductal administration of a recombinant transferrin receptor-directed immunotoxin clears ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer

Abstract Background: For DCIS, a preneoplastic lesion with a 15-50% chance of recurrence or progression, there is a reappraisal of the necessity of aggressive treatment with its attendant toxicities. HB21(Fv)-PE40, a conjugate of an Fv fragment of the human transferrin-receptor (TFR) mAb and a 40 kDa fragment of bacterial Pseudomonas exotoxin (PE), has potent tumor cell killing activity. HB21(Fv)-PE40 binds to abundantly expressed TFR on cancer cells; the molecule is internalized, processed, and trafficked to the cytosol. Inside the cell, PE-toxin catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor 2 (EF2), inactivates EF2, and leads to an arrest in protein translation and cell death by apoptosis. However, due to its abundant expression in the liver, the recombinant immunotoxin (RIT) is potentially toxic. We hypothesized that RIT administration via the mammary ductal opening at the teat will destroy preneoplasias, both visible and occult, in the entire ductal tree, while systemic effects will be minimal. The intraductal approach could provide a direct, safe, and highly effective treatment for DCIS. Methods: We tested this concept in two models of DCIS using an ER/PR-, HER2+ cell line derived from a DCIS, SUM225-Luc, and in the ER/PR+, HER2- breast cancer cell line, MCF-7-Luc. In both cell lines, the IC50 of the toxin was in the nM range. DCIS developed within one week in the MIND models following i.duc injection of tumor cells in the 4th pair of mammary glands of female NSG mice. On day 7, the tumor-bearing mice received vehicle, TFR mAb alone, or the HB21(Fv)-PE40 conjugate by the i.duc or i.v route three times at weekly intervals and were observed for 1-6 months. Within two weeks of the treatment, IVIS imaging signals were undetectable in the mammary gland treated with RIT i.duc in both the models (P<0.0001). Mammary gland whole mount analysis, histopathology, and immunohistochemistry for Ki67, Ku80, TFR and CD31 showed that i.duc toxin rendered more than 90% of the mice tumor-free. Pharmacokinetics studies were conducted in mice with the RIT administered i.v. or i.duc. RIT was undetectable by TFR-ELISA in the blood of i.duc-treated mice. Conclusions. Overall, we have shown that HB21(Fv)-PE40 has remarkable antitumor activity by i.duc administration in two MIND models of DCIS. RIT was undetectable in blood after i.duc injection by ELISA, suggesting that the toxin remains inside the ductal system during its 30 min half-life. This preclinical study provides a solid foundation for a clinical study. Dose escalation studies of HB21(Fv)-PE40 by the i.duc route could be performed in patients with DCIS and early breast cancer who will undergo mastectomy as part of their treatment regimen. This study design will allow a detailed examination of the local (breast) and systemic effects of i.duc HB21(Fv)-PE40. It will introduce a potent agent for DCIS treatment and cancer interception in women at high risk of developing breast cancer. The contribution of the immune system remains to be explored. Citation Format: Saraswati Sukumar. Intraductal administration of a recombinant transferrin receptor-directed immunotoxin clears ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR005.

Abstract P5-08-03: Intraductal administration of a Transferrin Receptor-directed immunotoxin eliminates ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer

Abstract Background: Increased detection of ductal carcinoma in situ (DCIS) has been a consequence of mammographic screening. Currently there is a reappraisal of the necessity of aggressive treatment, with their attendant toxicities for a preneoplastic lesion with 15-20% chance of recurrence or progression. The recombinant immunotoxin, TFR(fv)PE40, binds to the abundantly expressed transferrin receptors on cancer cells, is internalized, processed and trafficked to the cytosol. The released Pseudomonas exotoxin (PE) catalyzes the transfer of ADP-ribose from NAD to elongation factor 2 (EF2), inactivates EF2, and leads to an arrest in protein translation, and cell death by apoptosis. However, TFR(fv)PE40 is toxic to liver cells. We hypothesized that, when administered via the opening at the mammary teat, the immunotoxin will destroy preneoplasias in the entire ductal tree. Because of the closed nature of the ductal system, the toxic effects of the TFR(fv)PE40 will be confined to the tumor cells alone, and systemic effects will be minimal or absent. The intraductal approach, we proposed, will provide a direct, safe and effective treatment for DCIS. Methods: We tested this concept in two models of DCIS using two luc-tagged cell lines-SUM225-luc, derived from a human DCIS, and in MCF-7-luc. In both cell lines, the IC50 of the toxin was in the nM range. DCIS develop within one week in MIND models following intraductal (i.duc) injection of tumor cells (right and left 4th mammary gland) of ex-breeder NSG mice. DCIS progressed to invasive cancer in 2-3 weeks. At day 7, mice were administered vehicle, TFR mAb alone, or the HB21(fv)PE40 conjugate at two doses (0.15 or 1.5 ug per teat x 2) by the i.duc route, 3 times at weekly intervals, and observed for 1-3 months. Mice treated with the TFR-Ab showed no significant reduction in tumor size. Treated with HB21(fv)PE40 i.duc, by IVIS imaging, tumors were undetectable in both models (P<0.0001) within two weeks. No recurrence was observed during 3 months of followup. Mammary whole mount analysis and histopathology showed that i.duc toxin rendered more than 90% of the mice cancer-free. Immunohistochemical analysis of tumor sections for huTFR (CD71), Ki67, and CD31 supported the loss of TFR-expressing and proliferating cancer cells, and reduction of blood vessels. Pharmacokinetics studies showed that toxin was detectable by TFR-ELISA in blood 5, 30 and 60 min after i.p injection, but was undetectable in the blood of i.duc-treated mice. Conclusions: Overall, we have shown that i.duc HB21(fv)PE40 has remarkable antitumor activity in cell culture with an IC50 in the nM range, and in two MIND models of DCIS. Blood levels of the toxin after i.duc injection were undetectable, suggesting that the toxin remains inside the ductal system during its 30 min half-life, and its effect occurs during this time. This preclinical study provides a strong basis for conducting human trials. The toxicity of HB21(Fv)PE40 by the i. duc route of drug administration could be determined in dose escalation studies in patients with DCIS and early breast cancer who will undergo mastectomy as part of their treatment regimen, using a previously successful study design (PMID:2230751). This design will allow detailed examination of the local and systemic effects of HB21(fv)PE40 administered i.duc and reintroduce a very potent agent into DCIS treatment, and in the future, cancer prevention in women at high risk of developing breast cancer. Citation Format: Saraswati Sukumar, Guannan Wang, Alok Kumar, Wanjun Ding, Preethi Korangath, Priya Pai, Kathleen Gabrielson, Ira Pastan. Intraductal administration of a Transferrin Receptor-directed immunotoxin eliminates ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-03.

S0027 Predictors of Technical Success and Lower Procedure Burden for Single Operator Pancreatoscopy With Intraductal Lithotripsy vs Extracorporeal Shock Wave Lithotripsy Approaches for Large Main Pancreatic Duct Stones

S0027 Predictors of Technical Success and Lower Procedure Burden for Single Operator Pancreatoscopy With Intraductal Lithotripsy vs Extracorporeal Shock Wave Lithotripsy Approaches for Large Main Pancreatic Duct Stones

Intraductal patient-derived xenografts of estrogen receptor α-positive breast cancer recapitulate the histopathological spectrum and metastatic potential of human lesions.

Estrogen receptor α‐positive (ER‐positive) or ‘luminal’ breast cancers were notoriously difficult to establish as patient‐derived xenografts (PDXs). We and others recently demonstrated that the microenvironment is critical for ER‐positive tumor cells; when grafted as single cells into milk ducts of NOD Scid gamma females, >90% of ER‐positive tumors can be established as xenografts and recapitulate many features of the human disease in vivo. This intraductal approach holds promise for personalized medicine, yet human and murine stroma are organized differently and this and other species specificities may limit the value of this model. Here, we analyzed 21 ER‐positive intraductal PDXs histopathologically. We found that intraductal PDXs vary in extent and define four histopathological patterns: flat, lobular, in situ and invasive, which occur in pure and combined forms. The intraductal PDXs replicate earlier stages of tumor development than their clinical counterparts. Micrometastases are already detected when lesions appear in situ. Tumor extent, histopathological patterns and micrometastatic load correlate with biological properties of their tumors of origin. Our findings add evidence to the validity of the intraductal model for in vivo studies of ER‐positive breast cancer and raise the intriguing possibility that tumor cell dissemination may occur earlier than currently thought. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Theoretical investigation of transgastric and intraductal approaches for ultrasound-based thermal therapy of the pancreas

BackgroundThe goal of this study was to theoretically investigate the feasibility of intraductal and transgastric approaches to ultrasound-based thermal therapy of pancreatic tumors, and to evaluate possible treatment strategies.MethodsThis study considered ultrasound applicators with 1.2 mm outer diameter tubular transducers, which are inserted into the tissue to be treated by an endoscopic approach, either via insertion through the gastric wall (transgastric) or within the pancreatic duct lumen (intraductal). 8 patient-specific, 3D, transient, biothermal and acoustic finite element models were generated to model hyperthermia (n = 2) and ablation (n = 6), using sectored (210°–270°, n = 4) and 360° (n = 4) transducers for treatment of 3.3–17.0 cm3 tumors in the head (n = 5), body (n = 2), and tail (n = 1) of the pancreas. A parametric study was performed to determine appropriate treatment parameters as a function of tissue attenuation, blood perfusion rates, and distance to sensitive anatomy.ResultsParametric studies indicated that pancreatic tumors up to 2.5 or 2.7 cm diameter can be ablated within 10 min with the transgastric and intraductal approaches, respectively. Patient-specific simulations demonstrated that 67.1–83.3% of the volumes of four sample 3.3–11.4 cm3 tumors could be ablated within 3–10 min using transgastric or intraductal approaches. 55.3–60.0% of the volume of a large 17.0 cm3 tumor could be ablated using multiple applicator positions within 20–30 min with either transgastric or intraductal approaches. 89.9–94.7% of the volume of two 4.4–11.4 cm3 tumors could be treated with intraductal hyperthermia. Sectored applicators are effective in directing acoustic output away from and preserving sensitive structures. When acoustic energy is directed towards sensitive structures, applicators should be placed at least 13.9–14.8 mm from major vessels like the aorta, 9.4–12.0 mm from other vessels, depending on the vessel size and flow rate, and 14 mm from the duodenum.ConclusionsThis study demonstrated the feasibility of generating shaped or conformal ablative or hyperthermic temperature distributions within pancreatic tumors using transgastric or intraductal ultrasound.

Abstract ES5-3: ES5-3 Targeting the breast, locally: Old and new approaches

Abstract The oldest local approach to breast cancer prevention is surgery. The largest and most rigorous description of this procedure in a high risk population comes from the Mayo Clinic, where 639 women underwent bilateral mastectomy for prevention between 1960 and 1993. Of these procedures, 90% were subcutaneous mastectomies, i.e. ∼90% of the breast was removed, with preservation of the nipple and areola. The breast cancer incidence in this population, with 13 year follow-up, was 90% of the expected incidence. A subset was tested for BRCA1/2 mutations, and among 26 mutations carriers, no cancers were observed. Subsequent studies have focused on more defined high risk populations, mainly carriers of BRCA1/2 mutations. In one recent meta-analysis, the pooled relative risk (RR) was 0.11; 95% CI, 0.04-0.32. However, all-cause mortality was unaffected by mastectomy. Nipple-sparing mastectomy has emerged as an option for mutation carriers recently, and data are beginning to accumulate that overall satisfaction with reconstruction results, and sexual well-being, are greater with the nipple-sparing procedures. The long-term cancer risk data following nipple-sparing procedures are not yet mature. However, mastectomy carries a large physical and emotional cost, which could be avoided if effective non-surgical prevention options were available. Breast cancer prevention in high risk women and those with carcinoma in situ requires that the breast be exposed to an effective agent; systemic exposure is unnecessary, and its harms lead many eligible women to decline preventive therapy. Local transdermal therapy (LTT) to the breast involves the application of active drugs to the breast skin, resulting in high concentrations in the breast but low systemic exposure. It is non-invasive, self-delivered, and not dependent on hepatic metabolism. Existing data on LTT include two pre-surgical window trials of topical 4-hydroxytamoxifen (an active tamoxifen metabolite) in women with invasive breast cancer, and duct carcinoma in situ (DCIS). These demonstrate that LTT with 4-OHT decreases proliferation of invasive and non-invasive cancer cells to a similar degree as oral tamoxifen, with low systemic levels, and no effect on coagulation proteins. These data are promising regarding the use of LTT with 4-OHT for breast cancer prevention, and for therapy of DCIS, but also suggest that LTT could be developed for any small, lipophilic molecule with good dermal permeation, thus greatly expanding the menu of drugs that could be tested for breast cancer prevention. Another locally targeted approach involves the intraductal delivery of active drugs, using a catheter to cannulate the nipple duct orifice. Preliminary studies using Doxil in rodents and in humans have shown this is feasible, but concerns have been raised regarding carcinogenicity of cytotoxic drugs. More recently, RNA interference therapy, and trastuzumab conjugated to a cytotoxic alpha-emitter 225Ac, have been tried in mouse models of DCIS with some success. A trial of intraductal fulvestrant is on-going. Both transdermal and intraductal local therapy approaches hold promise, but are challenging in distinct ways. Nevertheless, ongoing research suggests that local therapy options for breast cancer prevention can move beyond surgical resection of breast tissue. Citation Format: Khan SA. ES5-3 Targeting the breast, locally: Old and new approaches [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr ES5-3.

The mammary ducts create a favourable microenvironment for xenografting of luminal and molecular apocrine breast tumours.

There is a paucity of models for hormone receptor-positive (HR+) breast cancer because of the difficulty of establishing xenografts from these tumours. We show that this obstacle can be overcome by injecting human tumour cells directly into the mammary ducts of immunodeficient mice. Tumours from 31 patients were infected overnight with a lentiviral vector expressing tdTomato and injected through the nipple into the mammary ducts of NOD-SCID-IL2RG-/- mice. Tumours formed in the mice in 77% of cases after the first injection (6/8 luminal A, 15/20 luminal B, and 3/3 molecular apocrine). Four luminal A and one molecular apocrine graft were tested in secondary and tertiary grafts: all were successfully passaged in secondary and 4/5 in tertiary grafts. None of the samples engrafted when injected subcutaneously. The morphology, oestrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), and Ki-67 profiles of the clinical samples were maintained in the tertiary grafts. We also show that the intraductal approach can be used to test the response to targeted therapy with fulvestrant and palbociclib, using a genetically defined ER+ model. We conclude that the mammary ducts create a microenvironment that is uniquely favourable to the survival and growth of tumours derived from mammary hormone-sensing cells. This approach opens the door to testing genomically targeted treatment of HR+ tumours in precision medicine programmes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Repeated nipple fluid aspiration: compliance and feasibility results from a prospective multicenter study.

BackgroundDespite intensive surveillance, a high rate of interval malignancies is still seen in women at increased breast cancer risk. Therefore, novel screening modalities aiming at early detection remain needed. The intraductal approach offers the possibility to directly sample fluid containing cells, DNA and proteins from the mammary ductal system where, in the majority of cases, breast cancer originates. Fluid from the breast can non-invasively be obtained by oxytocin-assisted vacuum aspiration, called nipple fluid aspiration (NFA). The goal of this feasibility study was to evaluate the potential of repeated NFA, which is a critical and essential step to evaluate its possible value as a breast cancer screening method.MethodsIn this multicenter, prospective study, we annually collected nipple fluid for up to 5 consecutive years from women at increased breast cancer risk, and performed a questionnaire-based survey regarding discomfort of the aspiration. Endpoints of the current interim analyses were the feasibility and results of 994 NFA procedures in 451 women with total follow-up of 560 person years of observation.ResultsIn this large group of women at increased risk of breast cancer, repetitive NFA appeared to be feasible and safe. In 66.4% of aspirated breasts, nipple fluid was successfully obtained. Independent predictive factors for successful NFA were premenopausal status, spontaneous nipple discharge, smaller breast size, bilateral oophorectomy and previous use of hormone replacement therapy or anti-hormonal treatment. The procedure was well tolerated with low discomfort. Drop-out rate was 20%, which was mainly due to repeated unsuccessful aspiration attempts. Only 1.6% of women prematurely declined further participation because of side effects.ConclusionsRepeated NFA in women at increased breast cancer risk is feasible and safe. Therefore, NFA is a promising method to non-invasively obtain a valuable source of potential breast cancer specific biomarkers.

The anatomy of fluid-yielding ducts in breast cancer

The concept of an intraductal approach to evaluate the breast microenvironment assumes direct access to the cancer-containing duct. Central duct access to the cancer-affected lobe is essential if cytology or cell markers are to be useful indicators of pre-malignant change. Access to the cancer-bearing lobe would be less important if field change effects of malignant change were predominantly supra-lobar. The aim of this study was to determine how often duct lavage fluid drains the breast cancer-affected segment. 58 patients undergoing mastectomy for breast cancer were recruited among which 47 had at least one fluid-yielding duct. Following duct lavage, fluid-yielding ducts were perfused ex vivo with Polyurethane Elastomer (PU4ii) resin. Specimens were sliced sagittally, and the extent of resin perfusion and anatomical relationship to the cancer-affected segment was recorded. Computed tomography (CT) scanning was performed on selected mastectomies before cut-up for a feasibility study of 3D duct reconstruction. The median number of fluid-yielding ducts cannulated per cancer-affected breast was 2 (range 1-4). 35/47 (74%) mastectomy specimens were successfully cannulated for resin perfusion. 29/35 (83%) showed tracing of the cancer-affected duct system, 6/35 resin perfusions traced duct systems unaffected by cancer and 12/35 perfusions extravasated. The proportion of sagittal breast slices perfused by resin was 13-68% (median 43%). Volume rendering CT showed it is feasible to produce a simulated image of the perfused ducts. Duct access to the cancer-containing segment is feasible in the majority of patients. Fluid-yielding ducts proportionately drain a significant volume of the breast. Large symptomatic cancers may cause obstruction with distal collapse. Further quantitative study of breast perfusion CT scans may be helpful for estimating the volume fraction of breast tissue perfused by fluid-yielding ducts. The intraductal approach is a valid concept for biomarker assessment of cancer-containing breast segments.

Mammary ductoscopy in the current management of breast disease

The majority of benign and malignant lesions of the breast are thought to arise from the epithelium of the terminal duct-lobular unit (TDLU). Although modern mammography, ultrasound, and MRI have improved diagnosis, a final pathological diagnosis currently relies on percutaneous methods of sampling breast lesions. The advantage of mammary ductoscopy (MD) is that it is possible to gain direct access to the ductal system via the nipple. Direct visualization of the duct epithelium allows the operator to precisely locate intraductal lesions, enabling accurate tissue sampling and providing guidance to the surgeon during excision. The intraductal approach may also have a role in screening individuals who are at high risk of breast cancer. Finally, in spontaneous nipple discharge (SND), as biopsy instruments improve and intraductal therapeutics, such as intraductal excision and laser ablation, become a possibility, normal or benign ductoscopic findings may help minimize surgery in selected patients. As MD technology is rapidly advancing, a comprehensive review of current practice will be a valuable guide for clinicians involved in the management of breast disease. This is a review of current ductoscopic practice based on an exhaustive literature search of Pubmed, Google Scholar, and conference proceedings. The search terms "ductoscopy", "duct endoscopy", "mammary", "breast," and "intraductal" were used. Duct endoscopes have become smaller in diameter with working channels and improved optical definition. Currently, the role of MD is best defined in the management of SND facilitating targeted surgical excision, potentially avoiding unnecessary surgery, and limiting the extent of surgical resection for benign disease. The role of MD in breast-cancer screening and breast conservation surgery has yet to be fully defined. Few prospective randomized trials exist in the literature, and these would be crucial to validate current opinion, not only in the benign setting but also in breast oncologic surgery.

Status of Intraductal Therapy for Ductal Carcinoma in Situ.

The intraductal approach is particularly appealing in the setting of ductal carcinoma in situ (DCIS), a preinvasive breast neoplasm that is thought to be entirely intraductal in its extent. Based on an emerging understanding of the anatomy of the ductal system as well as novel techniques to leverage the access accorded by the intraductal approach, researchers are actively exploring how ductal lavage, ductoscopy, and intraductal infusion of therapeutic agents may enhance breast cancer treatment. Both cytologic and molecular diagnostics continue to improve, and work is ongoing to identify the most effective diagnostic biomarkers for DCIS and cancer, although optimal targeting of the diseased duct remains an important consideration. Ductoscopy holds potential in detection of occult intraductal lesions, and ductoscopically guided lumpectomy could increase the likelihood of a more comprehensive surgical excision. Exciting pilot studies are in progress to determine the safety and feasibility of intraductal chemotherapy infusion. These studies are an important starting point for future investigations of intraductal ablative therapy for DCIS, because as our knowledge and techniques evolve, it is likely that DCIS may be the target most amenable to treatment by intraductal therapy. If such studies are successful, these approaches will allow an important and meaningful transformation in treatment options for women diagnosed with DCIS.

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

Introduction: The concept of the intraductal approach to evaluate the breast microenvironment assumes direct access to the breast cancer containing duct. 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.

Intracrinology of breast microenvironment: hormonal status in nipple aspirate fluid and its relationship to breast cancer

Breast cancer, a complex and multifactorial disease, is the most commonly diagnosed malignancy affecting women. Methods currently available for breast cancer detection have well-described limitations; in this respect, the intraductal approaches directly assess the microenvironment of the breast. Nipple aspirate fluid (NAF) can be noninvasively obtained from the breast in most women and represents a promising biological tool to assess metabolic, hormonal and molecular changes occurring in the cells lining the ducts, from which breast cancer arises. The aim of this review is to highlight the application of NAF studies in the field of biomarker discovery, which provide results useful for early detection and prevention of breast cancer risk; in fact, the analysis of NAF (mirroring the ductal–lobular microenvironment) is a reliable method for assessment of metabolic/hormonal pathways within the mammary gland, identifying biomolecular mechanisms of breast cancer initiation and progression. The intracrinology of breast microenvironment (i.e., hormonal status in NAF) may provide independent diagnostic/prognostic factors, highlighting the importance of early altered hormonal metabolism (e.g., aromatase, estrogen sulfotransferase and steroid sulfatase pathway) in relation to breast cancer initiation. The possible application of targeted therapies through the inhibition of intratumoral enzymes involved in steroid metabolism is also discussed. The intraductal approach to hormone analyses may provide a further panel of biomarkers providing clinical benefits and strengthening the armory against breast cancer.

Breast Ductoscopy and the Evolution of the Intra-Ductal Approach to Breast Cancer

Interest in breast endoscopy came from Oriental investigators in the early 1990s where bloody nipple discharge is a more common presentation of breast cancer. The early techniques using a single microfiber scope without ductal distension was successful in navigating only the first 1-3 cm of the ducts and fraught with technical problems such as scope breakage and poor image quality. In spite of these barriers there has been increasing use of this technology in Japan and more widespread acceptance as the technology of scope design improved. Dooley and others tested a new method of obtaining a rich cytologic specimen from the ducts of high-risk women known as ductal lavage recently. The success of this procedure was that it detected severe cytologic and malignant atypia in clinically and radiographically normal breasts. Reproducibly, the same breast duct could be cannulated and severely atypical cytology obtained. The problem arose in identifying the lesion within the breast, which was the source for the atypia. New American multi-fiber microendoscopes were applied to solve this problem in an initial series of patients with abnormal cytology to identify the lesions. Success of that series lead to wider application of the imaging technology and eventual adoption of this imaging modality help to guide during all non-mastectomy breast surgery where fluid could be elicited from the nipple to identify the duct connecting to the lesion for which surgery was being performed. Initial reports have demonstrated the types of operative findings in certain sub-populations early in the use of this technology.

State of the Science and the Intraductal Approach for Breast Cancer: Proceedings Summary of The Sixth International Symposium on the Intraductal Approach To Breast Cancer Santa Monica, California, 19–21 February 2009

Researchers are using the intraductal approach to advance breast cancer risk assessment, prevention, diagnosis, and treatment. Procedures and technologies that can access and interrogate the ductal-alveolar systems include nipple aspiration, ductal lavage and ductoscopy. Ductoscopic papillectomy, ductoscopic margin evaluation, and intraductal therapy are considered promising investigational and innovative treatments. These techniques are used to explore the biology of the normal breast; collect and analyze breast fluid and cells to identify biomarkers that can be used in breast cancer detection and risk assessment; and to identify new ways to find and administer therapeutic and/or preventive agents to the breast tissue. This report summarizes the latest research findings in these areas, presented at The 6th International Symposium on the Intraductal Approach to Breast Cancer in 2009.

Ducts in the human nipple: structure, epithelial immunophenotypes, and relationships with clear cells of nipple epidermis (toker cells)

It has been suggested that there are structurally distinct 'type A' and 'type B' ducts in human nipple. If so, properties such as their relative numbers, accessibility at the nipple surface, and relationships with the breast lobes they communicate with could have biological significance and practical relevance for the intraductal approach to breast cancer diagnosis and treatment. To investigate the two-duct-type hypothesis further we examined duct structure and duct epithelial immunophenotypes in the nipples of cancer mastectomies. Relationships between major nipple ducts and clear cells of nipple epidermis (Toker cells) were also explored. Sections through the nipple duct bundle at the base of the papilla in 10 mastectomy nipples were immunostained for oestrogen and progesterone receptor; proliferation markers Ki-67 and mcm-3; Cox-2; E-cadherin; basal epithelial markers p63, CD10, keratins 5, 14, 17 and luminal epithelial markers keratins 7/8 and 19. In 30 further cases sagittal (and some coronal) sections were immunostained for keratins 7/8, 14 and progesterone receptor. Duct perimeters were measured for all ducts in transverse sections of the nipple duct bundle in 15 cases. We did not find unambiguous evidence supporting the two-duct-type hypothesis. Duct perimeters at the base of the papilla were variable but did not apparently define separate populations of larger and smaller ducts; nor could separate duct populations be recognised on their epithelial immunophenotype. However, while some ducts open directly into a funnel-shaped infundibulum, others apparently taper to a pinpoint lumen, and may cluster around a common opening. Clear cells of nipple epidermis (Toker cells) were present in variable numbers in 31/40 cases, usually around duct openings on the nipple surface, sometimes show features suggesting possible locomotion, and are occasionally present in keratin plugs. Similarities between clear cells of nipple epidermis and mammary precursor cells have been noted previously; their location and possible motility suggest new avenues for research.

An intraductal approach to breast cancer risk assessment and screening in a diverse population

The Gail Risk model was developed based on data from Caucasian women. Little information is available regarding breast cancer risk assessment in women of other ethnicities. Our study invited asymptomatic women of color presenting for screening mammography, to be interviewed with a risk questionnaire and to undergo aspiration of nipple fluid (NAF) for cytology. The HALOR system was used to aspirate the nipple fluid. Thirty three patients participated in the study, including 24 African American, 7 Asian, and 2 Hispanic women all of whom completed the questionnaires. Twelve patients had NAF, and three of those patients had bilateral NAF aspirates, a total of 15 specimens. Ten of the twelve patients with NAF were African American, and all three of the patients with bilateral NAF were African American. The ages of the 33 patients in the study ranged from 40 to 72 years, with an average age of 53, while the patients with NAF were ages 40 to 60 years with an average age of 51 years. The age of menarche and prior use of oral contraceptives were similar in the NAF and Non-NAF groups. Only 7 of the 33 patients had a history of using any type of HRT. Only two patients had a history of fertility medications. No patients had DES exposure. Fourteen patients had a history of cigarette smoking, including 6 of the NAF patients and 8 of the non-NAF patients. Twelve patients had a family history of breast cancer, 5 with NAF and 7 without NAF, with a higher rate in the NAF group (71%) vs. the non-NAF group (50%). There were 20 patients who stated that they were postmenopausal, and 15 of these patients were in the non-NAF group. No patients with NAF had abnormal cytology. Eleven of these samples had no ductal epithelial cells present, and the other 4 samples had rare or scant numbers of epithelial cells present. All of the samples had Thin Prep ConcentrationR used for slide preparation, but only three samples had adequate fluid available for cell block. Three patients (one with NAF and two without NAF) had abnormal mammograms and are having additional imaging. We have shown that it is feasible to engage women of color in a study of breast cancer risk factors and aspiration of nipple fluid for cytology. Oakland, California is a very diverse community with multiple ethnic groups living in the city. One challenge with the Hispanic and Asian women was related to language barriers, and in a larger study a greater effort will be needed to be able to involve more patients from these communities. We were disappointed in the low number of patients with NAF samples and the adequacy of the samples, and are evaluating the issues that contributed to that problem.

Searching new biomarkers in nipple aspirate fluid: peroxidation status and adhesive molecules to early identify breast cancer

During the last two years (with the support of the DSLRF Grant Award 2007), we have mainly focused our attention on both 1) the involvement of oxidative stress in breast microenvironment and 2) the function of adhesion molecules in the breast cells of the ductal-lobular unit. 1) Starting from our previous evidence that active lipid peroxidation may have a physiologic role in the normal mammary gland and that lower levels of isoprostanes in NAF of BC patients suggest altered arachidonate metabolism (Mannello et al, Int J Cancer 2007;120:1971–6), we have recently discovered that in NAF Phospholipase A2-IIa activity (key enzyme regulating arachidonate metabolism) may play a dual role in breast microenvironment during cancer development and progression. It shows a beneficial role releasing low Arachidonate levels in physiologic status, while its up-regulation has detrimental functions allowing the conversion of high arachidonate levels into leukotrienes and prostaglandins, promoting BC progression. Noteworthy, the inhibition of this pathway through COX-inhibitors leads to induction of apoptosis and tumor growth reduction (Figure ​(Figure11). Figure 1 2) Based on our hypothesis that peculiar junctional complexes may differently seal breast ductal cells leading them to different evolution (Mannello et al, Breast Cancer Res Treat 2007;102:125), we have recently discovered that higher levels of soluble form of P-cadherin were found in NAF from cancer patients. We found a positive correlation of sP-cad with disease stages and tumor grade, and an inverse relationship with estrogen/progesterone receptor status. High levels of sP-cad in cancer NAF suggest its release via proteolytic processing, favouring cancer cell detachment from breast duct. 3) Moreover, we have found that breast epithelial cells floating in NAF of cancer patients contain higher concentrations of Erythropoietin which may be shed in NAF, triggering intracellular signal cascade with subsequent breast cancer initiation. These results may explain the controversies of preclinical/clinical studies about the use of Epo, (i.e., beneficial role improving patient' survival and negative impact promoting tumor growth progression) 4) We overviewed how Diet nutrients may regulate in breast microenvironment both NAF protein expression and cancer etiology. We also reviewed how the Intraductal approaches assessing metabolic changes in breast microenvironment may help to identify early breast cancer biomarkers in NAF. Finally, we critically overviewed and discussed how the Protein analyses of NAF (mirroring the breast microenvironment) may differentiate healthy women from breast cancer patients.