DNA promoter hypermethylation profiles in breast duct fluid.

DNA methylation of tumor-suppressor genes occurs early in the molecular transformation of precursor events to breast cancer and is therefore of interest to screening in high-risk women. The aim of this study was to use tumor-suppressor genes that have previously been shown to be cancer predictive in tissue to evaluate the potential of DNA methylation assays in cells from duct lavage (DL) fluid. The frequency of target gene DNA methylation in tissue and DL of cancer and healthy control patients was assessed, and an association of DNA methylation between different duct systems in the same breast was explored. The cancer and control groups were identified in the outpatient clinic when surgical treatment was finalized. Tumor, adjacent tissue and bilateral DL samples for comparative DNA methylation studies were obtained during surgery from women with cancer. In the healthy control group, samples of tissue and DL were collected. Reverse transcriptase methylation-specific PCR was conducted on modified DNA purified from 42 cancer biopsies, 41 benign excision cavity biopsies (internal control), 29 benign biopsies (external control), and 119 DL specimens. A validated panel of cancer predictive genes was analyzed in the study bank of tissue and DL samples from cancer and healthy patients. The sensitivity of DNA methylation in DL samples compared with matched cancer tissue was highest for SCGB3A1 (90 %), CDH13 (91 %), and RARB (83 %). The genetic algorithm selected RASSF1A, RARB, and IGFBP7 as the optimum predictor set for detecting DNA methylation in cancer tissue. The optimum area under the ROC curve for DNA methylation in cancer compared with internal control healthy tissue from excision margins was 0.84. The area under the ROC curve for DNA methylation in cancer DL compared with contralateral benign DL was 0.76. DL cytology was not a helpful predictor of breast cancer. This study shows that relative patterns of tumor-suppressor gene hypermethylation in breast cancer tissue are significantly reflected in the DL from the cancer affected breast. Using DL, nonconcordant patterns of DNA methylation between different duct systems confer independent oncologic potential for distinct breast lobes. The approach of DNA methylation in DL may be substantiated by a larger trial of breast cancer 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.

A review of the oncologic and surgical management of breast cancer in the augmented breast: diagnostic, surgical and surveillance challenges.

BACKGROUND: The challenges of managing breast cancer in women with augmented breasts include screening, diagnosis, oncologic and revisional surgery, and surveillance. In addition, women with augmented breasts frequently have greater expectations of cosmetic outcomes. More breast clinicians will be affected by these challenges as augmentation grows in popularity and women with implants reach the age range in which they are at higher risk of developing breast cancer. In the United States, more than 2 million women have undergone augmentation, making this the second most commonly performed cosmetic procedure. With a lifetime risk of developing breast cancer of 1 in 8, it is projected that more than 50,000 women who undergo augmentation each year in the United States will develop breast cancer at some point in their lives. METHODS: This is a review of current practice based on an exhaustive literature search of PubMed, Google Scholar, and conference proceedings. A series of case studies is presented to illustrate mammographic changes and cosmetic outcomes in augmented breasts that have required treatment for breast cancer. RESULTS: An evidence-based summary of recommendations has been produced to guide breast surgeons in managing this particular group of patients. CONCLUSIONS: Management of breast cancer in previously augmented breasts presents a unique range of challenges. Patients can be reassured that the presence of an implant does not increase the risk of breast cancer developing or affect the prognosis if breast cancer does develop. Clinical judgement is made balancing surgical and oncologic principles to provide the best cosmetic outcome.

Mammary ductoscopy in the current management of breast disease.

BACKGROUND: 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. METHODS: 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. RESULTS/CONCLUSIONS: 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.

Gene promoter hypermethylation in ductal lavage fluid from healthy BRCA gene mutation carriers and mutation-negative controls.

INTRODUCTION: Female germline BRCA gene mutation carriers are at increased risk for developing breast cancer. The purpose of our study was to establish whether healthy BRCA mutation carriers demonstrate an increased frequency of aberrant gene promoter hypermethylation in ductal lavage (DL) fluid, compared with predictive genetic test negative controls, that might serve as a surrogate marker of BRCA1/2 mutation status and/or breast cancer risk. METHODS: The pattern of CpG island hypermethylation within the promoter region of a panel of four genes (RAR-beta, HIN-1, Twist and Cyclin D2) was assessed by methylation-specific polymerase chain reaction using free DNA extracted from DL fluid. RESULTS: Fifty-one DL samples from 24 healthy women of known BRCA mutation status (7 BRCA1 mutation carriers, 12 BRCA2 mutation carriers and 5 controls) were available for methylation analysis. Eight of 19 (42.1%) BRCA mutation carriers were found to have at least one hypermethylated gene in the four-gene panel. Two BRCA mutation carriers, in whom aberrant methylation was found, also had duct epithelial cell atypia identified. No hypermethylation was found in DL samples from 5 negative controls (p = 0.13). CONCLUSION: We found substantial levels of aberrant methylation, with the use of a four-gene panel, in the fluid from the breasts of healthy BRCA mutation carriers compared with controls. Methylation analysis of free DNA in DL fluid may offer a useful surrogate marker for BRCA1/2 mutation status and/or breast cancer risk. Further studies are required for the evaluation of the specificity and predictive value of aberrant methylation in DL fluid for future breast cancer development in BRCA1/2 mutation carriers.

Pitfalls in Portacath location using the landmark technique: case report.

A 34 year old woman diagnosed with breast cancer and liver metastases underwent a left subclavian Portacath insertion. During the procedure, the clinical features and the findings of intra-operative investigations provided conflicting evidence of the catheter position. This report highlights the potential difficulties in establishing long-term central venous access, the limitations of common investigations and safety issues relating to the process of subclavian line insertion.

Loss of heterozygosity at the BRCA1 and BRCA2 loci detected in ductal lavage fluid from BRCA gene mutation carriers and controls.

Female BRCA gene mutation carriers are at increased risk for developing breast cancer. Ductal lavage is a novel method for sampling breast ductal fluid, providing epithelial cells for cytologic assessment and a source of free DNA for molecular analyses. Loss of heterozygosity (LOH) at the BRCA loci in ductal lavage fluid is a potential biomarker of breast cancer risk. The LOH rate was measured at the BRCA1/2 loci and compared with that at a control locus (APC) using free DNA from the ductal lavage fluid of BRCA carriers and predictive test negative controls. We evaluated the reproducibility of these analyses. Free DNA sufficient for PCR amplification was obtained from 33 ductal lavage samples of 17 healthy women of known BRCA status (14 BRCA carriers and 3 controls). LOH rates of 36.4% to 56.3% at the BRCA1 locus and 45% to 61.5% at the BRCA2 locus were found among BRCA carriers. The LOH rate at the APC locus was lower (18.5%). The interaliquot reproducibility for the D17S855 marker of the BRCA1 locus was 66.7%. Intraaliquot reproducibility was 90%. Although we successfully isolated sufficient free DNA from ductal lavage fluid for PCR amplification, the degree of reproducibility of these LOH studies raises questions about the robustness of this technique as a risk assessment tool in the evaluation of high-risk women. Further studies are required to evaluate the specificity and predictive value of LOH in ductal lavage fluid for breast cancer development.

Breast carcinoma and malignant melanoma metastasis within a single axillary lymph node.

A 58 year old lady presented with a right breast cancer and a prior history of malignant melanoma excised from the right chest wall eight years previously. An abnormal axillary lymph node resected contained features of both metastatic breast carcinoma and malignant melanoma. Following oncologic breast cancer management, the patient is well with no evidence of recurrence at three years.

A case-control study of treatment choices made by doctors diagnosed with early breast cancer.

INTRODUCTION: Doctors who are diagnosed with breast cancer form a small subset of women with unique insight into their disease. The aim of this study was to compare key treatment decisions in medically-qualified doctors with equivalent degree-educated, matched controls diagnosed with early breast cancer. METHODS: Patients diagnosed with breast cancer between 01/01/2006 and 31/12/2011 were included and screened for occupation by 2 independent investigators. Allied health professionals with a medical background (e.g. nurses, physiotherapists) were excluded. Patient controls were matched by age, nodal status and grade. If there were more than 5 possible matches then those patients with the closest tumour size were selected. RESULTS: 5259 of 8623 patients had signed the generic research consent form. 619 of these were either doctors, or had received tertiary level education and could form appropriate controls. After exclusions, 46 medically-qualified doctors and 230 matched control patients were included in the analysis. No statistical differences in age or tumour characteristics were identified between doctors with breast cancer and matched controls. No differences were identified between the two groups in the uptake of mastectomy, chemotherapy, immediate breast reconstruction or post-mastectomy radiotherapy. CONCLUSION: Medically-qualified patients diagnosed with early breast cancer are no more likely to opt for mastectomy, chemotherapy, immediate breast reconstruction or post-mastectomy radiotherapy than non-medically-qualified controls. The level of information generally provided to patients with breast cancer is adequate for a similar decision to be made by control patients with equivalent levels of education, independent of any knowledge of pathology or understanding of the medical system that medically-qualified patients may possess.

Immediate breast reconstruction using biodimensional anatomical permanent expander implants: a prospective analysis of outcome and patient satisfaction.

Immediate breast reconstruction is offered to the majority of women requiring a mastectomy for cancer treatment. Improvements in implant technology have seen the evolution of shaped, fixed-volume implants and permanent expanders. The theoretical benefits of biodimensional anatomical expander implants include better reconstructed breast form, the potential for achieving this at a single procedure, and the avoidance of a contralateral procedure as a result of improved ipsilateral cosmesis. The aim of the present study was to assess outcome after immediate breast reconstruction using the McGhan 150 permanent expander implant. A total of 107 consecutive patients (129 breast reconstructions) were studied over a 30-month period; 49 patients (68 reconstructions) had submuscular implant placement alone, and 58 patients (61 reconstructions) had an implant in conjunction with a latissimus dorsi flap. The mean patient age was 47 years (range, 22 to 72 years) and the mean follow-up was 18 months (range, 6 to 40 months). Outcome was assessed in the following four ways: geometric measurements, evaluation of photographs by four independent observers, linear numerical analogue scale, and a quality-of-life questionnaire. Geometric measurements after surgery showed median differences that were highly consistent with good symmetry. Transverse breast width difference between breasts was 0.35 cm, vertical breast height difference was 0.8 cm, sternal notch-to-nipple distance difference was 0.6 cm, midclavicular line-to-nipple distance difference was 0.5 cm, nipple-to-inframammary crease distance difference was 0.5 cm, midline-to-nipple distance difference was 0 cm, and projection difference was 0 cm. Photographic assessment by four observers who evaluated shape, cleavage, symmetry, and overall outcome produced correlation values of 0.73 to 0.81. More than 75 percent of each of these parameters was scored as good or excellent. A numerical analogue scale (from 1 to 10) assessing overall result by the surgeon and patient revealed good (7 to 8) or excellent (9 to 10) scores in 83 and 82 percent of respondents, respectively. Quality-of-life data showed that 81 and 88 percent of women felt "a little" or "not at all" less feminine, respectively, and 88 percent were satisfied with the appearance of their breasts. A total of 88 percent of women would also choose the same reconstructive procedure if faced with the same cancer diagnosis. Complication rates were low; infections occurred in 6.2 percent of reconstructions, hematoma occurred in 1.6 percent, and implant loss occurred in 3.9 percent. Only 24 of 107 patients (22 percent) elected to have a synchronous contralateral breast adjustment, and four of 107 (4 percent) chose to have a subsequent procedure for symmetry. Biodimensional expander implants used in immediate breast reconstruction are associated with high levels of patient and surgeon satisfaction. Optimum breast form can be achieved during a single operation with a low incidence of complications.

The feasibility of nipple aspiration and duct lavage to evaluate the breast duct epithelium of women with increased breast cancer risk.

AIM: Nipple aspiration (NA) and duct lavage (DL) are modalities for obtaining breast duct fluid for biomarker analyses. The aim of this study was to assess the feasibility of obtaining serial NA and DL samples at consecutive patient visits for cytology assessment and the creation of a biobank. METHODS: Seventy eligible subjects were enroled at a single institution in the United Kingdom as part of an international multicentre study. Entry criteria were based on a 5-year Gail model risk of ≥2% or Claus score lifetime risk of ≥26%. Women underwent NA and DL in an outpatient clinic under local anaesthesia. RESULTS: The mean patient age was 48 (range 41-69)years. Sixty seven out of 70 women (96%) attended three consecutive 6 monthly visits and follow-up for 2 years. Three women withdrew due to intolerance of the DL procedure. 56/67 (83%) women produced NA fluid from at least one duct. 204/264 (77%) of ducts declared by NA were cannulated for DL. 170/204 (83%) produced DL samples with adequate cellularity. By the final visit 52/67 (78%) women produced DL, 28/52 (54%) of whom were premenopausal and 24/52 (46%) were postmenopausal. 50/52 women (96%) underwent repeated DL of 81 ducts on 3 consecutive visits. CONCLUSION: NA and DL are well tolerated for repeated assessment to obtain material for cytology and to create a biobank for future biomarker studies in women at high breast cancer risk.

Biomarkers in the diagnosis of primary and recurrent breast cancer.

Despite the widespread use of mammography for breast cancer screening, breast cancer remains the most common cause of cancer-related mortality among women worldwide. The identification of biomarkers that identify cancers when they are small, localized and most treatable is an important aim of current breast cancer research. Biomarkers need to be sensitive, specific, reproducible and easily collected from patients from readily accessible tissue or body fluids. While conventional biomarker research has focused on soluble proteins, cell markers, proteomics and DNA methylation, much progress has also been made in the field of immunobiomarkers and multiparameter gene arrays. Currently, no one biomarker has demonstrated sufficient sensitivity and reproducibility for independent clinical and commercial use. This review summarizes the current state of breast cancer biomarker research and anticipated future directions.

Long-term quality-of-life assessment following one-stage immediate breast reconstruction using biodimensional expander implants: the patient's perspective.

BACKGROUND: Biodimensional devices may provide a superior breast form when an implant is considered for immediate breast reconstruction for breast cancer. The authors prospectively report patient perception of outcome using a permanent anatomical expander implant following a planned one-stage procedure. METHODS: The Quality of Life Questionnaire BR-23 was modified to measure body image perception, physical effects, cancer worry, and surgical choice. Responses were classified into four categories. A subset of patients completed the questionnaire 1 and 6 years after surgery. Data were compared using nonparametric analyses. RESULTS: One hundred ten patients were studied, with a mean follow-up of 63 months (range, 25 to 108 months); 46 patients had submuscular implant reconstruction and 64 had an implant-assisted latissimus dorsi flap. The mean patient age was 46 years (range, 20 to 76 years). There was no difference in patient perception of outcome between the two groups, although the latissimus dorsi group found it easier to fit into bras (p = 0.03, Mann-Whitney test). For patient perception of body image, the median score was within the most favorable category in four of six fields. Restricted arm movement and pain in the treated breast had resolved completely by 5 years after surgery. More than 80 percent of patients would choose the same surgical option if the cancer scenario presented itself again. CONCLUSIONS: A biodimensional permanent expander implant, when used appropriately, can achieve high levels of patient satisfaction. The authors' data provide long-term outcome measures with which to counsel patients about one-stage implant-assisted immediate breast reconstruction using an anatomical device.

Proteomic analysis of nipple aspirate fluid throughout the menstrual cycle in healthy pre-menopausal women.

A proteomic approach to nipple aspiration fluid (NAF) has been used in a number of studies comparing women with breast cancer and healthy women. However, to make useful comparisons between women with breast cancer and healthy women it is necessary to establish whether there is physiological variation in the proteomic profiles of NAF. The purpose of this study was, for the first time, to examine how the proteomic profile of NAF using surface-enhanced laser desorption ionisation time-of-flight mass spectrometry varies across the menstrual cycle in healthy pre-menopausal women. Twelve women were recruited and nipple aspiration was carried out weekly from both breasts of each subject for two menstrual cycles. Matching serum samples for luteinising hormone, follicle stimulating hormone and oestradiol were obtained at each aspiration attempt. Statistically significant peaks were found for three healthy volunteers (p < 0.05). However, the peaks that varied across the menstrual cycle were different from one healthy volunteer to another and the differences were small compared with the large variation in proteomic profiles between healthy volunteers. This study provides proof of concept that the NAF proteomic profile does not vary substantially during the menstrual cycle and that therefore it is valid to compare NAF profiles from pre-menopausal women that have been taken at different stages in the menstrual cycle.

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.

Evaluation of outcome after immediate breast reconstruction: prospective comparison of four methods.

BACKGROUND: Little is known about the association among different methods to assess outcome following breast reconstruction. The aim of this study was to comprehensively compare four distinct methods of outcome evaluation following immediate breast reconstruction. METHODS: There were 102 women who were prospectively evaluated: 46 patients had submuscular implants, and 56 patients had implant-assisted latissimus dorsi breast reconstruction. The mean patient age at the time of operation was 46 years (range, 22 to 72 years), with a mean follow-up of 18 months (range, 12 to 24 months). All patients were evaluated using geometric measurements, photographs, linear analogue scores, and a quality-of-life questionnaire. The Wilcoxon signed rank test, Spearman's correlation, and factor analysis statistics were used. RESULTS: Transverse breast width, vertical breast height, sternal notch-nipple, midclavicular line-nipple, nipple-inframammary crease, midline-nipple, internipple, and intermammary measurements were reproducible and reliable. Median differences of absolute vertical and horizontal differences were less than 1 cm and were associated with high levels of patient satisfaction. Assessors asked to score photographs without being specifically requested to consider shape, cleavage, or symmetry would focus mainly on vertical and horizontal differences. There was a high correlation between surgeon and patient linear analogue scores. Quality-of-life questionnaires provide valuable information on body image, physical effects, and continued cancer worry. Factor analyses failed to identify components to compress data fields to obtain equivalent information from fewer questions. CONCLUSIONS: Evaluation of immediate breast reconstruction is complex. Cumbersome data collection has to be balanced against practical variables that individual units can collect to evaluate outcome for audit and research.