Arnold Dk Hill

Lessons learned from 500 cases of lymphatic mapping for breast cancer.

OBJECTIVE To evaluate the factors affecting the identification and accuracy of the sentinel node in breast cancer in a single institutional experience. SUMMARY BACKGROUND DATA Few of the many published feasibility studies of lymphatic mapping for breast cancer have adequate numbers to assess in detail the factors affecting failed and falsely negative mapping procedures. METHODS Five hundred consecutive sentinel lymph node biopsies were performed using isosulfan blue dye and technetium-labeled sulfur colloid. A planned conventional axillary dissection was performed in 104 cases. RESULTS Sentinel nodes were identified in 458 of 492 (92%) evaluable cases. The mean number of sentinel nodes removed was 2.1. The sentinel node was successfully identified by blue dye in 80% (393/492), by isotope in 85% (419/492), and by the combination of blue dye and isotope in 93% (458/492) of patients. Success in locating the sentinel node was unrelated to tumor size, type, location, or multicentricity; the presence of lymphovascular invasion; histologic or nuclear grade; or a previous surgical biopsy. The false-negative rate of 10.6% (5/47) was calculated using only those 104 cases where a conventional axillary dissection was planned before surgery. CONCLUSIONS Sentinel node biopsy in patients with early breast cancer is a safe and effective alternative to routine axillary dissection for patients with negative nodes. Because of a small but definite rate of false-negative results, this procedure is most valuable in patients with a low risk of axillary nodal metastases. Both blue dye and radioisotope should be used to maximize the yield and accuracy of successful localizations.

Intradermal radiocolloid and intraparenchymal blue dye injection optimize sentinel node identification in breast cancer patients.

Background: Radiotracer and blue dye mapping of sentinel lymph nodes (SLN) have been advocated as accurate methods to stage the clinically negative axilla in breast cancer patients. The technical aspects of SLN biopsy are not fully characterized. In this study we compare the results of intraparenchymal (IP) and intradermal (ID) injection of Tc-99m sulfur colloid, to establish an optimal method for SLN localization.Methods: 200 consecutive patients had SLN biopsy performed by a single surgeon. Of these, 100 (Group I) had IP injection and 100 (Group II) had ID injection of Tc-99m sulfur colloid. All patients had IP injection of blue dye as well. Endpoints included (1) successful SLN localization by lymphoscintigraphy, (2) successful SLN localization at surgery, and (3) blue dye–isotope concordance (uptake of dye and isotope by the same SLN).Results: Isotope SLN localization was successful in 78% of Group I and 97% of group II patients (P < .001). When isotope was combined with blue dye, SLN were found in 92% of group I and 100% of Group II (P < .01). In cases where both dye and isotope were found in the axilla, dye mapped the same SLN as radiotracer in 97% of Group I and 95% of Group II patients.Conclusions: The dermal and parenchymal lymphatics of the breast drain to the same SLN in most patients. Because ID injection is easier to perform and more effective, this technique may simplify and optimize SLN localization.

Credentialing for breast lymphatic mapping: how many cases are enough?

OBJECTIVE To evaluate credentialing issues for sentinel lymphatic mapping for breast cancer. SUMMARY BACKGROUND DATA The sentinel lymph node (SLN) is defined as the first lymph node receiving lymphatic drainage from a tumor. The SLN accurately reflects the status of the axillary nodes in patients with early-stage breast cancer, and SLN mapping is gaining widespread acceptance. Few of the many published feasibility studies of lymphatic mapping for breast cancer have adequate numbers to assess credentialing issues for this new procedure. METHODS Five hundred consecutive SLN biopsies were performed at one institution, over a 20-month period, by eight surgeons, using isosulfan blue dye and technetium-labeled sulfur colloid. The authors reviewed each surgeons success rate in finding the SLN, and false-negative rate, relative to level of experience with the technique. RESULTS Lymphatic mapping performed by an experienced surgeon (surgeon A, B, or C) was associated with a higher success rate (94%) than when it was performed by one with less experience (86%). Ten failed mapping procedures occurred in the first 100 cases. For each of the ensuing 100 cases, there were eight, six, six, and four failed mapping procedures, suggesting that increasing experience does not eradicate failed mapping procedures completely. The false-negative rate among 104 patients in whom axillary dissection was planned in advance was 10.6% (5/47). Most false-negative results occurred early in the surgeons experience: when the first six cases of every surgeon were excluded, the false-negative rate fell to 5.2% (2/38). CONCLUSIONS With increasing experience, failed SLN localizations and false-negative SLN biopsies occur less often. Combined dye and isotope localization, enhanced histopathology, a backup axillary dissection, and judicious case selection are required to avoid the high false-negative rate of ones early experience.

Associations and Interactions between Ets-1 and Ets-2 and Coregulatory Proteins, SRC-1, AIB1, and NCoR in Breast Cancer

Purpose: Associations between p160 coactivator proteins and the development of resistance to endocrine treatment have been described. We hypothesized that nuclear receptor coregulatory proteins may interact with nonsteroid receptors. We investigated the mitogen-activated protein kinase–activated transcription factors, Ets, as possible interaction proteins for the coactivators SRC-1 and AIB1 and the corepressor NCoR in human breast cancer. Experimental Design: Expression and coexpression of Ets and the coregulatory proteins was investigated using immunohistochemistry and immunofluorescence in a cohort of breast tumor patients (N = 134). Protein expression, protein-DNA interactions and protein-protein interactions were assessed using Western blot, electromobility shift, and coimmunoprecipitation analysis, respectively. Results: Ets-1 and Ets-2 associated with reduced disease-free survival (P < 0.0292, P < 0.0001, respectively), whereas NCoR was a positive prognostic indicator (P < 0.0297). Up-regulation of Ets-1 protein expression in cell cultures derived from patient tumors in the presence of growth factors associated with tumor grade (P < 0.0013; n = 28). In primary breast tumor cell cultures and in the SKBR3 breast cell line, growth factors induced interaction between Ets and their DNA response element, induced recruitment of coactivators to the transcription factor-DNA complex, and up-regulated protein expression of HER2. Ets-1 and Ets-2 interacted with the coregulators under basal conditions, and growth factors up-regulated Ets-2 interaction with SRC-1 and AIB1. Coexpression of Ets-2 and SRC-1 significantly associated with the rate of recurrence and HER expression, compared with patients who expressed Ets-2 but not SRC-1 (P < 0.0001 and P < 0.0001, respectively). Conclusions: These data describe associations and interactions between nonsteroid transcription factors and coregulatory proteins in human breast cancer.

The accuracy of ultrasound, stereotactic, and clinical core biopsies in the diagnosis of breast cancer, with an analysis of false-negative cases.

Objective:Preoperative core biopsy in breast cancer is becoming the standard of care. The aim of this study was to analyze the various methods of core biopsy with respect to diagnostic accuracy and to examine the management and outcome of those patients with false-negative biopsies. Methods:All patients undergoing core biopsy for breast abnormalities over a 5-year period (1999–2003) were reviewed. The accuracy rates for each method of core biopsy, the histologic agreement between the core pathology and subsequent excision pathology, and the length of follow-up for cases of benign disease were studied. Patients whose biopsies were benign but who were subsequently diagnosed with cancer underwent detailed review. Results:There were 2427 core biopsies performed over the 5-year period, resulting in a final diagnosis of cancer in 1384 patients, benign disease in 954 patients, and atypical disease in 89 patients. Biopsy type consisted of 1279 ultrasound-guided cores, 739 clinically guided cores, and 409 stereotactic-guided cores. The overall false-negative rate was 6.1%, with specific rates for ultrasound-, clinical-, and stereotactic-guided cores of 1.7%, 13%, and 8.9%, respectively. False-negative biopsies occurred in 85 patients, and in 8 of these patients the diagnosis was delayed by greater than 2 months. In all other false-negative cases, “triple assessment” review allowed prompt recognition of discordant biopsy results and further evaluation. Conclusion:Ultrasound guidance should be used to perform core biopsies in evaluating all breast abnormalities visible on ultrasound. Adherence to principles of triple assessment following biopsy allows for early recognition of the majority of false-negative cases.

Expression of survivin and its splice variants survivin-2B and survivin-ΔEx3 in breast cancer

Alternative splicing of survivin mRNA gives rise to multiple isoforms, that is, survivin and 3 splice variants, survivin-2B, survivin-3B and survivin-ΔEx3. The aim of this study was to compare the expression of survivin, survivin-2B and survivin-ΔEx3 in normal breast tissue, fibroadenomas, primary breast cancer and axillary nodal metastases. Survivin, survivin-2B and survivin-ΔEx3 mRNA were measured using semiquantitative RT–PCR. In the primary carcinomas, we related mRNA for each form of survivin to both survivin protein and apoptosis. For each type of breast tissue, survivin was the predominant form detected, being present in 146 out of 156 (93.6%) primary breast carcinomas, 11 out of 11 (100%) axillary nodal metastases, 21 out of 31 (67.7%) fibroadenomas and five out of 22 (22.7%) specimens of normal breast tissue. Levels of the three forms of survivin were significantly higher in the carcinomas compared to normal breast tissue (P<0.0001). Levels of both survivin-2B and survivin-ΔEx3 but not survivin were significantly higher in nodal metastases than primary carcinomas. Survivin mRNA levels correlated significantly with survivin protein. Finally, both survivin and survivin-ΔEx3 but not survivin-2B correlated positively with apoptosis. Although survivin, survivin-2B and survivin-ΔEx3 were all detected in both malignant and nonmalignant breast tissue, the predominant form was survivin. Our results suggest that the different forms of survivin may have different roles in apoptosis in breast cancer.

ADAM-17 expression in breast cancer correlates with variables of tumor progression.

The ADAMs are a family of membrane proteins possessing a disintegrin and metalloprotease domain. One of their main functions is shedding of membrane proteins. The aim of this study was to test the hypothesis that ADAM-17 (also known as tumor necrosis factor-α converting enzyme) is involved in breast cancer progression. Overexpression of ADAM-17 in MCF-7 breast cancer cells increased in vitro invasion and proliferation, whereas down-regulation of ADAM-17 expression in MDA-MB-435 cells decreased invasion and proliferation. At both mRNA and protein levels, ADAM-17 expression was significantly up-regulated in breast cancer compared with normal breast tissue. Using Western blotting, ADAM-17 protein in breast cancer was shown to exist in two forms migrating with approximate molecular masses of 100 and 120 kDa. Based on their known molecular mass, these bands were taken to represent the active and precursor forms of ADAM-17, respectively. The proportion of active to total ADAM-17 increased progressively from normal breast tissue to primary breast cancer to lymph node metastases (P = 0.017, Kruskal-Wallis test). In primary cancers, the active form was expressed more frequently in node-positive compared with node-negative tumors (P = 0.034, χ2 test). Furthermore, in primary carcinomas, both forms of ADAM-17 correlated significantly (Spearman correlation analysis) with levels of urokinase plasminogen activator (precursor form: r = 0.246, P = 0.032, n = 83 and active form: r = 0.428, P = 0.0001, n = 83) and proliferating cell nuclear antigen (precursor form: r = 0.524, P < 0.0001, n = 73 and active form: r = 0.365, P = 0.002, n = 73). Our results support the hypothesis that ADAM-17 is involved in breast cancer progression.

Coassociation of Estrogen Receptor and p160 Proteins Predicts Resistance to Endocrine Treatment; SRC-1 is an Independent Predictor of Breast Cancer Recurrence

Purpose: This study investigates the role of the p160 coactivators AIB1 and SRC-1 independently, and their interactions with the estrogen receptor, in the development of resistance to endocrine treatments. Experimental Design: The expression of the p160s and the estrogen receptor, and their interactions, was analyzed by immunohistochemistry and quantitative coassociation immunofluorescent microscopy, using cell lines, primary breast tumor cell cultures, and a tissue microarray with breast cancer samples from 560 patients. Results: Coassociation of the p160s and estrogen receptor α was increased in the LY2 endocrine-resistant cell line following treatment with tamoxifen in comparison with endocrine-sensitive MCF-7 cells. In primary cultures, there was an increase in association of the coactivators with estrogen receptor α following estrogen treatment but dissociation was evident with tamoxifen. Immunohistochemical staining of the tissue microarray revealed that SRC-1 was a strong predictor of reduced disease-free survival (DFS), both in patients receiving adjuvant tamoxifen treatment and untreated patients (P < 0.0001 and P = 0.0111, respectively). SRC-1 was assigned a hazard ratio of 2.12 using a Cox proportional hazards model. Endocrine-treated patients who coexpressed AIB1 with human epidermal growth factor receptor 2 had a significantly shorter DFS compared with all other patients (P = 0.03). Quantitative coassociation analysis in the patient tissue microarray revealed significantly stronger colocalization of AIB1 and SRC-1 with estrogen receptor α in patients who have relapsed in comparison with those patients who did not recur (P = 0.026 and P = 0.00001, respectively). Conclusions: SRC-1 is a strong independent predictor of reduced DFS, whereas the interactions of the p160 proteins with estrogen receptor α can predict the response of patients to endocrine treatment.

Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase

IntroductionThe adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells.MethodsMCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation.ResultsJAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6 and the Rap1 activator PDZ-GEF2 in MCF7 cells and in primary cultures from breast cancer patients.ConclusionsOur findings provide compelling evidence of a novel role for JAM-A in driving breast cancer cell migration via activation of Rap1 GTPase and β1-integrin. We speculate that JAM-A over-expression in some breast cancer patients may represent a novel therapeutic target to reduce the likelihood of metastasis.

Surgical e-learning: validation of multimedia web-based lectures

Background  Distance learning has been advocated increasingly as a modern efficient method of teaching surgery. Efficiency of knowledge transfer and validity of web‐based courses have not been subjected to rigorous study to date.