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It is of highest importance to find proteins responsible for breast cancer dissemination, for use as biomarkers or treatment targets. We established and performed a combined nontargeted LC-MS/MS and a targeted LC-SRM workflow for discovery and validation of protein biomarkers. Eighty breast tumors, stratified for estrogen receptor status and development of distant recurrence (DR ± ), were collected. After enrichment of N-glycosylated peptides, label-free LC-MS/MS was performed on each individual tumor in triplicate. In total, 1515 glycopeptides from 778 proteins were identified and used to create a map of the breast cancer N-glycosylated proteome. Based on this specific proteome map, we constructed a 92-plex targeted label-free LC-SRM panel. These proteins were quantified across samples by LC-SRM, resulting in 10 proteins consistently differentially regulated between DR+/DR- tumors. Five proteins were further validated in a separate cohort as prognostic biomarkers at the gene expression level. We also compared the LC-SRM results to clinically reported HER2 status, demonstrating its clinical accuracy. In conclusion, we demonstrate a combined mass spectrometry strategy, at large scale on clinical samples, leading to the identification and validation of five proteins as potential biomarkers for breast cancer recurrence. All MS data are available via ProteomeXchange and PASSEL with identifiers PXD001685 and PASS00643.

Intraoperative frozen section (FS) margin evaluation is not common practice for patients undergoing breast conservation therapy (BCT), but offers a significant reduction in reoperation. In this study, a technique to allow for more effective freezing of breast tissue was developed to perform FS evaluation of lumpectomy margins (FSM) for all patients undergoing BCT at an ambulatory surgery center. FS evaluation of sentinel lymph node biopsy specimens was performed concurrently. One hundred eighty-one study and 188 control patients, with and without FS evaluation, were compared. Reexcision was reduced 34% (from 48.9% to 14.9%) and reoperation was reduced 36% (from 55.3% to 19.3%) with FS evaluation. Most of the decrease in reoperative rate was because of a decrease in the need for margin reexcision. The number of patients requiring 1, 2, or 3 operations to complete therapy was 84, 92, and 12, respectively, in the control group, and 146, 33, and 2, respectively, in the study group. Lobular subtype, multifocal disease, and larger tumor size (≥2 cm) were significantly associated with failure of FSM to prevent reoperation, but reoperation rates were still significantly decreased in this subgroup of patients (from 75.5% to 43.8%) with FSM. This study highlights an innovative yet simple and adaptable FS approach that resulted in a nearly 3-fold reduction in reoperation for patients undergoing BCT.

Despite the low likelihood of malignancy, it is recommended that all women with pathologic nipple discharge undergo duct excision based on the inadequate sensitivity of diagnostic modalities. However, these data originates prior to recent improvements in breast imaging. We performed a retrospective review of patients evaluated in the setting of modern diagnostic breast imaging. Of 175 women referred to our breast clinic with a primary complaint of nipple discharge, 142 (81%) had suspicious discharge. Of the 23 patients who opted for observation over duct excision, with a mean follow-up of 3.3 years, none have been diagnosed with cancer. Among patients who proceeded with surgery, cancer was diagnosed in seven patients (5%). Six of the seven patients had either an abnormal mammogram or ultrasound. Among 46 patients with suspicious nipple discharge, a normal physical exam and normal diagnostic mammogram/ultrasound, only one malignancy (2%) was identified in a 79-year-old patient with a personal history of breast cancer. In selected patients with suspicious nipple discharge, but normal physical exam and diagnostic imaging, short-term observation with repeat evaluation seems reasonable for patients who do not desire duct excision., (© 2011 Wiley Periodicals, Inc.)

Background: Negative surgical margins minimize the risk of local recurrence after breast-conserving surgery. Intraoperative frozen section analysis (FSA) is one method for margin evaluation. We retrospectively analyzed records of patients who received breast-conserving therapy with intraoperative FSA of the lumpectomy cavity to assess re-excision rates and local control., Methods: Records were retrospectively reviewed for individuals who underwent breast-conserving surgery for ductal carcinoma in situ (DCIS) or invasive carcinoma between 1993 and 2003. Inclusion criteria were a minimum of 2 years follow-up and intact tumor at the time of operation. The major outcome measure was local recurrence. The Kaplan-Meier test was used to evaluate local recurrence rates between groups., Results: 290 subjects with an average age of 57.2 years (range 27-89) underwent 292 lumpectomies with FSA. 11.3% had DCIS, 73.3% had infiltrating ductal, 5.8% had infiltrating lobular, and 9.6% exhibited other forms of invasive carcinoma. 70 subjects underwent additional resection at the time of breast surgery, 16 underwent subsequent re-excision, and 17 underwent subsequent mastectomy. At a median follow-up of 53.4 months (range 5.8-137.8), there were six local recurrences (2.74%) in patients who had breast-conserving procedures and two local recurrences in patients who underwent mastectomy. There were no statistically significant associations among local recurrence rate, tumor size, nodal status, or overall stage. Local recurrences were higher in patients with DCIS compared with invasive carcinoma, and tumors >2cm., Conclusions: Intraoperative FSA allows resection of suspicious or positive margins at the time of lumpectomy and results in low rates of local recurrence and re-excision. The low local recurrence rate reported here is comparable to those reported with other margin assessment techniques.

Background: Signal transduction pathways convey information from the outside of the cell to transcription factors, which in turn regulate gene expression. Our objective is to analyze tumor gene expression data from microarrays in the context of such pathways., Results: We use pathways compiled from the TRANSPATH/TRANSFAC databases and the literature, and three publicly available cancer microarray data sets. Variation in pathway activity, across the samples, is gauged by the degree of correlation between downstream targets of a pathway. Two correlation scores are applied; one considers all pairs of downstream targets, and the other considers only pairs without common transcription factors. Several pathways are found to be differentially active in the data sets using these scores. Moreover, we devise a score for pathway activity in individual samples, based on the average expression value of the downstream targets. Statistical significance is assigned to the scores using permutation of genes as null model. Hence, for individual samples, the status of a pathway is given as a sign, + or -, and a p-value. This approach defines a projection of high-dimensional gene expression data onto low-dimensional pathway activity scores. For each dataset and many pathways we find a much larger number of significant samples than expected by chance. Finally, we find that several sample-wise pathway activities are significantly associated with clinical classifications of the samples., Conclusion: This study shows that it is feasible to infer signal transduction pathway activity, in individual samples, from gene expression data. Furthermore, these pathway activities are biologically relevant in the three cancer data sets.

Purpose: Sentinel lymph node (SLN) biopsy has proved to be an accurate method for detecting nodal micrometastases in previously untreated patients with early-stage breast cancer. We investigated the accuracy of this technique for patients with more advanced breast cancer after neoadjuvant chemotherapy., Patients and Methods: Patients with stage II or III breast cancer who had undergone doxorubicin-based neoadjuvant chemotherapy before breast surgery were eligible. Intraoperative lymphatic mapping was performed with peritumoral injections of blue dye alone or in combination with technetium-labeled sulfur colloid. All patients were offered axillary lymph node dissection. Negative sentinel and axillary nodes were subjected to additional processing with serial step sectioning and immunohistochemical staining with an anticytokeratin antibody to detect micrometastases., Results: Fifty-one patients underwent SLN biopsy after neoadjuvant chemotherapy from 1994 to 1999. The SLN identification rate improved from 64.7% to 94.1%. Twenty-two (51.2%) of the 43 successfully mapped patients had positive SLNs, and in 10 of those 22 patients (45.5%), the SLN was the only positive node. Three patients had false-negative SLN biopsy; that is, the sentinel node was negative, but at least one nonsentinel node contained metastases. Additional processing revealed occult micrometastases in four patients (three in sentinel nodes and one in a nonsentinel node)., Conclusion: SLN biopsy is accurate after neoadjuvant chemotherapy. The SLN identification improved with experience. False-negative findings occurred at a low rate throughout the series. This technique is a potential way to guide the axillary treatment of patients who are clinically node negative after neoadjuvant chemotherapy.

Sentinel lymph node (SLN) biopsy has been shown to predict axillary metastases accurately in early stage breast cancer. Some patients with locally advanced breast cancer receive preoperative (neoadjuvant) chemotherapy, which may alter lymphatic drainage and lymph node structure. In this study, we examined the feasibility and accuracy of SLN mapping in these patients and whether serial sectioning and keratin immunohistochemical (IHC) staining would improve the identification of metastases in lymph nodes with chemotherapy-induced changes. Thirty-eight patients with stage II or III breast cancer treated with neoadjuvant chemotherapy were included. In all patients, SLN biopsy was attempted, and immediately afterward, axillary lymph node dissection was performed. If the result of the SLN biopsy was negative on initial hematoxylin and eosin-stained sections, all axillary nodes were examined with three additional hematoxylin and eosin sections and one keratin IHC stain. SLNs were identified in 31 (82%) of 38 patients. The SLN accurately predicted axillary status in 28 (90%) of 31 patients (three false negatives). On examination of the original hematoxylin and eosin-stained sections, 20 patients were found to have tumor-free SLNs. With the additional sections, 4 (20%) of these 20 patients were found to have occult lymph node metastases. These metastatic foci were seen on the hematoxylin and eosin staining and keratin IHC staining. Our findings indicate that lymph node mapping in patients with breast cancer treated with neoadjuvant chemotherapy can identify the SLN, and SLN biopsy in this group accurately predicts axillary nodal status in most patients. Furthermore, serial sectioning and IHC staining aid in the identification of occult micrometastases in lymph nodes with chemotherapy-induced changes.

Experimental and clinical evidence suggests that breast neoplasia appears to be a hormone-dependent process that may also be influenced by dietary factors in many women. Conflicting reports on the relationship between exogenous hormones and the development, progression, and recurrence of breast cancer are critically examined in this report. The absolute breast cancer risk associated with either hormone replacement therapy or oral contraceptive use has not been clearly defined. Data from some large prospective studies have actually documented lower mortality rates for women taking hormone replacement compared with those for women who did not have hormone replacement therapy. In this regard, age, duration of use, and preexisting breast cancer risk factors must be taken into account. Although the results of two major prospective clinical trials addressing the role of timing of surgery within the menstrual cycle are forthcoming, the majority of studies have found no consistent association between timing of surgery and breast cancer survival. Recently reported prospective randomized data showing that selective-estrogen-receptor-modulators can act as effective chemoprevention agents in women at increased risk for breast cancer development are presented. Finally, information regarding the effect of dietary manipulation on breast cancer risk and survival is reviewed., (Copyright 1999 Wiley-Liss, Inc.)

Objective: To determine the incidence and prognostic significance of documented eradication of breast cancer axillary lymph node (ALN) metastases after neoadjuvant chemotherapy., Summary Background Data: Neoadjuvant chemotherapy is the standard of care for patients with locally advanced breast cancer and is being evaluated in patients with earlier-stage operable disease., Methods: One hundred ninety-one patients with locally advanced breast cancer and cytologically documented ALN metastases were treated in two prospective trials of doxorubicin-based neoadjuvant chemotherapy. Patients had breast surgery with level I and II axillary dissection followed by additional chemotherapy and radiation treatment. Nodal sections from 43 patients who were originally identified as having negative ALNs at surgery were reevaluated and histologically confirmed to be without metastases. An additional 1112 sections from these lymph node blocks were obtained; half were stained with an anticytokeratin antibody cocktail and analyzed. Survival was calculated using the Kaplan-Meier method., Results: Of 191 patients with positive ALNs at diagnosis, 23% (43 patients) were converted to a negative axillary nodal status on histologic examination (median number of nodes removed = 16). Of the 43 patients with complete axillary conversion, 26% (n = 11) had N1 disease and 74% (n = 32) had N2 disease. On univariate analysis, patients with complete versus incomplete histologic axillary conversion were more likely to have initial estrogen-receptor-negative tumors, smaller primary tumors, and a complete pathologic response in the primary tumor. The 5-year disease-free survival rates were 87% in patients with preoperative eradication of axillary metastases and 51% for patients with residual nodal disease after neoadjuvant chemotherapy. Of the 39 patients with complete histologic conversion for whom nodal blocks were available, occult nodal metastases were found in additional nodal sections in 4 patients (10%). At a median follow-up of 61 months, the 5-year disease-free survival rates were 87% in patients without occult nodal metastases and 75% in patients with occult nodal metastases., Conclusions: Neoadjuvant chemotherapy can completely clear the axilla of microscopic disease before surgery, and occult metastases are found in only 10% of patients with a histologically negative axilla. The results of this study have implications for the potential use of sentinel lymph node biopsy as an alternative to axillary dissection in patients treated with neoadjuvant chemotherapy.