Characterization of surface markers on extracellular vesicles isolated from lymphatic exudate from patients with breast cancer.

BACKGROUND: Breast cancer is the most common cancer, and the leading cause of cancer-related deaths, among females world-wide. Recent research suggests that extracellular vesicles (EVs) play a major role in the development of breast cancer metastasis. Axillary lymph node dissection (ALND) is a procedure in patients with known lymph node metastases, and after surgery large amounts of serous fluid are produced from the axilla. The overall aim was to isolate and characterize EVs from axillary serous fluid, and more specifically to determine if potential breast cancer biomarkers could be identified. METHODS: Lymphatic drain fluid was collected from 7 patients with breast cancer the day after ALND. EVs were isolated using size exclusion chromatography, quantified and detected by nanoparticle tracking analysis, electron microscopy, nano flow cytometry and western blot. The expression of 37 EV surface proteins was evaluated by flow cytometry using the MACSPlex Exosome kit. RESULTS: Lymphatic drainage exudate retrieved after surgery from all 7 patients contained EVs. The isolated EVs were positive for the typical EV markers CD9, CD63, CD81 and Flotillin-1 while albumin was absent, indicating low contamination from blood proteins. In total, 24 different EV surface proteins were detected. Eleven of those proteins were detected in all patients, including the common EV markers CD9, CD63 and CD81, cancer-related markers CD24, CD29, CD44 and CD146, platelet markers CD41b, CD42a and CD62p as well as HLA-DR/DP/DQ. Furthermore, CD29 and CD146 were enriched in Her2+ patients compared to patients with Her2- tumors. CONCLUSIONS: Lymphatic drainage exudate retrieved from breast cancer patients after surgery contains EVs that can be isolated using SEC isolation. The EVs have several cancer-related markers including CD24, CD29, CD44 and CD146, proteins of potential interest as biomarkers as well as to increase the understanding of the mechanisms of cancer biology.

Evaluation of intraoperative touch imprint cytology on axillary sentinel lymph nodes in invasive breast carcinomas, a retrospective study of 1227 patients comparing sensitivity in the different tumor subtypes.

BACKGROUND: Intraoperative evaluation of the axillary sentinel lymph node (SLN) in patients with breast carcinoma reduces the need of re-operations in cases where an axillary completion lymph node dissection (CLND) is indicated. Different methods have been used to determine the SLN status intraoperatively, e.g. frozen section histology (FS) and touch imprint cytology (TIC). The sensitivity of intraoperative TIC examination on SLN is not consistent between different studies and varies according to different tumor histologic subtypes, tumor size and the age of the patient. The aim of this study was to describe the specificity and sensitivity of TIC and to compare TIC sensitivity in the different histological subtypes of breast carcinoma. METHODS: A retrospective review was performed of 1227 consecutive clinically node negative breast cancer patients treated with sentinel lymph node biopsy (SLNB) with intraoperative TIC between the years 2003 and 2008. The SLN was bisected and stained using the May-Grünwald-Giemsa method and immunocytochemically with the antibody MNF-116. RESULTS: The overall sensitivity of the TIC test was 68.6% and the specificity was 99.8%. There was no statistically significant difference between the detection of SLN metastases from ductal carcinoma versus lobular carcinoma. The sensitivity improved over the period of the study. CONCLUSION: TIC is highly specific with an acceptable overall sensitivity. The sensitivity increased under the period of the study and it was higher in cases with larger size of the primary tumor. There was no difference in TIC sensitivity between the different histological subtypes.