Background: Estrogen receptor α (ERα) signaling is a defining and driving event in most breast cancers; ERα is detected in malignant epithelial cells of 75% of all breast cancers (classified as ER-positive breast cancer) and, in these cases, ERα targeting is the main therapeutic strategy. However, the biological determinants of ERα heterogeneity and the mechanisms underlying therapeutic resistance are still elusive, hampered by the challenges in developing experimental models recapitulative of intra-tumoral heterogeneity and in which ERα signaling is sustained. Ex vivo cultures of human breast cancer tissue have been proposed to retain the original tissue architecture, epithelial and stromal cell components and ERα. However, loss of cellularity, viability and ERα expression are well-known culture-related phenomena. Methods: BC samples were collected and brought to the laboratory. Then they were minced, enzymatically digested, entrapped in alginate and cultured for 1 month. The histological architecture, cellular composition and cell proliferation of tissue microstructures were assessed by immunohistochemistry. Cell viability was assessed by measurement of cell metabolic activity and histological evaluation. The presence of ERα was accessed by immunohistochemistry and RT-qPCR and its functionality evaluated by challenge with 17-β-estradiol and fulvestrant. Results: We describe a strategy based on entrapment of breast cancer tissue microstructures in alginate capsules and their long-term culture under agitation, successfully applied to tissue obtained from 63 breast cancer patients. After 1 month in culture, the architectural features of the encapsulated tissue microstructures were similar to the original patient tumors: epithelial, stromal and endothelial compartments were maintained, with an average of 97% of cell viability compared to day 0. In ERα-positive cases, fibers of collagen, the main extracellular matrix component in vivo, were preserved. ERα expression was at least partially retained at gene and protein levels and response to ERα stimulation and inhibition was observed at the level of downstream targets, demonstrating active ER signaling. Conclusions: The proposed model system is a new methodology to study ex vivo breast cancer biology, in particular ERα signaling. It is suitable for interrogating the long-term effects of anti-endocrine drugs in a set-up that closely resembles the original tumor microenvironment, with potential application in pre- and co-clinical assays of ERα-positive breast cancer.

Objective A new protocol for fixation and slide preservation was evaluated in order to improve the quality of immunocytochemical reactions on cytology slides. Methods The quality of immunoreactions was evaluated retrospectively on 186 cytology slides (130 direct smears, 56 cytospins) prepared from different cytology samples. Ninety‐three of the slides were air dried, stored at −20 °C and fixed in acetone for 10 minutes (Protocol 1), whereas the other 93 were immediately fixed in methanol at −20 °C for at least 30 minutes, subsequently protected with polyethylene glycol (PEG) and stored at room temperature (Protocol 2). Immunocytochemical staining, with eight primary antibodies, was performed on a Ventana BenchMark Ultra instrument using an UltraView Universal DAB Detection Kit. The following parameters were evaluated for each immunoreaction: morphology preservation, intensity of specific staining, background and counterstain. The slides were blinded and independently scored by four observers with marks from 0 to 20. Results The quality of immunoreactions was better on methanol‐fixed slides protected with PEG than on air‐dried slides stored in the freezer: = 14.44 ± 3.58 versus = 11.02 ± 3.86, respectively (P < 0.001). Conclusion Immediate fixation of cytology slides in cold methanol with subsequent application of PEG is an easy and straightforward procedure that improves the quality of immunocytochemical reactions and allows the storage of the slides at room temperature.

Background: Identification of errors in anatomic pathology is an important issue in medical practice. The main objective of this study was to determine the prevalence and characterize preanalytic errors in surgical pathology and cytology samples. We also intended to explore associations between error prevalence and procedures implemented in hospitals concerning the type of requisition forms, use of guidelines for case acceptance and existence of error notification system. Methods: We analyzed 10,574 cases in five Portuguese hospitals. The pathology laboratories recorded during 20 days all cases submitted with preanalytic errors, using an input form that allowed the identification of sample type, error description, action taken before error, the professional who detected the fail and the test cycle segment where it was identified. Subsequently, particular procedures in use for preanalytic phase were characterized for each hospital. Results: The prevalence of cases with error was 3.1% (330/10,574), 95% confidence interval: 2.8%–3.5%. Errors occurred in 4.1% (250/6079) of histology specimens and 0.9% (40/4477) of cytology specimens, and included errors in the requisition forms (2.6% error rate) and in the sample container (1.5% error rate). Acceptance of cases with error was the most frequent action (66.9%), followed by rejection (24.4%) and retention (8.7%). Conclusions: The existence of written norms for sample acceptance and error reporting systems to submitting services and patient safety department were proven to be associated to lower error prevalence.