Diagnostic Accuracy of Digital Staining ex vivo Confocal Microscopy for Diagnosing and Subtyping Basal Cell Carcinoma in Fresh Pretherapeutic Punch Biopsies: A Monocentric Prospective Study.

BACKGROUND: Ex vivo confocal microscopy using fusion mode and digital staining (EVCM) scans unfixed fresh tissue and produces rapidly digitally stained images of very similar quality to classical pathology. We investigated whether EVCM could represent an alternative to the standard histological examination of the pretherapeutic basal cell carcinoma (BCC) punch biopsies. OBJECTIVES: The objective of the study was to assess diagnostic accuracy of EVCM versus traditional histopathological examination for diagnosing and subtyping clinically suspicious lesions of BCC in 3-mm fresh and nonfixed punch biopsies. METHODS: In this prospective monocentric observational study, patients with clinically suspected BCC were consecutively enrolled. Punch biopsies were imaged using EVCM and subsequently processed for standard histologic examination (gold standard). EVCM images were examined by a dermatopathologist blinded to clinical aspect of the lesion and histopathological results. Concordance between the EVCM and histology analysis was calculated with Cohen's kappa (κ) statistic. RESULTS: Sixty-six patients were recruited, and 106 biopsies were analyzed. EVCM correctly diagnosed 70/73 BCCs and 31/33 non-BCC lesions, corresponding to a sensitivity of 96% and a specificity of 94% (positive predictive value = 97%, negative predictive value = 91%). The EVCM assessment led to over-staging and under-staging of BCC subtypes in 5% and 11% of cases, respectively. It led to over-staging and under-staging of BCC depths in 5% and 15%, respectively. The kappa coefficient for concordance was 0.78 (95% confidence interval [CI]: 0.69-0.88) when considering BCC subtypes and 0.81 (95% CI: 0.72-0.90) when considering BCC depths. CONCLUSIONS: These results render EVCM as a promising option for "real-time" pretreatment evaluation of clinically suspected BCC lesions. Further larger randomized studies are needed to assess the efficiency of EVCM versus standard care in patients with clinically suspected BCC.

Extracellular 20S proteasome secreted via microvesicles can degrade poorly folded proteins and inhibit Galectin-3 agglutination activity.

Proteasomes are major non-lysosomal proteolytic complexes localized in the cytoplasm and in the nucleus of eukaryotic cells. Strikingly, high levels of extracellular proteasome have also been evidenced in the plasma (p-proteasome) of patients with specific diseases. Here, we examined the process by which proteasomes are secreted, as well as their structural and functional features once in the extracellular space. We demonstrate that assembled 20S core particles are secreted by cells within microvesicles budding from the plasma membrane. Part of the extracellular proteasome pool is also free of membranes in the supernatant of cultured cells, and likely originates from microvesicles leakage. We further demonstrate that this free proteasome released by cells (cc-proteasome for cell culture proteasome) possesses latent proteolytic activity and can degrade various extracellular proteins. Both standard (no immune-subunits) and intermediate (containing some immune-subunits) forms of 20S are observed. Moreover, we show that galectin-3, which displays a highly disordered N-terminal region, is efficiently cleaved by purified cc-proteasome, without SDS activation, likely after its binding to PSMA3 (α7) subunit through its intrinsically disordered region. As a consequence, galectin-3 is unable to induce red blood cells agglutination when preincubated with cc-proteasome. These results highlight potential novel physio- and pathologic functions for the extracellular proteasome.