P63 Expression Can Be Used in Differential Diagnosis of Salivary Gland Acinic Cell and Mucoepidermoid Carcinomas

Differentiation of salivary gland acinic cell carcinoma from mucoepidermoid carcinoma can be diagnostically challenging as both may have prominent mucin production. P63 is a p53 homologue required for limb and epidermal morphogenesis. It is expressed in basal and myoepithelial cells of normal salivary gland tissues. In this immunohistochemical study, we examined the expression of p63 in salivary gland acinic cell and mucoepidermoid carcinomas (MEC) and its use in differentiating these two entities. A search was performed and appropriate cases were selected from Lifespan Hospital System archives as well as the consult archives of one author (DRG). 31 salivary gland acinic cell carcinomas (ACC) and 24 MEC were examined for p63 expression by immunohistochemistry. The nuclear immunoreactivity was examined by both authors and was graded semi-quantitatively with negative being less than 10 % of cells staining. Positive staining was graded as follows: 10–25 % of tumor cells staining was weakly positive, 26–75 % of tumor cells staining was moderately positive, and 76–100 % of tumor cells staining was strongly positive. Negative nuclear staining of the tumor cells was seen in 30/31 (96 %) of salivary gland ACC while 1/31 (3 %) showed diffuse nuclear staining of the tumor cells. This latter case was later reclassified as mammary analogue secretory carcinoma following confirmatory molecular testing for the ETV6-NTRK3 fusion gene. Strong positive nuclear staining of the tumor cells was seen in 24 (100 %) of salivary gland MEC cases. P63 is an immunohistochemical stain that can potentially aid in differentiating unusual ACC with prominent mucin production from MEC of the salivary gland. According to this study, acinic cell carcinoma is always negative for p63 immunoreactivity while mucoepidermoid carcinoma is always positive.     

Prognostic significance of p53 and p63 immunolocalisation in primary and matched lymph node metastasis in oral squamous cell carcinoma

This study evaluates the prognostic significance of p53 and p63 immunolocalisation in oral squamous cell carcinoma samples from 45 matched primary tumors (PT) and lymph node metastases (LNM). Data regarding patient age, gender, primary site, histological differentiation, metastasis, disease-free survival (DFS) and overall survival (OS) were available. p53 and p63 immunolabeling was detected in 17 (37.8%) and 23 (51.1%) of the PT, respectively. For LNM, there was p53 and p63 labeling in 23 (51.1%) and 26 (57.8%) cases, respectively. Most cases showed similar labeling in PT and the corresponding LNM (73.3% for p53 and 53.3% for p63, respectively). No statistically significant associations were found between p53 and p63 immunolabeling and histological differentiation; p63 positive tumors showed higher DFS (p=0.006) and OS (p=0.049); and p53-negative tumors had a higher DFS interval (p=0.009). Our findings suggest that initially p53-negative tumors and initially p63-positive tumors that retain this labeling pattern may follow less aggressive biological courses and present better prognoses.     

A unifying concept of trophoblastic differentiation and malignancy defined by biomarker expression

Several trophoblast phenotypes, including cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast, emerge during gestation. To clarify the lineage relationship between these subtypes, we profiled p63 localization in developing and term placental tissue, as well as in trophoblastic tumors, using antibodies specific to full-length (TAp63) and one against all p63 isoforms (TAp63 and DeltaNp63). Localization of p63 was compared with that of biomarkers of proliferation and trophoblastic differentiation, including mib-1, inhibin, and MelCAM. In early gestation, p63 was localized principally to villous cytotrophoblast after contact with the villous mesenchyme, absent in the trophoblast columns, and early implantation trophoblast. In the maturing placenta, intraplacental perivillous fibrin correlated with the emergence of a p63-positive "transitional" (vacuolated) extravillous trophoblast from cytotrophoblast, which differentiated further into a "mature" p63-negative extravillous trophoblast. The same lineage pathway emerged from entrapped villi on the chorionic membrane. Virtually all p63 immunopositivity was attributed to dominant-negative p63. The immunophenotypic patterns seen in the immature and mature placenta permit the resolution of all trophoblastic phenotypes within 3 lineage pathways of cytotrophoblast differentiation, including cytotrophoblast-to-trophoblast column/implantation site, cytotrophoblast-to-syncytiotrophoblast, and cytotrophoblast-to-mature extravillous trophoblast. In the latter pathway, a transitional (vacuolated) p63-positive extravillous trophoblast emerges from and links cytotrophoblast to mature extravillous trophoblast in intraplacental fibrin, chorionic membrane, and basal plate. The placental trophoblast is thus resolved within this continuum of differentiation. Terms such as transitional and mature extravillous trophoblast are proposed to reflect the differentiation phases of this unique epithelium. p63 staining patterns in trophoblastic tumors reflect timing of neoplastic transformation during trophoblastic differentiation.     

p63 - Key molecule in the early phase of epithelial abnormality in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common lung disease predisposing lung cancer. To clarify the early phase of epithelial abnormalities in IPF, we used an in vitro squamous metaplasia model, transforming growth factor beta1 (TGF beta1)-treated airway epithelial cells (BEAS-2B). The model repeated the expression of squamous epithelial character, such as involucrin, and keratin 6 and 14. DNA microarray analysis disclosed a unique expression signature in TGF beta1-treated airway epithelial cells, 20 specifically up-regulated genes including p63, jagged 1 (jag1) and the genes of structure proteins. Western blotting and RT-PCR analysis revealed that DeltaNp63alpha was the dominant isoform of p63 in our experimental model. Immunohistochemical analysis demonstrated the expression of p63 and jag1 in lung tissues of IPF. Inhibition of p63 with siRNA caused the down-regulation of jag1 expression, but not of involucrin, or keratin 6 and 14. Interestingly, the up-regulation of p63 was totally suppressed by N-acetyl-l-cysteine (NAC), but not by dexamethasone or pirfenidone. Thus, the p63-jag1 pathway may be up-regulated at an early phase of epithelial abnormalities in IPF, which can be overcome by NAC even in the TGF beta1-rich milieu.     

Penta-block copolymer microspheres: Impact of polymer characteristics and process parameters on protein release

Here, we aimed to develop protein loaded microspheres (MSs) using penta-block PLGA-based copolymers to obtain sustained and complete protein release. We varied MS morphology and studied the control of protein release. Lysozyme was used as a model protein and MSs were prepared using the solid-in-oil-in-water emulsion solvent extraction method. We synthesized and studied various penta-block PLGA-based copolymers. Copolymer characteristics (LA/GA ratio and molecular weight of PLGA blocks) influenced MS morphology. MS porosity was influenced by process parameters (such as solvent type, polymer concentration, emulsifying speed), whereas the aqueous volume for extraction and stabilizer did not have a significant effect. MSs of the same size, but different morphologies, exhibited different protein release behavior, with porous structures being essential for the continuous and complete release of encapsulated protein. These findings suggest strategies to engineer the morphology of MSs produced from PLGA-based multi-block copolymers to achieve appropriate release rates for a protein delivery system.     

Thrombomodulin Functional Domains Support Osteoblast Differentiation and Bone Healing in Diabetes in Mice

Thrombomodulin (TM) is a transmembrane glycoprotein that contains five functional domains. Soluble TM (sTM), comprising extracellular domains TMD1 (lectin-like), TMD2 (epidermal growth factor [EGF]-like repeat containing), and TMD3 (serine-threonine rich), can be shed from cells by the intramembrane protease rhomboid-like-2 (RHBDL2). TM is expressed by osteoblasts, yet its role there has not been determined. Herein we aimed to investigate the properties of TM and its domains in osteoblast function and bone repair following injury in diabetes. In response to a scratch injury of cultured osteoblast-like MG63 cells, expression of TM and RHBDL2 was enhanced, with increased release of sTM. Conditioned media from the injured cells promoted osteoblast migration, an effect that was lacking with conditioned media from MG63 cells in which TM was silenced by shRNA. Exogenous recombinant TMD1 had no effect on osteoblast activities or on bone repair in vivo. However, TM domains 2 and 3 (TMD2/3), induced MG63 cell migration, proliferation and mineralization in vitro, and when locally administered in mice, improved in vivo healing of injured calvarium. This beneficial effect of TMD2/3, mediated via fibroblast growth factor receptor (FGFR)/ERK signaling pathways, was also observed in vitro under high glucose conditions where endogenous TM expression was reduced, and in vivo in diabetic mice following tibia fracture or calvarium injury, where the osteoblastic response and healing were otherwise dampened. Taken together, osteoblast TM participates in bone healing, and recombinant TMD2/3 holds promise as a novel therapy for diabetic bone defect healing. © 2020 American Society for Bone and Mineral Research.