Expression of p15 in a spectrum of spitzoid melanocytic neoplasms.

BACKGROUND: Accurate classification of spitzoid melanocytic lesions is difficult due to overlapping clinical and histopathologic features between Spitz nevi, atypical Spitz tumors (ASTs), and spitzoid melanomas. Expression of p16 (CDKN2A) has been used as a marker of spitzoid lesions. However, its expression may be variable. p15 is a tumor suppressor encoded by CDKN2B, loss of which has been recently shown to promote transition from nevus to melanoma. We sought to determine whether p15 is a useful immunohistochemical marker to distinguish Spitz nevi from spitzoid melanomas and to compare p15 and p16 staining in this population. METHODS: Immunohistochemistry for p15 and p16 was performed on Spitz nevi (n = 19), ASTs (n = 41), and spitzoid melanomas (n = 17). Immunoexpression was categorized by a four-tiered system: 0 (negative), 1+ (weak), 2+ (moderate), 3+ (strong). RESULTS: 3+/strong p15 staining was observed in 68.4% of Spitz nevi, 34.2% of ASTs, and 17.7% of spitzoid melanomas. By contrast, we observed 3+ p16 staining in roughly equivalent percentages of Spitz nevi (57.9%), ASTs (56.1%), and spitzoid melanomas (58.8%). CONCLUSION: These data illustrate that p15 may be more useful than p16 as a biomarker to help distinguish benign from malignant spitzoid lesions.

Inflammatory features of frontal fibrosing alopecia.

INTRODUCTION: Frontal fibrosing alopecia (FFA) is a cicatricial alopecia typically occurring in postmenopausal women. The etiology and pathophysiology of FFA is poorly understood but thought to be immune mediated. This study aims to further explore the extent of fibrosis and the inflammatory microenvironment by characterizing Langerhans cells (LCs), helper T cells, cytotoxic T cells and B cells near hair follicles in FFA. METHODS: Eleven paraffin-embedded tissues from patients with a clinical and histopathologic diagnosis of FFA were selected for immunohistochemical studies using CD3, CD4, CD8, CD1a and CD20. The lymphocytes and LCs were counted around involved follicles. The CD4/CD8 T-lymphocyte ratios were calculated and compared to the CD4/CD8 T-lymphocyte ratios in uninvolved areas. RESULTS: On histopathologic review, at least 35% of follicles in each case were affected by the disease with concentric perifollicular fibrosis and a perifollicular lichenoid lymphocytic infiltrate around the infundibuloisthmic portion of the hair follicle. There was an increase of perifollicular LCs (mean of 18, SD of 5.5) and intrafollicular LCs (mean of 14, SD of 4.3) in involved follicles compared to uninvolved follicles (P < .0001). The involved follicles also showed a relative decrease in the CD4/CD8 ratio indicating increased numbers of CD8+ T cells; a finding distinct from the CD4-predominant population in uninvolved follicles (P < .0001). CONCLUSION: The inflammatory features of FFA show a CD8-biased T-cell infiltrate with increased numbers of LCs in the infundibuloisthmic region. The increased LCs may represent an aberrant immune reaction promoting a CD8+ T-cell response.

Rho GTPase signaling and PTH 3-34, but not PTH 1-34, maintain the actin cytoskeleton and antagonize bisphosphonate effects in mouse osteoblastic MC3T3-E1 cells.

Cytoskeletal elements are critical for cell morphology and signal transduction, and are involved in many cellular processes including motility, intracellular transport, and differentiation. Small GTP-binding proteins (G proteins) of the Ras family, such as RhoA, influence various elements of the cytoskeleton. RhoA stabilizes the actin cytoskeleton and promotes formation of focal adhesions. We found previously that RhoA is expressed in osteoblastic cells and is translocated to the plasma membrane and activated by PTH 1-34 as well as by Nleu(8,18) Tyr(34) PTH 3-34 amide, a PTH analog that does not increase cAMP. We therefore investigated effects of manipulating RhoA on the actin cytoskeleton of osteoblastic MC3T3-E1 cells. Three inhibitors were used: 1) GGTI-2166, a geranylgeranyl transferase I inhibitor that prevents the isoprenylation and membrane translocation of RhoA, 2) Y-27632, a Rho kinase inhibitor, and 3) alendronate, a nitrogen (N)-containing bisphosphonate that reduces intracellular geranylgeranylpyrophosphate through inhibiting farnesyl pyrophosphate synthase. To increase RhoA activity, we used the geranylgeranyl group donor geranylgeraniol (GGOH), and a constitutively active RhoA. The F-actin cytoskeleton and focal adhesions (FA) were visualized with rhodamine-phalloidin and fluorescent anti-vinculin antibodies, respectively. Cells were imaged with confocal microscopy. Actin stress fiber density, edge actin bundle density, focal adhesion density, cellular area and circularity (a morphological descriptor relating area and perimeter) were quantified by a program developed with Matlab software. GGTI-2166, Y-27632, and alendronate reduced actin stress fibers, FA density, and FA size, but had no effect on edge actin bundle density, cellular area, or circularity. GGOH completely antagonized the effects of alendronate, but did not significantly affect responses to GGTI-2166 or Y-27632. Constitutively active RhoA antagonized the effects of alendronate and GGTI-2166, but not those of Y-27632. The effects of alendronate were also antagonized by Nleu(8,18) Tyr(34) PTH 3-34 amide, but not by PTH 1-34. The results indicate that RhoA is involved in the maintenance of stress fibers and focal adhesions in osteoblastic cells, that PTH can affect this pathway independently of cAMP, and that a N-containing bisphosphonate can affect the actin cytoskeleton and focal adhesions through actions on geranylgeranyl groups and potentially through RhoA. In view of the importance of the actin cytoskeleton, the findings constitute evidence that N-containing bisphosphonates, when they attain certain concentrations, have effects on osteoblasts that could influence bone remodeling.