PURPOSE. This was a pilot study conducted to examine the expression of osteopontin in uveal melanoma and to determine whether serum osteopontin can be used in detecting metastatic uveal melanoma.METHODS. Osteopontin mRNA was measured in three uveal melanoma cell lines of various invasive potential by real-time PCR. Tissue sections of primary and metastatic uveal melanomas were stained for osteopontin. Serum osteopontin levels were measured by ELISA assays in 15 patients with metastatic uveal melanoma and in 37 patients who were disease-free for at least 10 years after treatment of the primary tumor. Paired serum samples drawn from eight patients before and after development of metastasis were analyzed.RESULTS. By real-time PCR, highly invasive primary and metastatic uveal melanoma cells expressed 6- and 250-fold excess osteopontin mRNA, respectively, compared with poorly invasive primary uveal melanoma cells. Tissue sections of primary uveal melanomas lacking looping vasculogenic mimicry patterns either did not stain for osteopontin or exhibited weak, diffuse staining. In primary melanomas containing looping vasculogenic mimicry patterns, strong osteopontin staining was detected in the tumor periphery where patterns were located. Diffuse strong expression of osteopontin was detected in eight samples of uveal melanomas metastatic to the liver. Serum osteopontin levels were significantly higher in patients with metastatic uveal melanoma than in patients who had been disease free for at least 10 years after treatment (P = 0.0001) or in age-matched control subjects. Serum osteopontin levels were significantly higher (P = 0.008) after metastasis than before the detection of metastasis in eight patients. When a cutoff of 10 ng/mL was used, the sensitivity and specificity of serum osteopontin in detecting metastatic melanoma was 87.5%, and the area under the receiver operator characteristic curve was 96%.CONCLUSIONS. Osteopontin is expressed diffusely in tissue sections of hepatic metastases from uveal melanoma, and increased serum osteopontin levels correlate with melanoma metastasis to the liver with high specificity and sensitivity.

Given that expression of many genes changes when cells become malignant or are placed in. different microenvironments, we asked whether these changes were accompanied by global reorganization of chromatin. We reasoned that sequestration or exposure of chromatin-sensitive sites to restriction enzymes could be used to detect this reorganization. We found that AM-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of AluI-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, AluI-sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, AluI-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces that regulate cellular and tissue architecture.

CONTEXT:Molecular analyses indicate that periodic acid-Schiff (PAS)-positive (laminin-rich) patterns in melanomas are generated by invasive tumor cells by vasculogenic mimicry. Some observers, however, consider these patterns to be fibrovascular septa, generated by a stromal host response.OBJECTIVE:To delineate differences between vasculogenic mimicry patterns and fibrovascular septa in primary uveal melanomas.DESIGN:Frequency distributions, associations with outcome, and thicknesses of trichrome-positive and PAS-positive looping patterns were determined in 234 primary uveal melanomas. Sequential sections of 13 additional primary uveal melanomas that contained PAS-positive/trichrome-negative looping patterns were stained for type I and type IV collagens, laminin, and fibronectin. Real-time quantitative polymerase chain reaction was performed on RNA from cultured uveal melanoma cells for the expression of COL1A1, COL4A2, and fibronectin.RESULTS:Trichrome-positive loops were encountered less frequently than PAS-positive loops (10% vs 56%, respectively). Death from metastatic melanoma was strongly associated with PAS-positive (P < .001) but not with trichrome-positive (P = .57) loops. Trichrome-positive loops were significantly thicker than PAS-positive loops (P < .001). The PAS-positive patterns stained positive for laminin, type I and type IV collagens, and fibronectin. Type I collagen was detected within melanoma cells and focally within some PAS-positive patterns. Real-time quantitative polymerase chain reaction revealed 3-fold, 25-fold, and 97-fold increases, respectively, in expression of COL4A2, fibronectin, and COL1A1 by invasive pattern-forming primary melanoma cells compared with poorly invasive non-pattern-forming cells.CONCLUSIONS:Fibrovascular septa are rare and prognostically insignificant in uveal melanomas, whereas vasculogenic mimicry patterns are associated with increased mortality. Type I collagen, seen focally in some vasculogenic mimicry patterns, may be synthesized by tumor cells, independent of a host stromal response.

A Bluetooth radio in 0.18 /spl mu/m CMOS technology works on 2.5-3.0 V, dissipating 75 mW in RX and 90 mW in TX. RX uses a 2 MHz IF with an active poly-phase bandpass filter. The fractional-N PLL uses a VCO running at 5 GHz. TX uses IQ modulation. Special attention is paid to Si-crosstalk because this radio is combined with baseband circuitry. Silicon area is 5.5 mm/sup 2/.