Purpose: This study was designed to provide a comprehensive assessment on the role of h1,6-branched oligosaccharides in the metastasis and outcome of breast carcinoma. Generation of these structures on N-glycans is initiated by h1,6-N-acetylglucosaminyltransferase V and used by both myeloid cells and cancer cells in systemic migration.Experimental Design: Tissue microarrays of >700 tumors (>400 patients; 30-year follow-up data) were stained through lectin histochemistry with leukocytic phytohemagglutinin (LPHA), a selective marker for h1,6-branched oligosaccharides. Node-negative and node-positive primary tumors and patient-matched lymph node metastases were scored by blinded observers. Results: Metastases stained at significantly greater intensities than did the patient-matched primary tumors (P < 0.0001), demonstrating for the first time that the abundance of h1,6-branched oligosaccharides was directly associated with breast carcinoma nodal metastasis. Multivariate analyses revealed that h1,6-branched oligosaccharides in primary tumors were a predictor of poor outcome, most notably in node-negative tumors, where an LPHA staining score of 3+ gave a risk factor of 3.3, independent of tumor size, nuclear grade, or patient age (P = 0.007). Conclusions:The data firmly establish a role for h1,6-N-acetylglucosaminyltransferase V activity and h1,6-branched oligosaccharides in breast carcinoma metastasis, and reemphasize the involvement, although poorly understood, of aberrant glycosylation in tumor progression.h1,6-N-acetylglucosaminyltransferase V (GnT-V, E.C.2.4.1.155) is a key enzyme in the production of tri-or tetra-antennary glycans, and catalyzes the transfer of N-acetylglucosamine to a-1,6-mannose in the pentasaccharide core of acceptor glycans (1-3). This forms a h1,6 branch point, whose branches typically consist of polylactosamine antennae, carriers of the fucosyl-based antigens, Lewis x and Lewis a . These are used by both normal leukocytes and tumor cells in selectin binding (4-9). h1,6 branching also affects numerous cellular pathways for adhesion, motility, angiogenesis, and apoptosis (see Discussion). Thus, the h1,6 branch point represents a potential rate-limiting step in systemic migration. This branch point can be identified through lectin histochemistry with the plant lectin, leukocytic phytohemagglutinin (LPHA), which exhibits high specificity for h1,6 branching on N-glycans (10, 11) and can be used in formalin-fixed, paraffin-embedded tissues (12). LPHA binding to histologic sections of melanomas was resistant to the strong oxidative bleaching procedures necessary to decolorize melanin (12), indicating the h1,6 branches were stable to oxidation, a major cause of antigen loss on long-term storage of paraffin-embedded tissues (13). LPHA binding was markedly reduced when histologic sections of renal cell carcinomas, melanomas, and breast carcinomas were preincubated with glycosidase F, indicating that the h1,6 branch points were associated with N-glycans (asparagine linked) in these tissues ...

We document an unusual tattoo reaction presenting as verrucous plaques, which on histopathologic examination showed marked pseudoepitheliomatous epidermal hyperplasia. The patient is a 27-year-old female who presented to her dermatologist complaining of itchy overgrowth of her tattoo. Her symptoms began 2 months after tattoo placement approximately 1 year ago. Physical examination revealed verrucous plaques in the purple areas of the tattoo, suggesting a clinical diagnosis of a granulomatous tattoo reaction. A superficial biopsy showed epidermal hyperplasia somewhat reminiscent of a regressing keratoacanthoma. No tattoo was identified. A repeat shave biopsy demonstrated marked epidermal hyperplasia with focal keratin filled cystic dilatations, and local mild reactive keratinocytic atypia. In the surrounding dermis, there was dense chronic inflammation, fibrosis, and granules of dark red pigment. These findings suggest marked pseudoepitheliomatous hyperplasia secondary to the tattoo. Different reaction patterns have been described in association with tattoos, such as granulomatous and/or perivascular lymphocytic inflammation. However, there have been few cases reported of pseudoepitheliomatous hyperplasia arising at a tattoo site. Therefore, we encourage physicians to consider massive epidermal hyperplasia in the differential diagnosis of a verrucous tattoo reaction.

Tumor-associated macrophages (TAMs) play multiple roles in tumor initiation and progression. Tumors frequently appear in areas of chronic inflammation. This is likely aided by the mutagenic actions of macrophages. Tumor growth and progression is supported by macrophage-induced neoangiogenesis and stroma production, and macrophages produce tumor-stimulating growth factors. In most cancers a high density of TAMs predicts poor outcome. But not only do cancer cells depend upon macrophages for growth and invasion, they also co-opt macrophage traits. These include a wide diversity of molecules and pathways regulating adhesion, matrix alterations, neoangiogenesis, motility, chemotaxis, immune signaling pathways and even multidrug resistance proteins. Evidence is presented that these traits could be generated through macrophage-tumor cell fusion. Fusion has been reported in numerous animal tumor models and was recently documented in 2 human cases. Fusion could also account for the high degree of aneuploidy and plasticity in cancer, and for immune evasion. One common trait of myeloid-tumor fusion is the high expression of Beta1,6-branched N-glycans, used by macrophages in systemic migration. Beta1,6-branched oligosaccharides have long been associated with metastasis in animal models and were recently found to be common in a wide diversity of human cancers. We suggest that Beta1,6-branched oligosaccharides in human cancer may reflect widespread tumor cell fusion. Viewing the cancer cell as a myeloid hybrid provides new approaches towards understanding and treating this complex disease.

Fusion hybrids between normal macrophages and Cloudman S91 melanoma cells were shown earlier to have increased metastatic potential, along with high expression of beta1,6-N-acetylglucosaminyltransferase V and beta1,6-branched oligosaccharides. Curiously, hybrids, but not parental melanoma cells, also produced 'coarse melanin'- autophagic vesicles with multiple melanosomes. As beta1,6-branched oligosaccharides were known to be associated with metastasis, and coarse melanin had been described in invasive human melanomas, we looked for potential relationships between the two. Using lectin- and immunohistochemistry, we analyzed cell lines producing coarse melanin for beta1,6-branched oligosaccharides: gp100/pmel-17 (a melanosomal structural component) and CD63 (a late endosome/lysosome component associated with melanoma and certain other human cancers). Cell lines used in this study were (i) hybrid 94-H48, a highly metastatic, macrophage-melanoma experimental fusion hybrid; (ii) 6(neo) mouse melanoma cells, the weakly metastatic, parental fusion partner; and (iii) SKmel-23, a human melanoma cell line derived from a metastasis. Coarse melanin granules were prominent both in hybrids and in SKmel-23 cells, and co-localized with stains for beta1,6-branched oligosaccharides, gp100/pmel 17, and CD63. This is the first report of this phenotype being expressed in vitro, although co-expression of beta1,6-branched oligosaccharides and coarse melanin was recently shown to be a common and pervasive characteristic in archival specimens of human melanomas, and was most prominent in metastases. The results suggest that pathways of melanogenesis in melanoma may differ significantly from those in normal melanocytes. In vitro expression of this phenotype provides new biological systems for more detailed analyses of its genesis and regulation at the molecular genetic level.