Evidence for glycosylation as a regulator of the pigmentary system: key roles of sialyl(α2-6)gal/GalNAc-terminated glycans in melanin synthesis and transfer.

UNLABELLED: The major regulators of melanogenesis are glycoproteins, however no role for glycosylation in the pathway has yet been described. We stained skin biopsies and melanocyte-keratinocyte co-cultures with a panel of 20 lectins as oligosaccharide markers. Notably, the Elderberry Bark Lectin (EBL/SNA) stained melanocytes in both systems. EBL binds the sequence Neu5Ac(α(2-6)Gal/GalNAc)- at the termini of some oligosaccharide antennae. We used inhibitors of synthesis and/or binding of this sequence to assess effects on pigmentation. METHODS: Cell culture, lectin histochemistry, siRNA transfection, and assays for dopa oxidase and melanin were carried out by standard techniques. RESULTS: 6'-sialyllactose, a short homolog of the sequence in question, anti-sialyltransferase 6 (ST6) siRNA, and cytidine, a sialyltransferase (ST) inhibitor, each inhibited EBL binding, melanogenesis and melanosome transfer. Unexpectedly, 3'-sialyllactose and siRNA for ST3, chosen as a negative controls, also inhibited these processes. Though strong inhibitors of melanization, none of the agents affected tyrosinase/dopa oxidase activity, indicating previously unrecognized post-tyrosinase regulation of melanization. CONCLUSIONS: We report for the first time that Neu5Ac (α(2-6)Gal/GalNAc)- and possibly Neu5Ac(α(2-3)Gal/GalNAc)-terminated oligosaccharides play multiple roles in melanin synthesis and transfer.

Comparison of the expression of vimentin and actin in spitz nevi and spitzoid malignant melanomas.

INTRODUCTION: The differentiation between Spitz nevi (SN) and Spitzoid malignant melanomas (SMM) represents a challenge to dermatopathologists. We recently demonstrated differential expression of vimentin and Actin in SN and SMM by mass spectrometry (MS). We sought to investigate whether this differential expression could be detected using conventional immunohistochemistry or automated quantitative analysis (AQUA) of histological sections. METHODS: Cases of SN and SMM, which were previously studied by MS and have readily available blocks and enough material in the block, were selected from the Yale Spitzoid Neoplasm Repository. The cases were stained for vimentin and muscle-specific actin using standard protocols. H-scores were calculated by multiplying the percentage of cells staining and the intensity of staining. Selected cases were also studied for quantitative immunofluorescent staining using the AQUA method. RESULTS: All 21 cases of SN showed strong and diffuse staining for vimentin; 19 of 21 (91%) cases had an H-score of 300 (average, 294). Similar staining results were observed in SMM; 13 of 14 (93%) cases had an H-score of 300 (average, 297). Muscle-specific actin was weakly and focally positive in 5 of 21 (24%) SN (H-score = 3.3) and 5 of 14 (39%) SMM (H-score = 3.5). The AQUA method showed no significant difference in the staining intensity of SN and SMM for both vimentin and actin. CONCLUSIONS: There was no difference in the expression of vimentin and actin in SN and SMM shown by conventional immunohistochemistry or the AQUA method. This study shows that MS has much grater sensitivity in detecting the differential expression of these proteins.

Targeting ER stress-induced autophagy overcomes BRAF inhibitor resistance in melanoma.

Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.

A Melanoma Brain Metastasis with a Donor-Patient Hybrid Genome following Bone Marrow Transplantation: First Evidence for Fusion in Human Cancer.

BACKGROUND: Tumor cell fusion with motile bone marrow-derived cells (BMDCs) has long been posited as a mechanism for cancer metastasis. While there is much support for this from cell culture and animal studies, it has yet to be confirmed in human cancer, as tumor and marrow-derived cells from the same patient cannot be easily distinguished genetically. METHODS: We carried out genotyping of a metastatic melanoma to the brain that arose following allogeneic bone-marrow transplantation (BMT), using forensic short tandem repeat (STR) length-polymorphisms to distinguish donor and patient genomes. Tumor cells were isolated free of leucocytes by laser microdissection, and tumor and pre-transplant blood lymphocyte DNAs were analyzed for donor and patient alleles at 14 autosomal STR loci and the sex chromosomes. RESULTS: All alleles in the donor and patient pre-BMT lymphocytes were found in tumor cells. The alleles showed disproportionate relative abundances in similar patterns throughout the tumor, indicating the tumor was initiated by a clonal fusion event. CONCLUSIONS: Our results strongly support fusion between a BMDC and a tumor cell playing a role in the origin of this metastasis. Depending on the frequency of such events, the findings could have important implications for understanding the generation of metastases, including the origins of tumor initiating cells and the cancer epigenome.

Neuropilin-2 as a useful marker in the differentiation between Spitzoid malignant melanoma and Spitz nevus.

BACKGROUND: Spitzoid malignant melanoma (SMM) shares many histopathologic features with Spitz nevus (SN). The distinction between SMM and SN remains one of the most difficult diagnostic problems in dermatopathology. Neuropilin-2 (NRP2) is a cytoplasmic/cell surface protein that is a mediator of melanoma-endothelial cell interaction. OBJECTIVE: The aim of this study was to evaluate NRP2 expression in SMM and SN and to determine whether it can reliably differentiate between the 2 groups. METHODS: We studied the expression of NRP2 in 19 cases of SMM and 19 cases of SN from Yale Spitzoid Neoplasm Repository, New Haven, Conn. RESULTS: All 19 cases of SMM (100%) expressed NRP2. Most SMM showed moderate- and high-intensity staining in the majority of the melanoma cells. Most of the SN (14/19, 74%) were negative for the marker. NRP2 labeled only 5 of 19 SN (26%) and all of them demonstrated mild staining intensity. NRP2(+) staining was statistically significant in differentiating SMM from SN (P < .05). LIMITATIONS: Small study size is a limitation. CONCLUSIONS: NRP2 expression in SMM and SN may be a useful adjunct marker, in addition to histopathologic evaluation, in the differentiation between these 2 entities.

Detecting HPV in cutaneous lesions using anti-HPV antibody immunohistochemistry.

Most condyloma are diagnosed clinically (without a biopsy) or histopathologically (if biopsied) without any ancillary testing. In some cases, additional confirmation of productive infection by human papillomavirus (HPV) or typing of HPV is desired, and in situ hybridization (ISH) is the most commonly used test. However, ISH is not readily available in most laboratories and only detects certain genital subtypes of HPV. The aim of this study was to evaluate the sensitivity and specificity of an anti-HPV antibody, in 25 lesions (both HPV induced and non-HPV induced) mostly from the genital region, with comparison to results with ISH and findings on hematoxylin and eosin staining. The sensitivity and specificity for the anti-HPV antibody used in this study are 90.9% and 85.7%, respectively, compared with ISH. Immunohistochemistry with this anti-HPV antibody, like ISH, was generally positive in cases showing koilocytes/koilocytotic atypia (86%). Immunohistochemical staining also detected productive infection with HPV in 23% (3 of 13) of cases without koilocytes/koilocytotic atypia. Thus, although staining is generally positive in cases with diagnostic findings of koilocytes/koilocytotic atypia in hematoxylin and eosin sections, immunohistochemistry can detect HPV in some cases without koilocytes/koilocytotic atypia.

Expression of p16 alone does not differentiate between Spitz nevi and Spitzoid melanoma.

BACKGROUND: Spitz nevi and Spitzoid melanomas show overlapping histopathologic features, often making the diagnosis challenging. The p16 protein functions as a tumor suppressor and loss of its expression may be seen in some melanomas. METHODS: We evaluated 18 Spitz nevi and 19 Spitzoid melanomas from the Yale Spitzoid Neoplasm Repository for p16 expression. A staining intensity score (SIS) was calculated by multiplying a score for the percentage of stained cells (0-3) by a score for staining intensity (0-3). RESULTS: Staining with p16 was positive in 15/18 (83%) Spitz nevi and 15/19 (79%) Spitzoid melanomas (p = 0.73). Both Spitz nevi and Spitzoid melanomas had a similar SISs, 4.9 and 3.8, respectively (p = 0.057). All 19 patients with Spitzoid melanomas had poor outcome with either death (6 patients) or metastases (13 patients) at a median (3 years) and mean (5.4 years) follow up. In contrast, all 18 patients with Spitz nevi had a benign course with no adverse events at a median (4 years) and mean (4 years) follow up. CONCLUSIONS: We found no significant difference in p16 staining in Spitz nevi and Spitzoid melanomas. We conclude that p16 does not appear to be a useful immunohistochemical marker in distinguishing between Spitz nevi and Spitzoid melanomas.

Punctate LC3B expression is a common feature of solid tumors and associated with proliferation, metastasis, and poor outcome.

PURPOSE: Measurement of autophagy in cancer and correlation with histopathologic grading or clinical outcomes has been limited. Accordingly, we investigated LC3B as an autophagosome marker by analyzing nearly 1,400 tumors from 20 types of cancer, focusing on correlations with clinical outcomes in melanoma and breast cancer. EXPERIMENTAL DESIGN: Staining protocols were developed for automated quantitative analysis (AQUA) using antibodies versus LC3 isoform B (LC3B) and Ki-67. Clinically annotated breast and melanoma tissue microarrays (TMA) and a multitumor array were used. An AQUA program was developed to quantitate LC3B distribution in punctate and diffuse compartments of the cell. RESULTS: LC3B staining was moderate to high in the large majority of tumors. The percentage of area occupied by punctate LC3B was elevated by 3- to 5-fold at high LC3B intensities. In breast cancer and melanoma TMAs, LC3B and Ki-67 showed strong correlations (P < 0.0001), and in multitumor TMAs, mitotic figures were most often seen in tumors with the highest LC3B expression (P < 0.002). In breast cancer, LC3B expression was elevated in node-positive versus node-negative primaries and associated with increased nuclear grade and shortened survival. In a melanoma TMA with no survival data, LC3B levels were highest in nodal, visceral, and cutaneous metastases. CONCLUSIONS: The results reveal a common expression of LC3B in malignancy and support emerging evidence that autophagy plays a significant role in cancer progression. High LC3B was associated proliferation, invasion and metastasis, high nuclear grade, and worse outcome. Thus, autophagy presents a key target of therapeutic vulnerability in solid tumors.

Autophagy in cutaneous malignant melanoma.

We show that malignant melanoma cells display high levels of autophagy, a cytoplasmic process of protein and organelle digestion that provides an energy source in times of nutrient deprivation. In a panel of 12 cases of cutaneous malignant melanoma of the superficial spreading type, cells in florid melanoma in situ (MIS) and invasive cells in the dermis appeared to be undergoing autophagy. Autophagosomes were detected through immunohistochemistry using the marker LC3B (microtubule-associated light chain 3B), and by electron microscopy. Some autophagosomes contained melanized melanosomes, accounting for the phenomenon of 'coarse melanin' in malignant melanoma. Autophagosomes also contained the Golgi 58k protein, a structural component of the Golgi apparatus, and beta1,6-branched oligosaccharides, indicating that at least some of the autophagosomal proteins were glycosylated with these structures. The findings suggest that autophagy could be a constitutive metabolic state for invasive and metastatic melanoma cells. Interestingly, a similar phenotype was also expressed by tumor-associated melanophages. The findings are consistent with previous reports that endoplasmic reticulum (ER) stress drives melanoma progression, since ER stress is known to trigger autophagy. The results suggest that therapies inhibiting autophagy may be effective for the treatment of malignant melanoma by depriving cells of an important energy source.