Stem cell‐like cells have recently been identified in melanoma cell lines, but their relevance for melanoma pathogenesis is controversial. To characterize the stem cell signature of melanoma, expression of stem cell markers BMI‐1 and nestin was studied in 64 cutaneous melanomas, 165 melanoma metastases as well as 53 melanoma cell lines. Stem cell renewal factor BMI‐1 is a transcriptional repressor of the Ink4a/Arf locus encoding p16^ink4a and p14^Arf. Increased nuclear BMI‐1 expression was detectable in 41 of 64 (64%) primary melanomas, 117 of 165 melanoma metastases (71%) and 15 of 53 (28%) melanoma cell lines. High nestin expression was observed in 14 of 56 primary melanomas (25%), 84 of 165 melanoma metastases (50%) and 21 of 53 melanoma cell lines (40%). There was a significant correlation between BMI‐1 and nestin expression in cell lines (p = 0.001) and metastases (p = 0.02). These data indicate that cells in primary melanomas and their metastases may have stem cell properties. Cell lines obtained from melanoma metastases showed a significant higher BMI‐1 expression compared to cell lines from primary melanoma (p = 0.001). Further, primary melanoma lacking lymphatic metastases at presentation (pN0, n = 40) was less frequently BMI‐1 positive than melanomas presenting with lymphatic metastases (pN1; n = 24; 52% versus 83%; p = 0.01). Therefore, BMI‐1 expression appears to induce a metastatic tendency. Because BMI‐1 functions as a transcriptional repressor of the Ink4a/Arf locus, p16^ink4a and p14^Arf expression was also analyzed. A high BMI‐1/low p16^ink4a expression pattern was a significant predictor of metastasis by means of logistic regression analysis (p = 0.005). This suggests that BMI‐1 mediated repression of p16^ink4a may contribute to an increased aggressive behavior of stem cell‐like melanoma cells.

Lymph node (LN) status is an important prognostic factor in melanoma patients. p16 expression and proliferation rate (MIB‐1) of primary melanomas have been suggested as a marker of metastatic potential. In this study, the correlation of p16 expression and the proliferation rate (MIB‐1) with LN status and tumor‐specific survival was investigated in primary melanomas. MIB‐1 and p16 expression were analyzed by immunohistochemistry in 64 patients with primary cutaneous melanoma. Thirty four nevi were used as control. All patients underwent sentinel lymph node staging. Three different p16 staining patterns were observed: a combination of nuclear and cytoplasmic staining, only cytoplasmic staining and absence of p16 expression. All 34 nevi displayed a nuclear and cytoplasmic p16 staining, whereas p16 was negative in 14 of 64 (22%) melanomas. The level of p16 expression gradually decreased from benign nevi to melanoma without metastasis to melanoma with metastasis. There was a significant correlation between cytoplasmic p16 expression and absence of metastasis (p < 0.05). Death of disease correlated with absence of p16 immunostaining (p = 0.01). MIB‐1 expression was not associated with survival. These results confirm the relevance of p16 expression as a prognostic marker in melanoma patients. In addition, it was shown that cytoplasmic immunostaining for p16 in primary melanoma might serve as a predictor of the LN status. Therefore, immunohistochemical evaluation for p16 expression is of potential value for treatment planning in melanoma surgery.

Recent molecular studies provide evidence for a significant transcriptional plasticity of tumor cell subpopulations that facilitate an active contribution to tumor vasculature. This feature is accompanied by morphological changes both in vitro and in vivo. Herein, we investigated the morphological plasticity of tumor cells with special focus on vasculogenic mimicry and neovascularisation in human melanoma and mouse xenografts of human melanoma cell lines. In melanoma xenograft experiments, different vessel markers and green fluorescent protein expression were used to show how melanoma cells contribute to neovascularization. Additionally, we analyzed neovascularization in 49 primary melanomas and 175 melanoma metastases using immunostaining for blood (CD34) and lymphatic (D2–40) vessel-specific markers. We found significantly more lymphatic vessels in primary melanomas than in melanoma metastases (p<0.0001). In contrast to the near absence of lymphatic vessels within metastases, we found extensive blood micro-neovascularization. Blood micro-neovascularization was absent in micro metastases (less than 2 mm). A significant inverse correlation between Glut-1 expression (implying local hypoxia) and the presence of microvessels indicates their functional activity as blood vessels (p<0.0001). We suggest that the hypoxic microenvironment in metastases contributes to a phenotype switch allowing melanoma cells to physically contribute to blood vessel formation.

Immune checkpoint inhibitory (ICI) therapy represents a novel approach in a variety of cancers, with impressive survival benefit. With ICIs, however, a new spectrum of immune related adverse events (irAE) including life threatening hypohysitis has emerged. This autopsy study aimed to investigate inflammatory cells, PD-1 and PD-L1 expression in cases of patients who developed hypophysitis and involvement of other organs. We analysed 6 patients, who were treated with ICIs and developed hypophysitis. Two received an additional MAP-kinase inhibitor, MEK-inhibitor and cytotoxic chemotherapy. Besides the pituitary gland, all investigated adrenal glands (5/5) were affected; three cases had other organs involved (liver (2/6), thyroid (2/6), lung (1/6), myocardium (1/6), colon (1/6). The inflammatory cells of involved organs were further specified and PD1 and PDL-1 expression was analyzed using immunohistochemistry. We observed that patients treated with ICIs alone showed T-cell predominant lymphocytic infiltrates, whereas patients receiving additional therapies demonstrated an increase in B- and T-lymphocytes. Surprisingly, the dominant inflammatory population was not T-cell, but type 2 macrophages. CD25 positive T-regs were sparse or absent. Our study suggests that T cell activation is only partially responsible for irAE. ICI therapy interaction with CTLA-4, PD-1 and PDL-1 in type 2 macrophages appears to result in disturbance of their control. Furthermore, depletion of T-regs seems to contribute significantly. Our findings with simultaneous pituitary and adrenal gland involvement underlines the systemic involvement as well as the importance of monitoring cortisol levels to avoid potentially life threatening hypocortisolism.

The following communication summarizes the proceedings of a 1-day Workshop of the International Melanoma Pathology Study Group, which was devoted to thin melanoma. The definitions and histologic criteria for thin melanoma were reviewed. The principal differential diagnostic problems mentioned included the distinction of thin melanoma from nevi, especially from nevi of special site, irritated nevi, inflamed and regressing nevi, and dysplastic nevi. Histologic criteria for this analysis were discussed and the importance of clinico-pathologic correlation, especially in acral sites, was emphasized. Criteria for the minimal definition of invasion were also discussed. In addition, a new technique of m-RNA expression profiling with 14 genes was presented and facilitated the distinction of thin melanomas from nevus in histologically obvious cases. However, for particular nevi, it was not obvious why the results indicated a malignant lesion. Despite many molecular and other ancillary investigations, Breslow thickness remains the most important prognostic factor in thin melanoma. The prognostic significance of radial (horizontal) and vertical growth phases, Clark level, regression, and mitotic rate were also discussed. Because of the increasing frequency of thin melanomas, there is a great need to develop more refined predictors of thin melanomas with worse clinical outcome.

Brain metastasis is a common endpoint in patients suffering from malignant melanoma. However, little is known about factors that predispose to brain metastases. We performed a retrospective clinical and pathological investigation of melanoma patients with brain metastases in order to better characterise this patient population. 193 melanoma patients with brain metastasis histologically diagnosed between 1990 and 2015 at the University Hospital Zurich were retrospectively identified and further specified for sex, age at diagnosis and detection of brain metastasis, and localisation. In addition, data were extracted regarding the subtype of primary melanoma, Breslow tumour thickness, Clark Level, mutation status, extent of metastatic spread and history of a second melanoma. We found a significant male predominance (n = 126/193; 65%; p < 0.001). Breslow tumour thickness showed a wide range from 0.2 to 12.0 mm (n = 99; median 2.3 mm). 14 of 101 melanomas (14%) were classified as T1, thereof 11 (79%) were found in men. In 32 of 193 patients (17%), the primary melanoma was unknown. Of special interest in our series is the high incidence of male predominance (79%) in cases of thin metastasing melanoma (14%), implicating genetic or epigenetic (hormonal) gender differences underlying tumour progression. Additionally, the high percentage of unknown primary melanoma (17%), at least partly representing completely regressed melanomas, indicates the importance of immune surveillance in melanoma progression.