Concordance in BRAF V600E status over time in malignant melanoma and corresponding metastases.

AIMS: The aims of the present study were to analyse the usability of an immunohistochemical (IHC) analysis as compared with a frequently used mutation detection analysis, and to examine the extent of intratumour and intertumour heterogeneity of BRAF V600E in primary tumours and their corresponding metastases. In the development of intertumour heterogeneity between the primary tumour and the corresponding metastases, time as a factor was also investigated. METHODS AND RESULTS: In total, 227 samples from 224 melanoma patients were analysed with both the Cobas 4800 BRAF V600 Mutation Test and IHC anti-BRAF V600E staining. In 82 primary tumours and 224 corresponding metastases, the extents of intertumour and intratumour heterogeneity were investigated with IHC staining. In 15 cases, disagreement between IHC analysis and the Cobas test was seen. In all but one of the examined patients, homogeneity between the primary tumour and the corresponding metastasis was found. Except for this one case, no heterogeneity developed over longer periods. CONCLUSION: IHC analysis can be safely used as a BRAF pretreatment screening tool, and no additional test is needed when staining is positive. However, if stains are negative, additional tests are essential for detection of other BRAF mutations. We suggest that using primary melanoma tissues is just as safe as using metastatic tissue for detection of BRAF V600E, as BRAF intertumour heterogeneity is extremely rare. In addition, the time between diagnosis of the primary tumour and diagnosis of the corresponding metastasis seems not to increase the risk of intertumour heterogeneity.

Loss of E-cadherin as Part of a Migratory Phenotype in Melanoma Is Associated With Ulceration.

It has been suggested that embryogenic properties of migratory cells are reactivated during wound healing and metastasis in adults. This might explain the association between wound-induced inflammation and poor survival in patients with ulcerated melanoma. Linking inflammation with a migratory phenotype, we characterize the infiltration of innate inflammatory cells, loss of cell-to-cell adhesion (E-cadherin), factors associated with extracellular matrix degradation [matrix metalloproteinase-9 (MMP-9), and neutrophil elastase (NE)], and spindle-shaped cell morphology, between ulcerated (n = 179) and nonulcerated (n = 206) melanoma. In addition, the presence of "extravascular migratory metastasis" (angiotropism) and tumor-vessel density were evaluated as important factors for tumor cell dispersal in ulcerated melanoma. We showed a correlation between expression of the granulocyte marker cd66b+ and the expression of NE and MMP-9, reflecting activated neutrophils. Ulcerated melanoma correlated with a low global E-cadherin score (P = 0.041) and weak-spot score (P = 0.0004). Thus, 28% of the nonulcerated, 42% of the minimally/moderately ulcerated melanoma, and 53% of the excessively ulcerated melanoma presented low scores as opposed to a high E-cadherin score. In addition, the presence of ulceration was correlated with angiotropism (P < 0.0001) and spindle-shaped morphology (P = 0.021). There were no differences in MMP-9 expression or intratumoral vessel density between the ulcerated and nonulcerated group. In conclusion, expression of migratory cell properties showed a highly heterogeneous pattern, which was associated with ulcerated areas and inflammatory cells, in general and with neutrophils in particular. We, therefore, suggest that wound-associated inflammation may be involved in the induction of migratory cell transition and tumor cell dispersal in ulcerated melanoma.

MelanA-negative spindle-cell associated melanoma, a distinct inflammatory phenotype correlated with dense infiltration of CD163 macrophages and loss of E-cadherin.

MelanA is a known melanocyte marker and is important in melanoma diagnostics. Some tumours, however, show loss of MelanA expression and may therefore be difficult to distinguish from tumours of mesenchymal origin. Pure spindle-cell melanoma is a rare event, and little is known about its biological background and prognosis. However, morphological changes towards a more mesenchymal shape and cellular dedifferentiation may correlate with reactivation of important developmental programmes (epithelial-to-mesenchymal transition) and disseminative tumour cell properties. Inflammation and CD163+ macrophages have been shown to be important inducers of E-cadherin and cell-to-cell adhesion loss, a pivotal and final event of epithelial-to-mesenchymal transition. In a cohort of 385 patients with melanoma, we located nine tumours with a clonal MelanA expression, defined as a tumour section with a distinct MelanA-negative clone next to a MelanA-positive clone. Interestingly, MelanA-negative clones correlated significantly with an augmented inflammatory response of tumour-infiltrating macrophages (CD163+), complete loss of E-cadherin and a spindle-shaped morphology, irrespective of ulcerated status. These cases show the inflammatory heterogeneity of melanoma, which may have important diagnostic, prognostic and therapeutic implications for the patients. We show that melanomas harbour cell clones that bear strong resemblance to tumour-associated macrophages, a pivotal player in a tumour-supporting microenvironment. Interestingly, this distinct inflammatory phenotype is associated with loss of MelanA expression, the presence of spindle-shape morphology and complete loss of E-cadherin, considered as possible markers of poorly differentiated and more invasive tumour cells.

Prognostic stratification of ulcerated melanoma: not only the extent matters.

OBJECTIVES: For patients with melanoma, ulceration is an important prognostic marker and interestingly also a predictive marker for the response of adjuvant interferon. A consensual definition and accurate assessment of ulceration are therefore crucial for proper staging and clinical management. We evaluated the prognostic impact of the extent and type of ulceration and the epidermal involvement theoretically preceding it (consumption of epidermis and cleft formation) or seen subsequent to the inflammation (reepithelialization and reactive epidermal hyperplasia), aiming for better prognostic stratification of ulcerated lesions. METHODS: From H&E-stained sections, the status (presence vs absence), extent (percentage of the total tumor length), and type (infiltrative vs attenuative) of ulceration and epidermal involvement were evaluated from 385 patients with cutaneous melanoma. RESULTS: The presence of ulceration (hazard ratio [HR], 1.83), an attenuative type of ulceration (HR, 3.02), and excessive ulceration (HR, 3.57) were independent predictors of poor melanoma-specific survival. Further subdivision of minimal/moderate ulceration showed independent prognostic value only for lesions with epidermal involvement of the surrounding epidermis (HR, 1.78). CONCLUSION: The extent and type of ulceration and involvement of the surrounding epidermis provided more accurate prognostic information than the mere absence or presence and may be useful markers allowing better stratification of ulcerated lesions.