Prognostic value of uPAR expression and angiogenesis in primary and metastatic melanoma.

Angiogenesis is important for the progression of cutaneous melanoma. Here, we analyzed the prognostic impact of the angiogenic factor urokinase plasminogen activator resecptor (uPAR), vascular proliferation index (VPI) and tumor necrosis as a measure of hypoxia in a patient series of nodular melanomas (n = 255) and matched loco-regional metastases (n = 78). Expression of uPAR was determined by immunohistochemistry and VPI was assessed by dual immunohistochemistry using Factor-VIII/Ki67 staining. Necrosis was recorded based on HE-slides. As novel findings, high uPAR expression and high VPI were associated with each other, and with increased tumor thickness, presence of tumor necrosis, tumor ulceration, increased mitotic count and reduced cancer specific survival in primary melanoma. In matched cases, VPI was decreased in metastases, whereas the frequency of necrosis was increased. Our findings demonstrate for the first time the impact on melanoma specific survival of uPAR expression and VPI in primary tumors, and of increased necrosis as an indicator of tumor hypoxia in loco-regional metastases. These findings support the importance of tumor angiogenesis in melanoma aggressiveness, and suggest uPAR as an indicator of vascular proliferation and a potential biomarker in melanoma.

Prognostic impact and concordance of TERT promoter mutation and protein expression in matched primary and metastatic cutaneous melanoma.

BACKGROUND: TERT promoter mutations are frequent in melanoma. Here we analysed the concordance and prognostic impact of TERT mutation and telomerase reverse transcriptase (TERT) protein expression in a large melanoma series. METHODS: In 194 primary nodular melanomas with 72 matched loco-regional metastases, TERT promoter mutation status was assessed by Sanger sequencing and TERT protein expression by immunohistochemistry. RESULTS: TERT mutations were found in 68% of primary melanomas and 64% of metastases, and the mutation status was discordant between primary tumour and metastasis in 24% of the cases. 6 of the 10 cases with discordant and wild-type metastases were also TERT wild type when re-tested in other intra-tumour regions, whereas 4 cases were mutation positive. TERT-mutated tumours tended to be thicker, have a higher mitotic count and higher patient age than TERT wild-type cases, but there was no significant association with reduced survival. TERT protein expression did not correlate with mutation status, but showed a similar discordancy between the primary and first metastatic lesion, and was significantly associated with reduced survival. CONCLUSIONS: TERT promoter mutations showed inter- and intra-tumoural discordancy, whereas only expression of TERT protein was associated with reduced patient survival.

BRAF-V600E expression in primary nodular melanoma is associated with aggressive tumour features and reduced survival.

BACKGROUND: Around 50% of primary melanomas harbour BRAF mutations, but their prognostic impact has not been clear. Recently, a BRAF-V600E mutation-specific antibody has become available for immunohistochemistry. Here, we investigated for the first time the prognostic impact of BRAF-V600E protein expression in primary melanoma. METHODS: In a patient series of 248 nodular melanomas, BRAF-V600E and total BRAF expression were assessed by immunohistochemistry using tissue microarray sections of paraffin-embedded archival tissue. Mutation status was assessed by real-time PCR in cases with sufficient tumour tissue (n=191). RESULTS: Positive BRAF-V600E expression was present in 86 (35%) of the cases, and was significantly associated with increased tumour thickness, presence of tumour ulceration and reduced survival. Further, BRAF-V600E expression was an independent prognostic factor by multivariate analysis, whereas BRAF mutation status was not significant. There was 88% concordance between BRAF-V600E expression and mutation status. CONCLUSIONS: Our findings indicate that BRAF-V600E expression is a novel prognostic marker in primary melanoma.

Nestin expression is associated with aggressive cutaneous melanoma of the nodular type.

The intermediate filament nestin, a neural stem-cell marker, is reported to be expressed more strongly in melanomas compared with benign melanocytic lesions, and increasingly expressed in advanced melanoma stages. However, the prognostic impact of nestin on melanoma has not been well elucidated. The aim of the present study was to evaluate the prognostic influence of nestin expression in cutaneous melanoma in comparison with standard clinico-pathologic variables. In a large series of nodular cutaneous melanoma (n=348), nestin expression was assessed by immunohistochemistry using tissue microarray (TMA) sections. For comparison, nestin staining in corresponding metastases as well as in superficial spreading melanomas and benign nevi was also examined. Nestin was expressed to varying degrees in a majority of nodular melanomas (92%), and was significantly associated with increased tumor thickness, high mitotic count, and the presence of ulceration and tumor necrosis. Also, expression was stronger in the nodular type than in superficial spreading melanomas and benign nevi, but without significant difference when compared with matched metastases from the former. Importantly, strong expression of nestin was significantly associated with reduced survival in multivariate analysis. In conclusion, increased nestin expression was associated with aggressive melanoma features, with independent prognostic impact on multivariate survival analysis when compared with clinico-pathologic factors.

Prognostic importance of the mitotic marker phosphohistone H3 in cutaneous nodular melanoma.

Mitotic count is a known prognostic predictor in cutaneous melanoma, and is included in the current American Joint Committee on Cancer tumor-node-metastasis (TNM) staging system. The mitotic marker phosphohistone H3 (PHH3) is considered to facilitate counting of mitosis, and the purpose of this study was to evaluate the prognostic significance and strength of PHH3 in comparison with standard mitotic counting in cutaneous malignant melanoma. A total of 457 consecutive cases of nodular cutaneous melanoma were initially included in this series. The mitotic count was assessed on hematoxylin and eosin sections, and PHH3 was then examined by immunohistochemistry on standard sections of paraffin-embedded tumor tissue. Both the mitotic count and the number of PHH3-stained mitotic figures were recorded in a minimum area of 1 mm(2). Increased mitotic count and PHH3 value were both associated with unfavorable features like tumor thickness and presence of ulceration. Univariate survival analysis showed a highly significant prognostic impact of mitotic count and PHH3, whereas multivariate analysis indicated PHH3 to be a stronger prognostic indicator than mitotic count. Assessment of mitotic activity by PHH3 immunostaining might have important practical advantages, and should be further studied to consider a place in routine examination of all cutaneous melanomas.

Ki-67 expression is superior to mitotic count and novel proliferation markers PHH3, MCM4 and mitosin as a prognostic factor in thick cutaneous melanoma.

BACKGROUND: Tumor cell proliferation is a predictor of survival in cutaneous melanoma. The aim of the present study was to evaluate the prognostic impact of mitotic count, Ki-67 expression and novel proliferation markers phosphohistone H3 (PHH3), minichromosome maintenance protein 4 (MCM4) and mitosin, and to compare the results with histopathological variables. METHODS: 202 consecutive cases of nodular cutaneous melanoma were initially included. Mitotic count (mitosis per mm2) was assessed on H&E sections, and Ki-67 expression was estimated by immunohistochemistry on standard sections. PHH3, MCM4 and mitosin were examined by staining of tissue microarrays (TMA) sections. RESULTS: Increased mitotic count and elevated Ki-67 expression were strongly associated with increased tumor thickness, presence of ulceration and tumor necrosis. Furthermore, high mitotic count and elevated Ki-67 expression were also associated with Clark's level of invasion and presence of vascular invasion. High expression of PHH3 and MCM4 was correlated with high mitotic count, elevated Ki-67 expression and tumor ulceration, and increased PHH3 frequencies were associated with tumor thickness and presence of tumor necrosis. Univariate analyses showed a worse outcome in cases with elevated Ki-67 expression and high mitotic count, whereas PHH3, MCM4 and mitosin were not significant. Tumor cell proliferation by Ki-67 had significant prognostic impact by multivariate analysis. CONCLUSIONS: Ki-67 was a stronger and more robust prognostic indicator than mitotic count in this series of nodular melanoma. PHH3, MCM4 and mitosin did not predict patient survival.

Tumor necrosis is associated with increased alphavbeta3 integrin expression and poor prognosis in nodular cutaneous melanomas.

BACKGROUND: Tumor necrosis and apoptotic activity are considered important in cancer progression, but these features have not been much studied in melanomas. Our hypothesis was that rapid growth in cutaneous melanomas of the vertical growth phase might lead to tissue hypoxia, alterations in apoptotic activity and tumor necrosis. We proposed that these tumor characteristics might be associated with changes in expression of cell adhesion proteins leading to increased invasive capacity and reduced patient survival. METHODS: A well characterized series of nodular melanoma (originally 202 cases) and other benign and malignant melanocytic tumors (109 cases) were examined for the presence of necrosis, apoptotic activity (TUNEL assay), immunohistochemical expression of hypoxia markers (HIF-1 alpha, CAIX, TNF-alpha, Apaf-1) and cell adhesion proteins (alphavbeta3 integrin, CD44/HCAM and osteopontin). We hypothesized that tumor hypoxia and necrosis might be associated with increased invasiveness in melanoma through alterations of tumor cell adhesion proteins. RESULTS: Necrosis was present in 29% of nodular melanomas and was associated with increased tumor thickness, tumor ulceration, vascular invasion, higher tumor proliferation and apoptotic index, increased expression of alphavbeta3 integrin and poor patient outcome by multivariate analysis. Tumor cell apoptosis did also correlate with reduced patient survival. Expression of TNF-alpha and Apaf-1 was significantly associated with tumor thickness, and osteopontin expression correlated with increased tumor cell proliferation (Ki-67). CONCLUSION: Tumor necrosis and apoptotic activity are important features of melanoma progression and prognosis, at least partly through alterations in cell adhesion molecules such as increased alphavbeta3 integrin expression, revealing potentially important targets for new therapeutic approaches to be further explored.