High ALDH1 expression correlates with better prognosis in tumorigenic malignant melanoma.

Aldehyde dehydrogenase 1 (ALDH1) has been proposed as biomarker of stem cells for certain human cancers. ALDH1 expression has been correlated with poor patient outcomes in a variety of malignancies but better patient outcomes in others, and its prognostic significance in malignant melanoma is unclear. Thus, 68 melanoma patients with comprehensive clinical and pathologic follow-up data were used to construct a tissue microarray. A modified histological score (H-score) with a maximum score of 300 was used to quantify immunohistochemical staining for ALDH1. Survival time was defined as the time between diagnosis and melanoma-specific death. Using univariate logistic regression, a low (<80 H-score) ALDH1 score showed 3.7-fold increase in risk for melanoma-specific death within 10 years when compared with high (>80) ALDH1 levels (P=0.017). Odds of MSD were lower by a factor of ~0.9 for each 10-point increase in H-Score. Median survival time was 44.1 months and 180.9 months for patients with low and high ALDH1 expression, respectively. Using multivariate analysis, ALDH1 H-score was found to be an independent prognostic factor. These findings suggest that ALDH1 expression in malignant melanoma has a favorable effect on patient survival. Further study is needed elucidate the function of this enzymatic protein in melanoma progression.

High lymphatic vessel density and lymphatic invasion underlie the adverse prognostic effect of radial growth phase regression in melanoma.

Regression in the radial growth phase (RGP) of primary cutaneous melanomas is common and has been shown to be an adverse prognostic factor. However, the underlying mechanism is unclear. We performed dual immunohistochemical staining of podoplanin and S-100 on paraffin tissues from 321 patients with vertical growth phase primary melanomas, who had 10 years or more of follow-up. Lymphatic vessel density (LD) and lymphatic invasion (LI) were quantified and documented. The time to first metastasis and melanoma-specific death (MSD) from the date of definite treatment were analyzed using univariate and multivariate Cox models. Among the 116 vertical growth phase melanomas that had regression in the adjacent RGP, 75 (23%) were classified as complete and 41 (13%) were classified as partial. LD was significantly higher (P<0.001) in the 75 lesions with complete regression (mean±SD, 23.7±12.3/mm²) compared with the 41 lesions with partial regression (15.5±7.1/mm²) and was lower in 155 areas of the adjacent normal dermis (7.3±3.5/mm²) and 69 areas of the distant normal dermis (5.5±2.6/mm²). Patients whose lesions had areas of complete regression with LI and either high or low LD or had no LI with high LD, had shorter time to first metastasis (hazard ratio=2.5, 3.8, and 2.5, respectively) and increased risk of melanoma-specific death (hazard ratio=3.1, 1.3 and 3.0, respectively) than those with no LI, and low LD or those without areas of complete regression. These data indicate that complete RGP regression is associated with significantly increased LD. In addition, the adverse prognostic effect of RGP regression is at least partially mediated through lymphangiogenesis and LI in this area.

The role of BRAF mutation and p53 inactivation during transformation of a subpopulation of primary human melanocytes.

Melanocytic nevi frequently harbor oncogenic BRAF mutations, but only a minority progress to melanoma. In human melanocytes, persistent BRAF(V600E) expression triggers oncogene-induced senescence, which implies that bypass of oncogene-induced senescence is necessary for malignant transformation of melanocytes. We show that a subpopulation of primary human melanocytes with persistent expression of BRAF(V600E) do not enter oncogene-induced senescence, but instead survive despite heightened MAPK activity. Disruption of the p53 pathway using short-hairpin RNA initiated rapid growth of these V600E(+) melanocytes in vitro. The resultant V600E(+)/p53(sh) melanocytes grew anchorage-independently in soft agar, formed pigmented lesions reminiscent of in situ melanoma in artificial skin reconstructs, and were weakly tumorigenic in vivo. Array comparative genomic hybridization analysis demonstrated that the transformed melanocytes acquired a substantial deletion in chromosome 13, which encodes the Rb1 tumor suppressor gene. Gene expression profiling study of nevi and melanomas showed that p53 target genes were differentially expressed in melanomas compared with nevi, suggesting a dysfunctional p53 pathway in melanoma in vivo. In summary, these data demonstrate that a subpopulation of melanocytes possesses the ability to survive BRAF(V600E)-induced senescence, and suggest that p53 inactivation may promote malignant transformation of these cells.

Increased cyclin D1 expression can mediate BRAF inhibitor resistance in BRAF V600E-mutated melanomas.

Recent studies have shown that there is a considerable heterogeneity in the response of melanoma cell lines to MEK and BRAF inhibitors. In the current study, we address whether dysregulation of cyclin-dependent kinase 4 (CDK4) and/or cyclin D1 contribute to the BRAF inhibitor resistance of melanoma cells. Mutational screening identified a panel of melanoma cell lines that harbored both a BRAF V600E mutation and a CDK4 mutation: K22Q (1205Lu), R24C (WM39, WM46, and SK-Mel-28), and R24L (WM902B). Pharmacologic studies showed that the presence of a CDK4 mutation did not alter the sensitivity of these cell lines to the BRAF inhibitor. The only cell line with significant BRAF inhibitor resistance was found to harbor both a CDK4 mutation and a CCND1 amplification. Array comparative genomic hybridization analysis showed that CCND1 was amplified in 17% of BRAF V600E-mutated human metastatic melanoma samples, indicating the clinical relevance of this finding. As the levels of CCND1 amplification in cell lines are lower than those seen in clinical specimens, we overexpressed cyclin D1 alone and in the presence of CDK4 in a drug-sensitive melanoma line. Cyclin D1 overexpression alone increased resistance and this was enhanced when cyclin D1 and CDK4 were concurrently overexpressed. In conclusion, increased levels of cyclin D1, resulting from genomic amplification, may contribute to the BRAF inhibitor resistance of BRAF V600E-mutated melanomas, particularly when found in the context of a CDK4 mutation/overexpression.

A phase I trial of the oral, multikinase inhibitor sorafenib in combination with carboplatin and paclitaxel.

PURPOSE: This study evaluated the safety, maximum tolerated dose, pharmacokinetics, and antitumor activity of sorafenib, a multikinase inhibitor, combined with paclitaxel and carboplatin in patients with solid tumors. PATIENTS AND METHODS: Thirty-nine patients with advanced cancer (24 with melanoma) received oral sorafenib 100, 200, or 400 mg twice daily on days 2 to 19 of a 21-day cycle. All patients received carboplatin corresponding to AUC6 and 225 mg/m(2) paclitaxel on day 1. Pharmacokinetic analyses were done for sorafenib on days 2 and 19 of cycle 1 and for paclitaxel on day 1 of cycles 1 and 2. Pretreatment tumor samples from 17 melanoma patients were analyzed for BRAF mutations. RESULTS: Sorafenib was well tolerated at the doses evaluated. The most frequent severe adverse events were hematologic toxicities (grade 3 or 4 in 33 patients, 85%). Twenty-seven (69%) patients had sorafenib-related adverse events, the most frequent of which were dermatologic events (26 patients, 67%). Exposure to paclitaxel was not altered by intervening treatment with sorafenib. Treatment with sorafenib, paclitaxel, and carboplatin resulted in one complete response and nine partial responses, all among patients with melanoma. There was no correlation between BRAF mutational status and treatment responses in patients with melanoma. CONCLUSIONS: The recommended phase II doses are oral 400 mg twice daily sorafenib, carboplatin at an AUC6 dose, and 225 mg/m(2) paclitaxel. The tumor responses observed with this combined regimen in patients with melanoma warrant further investigation.

Lymphatic invasion revealed by multispectral imaging is common in primary melanomas and associates with prognosis.

Lymphatic invasion by tumor cells has been noted infrequently in primary melanomas. Our primary hypotheses were that using immunohistochemical markers of lymphatic vessels and of tumor cells would improve detection of lymphatic invasion and that lymphatic invasion would correlate with regional nodal metastatic disease. This study included 106 patients who were diagnosed between 1972 and 1991 and who had 10 years or more of follow-up. We performed dual immunohistochemical stains for podoplanin (for lymphatic vessels) and S-100 (for melanoma cells). Lymphatic invasion was identified by light microscopy and confirmed by multispectral imaging analysis. Lymphatic invasion was detected by morphology alone in 5 cases (4.7%) in contrast to immunohistochemical staining augmented by multispectral imaging analysis where 35 cases (33%) were identified (P < .0001). Lymphatic invasion was significantly associated with time to regional nodal metastatic disease, as well as first metastasis and melanoma-specific death. "Local metastasis," defined by immunohistochemistry-detected lymphatic invasion, satellites, or neural invasion, identified 64% of those who had regional nodal metastatic disease within 5 years of diagnosis. Lymphatic invasion is an underobserved phenomenon in primary melanomas that can be better detected by immunohistochemical staining. The presence of lymphatic invasion may be a clinically useful predictor of regionally metastatic disease.

Shared MHC class II-dependent melanoma ribosomal protein L8 identified by phage display.

Antigens recognized by T helper (Th) cells in the context of MHC class II molecules have vaccine potential against cancer and infectious agents. We have described previously a melanoma patient's HLA-DR7-restricted Th cell clone recognizing an antigen, which is shared among melanoma and glioma cells derived from various patients. Here, this antigen was cloned using a novel antigen phage display approach. The antigen was identified as the ribosomal protein L8 (RPL8). A peptide of RPL8 significantly stimulated proliferation and/or cytokine expression of the Th cell clone and lymphocytes in four of nine HLA-DR7(+) melanoma patients but not in healthy volunteers. The RPL8 antigen may represent a relevant vaccine target for patients with melanoma, glioma, and breast carcinoma whose tumors express this protein.

Preferential involvement of CX chemokine receptor 4 and CX chemokine ligand 12 in T-cell migration toward melanoma cells.

Our previous analysis of the role of chemokines in T lymphocyte trafficking toward human tumor cells revealed the migration of a melanoma patient's cytotoxic T lymphocytes (CTL) toward autologous tumor cells, resulting in tumor cell apoptosis, in an organotypic melanoma culture. CTL migration was mediated by CX chemokine receptor (CXCR) 4 expressed by the CTL and CX chemokine ligand (CXCL) 12 secreted by the tumor cells, as evidenced by blockage of CTL migration by antibodies to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. Here, we present the results of T cell migration in one additional melanoma patient and T cell and tumor cell analyses for CXCR4 and CXCL12 expression, respectively, in 12 additional melanoma patients, indicating the preferential role of CXCR4 and CXCL12 in CTL migration toward melanoma cells. These studies add to the increasing body of evidence suggesting that CXCL12 is a potent chemoattractant for T cells.

Human leukocyte antigen-A2-restricted CTL responses to mutated BRAF peptides in melanoma patients.

Mutated BRAF (BRAF(V600E)) is a potential immunotherapeutic target for melanoma because of its tumor specificity and expression in the majority of these lesions derived from different patients. BRAF(V600E) is expressed intracellularly and not on the cell surface, therefore providing a target for T cells but not B cells. Demonstration of patients' T cell responses to BRAF(V600E) would suggest the feasibility of active specific immunotherapy targeting the mutation in these patients. In the present study, BRAF(V600E) peptides with putative binding sites for human leukocyte antigen (HLA)-A2 were used to stimulate T lymphocytes of HLA-A2-positive melanoma patients. Four of five patients with BRAF(V600E)-positive lesions showed lymphoproliferative responses to BRAF(V600E) peptide stimulation. These responses were specific for the mutated epitope and HLA-A2 was restricted in three patients. Lymphocytes from these three patients were cytotoxic against HLA-A2-matched BRAF(V600E)-positive melanoma cells. None of the four patients with BRAF(V600E)-negative lesions and none of five healthy donors had lymphoproliferative responses specific for the mutated epitope. The high prevalence (approximately 50%) of HLA-A2 among melanoma patients renders HLA-A2-restricted BRAF(V600E) peptides attractive candidate vaccines for these patients.

Biologic and prognostic significance of dermal Ki67 expression, mitoses, and tumorigenicity in thin invasive cutaneous melanoma.

PURPOSE: Tumor cell proliferation is a central feature of melanoma progression. In this study, we characterized three biomarkers of proliferation (Ki67 expression, dermal mitotic rate [MR], and tumorigenicity) in thin (< or = 1.00 mm) primary cutaneous melanomas and examined their association with prognosis. PATIENTS AND METHODS: We used immunohistochemistry to determine Ki67 expression using the monoclonal antibody MIB-1 in lesions from a prospective cohort that included 396 patients with thin invasive primary melanomas seen between 1972 and 1991. A multivariate Cox proportional hazards model was used to define independent prognostic factors, and recursive partitioning was used to develop a prognostic tree identifying risk groups. RESULTS: Dermal Ki67 expression was lower than epidermal Ki67 expression in radial growth phase (RGP) melanomas (n = 171), and dermal Ki67 expression and MR were higher in tumorigenic vertical growth phase (VGP) melanomas (n = 193) compared with RGP and nontumorigenic VGP melanomas (n = 42). Dermal Ki67 expression, MR greater than 0, growth phase, thickness, ulceration, tumor-infiltrating lymphocytes, and sex were associated with metastasis at 10 years, however, only dermal Ki67 expression, MR greater than 0, and sex were independent prognostic factors. Two high-risk groups were identified: men and women with dermal MR greater than 0 and dermal Ki67 expression > or = 20% in tumor cells and men with MR greater than 0 and Ki67 expression less than 20%, with 10-year metastasis rates of 39% and 20%, respectively. CONCLUSION: Proliferation slows as melanoma cells enter the dermis and then increases with the onset of tumorigenic VGP. Ki67 expression and dermal MR provide independent prognostic information that can potentially be used in risk-based management of patients.

A tumorigenic subpopulation with stem cell properties in melanomas.

Recent studies suggest that cancer can arise from a cancer stem cell (CSC), a tumor-initiating cell that has properties similar to those of stem cells. CSCs have been identified in several malignancies, including those of blood, brain, and breast. Here, we test whether stem cell-like populations exist in human melanomas. In approximately 20% of the metastatic melanomas cultured in growth medium suitable for human embryonic stem cells, we found a subpopulation of cells propagating as nonadherent spheres, whereas in standard medium, adherent monolayer cultures were established. Individual cells from melanoma spheres (melanoma spheroid cells) could differentiate under appropriate conditions into multiple cell lineages, such as melanocytic, adipocytic, osteocytic, and chondrocytic lineages, which recapitulates the plasticity of neural crest stem cells. Multipotent melanoma spheroid cells persisted after serial cloning in vitro and transplantation in vivo, indicating their ability to self-renew. Furthermore, they were more tumorigenic than adherent cells when grafted to mice. We identified similar multipotent spheroid cells in melanoma cell lines and found that the stem cell population was enriched in a CD20+ fraction of melanoma cells. Based on these findings, we propose that melanomas can contain a subpopulation of stem cells that contribute to heterogeneity and tumorigenesis. Targeting this population may lead to effective treatments for melanomas.

Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation.

Dysregulated activation of Ras or its downstream effectors such as mitogen-activated protein kinase kinase and ERK has been shown to play a critical role in tumorigenesis of many cancer types. However, in melanoma, activating mutations in Ras are rarely observed and are limited to N-Ras in UV-exposed cells. In this study, we identify constitutively activated ERK in almost all melanoma cell lines and in tumor tissues tested, which is in contrast to normal melanocytes and several early stage radial growth phase melanoma lines where ERK can be activated by serum or growth factors. Constitutive activation of ERK is preceded by phosphorylation of mitogen-activated protein kinase kinase and c-RAF. In all of the melanoma cell lines tested, Ras is constitutively activated without underlying mutations. On the contrary, activating mutations in the kinase domain of BRAF are present in the majority of the cell lines tested. Furthermore, ERK activation can be partially inhibited from the cell surface using inhibitors of fibroblast growth factor and hepatocyte growth factor but not interleukin 8 signaling pathways. These data suggest that melanoma growth, invasion, and metastasis are attributable to constitutively activated ERK apparently mediated by excessive growth factors through autocrine mechanisms and BRAF kinase activation.