Novel inhibitors in the treatment of metastatic melanoma

Metastatic melanoma continues to be a difficult disease to treat. Recent efforts have focused on developing novel, target-directed therapeutic agents. In this review, we discuss the RAS-RAF-MAP kinase and the RAS-PI3K-AKT pathway in detail, as up to 80% of cutaneous melanomas exhibit a BRAF mutation. The preclinical and clinical data regarding BRAF inhibition is reviewed. Other potential targets in these pathways are also discussed. Preclinical data have recently emerged, suggesting that the following subsets of patients have a lower frequency of BRAF mutations: acral, mucosal and cutaneous melanomas with chronic sun-induced damage. These lesions have a higher frequency of KIT mutations. However, cutaneous melanomas without chronic sun damage have a higher frequency of BRAF mutations and are not noted to have KIT mutations. It is possible that the appropriate subset of patients may respond differently to available targeted therapies and clinical trials are in development to assess the utility of KIT inhibition in these patients.     

Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients

Malignant pleural mesothelioma (MPM) is an asbestos-related malignancy that is highly resistant to current therapeutic modalities. We established four MPM cell lines (ACC-MESO-1, ACC-MESO-4, Y-MESO-8A and Y-MESO-8D) from Japanese patients, with the latter two from the same patient with biphasic-like characteristics of MPM, showing epithelial and sarcomatous phenotypes, respectively, in cell culture. These cells grew well in RPMI-1640 medium supplemented with 10% fetal bovine serum under 5% CO2. Mutation and expression analyses demonstrated that the tumor suppressor gene NF2, which is known to be one of the most frequently mutated in MPM, is mutated in ACC-MESO-1. We detected homozygous deletion of p16INK4A/p14ARF in all four MPM cell lines. However, mutations of other tumor suppressor genes, including TP53, and protooncogenes, including KRAS, NRAS, BRAF, EGFR and HER2, were not found in these cell lines. Polymerase chain reaction amplification of the simian virus 40 sequence did not detect any products. We also analyzed genetic alterations of six other MPM cell lines and confirmed frequent mutations of NF2 and p16INK4A/p14ARF. To characterize the biological differences between Y-MESO-8A and Y-MESO-8D, we carried out cDNA microarray analysis and detected genes that were differentially expressed in these two cell lines. Thus, our new MPM cell lines seem to be useful as new models for studying various aspects of the biology of human MPM as well as materials for the development of future therapies.     

The decreased susceptibility of metastatic melanoma cells to killing involves an alteration of CTL reactivity

Metastases are known to be more resistant to therapy than matching primary tumors, in particular they are less prone to apoptosis. In this study we investigated the functional interaction of a CTL clone (LT12) specific for a melanoma TA with the primary tumor (T1) versus its metastatic counterpart (G1). The CTL clone (LT12) was shown to lyse the primary T1 cells more efficiently in a classical cytotoxicity test. This differential susceptibility was not associated with MHC class I down-regulation and conjugate formation but correlated with a differential increase in Ca++ flux in the LT12 CTL when stimulated with the primary versus the metastatic tumor cells. Since LT12 uses perforin/granzyme B to kill its autologous target we analysed perforin and granzyme B mRNA expression in the CTL in the presence of either primary and metastatic melanoma cells. Quantitative PCR analysis showed an increased expression of granzyme B and perforin mRNA levels in LT12 when cocultured in the presence of the primary tumor. However, a similar level of (cytotoxic molecule) degranulation as revealed by CD107 expression was observed when LT12 was stimulated with T1 or G1 cells. These data suggest that the differential susceptibility of primary and metastatic melanoma cells involves at least in part their distinct potential to induce autologous CTL reactivity and the subsequent triggering of granzyme B and perforin in these cells.     

Ganglioside signatures of primary and nodal metastatic melanoma cell lines from the same patient

The primary cutaneous melanoma initially migrates to the regional lymph nodes (LNs). Human melanoma overexpresses gangliosides, the sialylglycosphingolipids. The ganglioside signatures may differ between primary and LN melanomas owing to the differences in the tumor microenvironments. The melanoma cells obtained from the primary and LN of the same patient might be useful to evaluate the above hypothesis. For this purpose, the cryopreserved cell lines from a primary cutaneous melanoma (IGR-39) and its nodal metastasis (IGR-37) from the same patient were used. We have also compared the ganglioside signatures of freshly obtained melanoma cells from primary, LN and organ metastases from different patients. Gangliosides were extracted, purified and identified by resorcinol and specific murine monoclonal antibodies. Comparison of the primary cell line with the nodal metastatic line obtained from the same patient distinctly showed the following features: (i) an increased production of gangliosides, (ii) O-acetylation of GM2 and GD3, (iii) an increased and altered O-acetylation of GD2 and (iv) possibly de-N-acetylation of GD3. These findings suggest that the nodal microenvironment might favor activation of O-acetyl-transferases capable of O-acetylating both alpha2, 3 and alpha2, 8 sialic acids of gangliosides. Supporting this, the primary melanoma cells obtained from different patients, showed no O-acetylation of GD3 or GD2. The cell line from groin LN showed the presence of O-acetyl (O-Ac)GD3. The cell lines from thyroid, spleen and jejunum expressed O-AcGD2. In all metastatic melanoma cell lines GD1a is more prevalent than GD3, suggesting that GD1a may be a major melanoma-ganglioside.     

Centrosomal dysregulation in human metastatic melanoma cell lines

Correct partitioning of the replicated genome during mitosis is orchestrated by centrosomes, and chromosomal instability is a commonly reported feature of human cancer. Melanomas are notorious for their genetic instability and rapid clonal evolution that may be manifested as aggressive growth and facile generation of therapy-resistant variants. We characterized the centrosomal status, ploidy, and gene status (TP53, CDKN2A/B, BRAF, and NRAS) of 15 human metastatic melanoma cell lines. Cells were labelled for pericentrin (a centrosomal marker), DNA and α-tubulin, and scored for centrosome morphology, supernumerary centrosomes, and mitotic symmetry. The incidence of supernumerary centrosomes correlated with that of gross centrosomal abnormalities (r = 0.90), mitotic asymmetry (r = 0.90), and, surprisingly, increased content of G/M cells (r = 0.79). Centrosomal numerical dysregulation, observed in all cell lines, was found not to be specifically related to the status of any of the characterized gene mutations that were found in 13/15 cell lines. We conclude that centrosomal dysregulation may arise from multiple mechanisms and may drive the generation of genetic and phenotypic diversity in melanoma.     

Adhesion characteristics of human melanoma cell lines of varying metastatic potential

The adhesive behaviour of a series of human melanoma cell lines, of varying metastatic potential, to basement membrane and stromal components was investigated in vitro. Experimental metastatic propensity was assessed from the number of pulmonary nodules formed after i.v. injection of cells into BALB/c nude mice. All cell lines showed similar kinetics of attachment when tested on plastic, type-I collagen films, type-I collagen hydrated gels, fibronectin, laminin type-IV collagen substrates and bovine aortic endothelial monolayers. Fibronectin-coated plastic compared to plastic alone produced increased cell attachment and spreading to the same extent in all the cell lines. The melanoma lines attached preferentially to cryostat sections of lung compared to other organs reflecting the pattern of organ involvement of metastasis in vivo. However, no significant quantitative differences in attachment to lung sections were seen between melanoma variants of differing metastatic capacities. Cells labelled with [125I]iododeoxyuridine to determine their initial organ distribution following i.v. injection showed that tumour-cell arrest was not significantly changed enough to explain the differing metastatic capacities. Thus it appears that adhesive properties of these melanoma cells are not correlated with their capacity to form metastases in vivo.     

Establishment and characterization of human ocular melanoma cell lines

Five continuous cell lines have been established from 29 ocular melanomas and maintained for periods ranging from 3 to 9 years in medium identical to that in which 3 concomitantly studied lines of cutaneous melanoma cells were cultured as controls. The long-term problems to be overcome in establishing uveal cell lines are related to cell-doubling times which ranged from 72 to 432 hr, and plating efficiency, which ranged from 0.5%–6.5%. Tumors and cell lines were found to contain melanosomes. The morphology of uveal cells during the early subcultures exhibited multiple changes. Two different established cell lines were obtained from one ciliary-body tumor. Biochemical studies revealed markers of melanogenesis and neuroendocrine compounds. Cytogenetic studies revealed chromosomal abnormalities that differed between uveal and conjunctival melanomas.     

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.     

Enrichment of circulating melanoma cells (CMCs) using negative selection from patients with metastatic melanoma

Circulating tumor cells have emerged as prognostic biomarkers in the treatment of metastatic cancers of epithelial origins viz., breast, colorectal and prostate. These tumors express Epithelial Cell Adhesion Molecule (EpCAM) on their cell surface which is used as an antigen for immunoaffinity capture. However, EpCAM capture technologies are of limited utility for non-epithelial cancers such as melanoma.We report a method to enrich Circulating Melanoma Cells (CMCs) that does not presuppose malignant cell characteristics. CMCs were enriched by centrifugation of blood samples from healthy (N = 10) and patient (N = 11) donors, followed by RBC lysis and immunomagnetic depletion of CD45-positive leukocytes in a specialized magnetic separator. CMCs were identified by immunocytochemistry using Melan-A or S100B as melanoma markers and enumerated using automated microscopy image analyses. Separation was optimized for maximum sensitivity and recovery of CMCs.Our results indicate large number of CMCs in Stage IV melanoma patients. Analysis of survival suggested a trend toward decreased survival with increased number of CMCs. Moreover, melanoma-associated miRs were found to be higher in CMC-enriched fractions in two patients when compared with the unseparated samples, validating this method as applicable for molecular analyses.Negative selection is a promising approach for isolation of CMCs and other EpCAM -negative CTCs, and is amenable to molecular analysis of CMCs. Further studies are required to validate its efficacy at capturing specific circulating cells for genomic analysis, and xenograft studies.     

Inhibition of macrophage Ia antigen expression by shed plasma membrane vesicles from metastatic murine melanoma lines

Shed plasma membrane-derived vesicles from metastatic variants of the murine B16 melanoma were examined for their ability to inhibit the induction of murine immune region-associated (Ia) antigen expression on macrophages, the initial step in the formation of an immune response. Membrane material that appears as a greater than 50 million-dalton fraction on column chromatography is found only in conditioned media from tumor cells and not in culture media from normal cells, such as murine 3T3 cells. Membrane vesicles from both metastatic variants B16-F1 (low lung colonizing) and B16-F10 (high lung colonizing) were taken up by macrophages; however, only membrane vesicles isolated from the B16-F10 cultures exhibited significant inhibitory activity for Ia induction. This inhibition appears to result from enhanced prostaglandin synthesis, since treatment with aspirin can reverse the membrane vesicle-induced inhibition. The inhibitory component(s) released into the media was demonstrated to be predominantly associated with membrane vesicles; however, the component(s) retained its activity after Triton X-100 treatment, indicating that the intact membrane vesicle was not necessary for the action of the inhibitory material. Treatments with heat (65 degrees C) and proteases (papain) indicated that the inhibitory component(s) is a heat-labile protein.     

Malignant melanoma metastatic to the gastrointestinal tract

A retrospective study of 385 melanoma patients was performed, with the goal of evaluating the clinical characteristics, the role of imaging and the impact of treatment on patients with gastrointestinal (GI) metastases. Eighteen patients (4.7%) had GI tract metastases. In 50% the primary lesion was on the lower extremities (P< 0.01), while 61.1% had nodular melanomas (P < 0.01). Imaging and/or endoscopy were undertaken in 72.2% of the patients, yielding positive results in all. Eight patients underwent curative surgery, two received no treatment, while the remaining eight patients had chemotherapy or immunochemotherapy. Long-term palliation was offered to 87.5% of the surgical patients compared with 50% of the patients treated medically. Median survival in the patients treated with surgery was 47.5 months compared with 5.8 months in the medical group (P < 0.01). GI tract metastases were more common in patients with nodular melanoma of the lower extremities. To our knowledge, this is the first study correlating the primary lesion's characteristics with the development of GI tract metastases. Imaging is effective in the diagnosis of GI tract involvement. Melanoma patients with GI tract metastases can benefit from palliation by surgical resection. Survival is improved when such patients are treated with curative surgery.     

Generation of drug-resistant variants in metastatic B16 mouse melanoma cell lines

Genetic instability is recognized as an important aspect of the development of tumor heterogeneity and malignancy. In a previous study [Hill et al. Science (Wash. DC), 244:998-1001, 1984], we demonstrated that metastatic variants are generated at a more rapid rate in the highly metastatic B16F10 mouse melanoma cell line than in the less metastatic B16F1 cell line. The metastatic variants were phenotypically unstable, being generated and lost at high rates; consequently, we proposed a dynamic heterogeneity model of tumor metastasis which describes these properties quantitatively. As an extension of this work, we have examined the ability of these two melanoma cell lines to generate variants resistant to the drugs methotrexate and N-(phosphonacetyl)-L-aspartate. We observed that the highly metastatic B16F10 cell line generated variants resistant to a given concentration of methotrexate or N-(phosphonacetyl)-L-aspartate at higher rates than the B16F1 cell line. We conclude that B16F10 cells are genetically less stable than B16F1 cells and since resistance to methotrexate and N-(phosphonacetyl)-L-asparate usually results from gene amplification that B16F10 cells possess increased ability to amplify DNA. This higher rate of generation of drug-resistant variants corresponds to the higher rate of generation of metastatic variants we observed previously and suggests that a gene amplification mechanism may be involved in the generation of a metastic phenotype in B16 melanoma cells.     

Revisiting the role of systemic therapies in patients with metastatic melanoma to the CNS

The CNS is a common site of metastasis in patients with malignant melanoma. Locoregional control either with surgery or radiotherapy is first-line treatment for patients with brain metastasis should they be suitable candidates. For those patients who are not and those who progress after previous treatment, there is an unmet clinical need for effective systemic therapies. Systemic cytotoxics, such as temozolamide and fotemustine, have only modest activity, resulting in a median progression-free survival ranging from 1–2 months, in patients with metastatic melanoma to the brain. Newer systemic treatments such as vemurafenib and ipilimumab have been approved for the treatment of melanoma, but evidence regarding their activity in brain metastases is inconclusive due to the limited access of patients to clinical trials. This is now being revised and more data are emerging supporting the inclusion of patients with brain metastasis in trials. In this review, the authors present data regarding the efficacy of systemically administered therapies in patients with metastatic melanoma to the brain.     

Establishment and characterization of two new cell lines derived from human metastatic breast carcinomas

Two human cancer cell lines (MA 2 and MA 3) were established from pleural effusions of infiltrating ductal carcinomas of the breast.The lines were maintained in continuous monolayer culture withdoubling times of 70 (MA 2) and 78 (MA 3) hr for more than two years and possessed extensively rearranged abnormal karyotypeswith modal chromosome number of 83 (MA 2) and 81 (MA 3) and DNA index values of 1.65 and 1.77, respectively. No amplifications or rearrangements were evident in the c-myc, int-2, c-erb B2, c-Ha-ras, or hst 1 genes in MA 2 and MA 3 cell lines.The clinical histories of the patients from whom the cell lines were derived are reported and compared with the results observed inthe cell lines in vitro. The presence of CEA, CA 15-3, and MCA tumormarkers observed in the primary tumor tissues was retained by the established cell lines. While the primary tumor tissueswere ER+/PgR borderline + (MA 2) and ER–/PgR+(MA 3), the MA 2 line was ER+/PgR– and the MA 3 line remained ER–/PgR+.The MDR P-glycoprotein was not expressed either in primary tumor tissues or in the respective cell lines. High expression of cytokeratins7, 18, and 19 was evident by immunohistochemical analysis in each cell line, whereas cytokeratins 8 and 17 were poorly or not at allexpressed. The treatment history of the patients fromwhom the cell lines were derived involved CMF followed six monthslater by novantrone and cisplatin plus VP 16 (MA 2) and FEC followedfour years later by CMF (MA 3). The chemosensitivitypattern assay of the cell lines indicated that the MA 2 linewas sensitive to doxorubicin, cisplatin, and vinblastine, whereas theMA 3 line was sensitive to doxorubicin and cisplatin.The characteristics of these cell lines indicate them to be a goodexperimental model to investigate breast cancer biology and anticancerdrug response.     

Management of metastatic melanoma patients with brain metastases

Brain metastases seem to be an almost inevitable complication in patients with metastatic melanoma. Except for the rare patients who can undergo successful surgical resection of brain metastases, current management strategies do not appear adequate and result in a poor outcome (median survival, 2–4 months). In recent small series, stereotactic radiosurgery or gamma-knife treatment has suggested improvement in local control compared with whole brain radiation therapy. We have recently shown prolonged survival (11.1 months) using a multimodality treatment approach in 44 sequential patients with melanoma brain metastases. A subsequent study demonstrated that the outcome of biochemotherapy for metastatic melanoma is not affected by the presence or absence of brain metastases. Our results suggest that the outcome of patients with melanoma brain metastases can be improved using a multidisciplinary management strategy.     

Lymphocyte function and response to chemo-immunotherapy in patients with metastatic melanoma

Thirty-eight patients with metastatic melanoma were investigated for lymphocyte function immediately prior to chemo-immunotherapy. The pre-treatment immune tests were compared with normal control values and with response to therapy. The "non-responder" group (but not "responder") had significantly reduced values for lymphocyte, null-cell and E-rosette-cell counts compared with controls. Lymphocytoxicity ( using a Chang target cell) showed the same pattern, with depression of direct and K-cell cytotoxic capacity in non-responders compared with controls. Eight patients were studied sequentially whilst on treatment, and demonstrated considerable change (not statistically significant) in lymphocytotoxicity, an untreated "control" patient showed little variation. "Recall"-antigen skin testing showed no statistically significant difference between the patient groups. The data indicate that "non-T-cell activity" may be associated with response to chemo-immunotherapy.