Molecular markers in melanoma.

The last few years have witnessed the dawn of the molecular era in melanoma treatment. With the advent of successful therapy targeting mutant BRAF, melanoma is leading the field of cancer research in the molecular approach to therapy of advanced disease. Attempting to keep pace with advances in therapy are advances in the molecular assessment of melanoma progression, facilitated by the availability of genome-wide approaches to interrogate the malignant phenotype. At the DNA level, this has included approaches such as comparative genomic hybridization. At the RNA level, this has consisted of gene expression profiling using various assay methodologies. In certain instances, markers identified using these platforms have been further examined and developed using fluorescence in situ hybridization and immunohistochemical analysis. In this article, we will review recent progress in the development of novel molecular markers for melanoma that are nearing clinical application. We will review developments in the molecular classification of melanoma, in the molecular diagnosis of melanoma, and in the molecular assessment of melanoma prognosis.

MicroRNA-mediated regulation of melanoma.

Melanoma is one of the most aggressive and deadly skin cancers, and, in its advanced stages, accounts for > 80% mortality. The incidence of melanoma is increasing worldwide; however, beyond surgical removal of the tumour, there is currently no curative therapy available, especially for its advanced stages. This may, in part, be owing to incomplete understanding of the molecular mechanisms that regulate the initiation and/or progression of melanoma to metastasis. The molecular mechanisms leading to the development and progression of melanoma are the focus of intense investigation, and many genetic/epigenetic alterations affecting melanoma progression and development have been identified. microRNAs (miRNAs) are emerging as important causal modulators in the development and progression of melanoma. The understanding of miRNA-mediated regulation of tumours has grown immensely over the last few years, as it has been understood to regulate most biological processes. Here, we review the currently available data on miRNAs associated with melanoma, highlighting those deregulated miRNAs that target important genes and pathways involved in the progression of melanocytes to primary and metastatic melanoma. We also review their potential clinical utility as biomarkers and potential use in targeted therapy.

Growth inhibition efficacy of an adenovirus expressing dual therapeutic genes, wild-type p53, and anti-erbB2 ribozyme, against human bladder cancer cells.

The altered expression of both p53 and erbB2 is strongly related to the disease status and the outcome of bladder cancers. We examined the antitumor efficacy by the modulation of these genetic alterations with a newly designed dual-gene-expressing adenovirus (Ad-p53/erbB2Rz), which expresses p53 and anti-erbB2 ribozyme simultaneously in human bladder cancer cells. Cell growth inhibition efficacy along with biological responses of this virus was compared with other viral vectors (Ad-p53, which expresses wild-type p53 cDNA, and Ad-erbB2Rz, which expresses anti-erbB2 ribozyme, solely or in combination). Sufficient transgene expression in targeted cells and the altered expression of the targeted genes and their encoded proteins were obtained by each therapeutic vector. Each of the three therapeutic viral vectors inhibited bladder cancer cell growth, and the putative additive antitumor effect was shown by the combination of two of the therapeutic vectors. Furthermore, Ad-p53/erbB2Rz had superior therapeutic efficacy when the same titers of viruses were infected. Nonspecific vector-related toxicity was minimized by reducing the total amount of viral titers by using the dual-gene-expressing adenovirus. Modulation of multiple genetic abnormalities might enhance the therapeutic efficacy, and vector-related toxicity could be minimized when the total amount of viral titers are reduced.

Suppression of the neoplastic phenotype in vivo by an anti-ras ribozyme.

In this study, the efficacy of an anti-ras ribozyme in reversing the neoplastic phenotype was investigated. Murine NIH3T3 cells were transfected with cellular DNA from the FEMX-I human melanoma cell line expressing the activated H-ras gene. The transformed cells displayed the neoplastic phenotype in vitro and were tumorigenic in nude mice in vivo. When the transformants were transfected by a ribozyme designed to cleave only activated H-ras RNA, the transformed phenotype was abrogated. In contrast, expression of a mutant ribozyme, essentially acting only as antisense, into the transformed cells resulted in less dramatic changes in cell growth and tumorigenicity. These results reinforce the potential role of anti-oncogene ribozymes as suppressors of neoplastic growth, with possible implications for gene therapy.

Detection of drug resistance in human tumors by in vitro enzymatic amplification.

Both acquired and natural resistance to chemotherapy agents have proved problematic in the treatment of neoplasia. Thymidylate synthase, which catalyzes the synthesis of thymidine precursors, has been shown to be amplified in response to a variety of chemotherapeutic agents. The detection of such amplification could prove beneficial in the development of alternative clinical protocols. In this study we report the use of existing enzymatic amplification methods in order to detect incipient amplification of the thymidylate synthase gene upon resistance to cisplatin. The assay utilizes a modification of the polymerase chain reaction in which a sequence of the thymidylate synthase gene is amplified including two flanking oligonucleotides acting as primers for DNA synthesis. This method exhibits greater sensitivity than conventional nucleic acid detection methods and requires less than 100 ng of total RNA from patient tumors and no in vitro culturing of patient cells.

Gene amplifications characterize acral melanoma and permit the detection of occult tumor cells in the surrounding skin.

Acral melanoma (AM) is commonly distinguished from superficial spreading melanoma (SSM), the most common type of melanoma, by its clinical presentation as well as its ethnic distribution. However, justification for such a distinction is controversial because of histological overlap and lack of prognostic significance. We analyzed chromosomal aberrations of 15 AMs and 15 SSMs that were comparable for tumor thickness and patient age, using comparative genomic hybridization. All AMs had at least one (mean, 2.0) gene amplification, significantly more than the SSMs, in which only 2 of 15 (13%) had one amplification each (P < 0.0001). At least 15 different genomic regions were amplified in AM. These involved small portions of chromosomal arms, sometimes including known oncogenes implicated in melanoma. The most frequently amplified regions in AMs occurred at 11q13 (47%), 22q11-13 (40%), and 5p15 (20%). Comparison of the amplification levels of invasive and noninvasive portions of the tumors using fluorescence in situ hybridization suggested that amplifications occurred before the formation of the invasive portion. The finding of amplifications of 11q13 in three of five additional cases of AM in situ further supports the notion that amplifications arise early in the progression of AM. Very significantly, we found isolated melanocytes with amplifications in the epidermis up to 3 mm beyond the histologically recognizable extent of the melanomas in 5 of 15 invasive AMs. In conclusion, our data show that AM is a distinct type of melanoma characterized by focused gene amplifications occurring early in tumorigenesis, and that malignant cells are present beyond the histologically detectable boundary, thereby revealing one mechanism of local recurrence.

Cisplatin resistance in human cancers.

Cancer chemotherapeutic agents primarily act by damaging cellular DNA directly or indirectly. Tumor cells, in contrast to normal cells, respond to cisplatin with transient gene expression to protect and/or repair their chromosomes. Repeated cisplatin treatments results in a stable resistant cell line with enhanced gene expression but lacking gene amplification for the proteins that will limit cisplatin cytotoxicity. Recently, several new human cell lines have been characterized for cisplatin resistance. These cell lines have led to a better understanding of the molecular and biochemical basis of cisplatin resistance. The c-fos proto-oncogene, a master switch for turning on other genes in response to a wide range of stimuli, has been shown to play an important role in cisplatin resistance both in vitro and in patients. Based on these studies, new strategies have been developed to circumvent and/or exploit clinical cisplatin resistance.

Therapeutic applications of ribozymes.

The demonstration that RNA can be cleaved by cis or trans ribozymes (catalytic RNAs, RNA enzymes) has potentially important therapeutic implications. Since their discovery in the 1980s, the biochemistry and conserved sequences of ribozymes have been well characterized. Ribozymes are effective modulators of gene expression because of their simple structure, sitespecific cleavage activity, and catalytic potential. The targets of ribozyme-mediated gene modulation have ranged from cancer cells to foreign genes that cause infectious diseases. Additional target sites for ribozymes are in initial phases of development and design. Ribozymes have been targeted against a myriad of genes, including oncogenes (ras, BCR-ABL, c-fos) and drug resistance genes, as well as the human immunodeficiency virus-type I genome. These ribozymes have cleaved the target RNAs in vitro and altered the cellular pathology. Currently, the therapeutic application of ribozymes to human diseases is limited by gene transfer systems. It is anticipated that ribozymes ultimately will play an important role in human gene therapy.

Suppression of the malignant phenotype of melanoma cells by anti-oncogene ribozymes.

The activation of signal transduction pathways by mutation or overexpression of cellular oncogenes has been associated with neoplastic transformation. In this study, we addressed the therapeutic potential of ribozymes targeted against the activated H-ras oncogene as well as against the nuclear proto-oncogenes c-fos and c-myc in the FEM human melanoma cell line containing a H-ras mutation. FEM cells transfected with the anti-ras ribozyme were shown to have the longest doubling time, the least DNA synthesis, and the fewest colonies in soft agar when compared with transfectants with ribozymes against c-fos or c-myc mRNA. Furthermore, anti-ras ribozyme clones showed a dendritic appearance in monolayer culture that was associated with enhanced melanin synthesis. These results suggest that the anti-ras ribozyme could affect not only the proliferation but also the differentiation process of human melanoma cells in vitro. They also reinforce the role of anti-oncogene ribozymes as suppressors of the neoplastic phenotype of melanoma cells.

Topical gene delivery to murine skin.

We topically applied naked plasmid DNA containing the luciferase or chloramphenicol acetyltransferase cDNA directly to mouse skin. Gene expression was detected in skin samples as early as 4 h after DNA application, plateaued from 16 to 72 h post-application, and had decreased significantly by 7 d post-application. Reporter gene activity following topical DNA delivery was comparable with that produced by intradermal injection of DNA. Plasmid DNA at concentrations > or =0.25 microg per microl were required to achieve maximal expression levels. Reporter gene expression following topical administration was largely confined to the superficial layers of the epidermis and to hair follicles. Surprisingly, certain cationic liposomes inhibited the efficiency of cutaneous gene transfer. This technique provides a simple, clinically relevant approach to deliver genes to the skin, with potential application in treating a variety of cutaneous disorders.

Efficient gene expression in skin wound sites following local plasmid injection.

Transfection of the skin by local gene delivery, as well as widespread transfection of systemic tissues following intravenous injection of cationic liposome/DNA complexes have been reported. Here, we show that surgically wounded mouse skin can be transfected either by local injection of DNA alone or by intravenous injection of optimized cationic liposome/DNA complexes; however, direct cutaneous injection produces much higher levels of gene expression in the skin, which is targeted to dermal and subdermal layers. High levels of chloramphenicol acetyltransferase activity were present from 3 h to 2 wk following direct injection of a gene expression plasmid into wounded skin and were maintained at detectable levels up to 8 wk after injection. Expression of transferred chloramphenicol acetyltransferase as well as beta-GAL genes was localized to fibroblasts, macrophages, and adipocytes as determined by histochemistry and immunohistochemistry. Further- more, local injection of a human granulocyte- colony-stimulating factor gene expression plasmid produced high levels of the biologically relevant human granulocyte-colony-stimulating factor protein in wounded mouse skin. This efficient and simple method of site-specific gene transfer into wounds may lead to the development of cutaneous gene therapy directed against disorders of abnormal cutaneous wound healing.

Differential oncogene amplification in tumor cells from a patient treated with cisplatin and 5-fluorouracil.

Peritoneal cells were derived from a patient (PK) with adenocarcinoma of the colon during the course of cisplatin/5-fluorouracil (5-FUra) treatment. Resistance to cisplatin and 5-FUra, characterized by a lack of response to chemotherapy and continued growth of the tumor, was concomitantly associated with a 2-4-fold increase in DNA copy number for dTMP synthase and dihydrofolate reductase. There was a corresponding amplification in DNA copy number of the c-myc (2X), H-ras (4X), and c-fos (15X) oncogenes. Cytogenetic studies revealed an iso (13q) chromosome, but failed to show any double minutes or homogeneously staining regions. In addition, drug-resistant tumor cells from PK and another patient (HG) displayed enhanced expression of dTMP synthase, c-fos and DNA polymerase beta when compared to normal colon tissue and the HCT8 human colon carcinoma cell line. These results suggest that elevated oncogene DNA and gene expression may be involved in the development of cisplatin resistance.

Mechanisms of cross-resistance to methotrexate and 5-fluorouracil in an A2780 human ovarian carcinoma cell subline resistant to cisplatin.

Some properties of the human ovarian carcinoma line A2780 and a subline three times more resistant than the parent line to cisplatin are compared in this report. The rates of uptake and release of cisplatin were similar in the two cell lines. Resistance to cisplatin was associated with: (a) cross-resistance to 5-fluorouracil and methotrexate; (b) a 2.5-fold increase in thymidylate synthase, as measured by both enzyme activity and the capacity to complex 5-fluorodeoxyuridylate; and (c) an increase in the intracellular pools of 5,10-methylenetetrahydrofolate and tetrahydrofolate. These data suggest that cross-resistance to 5-fluorouracil and methotrexate in A2780 cells may be a consequence of increases in their respective target enzymes.

Suppression of H-ras-mediated transformation in NIH3T3 cells by a ras ribozyme.

Murine NIH3T3 cells were used to study the effect of ribozymes on H-ras-mediated transformation. Parental 3T3 cells were transfected with the activated H-ras gene. H-ras-transformed cells had altered morphology and increased colony formation in soft agar in contrast to untransfected 3T3 cells. A hammerhead ribozyme (site-specific ribonuclease) designed to cleave codon 12 (GUC) of the activated H-ras RNA was expressed in transformed cells. 3T3 clones expressing the ras ribozyme displayed decreased expression of activated H-ras RNA. The ras ribozyme reversed the transformed phenotype to resemble that of untransfected 3T3 cells. Furthermore, 3T3 cells containing the ras ribozyme were shown to suppress transformation when they were subsequently transfected with activated H-ras. Insertion of a mutant ribozyme largely devoid of cleaving capacity into H-ras-transformed cells resulted in smaller reductions in H-ras gene expression and colony formation in soft agar when compared with the ras ribozyme. Finally, the ras ribozyme alone did not perturb normal 3T3 cell growth. This study suggests the possible utility of anti-oncogene ribozymes as suppressors of tumor cell growth as well as inhibitors of cellular transformation.

Optimal selective sentinel lymph node dissection in primary malignant melanoma.

OBJECTIVE: To determine the optimal approach of selective sentinel lymph node (SLN) dissection in primary malignant melanoma. DESIGN: Consecutive patient study. Prior to selective SLN dissection and wide local excision of the primary melanoma biopsy site, technetium Tc 99m sulfur colloid was injected intradermally around the primary melanoma or biopsy site to mark the SLN. Isosulfan blue (Lymphazurin, Hirsch Industries Inc, Richmond, Va) was injected at the primary biopsy site immediately before the surgical procedure. SETTING: Teaching hospital tertiary care referral center. MAIN OUTCOME MEASURES: Successful identification of SLNs being defined as positive for microscopic metastatic melanoma by blue dye staining, radioisotope uptake, or both. RESULTS: Selective intraoperative mapping by gamma probe and visualization of blue dye-stained SLN(s) resulted in a 98% (160/163) successful identification rate. Thirty patients (18.4%) had microscopic metastatic melanoma of the SLN(s), 22 of whom had subsequently completed lymphadenectomy. In 4 (18.2%) of these 22 patients, further microscopic metastatic disease was found in 1 of 8 nodes, 1 of 8 nodes, 1 of 28 nodes, and 1 of 9 nodes. No notable complications were encountered. Five recurrent cases from patients with SLNs without microscopic metastatic melanoma (3.8%) and 2 from patients with SLNs with microscopic metastatic melanoma (6%) were found during a median follow-up period of 463 days. A second primary melanoma developed in 2 patients; neither had no local recurrence. CONCLUSIONS: Sequential combination of preoperative lymphoscintigraphy and intraoperative mapping is a reliable way to identify regional SLN. The frequency of microscopic metastatic melanoma of the SLN(s) is 18.4%. Gamma-probe--guided resection minimizes the extent of lymph node dissection. Further follow-up is needed to assess the outcome of this group of patients for regional and systemic recurrences.

The role of methionine in methotrexate-sensitive and methotrexate-resistant mouse leukemia L1210 cells.

A mouse L1210 leukemia cell line was made 25-fold resistant to methotrexate (MTX) and had altered methionine transport and metabolism. L1210 cells resistant to methotrexate also had a 50-fold decrease in the exogenous methionine requirement for optimal cell growth compared to the parent cells. This change in methionine requirement was associated with differences in methionine metabolism between MTX-sensitive and MTX-resistant cell lines. Analysis of amino acid transport systems revealed different Kt and Vmax properties of methionine and nonmetbolizable amino acid analogues. There was a greater than twofold decrease in the initial sodium-dependent uptake of methionine in the resistant cells. Amino acid competition experiments revealed altered substrate specificities in the resistant cells. The cellular alterations occurring upon resistance may result from methotrexate-membrane interactions, and have been previously observed in cisplatinum-resistant cells. Thus modulation of methionine metabolism may provide the biochemical basis for MTX and cisplatinum collateral resistance.

Inhibition of methionine uptake by methotrexate in mouse leukemia L1210 cells.

Methionine-auxotrophic L1210 cells were used to study the effect of methotrexate (MTX) on methionine uptake and metabolism. MTX was shown to inhibit amino acid transport systems and cause a decrease of methionine uptake into L1210 cells. Conversely, a nonmetabolizable amino acid analogue reduced MTX uptake into L1210 cells. MTX also blocked the transfer of the beta carbon from serine into methionine. Therefore, methionine deprivation may be an additional mechanism of action for MTX in methionine-auxotrophic tumor cells.

The utility of an anti-fos ribozyme in reversing cisplatin resistance in human carcinomas.

The results presented here demonstrate that expression of a fos ribozyme limits Fos protein synthesis and enhances sensitivity of A2780DDP cells to antineoplastic agents, including cisplatin. Moreover, the reversal of this resistance is associated with down-regulation of dTMP synthase, DNA polymerase beta, topoisomerase I and hMTII-A, genes previously linked to DNA synthesis and repair. Thus these studies further implicate the role of the c-fos gene in DNA synthesis through modulation of expression of dTMP syntase, DNA polymerase beta and topoisomerase I. Finally, the use of ribozymes to circumvent drug resistance suggests their potential utility as agents to inhibit tumor cell growth.

Topical corticosteroids for mycosis fungoides. Experience in 79 patients.

OBJECTIVE: To determine the effectiveness of topical corticosteroids in the management of mycosis fungoides. DESIGN: Prospective study. SETTING: Academic referral center, Veterans Affairs Medical Center, and private practice. PATIENTS: Seventy-nine patients with patch or plaque stage of mycosis fungoides. Fifty-one were stage T1 (less than 10% of skin involved) and 28 were stage T2 (10% or more of skin involved). Seventy-five had patch-stage and 4 had plaque-stage disease as determined by histological examination. INTERVENTION: Patients were treated with topical class I to III corticosteroids. Of the stage T1 patients, all used class I corticosteroids, and 4 (8%) also used class II or III corticosteroids. Of the stage T2 patients, 19 (68%) used class I and 12 (43%) used class II or III compounds. Some patients used more than 1 class of corticosteroid. Applications were almost always twice daily. Three stage T1 and 2 stage T2 patients used plastic film occlusion. Baseline and monthly morning serum cortisol levels were obtained during treatment. MAIN OUTCOME MEASURES: Response to treatment and side effects. RESULTS: The median follow-up period was 9 months. Thirty-two (63%) of stage T1 patients achieved complete remission and 16 (31%) achieved partial remission, for a total response rate of 48 (94%). The comparable figures for stage T2 patients were 7 (25%), 16 (57%), and 23 (82%), respectively. Responses were determined by clinical examination. Thirty-nine patients achieved clinical clearing. In 7 of these, posttreatment biopsy specimens were obtained, and all showed histological clearing. Reversible depression of serum cortisol levels occurred in 10 (13%). Minor skin irritation occurred in 2 patients and localized, reversible skin atrophy in 1. CONCLUSION: Topical corticosteroids, especially class I compounds, are an effective treatment for patch-stage mycosis fungoides.

Vascular involvement in the prognosis of primary cutaneous melanoma.

OBJECTIVE: To examine the role of vascular invasion as a prognostic factor in melanoma. DESIGN: Retrospective survival analysis. SETTING: Academic medical center. PATIENTS: A total of 526 patients with primary cutaneous melanoma from the University of California, San Francisco, Melanoma Center database with 2 years of follow-up or documented relapse. MAIN OUTCOME MEASURES: (1) Presence of vascular involvement defined as vascular invasion with tumor cells within blood or lymphatic vessels; or uncertain vascular invasion, with melanoma cells immediately adjacent to the endothelium. (2) Percentage with metastasis or death and relapse-free and overall survival. RESULTS: The presence of either type of vascular involvement significantly increased the risk of relapse and death and reduced the survival associated with melanoma. The impact of vascular involvement on these outcomes was similar to that of ulceration. In a multivariate analysis, vascular involvement was the second most important factor (after tumor thickness) in the primary tumor in predicting survival. CONCLUSIONS: Vascular involvement is an important independent predictor of metastasis and survival in melanoma. The phenomenon of uncertain vascular invasion describes an earlier step than definite vascular invasion in tumor progression.