Melanoma Biomarkershow To Find Them

Sample-based screening of melanoma tumors for desired markers can be performed with traditional immunohisto-chemistry (IHC) or with more sophisticated gene arrays, TMAs, or tumor protein lysate array.

Melanocytes and melanoma cells in vitro and in tumor samples from different stages of melanoma progression have been studied widely by immunohistochemistry to identify specific proteins to be used as biomarkers for prognosis. Markers which have shown prognostic value in restricted patient groups include S-100B, melanoma inhibitory activity protein (MIA), tyrosinase, certain matrix metalloproteinases, integrins, interleukin-8, and CD 44 (Ref. [8] and references therein). Some of these markers can also be measured in patient sera. The increased expression levels or bioactivity of these markers is usually associated with melanoma progression, recurrence, or poorer survival. However, the clinical use of these markers is limited to oncology departments with research activity.

In microarray studies thousands of marker genes can be simultaneously studied to diagnose melanoma, to classify patients into different prognostic subgroups, and to identify new targets for therapy.[9,10] DNA microarrays are based on high-density oligonucleotide or cDNA microarrays, which measure in parallel thousands of gene-specific mRNAs in a single RNA sample.[1] This kind of large-scale gene expression analysis has proved to be a valid strategy for developing sample-specific gene expression profiles.[1] DNA microarrays have potential application in clinical practice, but their application is limited by the requirement of fresh or frozen tissue for analysis (Fig. 1).

DNA microarrays determine the levels of target gene mRNA in studied tumors, but do not give information on gene function. Study of protein complement of the genome by functional proteomics can be used to screen for alterations in protein expression and posttranslational modifications under melanoma progression.[1]

Tissue microarrays are arrays in which hundreds of small core (0.6-2.0 mm) tissue sections are arrayed on a glass slide.[11] Tissue microarrays give a good impression of the whole tumor area and they can be used for IHC and in situ hybridization. Tissue microarrays are useful in screening of a desired marker rapidly in a large patient group.[11]

Tumor protein lysate array by proteomics is a new technique based on detection of a particular protein expression in patient samples. This array also requires frozen or fresh tissue samples.

■ " Primary diagnosis ZLl.rlJ Diagnosis of rare subgroups Prognosis

Requirement for adjuvant therapy Tissue-specific site of metastasis Therapy response Survival?

Prognosis Therapy response Drug discovery

Fig. 1 Putative clinical value of DNA or tissue microarrays of primary or metastatic melanoma specimen.

Molecular Profiling of Cutaneous Melanoma

Molecular profiling of melanomas by gene expression analysis has emerged as a new possibility to predict melanoma prognosis and treatment planning. Detailed genetic profiles found by microarray analyses are available on the internet (http://www.nhgri.nih.gov). Bittner et al.[9] compared the tumor cell mRNA with a single reference probe, providing normalized measures of the expression of each gene in each sample relative to the standard. They found several genes whose expression levels were altered giving a possibility to discriminate studied genes into different clusters.[9] Interestingly, the melanoma cells studied belonging to different groups also had different biological properties, i.e., their ability to migrate, form tubular networks, and contract collagen gels in vitro was different.[9] It has become evident that there are genes whose expression levels are elevated in aggressive melanoma cell lines or tumors, as well as genes whose expression levels are downregulated. The expression of several extracellular matrix components, such as fibronectin, g2 chain of laminin 5, and a2 chain of collagen IV, is upregulated in aggressive melanoma cells,[9] suggesting that invasive tumors have increased capacity to regulate cytoskeletal organization, cell movement, and invasion by modifying the composition of the surrounding extracellular matrix. Similarly, the expression level of Wnt5A, a gene involved in melanoma cell motility and invasion, shows correlation with tumor grade while inversely correlating with survival.[12] In addition, several studies have shown upregulation of invasion-promoting matrix-degrading proteinases, such as cathepsin Z.[12] Melanocyte-specific markers, such as tyrosinase and melan-A, are downregulated suggesting that highly aggressive tumor cells undergo dedifferentiation into less melanocyte-like cells. Several markers of angiogenesis, such as endothelial protein receptor TIE-1, vascular endothelial growth factor-C (VEGF-C), and vascular endothelial cadherin (VE-cadherin) have been found to be overexpressed in melanomas.[10] The expression of these genes is related to a phenomenon called vascular mimicry, in which highly aggressive tumor cells are able to undergo a genetic reversion to a pluripotent, more embryonic-like phenotype, which may lead to the formation of new vessels by tumor cells themselves. Neovascularization by tumor cells may explain the aggressiveness of metastatic melanoma and provide a basis for therapy targeting angiogenic molecules and signal transduction pathways.

The possible clinical use of these arrays in diagnosis, prognosis, and treatment planning is further discussed by describing recent findings in in vitro models as well as in patient-derived sample analysis.

■ " Primary diagnosis ZLl.rlJ Diagnosis of rare subgroups Prognosis

Requirement for adjuvant therapy Tissue-specific site of metastasis Therapy response Survival?

Prognosis Therapy response Drug discovery

Molecular Profiling of Sentinel Nodes

The importance of sentinel node evaluation and detection of micrometastases in lymph nodes has been shown in a systematic evaluation of 17,600 patients with cutaneous melanoma.[13] Patients with detected micrometastases have less favorable prognosis than those whose nodes have been found to be unaffected.[13] However, this implication may change to a more detailed, molecular analysis of dissected lymph nodes.[14] Kuo et al.[14] have shown the feasibility of PCR-based molecular multi-marker analysis of sentinel lymph nodes in the detection and prognostication of recurrence in patients with early-stage melanoma. The PCR-based multimarker analysis might even detect single metastatic melanoma cells in sentinel lymph nodes thought to be normal in microscopic IHC analysis.

CLINICAL IMPLICATIONS Diagnosis

Detailed analysis of the expression patterns of thousands of genes simultaneously has made it possible to identify novel disease genes, such as Wnt5A, RhoC, and BRAF, for cutaneous melanoma.[12] De Wit et al.[15] have been able to identify new melanoma-specific antigens, such as MMA-1a and MMA-1b, by using oligonucleotide array-based analysis. In addition to diagnosis, gene expression profiling can be used in tumor classification. On the basis of melanoma-specific biomarker listing it would be possible to molecularly distinguish atypical melanomas from nonmelanoma skin cancers.

Gene array and cluster analysis might also help in the characterization of rare melanoma subgroups. Tschentscher and coworkers1-16-1 have found new subtypes of uveal melanoma, and Segal et al.[17] have shown that clear-cell sarcoma is a distinct subtype of cutaneous melanoma.

Prognosis

Classification of tumors with similar histology into different prognostic subgroups is a clinically valuable benefit of DNA arrays. In a pronounced work by Clark et al.,[18] specific genes, such as RhoC, responsible for melanoma progression were identified. Alonso et al.[19] were able to identify distinct gene expression profiles distinguishing specific melanoma progression stages. Genes whose expression was reduced in advanced melanoma stages included p16, p27, and cyclin D, suggesting that losing cell-cycle control is essential in melanoma progression.1-19-1 Previous work with primary breast cancer and its node-positive counterparts has suggested that gene expression pattern does not differ much between primary tumor and its node metastasis.[20-This may also be true in melanoma, as at least early mutation status of NRAS and BRAF is maintained from dysplastic nevi to primary melanomas and their metasta-ses.[5- Thus detection of upregulation of genes responsible for dissemination in primary tumors would be clinically valuable as prognostic factor. Furthermore, DNA micro-array analyses performed in murine melanoma models have even defined genes which are consistently elevated in pulmonary metastases,1-21-1 suggesting that predictable, organ-specific metastasis gene expression profiles could be found and used in further patient surveillance. Similar hypothesis has been presented concerning breast cancer, where new findings suggest that primary tumors with metastatic capacity possess the signature for poor prognosis, but subpopulations of cells also display a tissue-specific expression profile predicting the site of metastasis.[22- According to these findings, the prevailing model of metastasis, where it is suggested to be a late and rare event in tumorigenesis, may change.

Direct analysis of melanocytic lesions by tissue microarrays has also helped in finding molecules which predict melanoma progression. Shen et al.[23] analyzed several benign and dysplastic nevi as well as different growth-phase melanomas to show that particular growth factor receptor tyrosine kinases are differently expressed in distinct stages of tumor development. They found that nearly 100% of dysplastic nevi and vertical growth-phase melanomas studied express c-kit and platelet-derived growth factor receptor p (PDGFR-p). Interestingly, metastases seemed to be associated with loss of c-kit and PDGFR-p expression, suggesting that patients with an earlier stage of disease might benefit from imatinib, a PTK inhibitor targeted toward these receptors.

Treatment

Different adjuvant treatment modalities, i.e., interferon-a (IFN-a) and vaccine therapy, have been extensively studied among melanoma patients. It is not known whether melanoma patients with high metastasis risk gain survival benefit from adjuvant therapy. Interferon-a is the most studied adjuvant therapy agent, although its routine use cannot be recommended in high-risk primary melanoma because of lack of phase III studies with evidence for survival benefit. Interferon-a exerts its effects through antiproliferative, apoptosis-inducing, and antiangiogenic effects in addition to immunological modulation. Certa et al.[24] have found differently regulated IFN-a responsive gene groups in IFN-a-

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