PMC:2948432 / 1796-8736 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2948432","sourcedb":"PMC","sourceid":"2948432","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2948432","text":"INTRODUCTION\nThe AJCC staging system for melanoma utilizes the Breslow thickness of primary melanoma, the presence of ulceration, and staging by sentinel node biopsy to give the best estimate of prognosis (Balch et al.,2001). Use of the staging system is associated with a range of estimates of outcome (Balch et al.,2001; Gimotty et al.,2005), so that a significant proportion of the variance in survival remains unexplained. Therefore, it is important to seek molecular markers of metastatic potential for use in clinical practice. In recent years, progress has taken place in understanding the somatic events that occur in melanoma primary tumors. However, there is comparatively little known of the correlation between these events and outcome, and it is therefore important to increase our understanding of the carcinogenetic process so that logical approaches to treatment can be developed.\nA number of signal transduction and cell cycle regulatory pathways have been implicated in the etiology and progression of melanoma, including the retinoblastoma (RB1) and p44/42 mitogen-activated protein kinase (MAPK) pathways. A key region is 9p21.3, which contains the CDKN2A and CDKN2B genes. These genes encode three separate tumor suppressor proteins. CDKN2A encodes both CDKN2A and, using a separate first exon and alternate reading frame, P14ARF. Although both transcripts use exons 2 and 3 of CDKN2A, the CDKN2A and P14ARF proteins share no homology at the amino acid level and have distinct tumor suppressor functions in the RB1 and TP53 pathways; CDKN2B encodes CDKN2B, which has its own open reading frame (Sharpless and DePinho,1999; Weber et al.,1999). The CDKN2A protein controls passage through the G1 checkpoint of the cell cycle by inhibiting the phosphorylation of the RB1 protein (Roussel,1999) and of the three tumor suppressors at this locus is the one longest recognized to have a significant role in melanoma. It is known to play a key role in normal melanocyte senescence (Ha et al.,2008). The P14ARF protein acts on the TP53 cell cycle control pathway by interaction with the human double minute (HDM2) protein to stabilize TP53 and allow cell cycle arrest at the G1/G2 phase (Weber et al.,1999). CDKN2A was identified first as a tumor suppressor gene commonly deleted/mutated in tumor cell lines (Kamb et al.,1994a) and subsequently its role as a high-risk susceptibility gene in melanoma families was elucidated (Kamb et al.,1994b). Germline mutations have been identified in ∼20% of tested melanoma families (Goldstein and Tucker,2001; Bishop et al.,2002). Some mutations impact on CDKN2A protein alone, some on P14ARF, and some on both proteins.\nMost melanoma cell lines show deletion/mutation of CDKN2A (Flores et al.,1996; Walker et al.,1998). The majority of primary tumors have allelic loss at microsatellite markers mapping to the CDKN2A locus, indicating that deletions are the principal genetic event in vivo (Flores et al.,1996; Rodolfo et al.,2004). More recent reports showed biallelic deletion in ∼45% of melanoma metastases, supporting the role of this locus in melanoma progression (Grafstrom et al.,2005) and we have shown that epigenetic silencing of P14ARF is also common in metastatic disease (Freedberg et al.,2008). There are few data from primary tumors on the role of deletion at the locus on outcome (Koynova et al.,2007) and none on the effect of deletion across the larger region, which we have addressed using Multiplex ligation-dependent probe amplification (MLPA) (Nygren et al.,2005) rather than by studying the small intragenic regions previously reported.\nMore recently there has been some suggestion that CDKN2B may also have tumor suppressor functions in melanoma. Krimpenfort et al. (2007) showed that mice null for CDKN2B, CDKN2A, and P14ARF were more tumor prone than CDKN2A/P14ARF null mice and developed a predominance of skin tumors (Krimpenfort et al.,2007). The authors suggested that CDKN2B might act as critical “back up” tumor suppressor in cells null for CDKN2A.\nActivating mutations of the NRAS and BRAF genes occur in ∼20 and 50% of malignant melanomas respectively, and are almost always mutually exclusive (Omholt et al.,2003; Garnett and Marais,2004). BRAF and NRAS mutations have also been found in benign nevi (Poynter et al.,2006) and are therefore thought to be involved early in melanoma carcinogenesis. In cultured human melanocytes, mutant BRAF protein has been shown to induce cell senescence by increasing the expression of CDKN2A (Michaloglou et al.,2005). It is postulated, therefore, that to become an invasive melanoma, arrest of the cell cycle caused by normal CDKN2A must subsequently be overcome by mutation or deletion of CDKN2A or by alterations to other cell cycle regulators. Moreover, a recent in vitro study showed that simultaneous knockdown of BRAF and expression of CDKN2A in melanoma cells led to potent growth inhibition and apoptosis, whereas knockdown of BRAF or expression of CDKN2A alone did not (Zhao et al.,2008). Studies of BRAF mutated nevi using the senescence marker SA-β-gal, however, revealed a marked mosaic induction of CDKN2A, which the authors suggested was indicative of a role for multiple tumor suppressors in the prevention of BRAF oncogenesis (Michaloglou et al.,2005).\nIn this study, we have investigated the gene dosage of multiple tumor suppressors at 9p21 in formalin-fixed, paraffin-embedded (FFPE) primary melanoma tumors. Furthermore, we have investigated the relationship between reduced gene dosage at the CDKN2A locus and BRAF/NRAS mutations using tumors from patients who have relapsed and from patients with similar tumors who have not relapsed, to determine the prognostic value of these events. The presence of ulceration is an important prognostic factor for melanoma even in stage III or metastatic disease (Balch et al.,2001) and, therefore, we also assessed the associations between CDKN2A deletion and BRAF/NRAS mutation and ulceration. In other series, mitotic rate has important prognostic significance (Elder and Murphy,2008), and therefore, we also examined genetic markers in relation to mitotic rate. CDKN2A is a cyclin D kinase (CDK) inhibitor and therefore loss of CDKN2A would likely be related to increased mitotic rate.\nThe methylthioadenosine phosphorylase (MTAP) gene is also located at 9p. It has been suggested that loss of expression of the gene has prognostic implications for melanoma (Behrmann et al.,2003) and codeletion of MTAP with CDKN2A has been investigated in a number of cancers (Chen et al.,1996). There is evidence that loss of MTAP results in an inhibition of STAT signalling pathways regulated by interferon, so it is of interest that response of melanoma patients to interferon used as an adjuvant therapy for this cancer has been reported to be related to MTAP status (Wild et al.,2007). We were able in this study to look at deletion of MTAP in primary 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