Alterations in the p53 pathway and p16INK4a expression predict overall survival in metastatic melanoma patients treated with dacarbazine.


TO THE EDITOR Malignant melanoma is one of the least chemosensitive human cancers. The cause of drug resistance in melanoma remains poorly understood. So far, no cytotoxic regimen has revealed superiority compared to dacarbazine monotherapy (Eggermont and Kirkwood, 2004). Although mutations inactivating the TP53 gene (encoding the p53 protein) are rare events in melanoma (Hartmann et al., 1996), p53 may be inactivated through other mechanisms. The murine double-minute oncogene (MDM2) binds to and ubiquitinates p53 (Piette et al., 1997), and MDM2 amplification has been found responsible for p53 inactivation in some malignancies (Momand et al., 1998). Recently, an MDM2 promoter polymorphism (SNP309T4G) increasing MDM2 expression through enhanced binding of the Sp1 transcriptional activator was shown to enhance carcinogenesis (Bond et al., 2004). p14ARF activates p53 by inhibiting MDM2 binding (Figure 1a); thus, loss of p14ARF may reduce p53 function (Efeyan and Serrano, 2007). Although p14ARF in general is activated in response to oncogenic stimulation, there is evidence that p14ARF may also be involved in response to genotoxic stress (Christophorou et al., 2006). p14ARF is encoded by ‘‘the alternative reading frame’’ of the CDKN2A gene. Although most CDKN2A mutations identified in melanoma affect the p16INK4a transcript only (Grafstrom et al., 2005; Knappskog et al., 2006; Goldstein et al., 2007), mutations affecting the 50 half of exon 2 may affect both transcripts. Here, we analyzed pretreatment tumor biopsies from 85 patients receiving dacarbazine for advanced malignant melanoma (Busch et al., unpublished data) for potential disturbances in p53, MDM2, p14ARF, or p16INK4a. Pretreatment biopsies from all patients were snap frozen in liquid nitrogen. Each patient provided written informed consent, and the study protocol was approved by the regional ethical committee. The study was conducted according to the Declaration of Helsinki Principles. Of the 85 patients, 75 were evaluable for clinical response according to the UICC-criteria (Busch et al., unpublished data). RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA), and cDNA was synthesized with reverse transcriptase (Roche Diagnostics, Basel, Switzerland). The tumors were screened for TP53 and CDKN2a mutations and MDM2 promoter status by PCR amplification and subsequent sequencing. In addition, intragenetic deletions or amplifications in CDKN2A or MDM2 were screened for using MLPA (MRC Holland, Amsterdam, the Netherlands; Schouten et al., 2002). mRNA expression analysis of p16INK4a and BMI-1 was performed in a duplex qRT-PCR system (LightCycler 480; Roche Diagnostics) using TaqMan probes and b-2-microglobulin as an internal reference. Dilutions of PCR products of these genes were included in each run to make a standard curve for calculation of relative concentrations. We considered the ‘‘p53 pathway’’ to be disturbed in tumors harboring one of the following events: TP53 or p14ARF mutations causing amino-acid changes, biallelic p14ARF deletions, MDM2 amplification, and/or homozygosity for the SNP309G haplotype. To evaluate the impact of p16INK4a expression levels, we compared tumors expressing p16INK4a above or below median of the analyzed samples. Potential correlations between molecular parameters and clinical outcome were tested using the Fisher’s exact test comparing objective responders (complete response or partial response) to nonresponders (progressive disease or stable disease) or stable disease þ complete response/partial response versus progressive disease. Univariate analyses were performed analyzing different factors impact on time to progression and overall survival (OS) using log-rank test. Variables with P-value o0.10 were entered into Abbreviations: BMI-1, polycomb ring finger oncogene; MDM2, mouse double-minute homolog; OS, overall survival

DOI: 10.1038/jid.2010.138

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@article{Busch2010AlterationsIT, title={Alterations in the p53 pathway and p16INK4a expression predict overall survival in metastatic melanoma patients treated with dacarbazine.}, author={Christian Busch and J{\"{u}rgen Geisler and Stian Knappskog and Johan Richard Lillehaug and Per Eystein L\onning}, journal={The Journal of investigative dermatology}, year={2010}, volume={130 10}, pages={2514-6} }