AIM1 and LINE-1 Epigenetic Aberrations in Tumor and Serum Relate to Melanoma Progression and Disease Outcome Sojun Hoshimoto, Christine T. Kuo, Kelly.

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AIM1 and LINE-1 Epigenetic Aberrations in Tumor and Serum Relate to Melanoma Progression and Disease Outcome Sojun Hoshimoto, Christine T. Kuo, Kelly K. Chong, Teh-Ling Takeshima, Yoshiki Takei, Michelle W. Li, Sharon K. Huang, Myung-Shin Sim, Donald L. Morton, Dave S.B. Hoon Journal of Investigative Dermatology Volume 132, Issue 6, Pages 1689-1697 (June 2012) DOI: 10.1038/jid.2012.36 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 LINE-1 hypomethylation in paraffin-embedded archival tissues. Individual and mean (solid horizontal line) long interspersed nucleotide element-1 (LINE-1) U-Index values for tissue specimens of (a) normal skin versus primary/metastatic melanoma, (b) normal skin versus primary melanoma versus metastatic melanoma and according to the (c) American Joint Committee on Cancer (AJCC) stage and (d) primary and metastatic paired samples. P-values were obtained by (a, d) Student's t-test, (b) Tukey's HSD (honest significance test), and (c) analysis of variance (ANOVA). NS, not significant; U, unmethylated. Journal of Investigative Dermatology 2012 132, 1689-1697DOI: (10.1038/jid.2012.36) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 AIM1 (absent in melanoma-1) promoter methylation and gene expression. (a) CpG methylation in the promoter region of AIM1 gene as assessed by PCR of three amplicons (A, B, and C) covering over 1,000bp of the promoter region. (b) AIM1 mRNA expression in melanoma cell lines. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBL, peripheral blood lymphocyte. (c) Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MassARRAY analysis of specific CpG sites as target region of methylation-specific PCR (MSP) primers for AIM1. Journal of Investigative Dermatology 2012 132, 1689-1697DOI: (10.1038/jid.2012.36) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Kaplan–Meier survival curves for melanoma patients using a combination of long interspersed nucleotide element-1 (LINE-1) and absent in melanoma-1 (AIM1) methylation status in paraffin-embedded archival tissue (PEAT). (a) Overall survival (OS) of patients with LINE-1 hypomethylation and/or AIM1 hypermethylation versus LINE-1 hypermethylation and AIM1 hypomethylation. M, hypermethylated; U, hypomethylated. (b) Disease-free survival (DFS) of stage I and II melanoma patients with LINE-1 hypomethylation and/or AIM1 hypermethylation versus LINE-1 hypermethylation and AIM1 hypomethylation. Journal of Investigative Dermatology 2012 132, 1689-1697DOI: (10.1038/jid.2012.36) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Kaplan–Meier survival curves for stage IV patients in serum studies. (a) Overall survival (OS) of stage IV patients with methylated- versus unmethylated-AIM1 (absent in melanoma-1) circulating cell-free DNA (cf-cDNA). (b) OS of stage IV patients with LINE-1 (long interspersed nucleotide element-1) unmethylated and/or AIM1 methylated cf-cDNA versus those with LINE-1 methylated and AIM1 unmethylated cf-cDNA. M, methylated; U, unmethylated. Journal of Investigative Dermatology 2012 132, 1689-1697DOI: (10.1038/jid.2012.36) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions