Genetic and epigenetic alterations in melanoma

Malignant melanoma is a disease that may arise in several different parts of the body, preferentially the skin, rarely in the mucosal membranes or the choroidal tissues of the eye. The incidence of cutaneous melanoma (CMM) is steadily increasing in the Caucasian populations, unlike uveal melanoma (UM) that shows a stable incidence. The increase is likely to be related to t UV-irradiation leading to genetic aberrations that allow skin melanocytes to develop unlimited growth and immortality and ultimately lead to metastases.

Paper I presents a genomic and epigenomic screening of 77 metastatic cutaneous melanoma metastases for the protein expression of p16INK4A in relation to 3 well-known causes of expression loss: truncating and non-synonymous mutations in CDKN2A, the gene for p16INK4A, transcriptional silencing of p16INK4A gene promoter and previously studied deletions in the CDKN2A loci encompassing p16INK4A. These aberrations were compared to p16INK4A expression in tumours and presence of mutations in BRAF and NRAS genes. Unexpectedly, a significant association between tumours carrying NRAS mutations and transcriptional silencing of p16INK4A promoter was observed.

Paper II was a case study of a family with multiple cases of uveal melanoma. Family members with were found to be negative for germ-line CDKN2A aberrations, why next generation sequencing was employed. The proband, the proband’s sister and both parents were analyzed. The final mapped and filtered variants were filtered against variants found in the DNA of the non-carrier mother. A germ-line, frame-shift, insertion in BAP1 (exon 3 c.75insG) was identified and validated by Sanger sequencing. The insertion leads to a truncation at codon 43 and was found to segregate with the disease.

Paper III is a retrospective study to evaluate the naturally occurring transcriptional silencing of DNA repair protein O6-methylguanine DNA methyltransferase, MGMT. MGMT activity counteracts the efficacy of alkylating chemotherapy. Two cohorts of patients mainly derived from Sweden (n=74) and Belgium (n=79) were included, in total encompassing 191 tumours. The hypermethylation of MGMT gene promoter was found in 21.5% of tumours successfully analyzed (28 positive, 130 total) and to be associated with a significantly longer progression free survival (PFS) and to be an independent variable in a multivariate analysis for PFS.

Paper IV is an in vitro melanoma study for combination therapy efficacy. The BRAFV600E- melanoma cell line A375 and a mutant BRAF inhibitor (BRAFi)-resistant subline were subjected to sequential and simultaneous exposures for BRAFi PLX4720 and temozolomide (TMZ). Administration order was found to influence the treatment outcome: administration of BRAFi followed by TMZ displayed a poorer efficacy compared to exposure simultaneously or administration in the reverse order. This effect was related to BRAFi induction of MGMT mRNA and protein, but also induction of the DNA damage marker H2AX by BRAFi and TMZ.

List of scientific papers

I. Jonsson, A., Tuominen, R., Grafstrom, E., Hansson, J., Egyhazi, S. (2010) High Frequency of p16 (INK4A) Promoter Methylation in NRAS-Mutated Cutaneous Melanoma. Journal of Investigative Dermatology 130(12): 2809-2817
https://doi.org/10.1038/jid.2010.216

II. Veronica Höiom, Daniel Edsgärd, Hildur Helgadottir, Hanna Eriksson, Charlotta All-Ericsson, Rainer Tuominen, Ivayla Ivanova, Joakim Lundeberg, Olof Emanuelsson and Johan Hansson(2013) Hereditary Uveal Melanoma: A Report of a Germline Mutation in BAP1. GENES, CHROMOSOMES & CANCER 52:378–384
https://doi.org/10.1002/gcc.22035

III. Rainer Tuominen, Rosalyn Jewell, Joost J.van den Oord, Pascal Wolter, Ulrika Stierner, Christer Lindholm, Carolina Hertzman Johansson, Diana Lindén, Hemming Johansson, Marianne Frostvik Stolt, Christy Walker, Helen Snowden, Julia Newton-Bishop , Johan Hansson and Suzanne Egyházi Brage (2014) MGMT promoter methylation is associated with temozolomide response and prolonged progression-free survival in disseminated cutaneous melanoma Int J Cancer epub ahead of print.
https://doi.org/10.1002/ijc.29332

IV. Alireza Azimi, Rainer Tuominen, Samaneh Ghashghaei, Hanif RassoolZadeh, Marianne Frostvik Stolt, Marianne Farnebo, Suzanne Egyházi Brage, Carolina Hertzman Johansson Induction of MGMT by BRAF inhibitors decreases the response to temozolomide in melanoma cells – DNA damage response central for the combination therapy outcome [Manuscript]