O6-methylguanine-DNA-methyltransferase and DNA mismatch repair in relation to drug resistance in malignant melanoma

Sammanfattning: Although the survival rate of patients with malignant melanoma has improved, treatment of the disease poses large problems since disseminated melanoma frequently shows primary resistance to chemotherapy. Resistance to chemotherapy is thus a major clinical problem and a cause of failure in the treatment of metastatic malignant melanoma. The primary aim of this thesis was to investigate whether the DNA repair protein 06-methylguanineDNA-metbyltransferase (MGMT) and the post-replication repair system DNA mismatch repair (MMR) have clinical relevance as drug resistance factors against DTIC-based chemotherapy. The results of the MGMT protein expression analyses demonstrate an inverse relationship between MGMT expression and clinical response to chemotherapy in patients with metastatic melanoma, indicating that MGMT is an important drug resistance factor against DTIC-based chemotherapy in melanoma. There was frequently considerable variation with respect to the proportion of MGMTpositive tumour cells in different melanoma biopsies from the same patient. It is therefore necessary to investigate MGMT expression in multiple metastases to obtain a correct evaluation of MGMT levels in patients with metastatic melanoma. We compared MGMT single nucleotide polymorphisms (SNPs) in melanoma patients belonging to families with a hereditary predisposition for melanoma and healthy individuals. In total 11 SNPs were detected, 5 in the 5'-noncoding region, I in exon 1, 2 in exon 3 and 3 in exon 5. Six of the alterations were novel polymorphisms, of which 5 were located in the 5'-noncoding region and one in exon 5. C575A as a single SNP was only present in melanoma patients (p < 0.072) indicating a possible association with familial melanoma. Moreover, only 12% of melanoma patients had the wild type genotype without SNPs compared with 20% of the healthy individuals. There was no significant correlation between MGMT polymorphisms in exons 3 or 5 and clinical response to DTIC-based chemotherapy in melanoma. Thus, MGMT expression levels seem to be more relevant for response to chemotherapy than these MGMT SNPs. We performed a functional analysis by in vitro mutagenesis in E. coli to investigate whether the MGMT I143V or I143V/K178R variants have an effect on MGMT activity. E. coli strain GWR111 carrying variant MGMT I143V or double variants I143V/K178R exhibited almost identical sensitivity to MNNG, as did GWR111 with wild type MGMT, and assays of MGMT expression and MGMT activity showed no decrease compared to the wild type protein. No evidence was thus found that variants MGMT I143V and I143V/K178R have a negative effect on MGMT protein activity in. The expression of the MMR proteins, hMSH2, hMSH6 and hMLH1, were analysed in melanoma metastases. More than half of the melanoma patients had tumours without nuclear staining for either hMSH2 or hMSH6, while all tumours showed positive nuclear staining for hMLH1. Moreover, hMSH2 and hMSH6 were frequently down-regulated together. There was, however, no significant correlation between MMR expression, alone or combined with MGMT levels, and clinical response to DTIC-based chemotherapy in these melanoma patients. To investigate whether genomic instability has an impact on response to chemotherapy the frequencies of LOH and MSI were studied in tumours from 11 responders and 17 nonresponders. MSI was observed in 13 (46%) of 28 cases, and LOH in 22 cases (79%). There was no significant correlation between MSI or LOH and clinical response to DTIC-based chemotherapy in melanoma. However, a higher frequency of LOH was observed at the specific loci, 3p23, in non-responders 5/9 (56%) vs. 1/7 (14%) responders, and 11q23, which occurred in 4/9 (44%) of non-responders compared to 115 (20%) of responders.