DNA repair mechanisms are essential for correcting post-replication errors. Impaired DNA repair is related to increases in mutation frequency, genomic instability and cell death. Aberrant DNA methylation and expression silencing is an important molecular alteration that is commonly detected in DNA repair genes in different types of cancer.
Because MLH1 promoter methylation was previously associated with MSI and a BRAF V600E mutation , we tested the methylation status of MLH1 in a series of thyroid tumours and correlated them with mutational status and MSI. Additionally, a previous study has reported that MGMT hypermethylation was associated with transitions in IDH1 and RAS. Thus, we assessed whether the presence of IDH1 and RAS mutations is associated with MGMT methylation and/or loss of MGMT expression.
Our study did not find significant differences in MLH1 and MGMT promoter methylation between the groups studied. This result suggests that mechanisms other than DNA methylation in the CpG island studied, e.g., methylation in other CpG islands, miRNAs and histone modifications, could be acting to silence the expression of the genes studied.
The putative relationships between MLH1 expression and BRAF V600E, RET/PTC and IDH1 genetic alterations were evaluated. Our results showed diminished expression of MLH1 in patients harbouring BRAF V600E mutations, RET/PTC rearrangements and transitions (IDH1 and NRAS). Although not significant, this result suggests a trend toward significance. Studying a larger set of samples may provide a more detailed understanding of this issue. In fact, when all samples with point mutations were grouped together, a significant association was found. To the best of our knowledge, this evidence is the first to indicate that the down-regulation of MHL1 is related to BRAF V600E mutations, RET/PTC rearrangements and transitions (IDH1 and NRAS) in patients with thyroid carcinoma.
We did not find a significant correlation between MGMT hypermethylation and/or loss of expression and its association with mutational status. A larger sample set may be necessary to reject the hypothesis that MGMT hypermethylation or loss of expression is not associated with IDH1 or RAS mutations. Interestingly, the primary mutation found in IDH1 brain tumours was at codon R132, while in thyroid carcinomas, non-R132 mutations were found .
Microsatellite instability is a hallmark of the mismatch repair (MMR) deficiency [21, 22]. Thus, to evaluate the MSI status of the thyroid samples, we used seven markers. The microsatellite markers demonstrating the highest frequency of MSI were D17S250, D2S346 and D2S123. The lowest frequencies were observed for BAT40, BAT26 and BAT25. Furthermore, mononucleotide markers (BAT40, BAT26 and BAT25) present the lowest frequency of instability among all microsatellite markers tested for both benign and malignant thyroid tumours [23, 24].
A significant relationship between MSI status (MSI-H) and histological subtypes was demonstrated in both PTC and FTC, and a higher frequency was found in patients with FTC. MSI is related to malignancy and the clinicopathological factors that indicate poor prognosis [23–25]. MSI does not act as an early event in thyroid tumourigenesis, but MSI is instead involved in tumour progression , indicating that MSI might play an important role in thyroid carcinogenesis, as previously described [23–26]. We evaluated the relationship between each mutation and the MSI pattern. Although the BRAF V600E mutation has been associated with sporadic MSI-H colorectal cancers , no relationship was found between BRAF V600E mutations, RET/PTC rearrangements and transitions (IDH1 and NRAS) and MSI status.
Although it has been suggested that in thyroid tumours, MSI is an important indicator of defects in the MMR system , the data presented in this study showed no relationship between MLH1 expression and MSI status. In addition, a previous study on colorectal cancer showed that methylation of another DNA repair gene, MGMT, is also linked to MSI [22, 29]. In thyroid samples, our data showed a relationship between promoter methylation and MSI phenotype.