Methylation of O6-methylguanine DNA methyltransferase promoter is a predictive biomarker in Chinese melanoma patients treated with alkylating agents

Introduction

Melanoma is a highly aggressive skin cancer with high treatment resistance. Although the U.S. Food and Drug Administration (FDA) has approved various targeted therapies for melanoma which includes BRAF and MEK mutation inhibitors, based on the improved responses observed among patients harboring such mutations, many patients acquire therapeutic resistance and therefore do not achieve persistent, long-term responses (1). Antibodies specific for immune checkpoints, such as those targeting CTLA-4 or the PD1/PDL1 interaction, have also been approved by the FDA and have become a standard of care for patients with unresectable or metastatic melanoma; however, these therapeutic antibodies provide benefits to only some patients (2).

Despite the above limitations, chemotherapy remains a useful first- or second-line treatment for advanced metastatic melanoma (3). Alkylating chemotherapeutic agents such as temozolomide (TMZ) and dacarbazine (DTIC), which are commonly used to treat melanomas, cause DNA double-strand breaks, subsequent termination of replication, and apoptosis in tumor cells (4,5). However, a previous study of TMZ monotherapy reported an extremely objective response rate (13–15%) and median overall survival (OS) (7.7–8.4 months) (6). These findings underscore the need to explore the chemorefractory mechanisms exploited by melanomas and to identify biomarkers that can predict the prognosis of patients treated with alkylating agents. Moreover, a better understanding of biomarkers might provide new combination therapy strategies.

The O6-methylguanine-DNA methyltransferase (MGMT) pathway is considered highly significant among the mechanisms of resistance to alkylating agents. MGMT is a DNA repair enzyme encoded by the MGMT gene at locus 10q26 (7). Largely anecdotal evidence suggests that MGMT plays a central role in preventing the transformation of a physiologic proliferation into malignancy (8). In addition to protecting normal cells from carcinogenesis, MGMT activity also protects tumor cells from the lethal effects of alkylating agents by removing methyl groups from the O6 position of guanine (9).

During the oncogenesis of several human cancers, MGMT was found to be transcriptionally silenced by promoter hypermethylation. The immediate consequence of this phenomenon is the loss of MGMT protein expression and a reduced DNA repair capacity, which results in increased sensitivity to alkylating agents and decreased tumor survival (10,11). Therefore, several studies have described the MGMT promoter methylation status as a predictive biomarker of drug efficacy and prognosis, especially among patients with glioblastoma (GBM) (12). For example, a phase III clinical trial conducted by the European Organization for Research and Treatment of Cancer (EORTC) and National Cancer Institute of Canada (NCIC) identified MGMT methylation as the strongest predictor of TMZ chemotherapy outcomes in patients with GBM; specifically, MGMT methylation was beneficial in terms of response, progression-free survival, and OS (12). Several additional studies have found associations of MGMT inactivation with improved outcomes in Caucasian patients with melanoma who were treated with alkylating agents (13-15). However, as Caucasian and non-Caucasian patients exhibit significant differences in terms of pathogenesis and pathologic subtypes (16,17), the predictive role of MGMT should also be determined in a large cohort of non-Caucasian patients with melanoma.

Many previous reports of ovarian, colorectal, and brain cancers, including malignant astrocytomas, have associated MGMT methylation with TP53 mutation, particularly the G:C to A:T transition (5,11,18-20). By contrast, however, other studies have not identified a relationship between these factors (21,22), indicating the need for further investigation of the potential correlation between MGMT promoter methylation and TP53 mutation and the corresponding effect on prognosis in patients with melanoma. Accordingly, this study aimed to investigate the role of MGMT promoter methylation and TP53 mutation as prognostic biomarkers prognosis in Chinese patients with melanoma who were treated with alkylating agents.

Methods

Patients and tumor tissue samples

Archived formalin-fixed, paraffin-embedded (FFPE) melanoma samples were collected from 205 patients (including 64 with mucosal melanomas, 107 with acral melanomas, and 34 with non-acral skin melanomas) who received DTIC/TMZ and cisplatin-based chemotherapy at the Peking Cancer Hospital & Institute (Beijing, China) between 2005 and 2016 were collected for this study. The samples were analyzed by hematoxylin and eosin staining and immunohistochemistry to confirm the diagnosis of melanoma. Clinical case data were also collected, including patient age and sex, TNM (tumor-node-metastasis) stage, thickness (Breslow), ulceration, and survival outcomes (for which patients were followed until loss to follow-up or death). This study was approved by the Medical Ethics Committee of the Beijing Cancer Hospital & Institute (2017KT21) and was conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants.

DNA isolation and bisulfite modification

Genomic DNA was isolated using the Omega FFPE DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA). Subsequently, the isolated genomic DNA was subjected to sodium bisulfite modification using the Methylamp^TM DNA Modification Kit (EpiGentek Inc., Farmingdale, NY, USA) according to the manufacturer’s protocol. The bisulfite-treated DNA was stored at −80 °C.

Detection of MGMT methylation

In each tumor sample, the methylation status of the MGMT promoter was determined using methylation-specific polymerase chain reaction (MS-PCR). Bisulfite-treated DNA was amplified using primers specific to either modified or unmodified DNA. The following primer sequences based on previous research (23,24) were used: 5'-TTTCGACGTTCGTAGGTTTTCGC-3' (forward) and 5'-GCACTCTTCCGAAAACGAAACG-3' (reverse) for the methylated reaction and 5'-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3' (forward) and 5'-AACTCCACACTCTTCCAAAAACAAAACA-3' (reverse) for the unmethylated modified reaction. The unmethylated reaction yielded a 91-base pair (bp) product, and the methylated reaction yielded an 81-bp product that included the PYRCpG3, PYRCpG4, and PYRCpG5 region. The PCRs were performed in total volume of 25 µL, and tumor DNA was amplified during 40 cycles at an annealing temperature of 66 °C. The A549 cell line, which is known to exhibit MGMT methylation, was used as a control for methylated MGMT, and DNA from the peripheral blood leukocytes of a healthy individual was used as a control for unmethylated MGMT. Template-free H₂O was used as a negative PCR control. The PCR products were analyzed by 3% agarose gel electrophoresis and ethidium bromide staining.

MGMT protein immunohistochemistry

FFPE tissue sections were examined by IHC with the monoclonal mouse anti-human MGMT antibody clone MT3.1 (Abcam, Cambridge, UK). The primary antibody was omitted from negative controls. Inflammatory cells and reactive stromal cells served as internal positive controls. Prior to staining, the tissue sections were deparaffinized with xylene for 30 min and rehydrated in decreasing concentrations of ethanol. Endogenous peroxidases were then blocked with 30% H₂O₂ for 20 min in phosphate-buffered saline (PBS). For antigen retrieval, the slides were heated in 0.01 M citrate buffer (pH 6.0) in a pressure cooker for 4 min and subsequently cooled to room temperature in the same buffer. After washing, the slides were incubated overnight with the primary antibody at 4 °C (dilution 1:100). The tumor sections were then stained using the Envision^TM Detection kit (Gene Tech, Shanghai, PR China) according to the manufacturer’s instructions and counterstained with hematoxylin. MGMT status was determined based on staining intensity {negative (0), low positive [1], strongly positive [2]} and percentage of positive tumor cells {0% (0), 1–50% [1], 51–100% [2]}. Only nuclear staining was regarded as positive staining.

TP53 mutation detection

Exons 5–8 of TP53 were amplified by a two-step PCR. The following primer sequences were used for step I amplification: 5'-GTTTCTTTGCTGCCGTCTTC-3' (forward) and 5'-CCTTCCACTCGGATAAGATG-3' (reverse) for exon 5, 5’-AGCACATGACGGAGGTTGTG-3' (forward) and 5'-TCTCATGGGGTTATAGGGAG-3' (reverse) for exon 6, 5'-GCCTCCCCTGCTTGCCACAG-3' (forward) and 5'-GAGAGGTGGATGGGTAGTAG-3' (reverse) for exon 7, and 5'-TACCTGGAGCTGGAGCTTAG-3' (forward) and 5'-GAAAGAGGCAAGGAAAGGTG-3' (reverse) for exon 8. The following primer sequences were used for step II amplification: 5'-CTTTATCTGTTCACTTGTGC-3' (forward) and 5'-CAATCAGTGAGGAATCAGAG-3' (reverse) for exon 5, 5'-CATGAGCGCTGCTCAGATAG-3' (forward) and 5'-TAGGGAGGTCAAATAAGCAG-3' (reverse) for exon 6, 5'-CATCTTGGGCCTGTGTTATC-3' (forward) and 5'-GAAGAAATCGGTAAGAGGTG-3' (reverse) for exon 7, and 5'-GACAGGTAGGACCTGATTTC-3' (forward) and 5'-AAGTGAATCTGAGGCATAAC-3' (reverse) for exon 8.

Clinical data analysis and statistical methods

OS was defined as the time from diagnosis to the death of the patient. For the patients who were still alive at the time of the study, OS was defined as the time from diagnosis to the current date. The patients’ clinicopathological characteristics were correlated with the MGMT promoter methylation status and MGMT protein expression, and the significance of these associations was determined using Pearson’s chi-square test, the continuity correction test, Fisher’s exact test, and the linear-by-linear association test as appropriate. Survival data were used to generate Kaplan-Meier curves, which were compared according to MGMT promoter methylation status using the log-rank test. A Cox regression model was used for both univariate and multivariate analyses. Age, sex, primary site, stage, MGMT promoter methylation, MGMT protein expression, TP53 mutation status, and the combination of MGMT promoter methylation and TP53 mutation status were included in the multivariate model, which used a stepwise method for variable selection. The level of statistical significance was set at P<0.05, and all tests were two-sided. The statistical analysis was performed using IBM SPSS statistical software (version 20.0; IBM, Inc., Armonk, NY, USA).

Results

Correlation of MGMT promoter methylation with clinicopathological features

Using MS-PCR, we found that the MGMT promoter was methylated in 97 (47%) of 205 tested melanomas (Table 1 and Figure 1). We next used the Pearson method to evaluate the associations of MGMT promoter methylation with different clinicopathological features (Table 1), and observed a significant association with tumor stage (P=0.017), but not with sex (P=0.735), age (P=0.647), or the primary tumor site (P=0.875).

Table 1 Correlation of MGMT promoter methylation with clinicopathological features
Full table

Figure 1 MS-PCR analysis of MGMT promoter methylation status in DNA from melanoma patients. PC, positive control; NC, normal control; NTC, no template control; M, methylated; U, unmethylated.

Association of MGMT protein expression with MGMT promoter methylation

We next used IHC to detect positive nuclear staining for the MGMT protein in 15 of 97 cases with MGMT promoter methylation and 42 of 108 cases without methylation (Table 2). As described in the Materials and Methods, IHC staining was scored from 0 to 2 on the basis of the staining intensity and percentage of positive tumor cells (Figure 2). Strongly positive nuclear MGMT protein staining (IHC score =2) was detected in 8 of 97 patients with methylation and 25 of 108 patients without methylation, whereas low positive nuclear staining (IHC score =1) was detected in 25 of 97 cases with methylation and 17 of 108 cases without methylation.

Table 2 Correlation of MGMT protein expression with clinicopathological features
Full table

Figure 2 Representative images of melanoma tumor cells of MGMT expression (IHC; cell scale bar: 100 µm). (A) score 0; (B) score 1; (C) score 2.

Using Pearson’s chi-square test, we identified a significant association between MGMT expression and MGMT promoter methylation in patients with melanoma (P=0.021). Notably, both strongly positive and weakly positive nuclear MGMT protein expression were significantly associated with the MGMT promoter methylation status in our melanoma samples (P=0.001 and 0.027, respectively). However, we observed no significant associations of MGMT protein expression with sex (P=0.278), age (P=0.332), stage (P=0.149), or primary tumor site (P=0.247).

Correlation of MGMT promoter methylation with TP53 mutation

To evaluate the potential association of MGMT promoter methylation with TP53 mutation in melanoma, we subjected DNA samples from the same 205 tumors to PCR followed by Sanger sequencing to examine TP53 mutations in exons 5–8, which are commonly affected. We identified 37 cases with TP53 mutations. Of these, the MGMT promoter was methylated in 29 cases (30% of 97 samples with methylation) and unmethylated in only 8 (7% of 108 samples without methylation) (Table 3). Accordingly, we found a significant association of MGMT promoter methylation with TP53 mutation (P=0.004), with an odds ratio of 4.463 [95% confidence interval (CI): 1.879–22.664] When separating the patient by the primary site (mucosal vs. acral vs. skin), there is a significant correlation between MGMT promoter methylation status and TP53 mutation (P=0.01).

Table 3 TP53 mutation and methylation status of the MGMT promoter
Full table

A further analysis revealed that the melanomas harbored different TP53 mutation types, such as T:G, A:G, and C:G. The G:C to A:T mutation at exon 5 was identified in 8 of 97 (8%) cases with a methylated MGMT promoter and none of the cases with an unmethylated MGMT promoter, indicating that this type of mutation tends to occur under conditions of MGMT promoter methylation (P<0.01). Moreover, the G:C to A:T mutation at exon 5 was detected in 8 of 148 (5%) patients with MGMT protein expression and none of the cases without MGMT protein expression, indicating an increased tendency of this mutation to occur in melanomas expressing MGMT protein. However, this latter association was not statistically significant (P=0.109).

Association of MGMT promoter methylation and TP53 mutation with OS among patients with melanoma following treatment with alkylating agents

In our univariate Cox analyses, we identified stage and MGMT promoter methylation as factors significantly associated with OS among melanoma patients treated with alkylating agents (Table 4). The multivariate Cox regression model subsequently identified both factors as significantly predictive of OS (Table 4).

Table 4 Cox regression analysis of MGMT promoter methylation and clinicopathologic factors with overall survival
Full table

The median OS time of all 205 patients was 38.9 months, and a Kaplan-Meier analysis showed that patients with MGMT promoter methylation had a significantly greater median OS, compared to those without MGMT promoter methylation (36.7 vs. 23.1 months, P=0.021, Figure 3A). Although patients without MGMT protein expression also had a greater OS than did those with MGMT protein expression, this difference was not significant (34.5 vs. 25.9 months, P=0.092). Similarly, when patients were grouped by MGMT IHC staining scores, those with scores of 0 had poorer OS relative to those with scores of 1 or 2, although this difference was not statistically significant (36.7 vs. 26.1 and 21.5 months, respectively, P=0.086, Figure 3B).

Figure 3 Overall survival of melanoma patients in relation to MGMT promoter methylation. (A) Kaplan-Meier curves showing the correlation of MGMT promoter methylation status with OS of melanoma patients (P=0.021); (B) Kaplan-Meier curves showing the correlation of MGMT expression with OS of melanoma patients (P=0.086).

Although patients harboring a TP53 mutation or the combination of a TP53 mutation and MGMT promoter methylation had a relatively better OS outcome, this difference was also not significant (33.4 vs. 28.5 months, P=0.247 and 35.1 vs. 27.2 months, P=0.259, respectively). Finally, the stratification of patients by primary tumor site (mucosal vs. acral vs. skin) revealed that patients without MGMT promoter methylation exhibited worse OS, compared to those with MGMT promoter methylation (P=0.015).

Discussion

Although the DNA alkylating agents TMZ and DTIC are considered standard components of chemotherapy for melanoma, patients who receive TMZ for stage IV melanoma have a low response rate (13–15%) and dismal prognosis (OS, 7.7–8.4 months) (6). In addition, only 5% of patients achieve a complete response with TMZ or DTIC treatment, and few achieve a sustained response to either drug (25,26). However, the factors that might better identify patients who may benefit from TMZ or DTIC chemotherapy remain largely unknown. Therefore, in this study, we aimed to evaluate the prognostic role of MGMT promoter methylation and TP53 mutation in a large cohort of Chinese patients treated with alkylating agents for melanoma. In our population of 205 patients, we observed a MGMT promoter methylation rate of 47%, which was demonstrably higher than the corresponding frequency among Caucasian patients (21.5–26%) (15,27). This discrepancy may reflect differences in the pathogenesis and pathological subtypes of melanoma between non-Caucasians and Caucasians.

Additionally, our analysis of correlations between MGMT promoter methylation and clinicopathological features found that MGMT methylation only associated significantly with tumor stage, but not with sex, age, or primary tumor site. Specifically, MGMT promoter methylation was significantly more frequent among high-grade (stages III, IV) melanomas, compared to low-grade (I, II) melanomas (51% vs. 24%, P=0.004). However, we note that the large difference in the numbers of low-grade and high-grade melanomas (34 vs. 171) included in this analysis might have led to statistical bias. Still, our findings are consistent with a previous study, which found that MGMT promoter methylation was more common in advanced-stage tumors relative to early-stage tumors (28). Taken together, these findings suggest that MGMT promoter methylation and consequent inactivation may play a crucial role in melanoma progression.

Immunohistochemistry revealed a lack of MGMT expression in 82 of 97 (85%) tumors exhibiting MGMT promoter methylation, compared to 66 of 108 (61%) tumors with an unmethylated MGMT promoter. This finding indicates differences in MGMT protein expression between melanomas that do and do not exhibit MGMT promoter methylation (P=0.021); specifically, promoter methylation is associated with a loss of protein expression. However, the high percentage of melanomas lacking both MGMT promoter expression and detectable MGMT protein expression suggests that the latter process may be regulated by additional factors. Additionally, technical issues and other factors, such as tumor heterogeneity, may have influenced the results, as a previous study indicated that the use of different methods to assess the MGMT promoter methylation status might affect the results (29). In a phase II study of extended-dose temozolomide in patients with melanoma, Petra performed MS-PCR to detect the MGMT promoter methylation rate of 26%, and Rainer and his colleague determined a MGMT promoter methylation frequency of 21.5% via methylation-sensitive high-resolution melting (15,27). In our cohort, we used MS-PCR to detect the MGMT promoter methylation status. Petra reported that IHC staining for MGMT could not predict the responses of melanoma patients treated with TMZ (30). However, Augustine profiled the expression of 38,000 genes using an oligonucleotide-based microarray platform, and found a significant correlation of MGMT expression with TMZ sensitivity in the context of melanoma (P≤0.0001) (31). In our cohort, we used IHC to evaluate the expression of MGMT protein in melanomas but found that this factor could not predict the prognosis of patients treated with alkylating agents. Therefore, future studies should focus on gene expression profiling. Regarding tumor heterogeneity, promoter hypermethylation heterogeneity has been frequently observed in melanomas and glioblastomas (32,33). However, we could not explore the issue of heterogeneity in this study because of an inability to acquiring tumor tissues from in multiple sites in the body.

We further investigated the correlation of TP53 mutation with MGMT expression, as such correlations have been previously identified in different cancer types, including melanoma and glioblastoma (34-36). In our cohort, we identified TP53 mutations in 18% of melanoma cases, and observed that this mutation was more frequent among patients with MGMT promoter methylation (30%) than among those without MGMT promoter methylation (7%). Several previous studies have indicated an association between MGMT promoter hypermethylation and G:C to A:T transition mutations of the TP53 gene in nervous system tumors and glioblastoma (37,38). Consistent with these earlier findings, we observed a significant incidence of TP53 G:C to A:T mutation in patients with MGMT promoter methylation, (8 of 97, 8%, P<0.01). However, the relationship between the MGMT promoter methylation status and TP53 mutation remains under debate (39-43). Our analysis indicated a significant correlation of MGMT promoter methylation with TP53 mutation (P=0.004), suggesting that the former may increase the occurrence of the latter in melanoma. It is therefore reasonable to surmise that MGMT promoter methylation and TP53 mutation are not independent in the carcinogenesis of melanoma. However, the underlying mechanism and functional aspects require further investigation.

We believe that the most crucial finding of the present study is the association of MGMT promoter methylation with a significantly longer OS among patients with melanoma. In our multivariate Cox analyses, we identified the MGMT promoter methylation status as an independent prognostic variable associated with a longer OS. This is the first study to demonstrate the prognostic value of MGMT promoter methylation in non-Caucasian melanoma population treated with alkylating agents.

Although previous studies have established that the MGMT promoter methylation status can predict the efficacy of TMZ treatment in patients with glioblastoma (44,45), randomized clinical trials of a combination regimen comprising the MGMT inhibitor lomeguatrib (LM) and TMZ for metastatic cutaneous melanoma have not demonstrated any significant improvements in patient responses or OS when compared to TMZ monotherapy in a Caucasian population (46). As we noted previously, however, epidemiologic factors differ greatly between Asian and Caucasian populations. The predictive value of MGMT promoter methylation regarding response to alkylating agents needs to be supported by further validation. Moreover, the existing evidence merits additional basic research and clinical trials of MGMT promoter methylation.

In summary, we have demonstrated the prognostic significance of MGMT promoter methylation in melanoma patients treated with alkylating agents. Notably, the presence of MGMT promoter methylation was associated with the occurrence of TP53 mutation in melanoma. Determination of the MGMT promoter methylation status might help to identify melanoma patients who will likely benefit from chemotherapy with alkylating agents.

Acknowledgments

Funding: This work was supported by grants from National Natural Science Foundation of China (81402264), Beijing Municipal Natural Science Foundation (7152033) and Beijing Municipal Administration of Hospitals Clinical medicine Development of special funding support (ZYLX201603).

Footnote

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr.2018.05.06). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by the Medical Ethics Committee of the Beijing Cancer Hospital & Institute (2017KT21) and was conducted according to the principles of the Declaration of Helsinki (as revised in 2013). Written informed consent was obtained from all participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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