Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.     

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: Correlation with physiological and pathological characteristics,and with biomarkers of one-carbon metabolism

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.     

DNA methylation-mediated silencing of neuronatin (NNAT) in pig parthenogenetic fetuses

It is generally believed that aberrant expression of imprinted genes participates in growth retardation of mammalian parthenogenesis. Neuronatin (NNAT), a paternally expressed gene, plays important roles in neuronal growth and metabolic regulation. Here we have compared the gene expression and promoter methylation pattern of NNAT between pig normally fertilized (Con) and parthenogenetic (PA) embryos. The results showed loss of NNAT expression (p < 0.001) and hypermethylation of NNAT promoter in PA samples. Additionally, partial methylation was observed in Con fetuses, while almost full methylation and unmethylation of NNAT promoter were apparent in Metaphase II (MII) oocytes and mature sperms, respectively, which identified the CpG promoter region as a putative differentially methylated region (DMR) of NNAT. The data demonstrate that promoter hypermethylation is associated with the silencing of NNAT in pig PA fetuses, which may be related to developmental failure of pig parthenogenesis at early stages.     

Epigenetic silencing of Na,K-ATPase β1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na⁺ and uptake of K⁺ across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β₁ (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients’ tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2′-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression.     

Wnt inhibitory factor-1, a Wnt antagonist, is silenced by promoter hypermethylation in malignant pleural mesothelioma

Wnt inhibitory factor-1 (WIF-1) is a secreted protein that antagonizes Wnt signaling. We recently demonstrated the importance of aberrant activation of the Wnt signaling pathway in various cancers including malignant pleural mesothelioma. In this study, we revealed downregulated WIF-1 expression in cell lines and primary tissue when compared to normal mesothelial cell lines and adjacent pleura, respectively. We observed hypermethylation in four of four mesothelioma cell lines, but not in two normal mesothelial cell lines. In primary tissue samples, we observed methylation in three paired tumor specimens compared to their adjacent normal pleura and methylation in eight of nine unpaired tumor tissue samples. Taken together, our studies suggest that WIF-1 silencing due to its promoter hypermethylation is an important mechanism underlying the constitutively activated Wnt signaling in mesothelioma. New therapies toward inhibition of the Wnt pathway through WIF-1 might be promising for the future treatment of malignant mesothelioma.     

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.     

Wnt inhibitory factor 1 suppresses cancer stemness and induces cellular senescence

Hyperactivation of the Wingless-type (Wnt)/β-catenin pathway promotes tumor initiation, tumor growth and metastasis in various tissues. Although there is evidence for the involvement of Wnt/β-catenin pathway activation in salivary gland tumors, the precise mechanisms are unknown. Here we report for the first time that downregulation of the Wnt inhibitory factor 1 (WIF1) is a widespread event in salivary gland carcinoma ex-pleomorphic adenoma (CaExPA). We also show that WIF1 downregulation occurs in the CaExPA precursor lesion pleomorphic adenoma (PA) and indicates a higher risk of progression from benign to malignant tumor. Our results demonstrate that diverse mechanisms including WIF1 promoter hypermethylation and loss of heterozygosity contribute to WIF1 downregulation in human salivary gland tumors. In accordance with a crucial role in suppressing salivary gland tumor progression, WIF1 re-expression in salivary gland tumor cells inhibited cell proliferation, induced more differentiated phenotype and promoted cellular senescence, possibly through upregulation of tumor-suppressor genes, such as p53 and p21. Most importantly, WIF1 significantly diminished the number of salivary gland cancer stem cells and the anchorage-independent cell growth. Consistent with this observation, WIF1 caused a reduction in the expression of pluripotency and stemness markers (OCT4 and c-MYC), as well as adult stem cell self-renewal and multi-lineage differentiation markers, such as WNT3A, TCF4, c-KIT and MYB. Furthermore, WIF1 significantly increased the expression of microRNAs pri-let-7a and pri-miR-200c, negative regulators of stemness and cancer progression. In addition, we show that WIF1 functions as a positive regulator of miR-200c, leading to downregulation of BMI1, ZEB1 and ZEB2, with a consequent increase in downstream targets such as E-cadherin. Our study emphasizes the prognostic and therapeutic potential of WIF1 in human salivary gland CaExPA. Moreover, our findings demonstrate a novel mechanism by which WIF1 regulates cancer stemness and senescence, which might have major implications in the field of cancer biology.     

Prognostic Significance of Promoter DNA Hypermethylation of cysteine dioxygenase 1 (CDO1) Gene in Primary Breast Cancer

Using pharmacological unmasking microarray, we identified promoter DNA methylation of cysteine dioxygenase 1 (CDO1) gene in human cancer. In this study, we assessed the clinicopathological significance of CDO1 methylation in primary breast cancer (BC) with no prior chemotherapy. The CDO1 DNA methylation was quantified by TaqMan methylation specific PCR (Q-MSP) in 7 BC cell lines and 172 primary BC patients with no prior chemotherapy. Promoter DNA of the CDO1 gene was hypermethylated in 6 BC cell lines except SK-BR3, and CDO1 gene expression was all silenced at mRNA level in the 7 BC cell lines. Quantification of CDO1 methylation was developed using Q-MSP, and assessed in primary BC. Among the clinicopathologic factors, CDO1 methylation level was not statistically significantly associated with any prognostic factors. The log-rank plot analysis elucidated that the higher methylation the tumors harbored, the poorer prognosis the patients exhibited. Using the median value of 58.0 as a cut-off one, disease specific survival in BC patients with CDO1 hypermethylation showed significantly poorer prognosis than those with hypomethylation (p = 0.004). Multivariate Cox proportional hazards model identified that CDO1 hypermethylation was prognostic factor as well as Ki-67 and hormone receptor status. The most intriguingly, CDO1 hypermethylation was of robust prognostic relevance in triple negative BC (p = 0.007). Promoter DNA methylation of CDO1 gene was robust prognostic indicator in primary BC patients with no prior chemotherapy. Prognostic relevance of the CDO1 promoter DNA methylation is worthy of being paid attention in triple negative BC cancer.     

巢式甲基化特异性PCR检测肺癌病人WIF-1基因启动子区异常甲基化

为了检测肺癌患者血浆中WIF-I基因启动子区的甲基化状态,收集肺癌患者及健康对照者的血浆标本,采用巢式甲基化特异性PCR（nMSP）法检测WIF-I基因启动子区甲基化状态,并与普通甲基化特异性PCR（MSP）法进行了比较,结果在58例肺癌患者血浆样品中经nMSP法发现20例WIF-I基因启动子的过甲基化,用MSP法只检出10例,有吸烟史组WIF-1基因的甲基化率高于无吸烟史组（P〈0．05）．而20例正常对照血浆中都未检测到形胆J基因启动子的过甲基化;表明利用巢式MSP（nMSP）法检测外周血血浆中WIF-1基因启动子的甲基化,可为非损伤性筛选和早期诊断肺癌提供有价值的信息．     

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.     

Promoter hypermethylation using 24-gene array in early head and neck cancer: Better outcome in oral than in oropharyngeal cancer

Silencing of tumor suppressor genes (TSGs) by DNA promoter hypermethylation is an early event in carcinogenesis and a potential target for personalized cancer treatment. In head and neck cancer, little is known about the role of promoter hypermethylation in survival. Using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) we investigated the role of promoter hypermethylation of 24 well-described genes (some of which are classic TSGs), which are frequently methylated in different cancer types, in 166 HPV-negative early oral squamous cell carcinomas (OSCC), and 51 HPV-negative early oropharyngeal squamous cell carcinomas (OPSCC) in relation to clinicopathological features and survival. Early OSCC showed frequent promoter hypermethylation in RARB (31% of cases), CHFR (20%), CDH13 (13%), DAPK1 (12%), and APC (10%). More hypermethylation (≥ 2 genes) independently correlated with improved disease specific survival (hazard ratio 0.17, P = 0.014) in early OSCC and could therefore be used as prognostic biomarker. Early OPSCCs showed more hypermethylation of CDH13 (58%), TP73 (14%), and total hypermethylated genes. Hypermethylation of two or more genes has a significantly different effect on survival in OPSCC compared with OSCC, with a trend toward worse instead of better survival. This could have a biological explanation, which deserves further investigation and could possibly lead to more stratified treatment in the future.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Epigenetic inactivation of SLIT2 in human hepatocellular carcinomas

Recent findings have shown that SLIT2 appears to function as a novel tumor suppressor gene. In addition, hypermethylation of its promoter region has been detected in various cancers, including breast and lung cancer, colorectal carcinoma, and gliomas. Here, we report for the first time that there is epigenetic silencing of SLIT2 in human hepatocellular carcinoma (HCC). Downregulation of SLIT2 was detected in 6 of 8 (75%) HCC cell lines by quantitative real-time RT-PCR (qRT-PCR), and the downregulation of SLIT2 was generally dependent on the degree of methylation at the promoter region. Furthermore, expression of SLIT2 was restored in relatively low-expressing cell lines after treatment with 5-aza-2-deoxycytidine (5-Aza-dC). Downregulation of SLIT2 expression was also detected in 45 of 54 primary HCC samples (83.3%), and the decrease in expression was significantly correlated with CpG island hypermethylation. This decrease of SLIT2 expression was also associated with lymph node metastasis in HCC. Moreover, overexpression of SLIT2 in SMMC-7721 cells induced by recombinant adenovirus suppressed cell growth, migration, and invasion, These results suggest that epigenetic inactivation of SLIT2 in HCC may be important in the development and progression of HCC. Thus, SLIT2 may be useful as a therapeutic target in the treatment of HCC.     

A reinvestigation of somatic hypermethylation at the PTEN CpG island in cancer cell lines

Background

PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.

Results

Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.

Conclusions

We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.     

BRCA1 promoter methylation in peripheral blood DNA was identified in sporadic breast cancer and controls

Objective Epigenetics, particularly DNA methylation, has recently been shown to be important in breast cancer initiation. We investigated the clinical and prognostic importance of whole blood breast cancer early onset gene 1 (BRCA1) DNA methylation in sporadic breast cancer. Methods Genomic DNA was extracted from the peripheral blood cells (PBCs) of 902 breast cancer patients at diagnosis, with no BRCA1 mutation, and 990 control women. DNA methylation was measured by quantitative analysis of methylated alleles (QAMA) to estimate the extent of methylation of 2 CpG sites in the promoter region of BRCA1 oncosuppressor. Results BRCA1 promoter methylation rate in PBCs was 47.1% with a 95% confidence interval [46.1; 48.1] in breast cancer patients, and 45.9% with a 95% confidence interval [45.0; 46.8] in controls. We found a trend toward BRCA1 promoter hypermethylation in PBCs of sporadic breast cancer patients compared with controls. Association between methylation and clinicopathological features was evaluated using statistical tests. BRCA1 promoter methylation in PBCs increased significantly in breast cancer patients compared with controls, for age over 70 years (p = 0.022), in post-menopausal status (p = 0.013), for a body mass index (BMI) <20 (p = 0.0095) or a waist-to-hip ratio (WHR) ≤76.8 (p = 0.0027). We also found an association of increased BRCA1 promoter methylation in PBCs with ACA/ACA genotype for the SNP Thr594Thr in ESR (estrogen receptor gene), known to be associated with breast cancer risk (p = 0.092), reflecting the reduced presence of this genotype in this breast cancer case-control study. Conclusion Analysis of site-specific DNA methylation in PBCs by QAMA provides quantitative DNA methylation values that may serve as important prognostic indicators.     

Characterisation of CpG methylation in the upstream control region of mouse Nat2: Evidence for a gene–environment interaction in a polymorphic gene implicated in folate metabolism

Human arylamine N-acetyltransferase 1 (NAT1), a polymorphic xenobiotic metabolising enzyme, has been investigated in relation to susceptibility and prognosis in certain types of cancer. Both human NAT1 and its murine equivalent NAT2 have previously been shown to play roles in the catabolism of folate, which is required for the synthesis of S-adenosylmethionine, the methyl donor for cellular methylation reactions. We have tested whether the expression of mouse Nat2 is subject to epigenetic regulation, specifically CpG methylation in the promoter region, by determining levels of 5-methylcytosine by bisulphite sequencing and methylation-specific PCR. Under normal conditions, methylation levels of the Nat2 promoter were low, and varied in different tissues. However, CpG methylation was significantly increased by dietary folate supplementation, and increased methylation corresponded to decreased use of the core promoter. Functional deletion of the Nat2 gene gave rise to a significant increase in Nat2 methylation, extending our previous observations that folate catabolism is decreased in Nat2 null mice. Mouse NAT2 is likely to influence epigenetic gene control, particularly of its own locus, and this is consistent with recent evidence associating aberrant mouse Nat2/human NAT1 gene expression with certain developmental malformations and cancers.     

Isocitrate dehydrogenase 1 Gene Mutation Is Associated with Prognosis in Clinical Low-Grade Gliomas

Isocitrate dehydrogenase 1 gene mutations are found in most World Health Organization grade II and III gliomas and secondary glioblastomas. Isocitrate dehydrogenase 1 mutations are known to have prognostic value in high-grade gliomas. However, their prognostic significance in low-grade gliomas remains controversial. We determined the predictive and prognostic value of isocitrate dehydrogenase 1 status in low-grade gliomas. The association of isocitrate dehydrogenase 1 status with clinicopathological and genetic factors was also evaluated. Clinical information and genetic data including isocitrate dehydrogenase 1 mutation, O 6-methylguanine DNA methyltransferase promoter methylation, 1p/19q chromosome loss, and TP53 mutation of 417 low-grade gliomas were collected from the Chinese Glioma Genome Atlas database. Kaplan–Meier and Cox proportional hazards regression analyses were performed to evaluate the prognostic effect of clinical characteristics and molecular biomarkers. Isocitrate dehydrogenase 1 mutation was identified as an independent prognostic factor for overall, but not progression-free, survival. Notably, isocitrate dehydrogenase 1 mutation was found to be a significant prognostic factor in patients with oligodendrogliomas, but not in patients with astrocytomas. Furthermore, O 6-methylguanine DNA methyltransferase promoter methylation (p = 0.017) and TP53 mutation (p < 0.001), but not 1p/19q loss (p = 0.834), occurred at a higher frequency in isocitrate dehydrogenase 1-mutated tumors than in isocitrate dehydrogenase 1 wild-type tumors. Younger patient age (p = 0.041) and frontal lobe location (p = 0.010) were significantly correlated with isocitrate dehydrogenase 1 mutation. Chemotherapy did not provide a survival benefit in patients with isocitrate dehydrogenase 1-mutated tumors. Isocitrate dehydrogenase 1 mutation was an independent prognostic factor in low-grade gliomas, whereas it showed no predictive value for chemotherapy response. Isocitrate dehydrogenase 1 mutation was highly associated with O 6-methylguanine DNA methyltransferase promoter methylation and TP53 mutation.