Elevated DNA methylation in malignant tumors of the sinonasal tract and its association with patient survival

Background. Epigenetic modifications have been recognized as an important mechanism underlying carcinoma progression. DNA methylation plays an important role in cancer biology and represents potentially heritable changes in gene expression that do not involve DNA sequence. The aim of this study was to investigate promoter methylation of selected genes in sinonasal carcinoma by comparison with noncancerous sinonasal tissue. Methods. To search for epigenetic events (methylation in 25 tumor suppressor genes) we used MS–MLPA (Methylationspecific multiplex ligation-dependent probe amplification) to compare methylation status of 59 formalin fixed, paraffin embedded tissue samples of sinonasal carcinomas with 18 control samples. The most important changes in methylation were confirmed using MSP (Methylation specific PCR). Detected alterations in methylation were compared with clinicopathological characteristics. Results. Using a 20% cut-off for methylation (MS-MLPA), we found significantly higher methylation in GATA5 (P=0.0005), THSB1 (P=0.0002) and PAX5 (P=0.03) genes in the sinonasal cancer group compared to the control group. Methylation in five or more genes was associated with impaired overall survival (P=0.017). Conclusion. These findings provide evidence that alterations in methylation profile may be one of the major mechanisms in sinonasal carcinogenesis. In addition, changes in methylation could potentially be used as prognostic factors of sinonasal carcinoma and may have implications for future individualized therapy based on epigenetic changes.


INTRODUCTION
Malignant tumors of the sinonasal tract are rare tumors of the head and neck area that account for approximately 3% to 5% of all upper respiratory tract malignancies 1 .In 2014, 49 new cases in men and 39 in women were diagnosed in the Czech Republic, giving the incidence rate 0.84/100,000 (ref. 2 ).These figures probably reflect the general status in Europe and worldwide [3][4] .The average age at which patients are diagnosed with sinonasal carcinomas is between 50-60 years.However, there are some risk factors including exposure to wood and leather dust, tabaco smoke exposure, contact with chemical substances such as formaldehyde, chrome pigment, nickel and asbestos and HPV infection 5 .Diagnosis and treatment of these tumors pose several problems due to their very low incidence, histological diversity and production of non-specific symptoms in the early stages that can closely mimic inflammatory conditions.Their prognosis largely depends on histology, location and staging 6 .
Sinonasal carcinoma has been considered as a disease driven by progressive genetic alterations, such as mutations involving oncogenes or tumor suppressor genes, as well as chromosomal abnormalities 5 .In addition, approximately 20-30% of these tumors harbor transcriptionally active high risk human papillomavirus infection 7 .More recently, it has been demonstrated that sinonasal cancer is also driven by epigenetic alterations [8][9] .Currently, DNA methylation is one of the most broadly studied and wellcharacterized epigenetic modifications.Our knowledge about it is dating back to 1969, when Griffith and Mahler suggested that DNA methylation may be important in long term memory function 10 .DNA methylation is mediated by DNA methyltransferases, which catalyze the covalent addition of a methyl group to the 5-carbon of the cytosine in CpG context dispersed throughout the genome or in DNA repetitive regions.Promoter DNA methylation at CpG sites represses gene expression by impeding access to transcription factors and inhibiting RNA polymerase II (ref. 11).In cancer, aberrant DNA methylation is typically observed in the promoter/exon1 regions of various tumor suppressor genes 12 , transcription factors 13 , DNA repair genes and cell cycle regulators leading to their transcriptional silence 14 .However, various tumor types have different patterns of hypermethylated genes.
As sinonasal carcinomas are group of aggressive tumors with poor prognosis, it is very important to know their molecular parameters to be able to establish diagnostic strategies and individualized therapies.Sinonasal malignancies are rare, which makes it difficult to conduct extensive methylation studies.In the present study we were able to gather, thanks to cooperation of three university hospitals in Czech Republic, a huge and unique group of sinonasal cancer patients.The aim of this study was to investigate promoter methylation of specific genes in sinonasal carcinoma by comparison with nonmalignant sinonasal tissue.We are fully aware of the fact that the absence of mRNA expression analysis may be the main weakness of this study.However, we are unable to provide relevant information about gene expression of selected genes, because we were limited to work only with FFPE tissue samples, which are generally not suitable for RNA isolation and subsequent gene expression analysis due to high fragmentation of RNA.

MATERIALS AND METHODS
Formalin-fixed, paraffin-embedded tissue samples of sinonasal carcinomas and noncancerous sinonasal tissue were obtained from 77 patients: 59 patients with sinonasal cancer, and 18 patients with noncancerous sinonasal tissue.Only tumors primarily originating from the nasal cavity, maxillary sinuses and ethmoid complex were included.No tumors were found in the frontal or sphenoid sinuses.The samples of noncancerous tissue (9 mucosal specimens from the nasal cavity and 9 from the maxillary sinus) were obtained from patients treated for chronic rhinitis and sinusitis.The paraffin blocks were retrieved from the archives of the Fingerland Department of Pathology, University Hospital Hradec Kralove; the Department of Pathology, General University Hospital, Prague, Czech Republic; and the Department of Pathology, University Hospital Olomouc, Czech Republic.All slides were reviewed by experienced head and neck pathologist (J.L.) and the carcinomas were classified according to the current WHO classification 15 .The study was approved by the Ethics Committee of University Hospital Hradec Kralove.The need for informed consent was waived by the review board in view of the retrospective nature of the study and long archival period of the formalin-fixed, paraffin-embedded tissue samples involved.
For every patient were recorded data such as gender, age at the time of diagnosis, smoking history (non-smoker vs. ex-smoker vs. current smoker), occupation (risky vs. non-risky), and tumor localization, including the nasal cavity, maxillary sinus, and ethmoid complex, laterality and pathological TNM.During the follow-up period local recurrence, regional recurrence, distant recurrence, death, and tumor-related death staging were recorded.When radical surgery was not performed, clinical TNM staging was used instead.Treatment modalities were radical surgery, radiotherapy, and chemotherapy in various combinations.
The tumor types included squamous cell carcinoma (SCC), lymphoepithelial carcinoma (LEC), sinonasal undifferentiated carcinoma (SNUC), adenocarcinoma, and neuroendocrine carcinoma.Vascular invasion, perineural spread, status of resection margins (in the case of radical surgery), and microscopic findings in the surrounding mucosa were also noted.
DNA for methylation analysis was extracted from formalin-fixed, paraffin-embedded tissue samples using a Qiagen DNA extraction kit (Hilden, Germany).

Methylation-specific polymerase chain reaction (MSP)
The most important genes showing highly significant methylation (P<0.001) in MS-MLPA (GATA4 and THSB1) were further tested by means of MSP for confirmation.MSP requires bisulfite treatment of genomic DNA, which is used for conversion of all unmethylated cytosines to uracils, leaving methylated cytosines unaffected.A total of 500 ng of genomic DNA was treated with bisulfite using the EZ DNA Methylation-Gold™ Kit according to the manufacturer's protocol (Zymo Research Corporation Irvine, CA, USA).MSP was performed on the Rotor-Gene Q (Qiagen, Hilden, Germany) in two types of reaction mixture within one run, for amplifying methylated and unmethylated DNA, respectively.Primers were designed using MethPrimer with consideration of the MS-MLPA probe locations and the FFPE DNA fragmentation.Primer sequences with amplicon lengths are listed in Table 1.MSP reaction mixture contained 10x PCR Buffer, MgCl 2 (25 mM), dNTPs solution Takara (2.5 mM), primers (10 µM), Platinum® Taq DNA Polymerase (Invitrogen by Thermo Fisher Scientific, Foster City, CA, USA), SYTO®9 Dye (0.05 mM), bisulfite converted DNA and water.Each run included a bisulfiteconverted universal methylated and unmethylated DNA (Qiagen, Hilden, Germany) and a no template control.Fluorescence data were analyzed using Rotor Gene Q software.Amplicon was considered as methylated when there was amplification in reaction mixture with methylated primers or both types of reaction mixture.When there was amplification only in mixture with primer pair for unmethylated DNA, the amplicon was considered as unmethylated.

Statistical analysis
Basic descriptive statistics were adopted for the analysis: median, mean, and 95% confidence interval for continuous data, and absolute and relative frequencies for categorical data.The relationship between gene methylation and other independent factors was analyzed using the chi-square test, Fisher's exact test, or Logistic regression analysis.Kaplan-Maier and Logrank tests were used for survival analysis; Cox regression analysis was used to determine the influence of gene methylation upon survival.We considered P<0.05 to be statistically significant.All statistical analyses were performed using STATISTICA Cz (data analysis software system) version 12 (StatSoft, Inc., Tulsa, OK, USA).

RESULTS
Using a 20% cut-off for methylation (MS-MLPA), we observed significantly higher methylation in GATA5 (P=0.0005),THBS1 (P=0.0002) and PAX5 (P=0.03)genes in the sinonasal cancer group compared to the control noncancerous group.For genes BRCA1, BRCA2, ATM, VHL, and RB1a the methylation rate did not exceed the 20% threshold; the other genes also showed relevant differences in methylation between samples with sinonasal carcinoma and control samples (see Fig. 1).Using MSP, we confirmed significantly higher methylation of GATA5 gene in samples of sinonasal carcinoma in comparison with noncancerous sinonasal tissue (P=0.03).Using MSP we weren't able to confirm the presence of methylation of THSB1 gene in cancer samples, but it could be the result of the assay strategy (6 CpGs in primers, compared to 2 CpG restriction sites in MS-MLPA probe).

Correlation with clinicopathological features
Clinicopathological data are listed below.Due to a few   From those in the carcinoma group with a known history, 25 patients were non-smokers, 9 former smokers, and 17 were current smokers.In only 5/51 patients, occupational exposure to wood dust or other air pollutants/ irritants was recorded.
As regards the whole study sample, most of the tumors arose in the nasal cavity, but SCCs were slightly more common in the maxillary sinus.The majority of the patients were diagnosed with advanced tumors and three patients had lung metastases (cM1) at the time of diagnosis.
The follow-up period ranged from 1 to 241 months (median 23 months).Local recurrence was found in 23/50 tumors, 5/50 recurred regionally, and 3/59 patients developed distant metastases in the lungs.During the follow-up period, 29/49 patients died, of whom 14/49 due to the tumor.
The methylation results from the carcinoma specimens were compared with clinicopathological characteristics mentioned above (Table 2).Presence of methylation in five or more genes was connected with worse overall survival (P=0.017),see Fig. 2. No correlation was found between DNA methylation and tumor type, stage, grade and smoking history.

DISCUSSION
A wide array of techniques is currently available to measure DNA methylation level.In our study we used MS-MLPA a semi-quantitative method for methylation profiling.MS-MLPA allows simultaneous assessment of aberrant promoter methylation of large sets of genes and requires only small quantities of short DNA fragments, making it very suitable for analysis of DNA isolated from FFPE tissue samples 16 .Using DNA methylation profiling as a biomarker has a number of advantages including DNA stability, relatively low cost of testing and restriction to limited specific regions of DNA.Moreover DNA methylation analysis can be performed from small biopsy samples obtained during the routine diagnostic work-up of patients.In our study we employed the idea of a candidate -gene approach for searching the potential prognostic biomarkers in sinonasal carcinoma.Recently, a successful example of methylation-based prognostic and predictive cancer biomarker is hypermethylation of MGMT promoter region, which is routinely analyzed and predicts response to temozolomide in glioblastoma patients and their clinical outcome 17 .Next promising biomarker is methylation of XRCC2 gene predicting the occurrence of late toxicity in radiotherapy-treated cervical cancer patients 18 .In head and neck cancer area methylation in PITX3, SHISA3 and FOXF2 in squamous cell types of carcinomas also represent promising prognostic biomarkers [19][20][21] .
In our study we observed significantly higher methylation in three genes: GATA5, THBS1 and PAX5.GATA5 methylation has been already found in hepatocellular carcinoma 22 , colorectal cancer 23 , glioblastomas 24 and ovarian cancer 25 .THBS1 methylation has been demonstrated in laryngeal squamous cell carcinoma 26 , glioblastomas 24 and ovarian cancer 25 and PAX5 methylation has been shown in hepatocellular carcinoma 27 and non-small cell lung cancer 28 .These findings supported the theory that methylation changes in these genes could lead to carcinogenesis also in sinonasal carcinoma.
Our experiments never showed methylation in genes BRCA1, BRCA2, ATM, VHL, and RB1a, suggesting that methylation of selected CpG loci of these tumor suppressor genes may not play an important role in carcinogenesis of the sinonasal area.On the other hand, other genes (see Fig. 1) did show promoter methylation to a varying extent above the 20% threshold.Methylation of TP53, WT1 and MSH6 was detected in more than 10% of control samples.Presence of this methylation could be associated with nature of the control samples.All control samples were obtained from patients with inflammation in sinonasal area.At sites of chronic inflammation, epithelial cells are exposed to high levels of reactive oxygen species and undergo cancer-associated DNA methylation changes, suggesting that inflammation may initiate epigenetic alterations 29 .These findings correlate with research Wang et al. (2014), where methylation of TIMP3, GSTP-1 and 14-3-3σ was found in patients with chronic inflammation and in cancer patients 30 .
Methylation changes may be connected with a more aggressive phenotype and thus be associated with unfavorable clinical outcome.We demonstrated correlation between presence of methylation in five or more genes and shorter overall survival time.These findings show that accumulation of changes in methylation during sinonasal cancer development could be associated with patient's prognosis and so has a potential to serve as a biomarker in the future.

CONCLUSION
Our observations provide evidence that changes in methylation of these genes may be one of the major mechanisms in sinonasal carcinogenesis.In addition, changes in methylation could potentially be used as prognostic markers of sinonasal cancer and may have implications for future individualized therapy based on epigenetic changes.Future work with a larger number and in various ethnic groups is warranted to confirm that methylation in selected genes is a reliable prognostic biomarker for SNC patients.

Fig. 1 :Fig. 1 .
Fig. 1: Methylation of specific genes in samples of sinonasal carcinoma and control samples

Fig. 2 .
Fig. 2. Kaplan-Meier survival analysis.Overall survival of patients according to the presence of methylation.Vertical hatch marks show censored data.

Table 1 .
MSP primers and amplicon information.