Diagnostic Pathology: Open Access
Open Access

Laryngeal Cancer (LC) is a second most common cancer of Head and Neck Cancer (HNC) group which develops from laryngeal squamous cell epithelium. It comprises 40% of all HNC and 1%-2% of all human malignancies [1]. The most important risk factors in LC development are tobacco smoking and excessive alcohol consumption comprising very important synergistic effect [2,3]. In past 20 years there is a slight decrease in the overall incidence of LC due to global anti-smoking campaign especially in USA and European countries. Unfortunately it has no significant influence on overall patient survival which is not markedly improved as there is still a great number of local and regional recurrences, advanced metastases and secondary primary tumors [4]. Patient prognosis depends mostly on staging status at presentation [5-7]. Last few decades it became obvious that chronic inflammation predisposes to different forms of cancer. Pathogens as bacteria and viruses activate innate immune mechanisms which enables their elimination from the organism [8]. Some pathogens can change innate immune mechanisms and lead to the chronic inflammation. Chronic inflammation has protumorigenic effect and enhances the risk of tumorigenesis [9]. Important examples are H. pylori infection and development of gastric carcinoma [10], Hepatitis B and C virus infection and hepatocellular carcinoma [11], Human papilloma virus in development of oropharyngeal carcinoma [12,13]. The profile of expression of different molecules engaged in activation and modulation of immune response and inflammation, their enhanced accumulation and decreased activation determine the balance between protumorigenic and antitumorigenic microenvironment [8]. Cytokines control the inflammation through activation of different signal pathways by inducing protumorigenic immunity (IL-12, TRAIL, IFNγ), tumor development (IL-6, IL-17, IL-23), tumor growth and survival (TRAIL, FasL, TNF-α, EGFR, TGF-β). Generally, we can say that carcinoma is result of the change in signal pathways which participate in the cell proliferation regulation. Key factors in relationship between chronic inflammation and carcinogenesis are transcription factors (NF-κB i STAT3) and proinflammation cytokines (IL-1β, IL-6, IL-23 i TNF-α) [14].

Innate immunity mechanism is the first line of organism defense and it is regulated by germline-encoded Pattern Recognition Receptors (PRRs) expressed by cells, including macrophages, monocytes, dendritic cells, neutrophils and epithelial cells. The most studied PRRs are membrane-bound Toll-Like Receptors (TLRs) which recognize Pathogen-Associated Molecular Patterns (PAMPs) and intracellular NOD-Like Receptors (NLRs) which recognize PAMPs as well as Danger-Associated Molecular Patterns (DAMPs).

Activation of those receptors lead to complex signal pathway activation with resulting transcriptional factors NF-κB i AP-1 activation and synthesis and producing of proinflammatory cytokines and chemokines. Activation of NLRs results in inflammasomes formation which enables inactive to active caspase transformation and pro-IL-1β maturation. NLRs and TLRs pathways are connected so activation of TLRs pathway enhance producing of pro-IL-1β which mature by NLRs signaling pathway activation [15].

There are two genes, ASC/TMS1 (Apoptosis-associated speck like protein containing a caspase recruiting domain/Target of methylation induced silencing-1) gene and MYD88 (Myeloid differentiation primary response 88) gene, which both encode adaptor molecules with important roles in signal pathways activated by organism response to inflammation or injury.

Adaptor molecule ASC is involved in signal pathway characterized by formation of molecular platforms inflammasomes which activate NLRs and control maturation and secretion of proinflammatory cytokines Interleukin-1β (IL-1β) and Interleukin-18 (IL-18).

From all NLRs NLRP3 is currently the most fully characterized in creation of NLRP3 inflammasomes. NLRP3 inflammasomes consists of NLRP3 scaffold, the ASC adaptor molecule and caspase-1. It is activated upon exposure to specific ligands like pathogens, toxins, crystals, PAMPs and DAMPs. Upon NLRP3 activation, NLRP3 oligomerization leads to the PYD domain clustering homotypic interaction with the PYD domain of adaptor molecule ASC, and interaction of the CARD domain of adaptor molecule ASC with CARD domain of the procaspase-1. Procaspase-1 permits auto cleavage and formation of the active caspase-1 which leads to the maturation and secretion of Interleukin-1β (IL-1β) [8,15,16].

From the literature it is known that the mechanisms involved in NLRP3 activation are not well established and still intensely debated but mostly acceptable are three main models. First model explain that extracellular ATP stimulates opening of ATP-dependent ion channel of the cell membrane and in that way pore formation in the cell membrane allows direct NLRP3 inflammasomes activation by NLRP3 cytosol agonists. Second model propose as activators particulate structures (urates, silica, asbestos) which activate phagocytes and by lysosomal damage release lysosomal contents consequently activating NLRP3 inflammasome formation. According to third model all NLRP3 agonists trigger the generation of Reactive Oxygen Species (ROS) and this pathway activate NLRP3 inflammasome formation [17].

On the other hand, adaptor molecule MYD88 has a key role in TLRs signaling pathway and enables signal transduction cascade with final activation of transcription factor NF-κB and secretion of proinflammatory cytokines like Interleukin-6 (IL-6), Tumor Necrosis Factor Alpha (TNF-α) and Interleukin-12 (IL-12). The signal transduction begins by biding adaptor protein MYD88 with intracellular TLR domain, activation of kinase IRAK-4, 2, and 1 (IL- 4,2,1 receptor associated kinase) and TRAF6 (receptor associated factor 6) phosphorylation, which subsequently activate TAK1 (TGF-β activated kinase 1). TAK1 phosphorylates IKKβ leading to degradation of IκB inhibitory protein dissociating it from NF-κB, thus allowing NF- κB to translocate to the nucleus and finally activate transcription [18].

It is well known that some epigenetic mechanisms, like DNA methylation, can change gene expression by methylation of CpG islands in the regulatory regions of the genome. As those islands, rich with CG dinucleotides (>55%) are situated in regulatory gene regions they affect the transcription of genes. If CpG islands are unmethylated genes are expressed and if they are hypermethylated the expression of the genes is repressed (gene silencing). The inflammation induce DNA Methyltransferase (DNMT) dependent DNA methylation which subsequently leads to silencing of the genes associated with carcinogenesis [19].

In this study, we hypothesized that the changes in the methylation status of promoter regions of ASC/TMS1 and MYD88 genes, responsible for activation and regulation of inflammation in healthy and laryngeal cancer tissue, might be related with development and progression of the laryngeal cancer. The aim of our study is to investigate is there a difference in the methylation status of this genes in healthy and tumor tissue and does it correlate with protein expression.

In recent literature the methylation status of this genes has not yet been analyzed in laryngeal cancer and the pyrosequencing method used to determinate methylation status of this genes has not been used in other cancers studies.

In this study, during routine surgical procedure in the treatment of laryngeal cancer, we have collected 36 samples of cancer tissue with related histologically healthy/not tumor involved tissue. All samples of cancer and healthy/not tumor involved tissue were analyzed histopathologically. Cancer tissue was analyzed to determine the value of cancer density and only cancer tissue with >70% of tumor cells was used for further analysis. Adjacent healthy/not tumor involved tissue was analyzed to confirm lack of malignant cells. We started research after applying and receiving approval from Ethics Committee of University Hospital Center Zagreb and all procedures followed were in the accordance with the ethical standards of the Committee. The study was performed in the Department of Otorhinolaryngology and Head and Neck Surgery University Hospital Center Zagreb and all molecular researches were done in the Department for Molecular Medicine, Laboratory for Advanced Genomics Ruđer Bošković Institute in Zagreb. All patients signed informed consent prior to a surgical intervention.

Analysis of DNA methylation by pyrosequencing

The methylation status of both genes promoters was analyzed by pyrosequencing method which as quantitative analysis of DNA CpG methylation enables precise expression of percentage of methylated DNA in the analyzed sample tissue. This method offers a robust, versatile platform with rapid quantitative results without the onerous time commitment, high costs and technical difficulty of alternative methods.

First step was isolation of genomic DNA of interest by using commercially available DNA isolation kit (Qiagen, Hilden, Germany; DNeasy Blood and Tissue Kit). Genomic DNA of interest was submitted to bisulfite conversion in which treating genomic DNA with sodium bisulfite selectively converts cytosine to uracil; however 5-methylcytosine was protected from deamination and the CG sequence was preserved in downstream reactions. Unmethylated cytosine through conversion by deamination into uracil was finally converted into thymine with epigenetic change in DNA sequence. Bisulfite conversion was performed by using commercially available kit (Qiagen, Hilden, Germany; Epitect^® 96 bisulfite kit). DNA sequence for Polymerase Chain Reaction (PCR) primers were acquired from free genetic sequence database GenBank (National Center for Biotechnology Information, NCBI). Identification of MYD88 gene in the database was NG_016964 and of ASC/TMS1 gene was AF184072.1. Primer designing started by determination of promoter DNA sequence of interest of ASC/TMS1 gene and MYD88 gene. After detecting Region Of Interest (ROI) of both genes primers were designed. Primers for PCR of bisulfite converted DNA and pyrosequencing were designed using PyroMark Assay Design Software 2-0 (Qiagen, Hilden; Germany). Following pyrosequencing, pyrograms were analyzed by Pyro Q-CpGTM program (Qiagen, Hilden; Germany). This program can automatically determine individual methylation frequencies for all CpG islands as well as to evaluate the assays quality. Pyrograms which did not satisfy by quality were excluded from the study.

The results of the pyrosequencing of DNA sequence showed percentage of DNA methylation rate in analyzed sequence by using paired t-test (p-values<0.05 were statistically significant) in GraphPad prism 5.0 program.

Extraction of total proteins from laryngeal cancer tissue and healthy/not tumor involved tissue

Proteins from cancer tissue and healthy/not tumor involved tissue were extracted by Passive Lysis 5x Buffer (Promega, Fitchburg, Wisconsin, USA). By Western Blotting procedure particular proteins were identified from the mixture of protein extracted from the samples. By SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) proteins were separated according their electrophoretic mobility. Following separation, proteins were transferred from gel to nitrocellulose membrane using Trans Blot TurboTM Transfer System (BIO-RAD, Hercules, California, USA). Finally, protein of interest were labeled by primary and secondary antibodies and the signal was detected by the method of chemiluminescence using reagent ClarityTM Western ECL Blotting Substrates (BIO-RAD, Hercules, California, USA). In order to quantify detected signal we used free ImageJ 1.49v software. The values of tested proteins (MyD88 and ASC/TMS1) were normalized with the values of control protein (vinkulin) from the adjacent gel. Normalized values were statistically analyzed in GraphPad Prism 5.0 program (GraphPad Software, San Diego, USA) by using 2-way Analysis of Variance (2way ANOVA test) and paired t–test. P-values less than 0,05 were regarded as statistically significant.

The total of 36 patients participated in this study; 33 male patients and 3 female patients. All of them had Squamous Cell Carcinoma (SCC) of the larynx with different grades of differentiation from grade I to grade III. As the most important variable for overall survival of those patients beside differentiation grade is tumor localization, tumor size, and existence or absence of tumor metastasis and in all patients the disease staging was defined using UICC TNM (Union for International Cancer Control Tumor Node Metastasis) classification.

Results of analysis of MYD88 gene methylation status in healthy/not tumor involved and tumor tissue

To accomplish differential methylation analysis of MYD88 gene in healthy/not tumor involved and tumor tissue, the methylation status of CpG islands of gene promoter region was performed (Table 1).

Table 1: The results of methylation status of promoter region of MYD88 gene by pyrosequencing of healthy/not tumor involved and tumor tissue.

According to nucleotide sequence of DNA in MYD88 gene 3 Regions of Interest (ROI) were defined. In region of interest 1 (ROI_1) we detected 4 CpG islands, in Region of Interest 2 (ROI_2) 3 CpG islands, and in Region of Interest 3 (ROI_3) 3 CpG islands. In promoter region of MYD88 gene in ROI_1 in CpG1 island we found that the average methylation status is slightly higher in healthy/not tumor involved tissue than in cancer tissue without statistically significant difference (p>0.05; p=0.276). In CpG2 island we found that the average methylation status is statistically significantly lower in cancer tissue than in healthy/not tumor involved tissue with statistical significance of p=0.0267 (p<0.05). In CpG3 and CpG4 islands of promoter region of MYD88 gene there was no statistically significant difference in methylation status between cancer and healthy/not tumor involved tissue of the larynx with p-values p=0.9803 (p>0.05) and p=0.2048 (p>0.05), respectively. In ROI_2 the methylation status of CpG5 island and CpG6 island showed there was no statistically significant difference between cancer and healthy/not tumor involved tissue of the larynx with p-values p=0.2302 (p>0.05) and p=0.1870 (p>0.05), respectively. In the same ROI_2 in the promoter region of MYD88 gene in CpG7 island we found statistically significant difference in the methylation status between cancer and healthy/not tumor involved tissue. We found that the methylation status of CpG7 in promoter region of MYD88 gene was statistically significantly lower in cancer tissue in relation to healthy/not tumor involved tissue according to p-value, p=0.0174 (p<0.05). In the ROI_3 in CpG8 and CpG9 islands of promoter region of MYD88 gene there was no statistically significant difference in the methylation status between cancer and healthy/not tumor involved tissue with p-values, p=0.1740 (p>0.05) and p=0.1255 (p>0.05), respectively. In the CpG10 island of promoter region of MYD88 gene we found that cancer tissue is statistically significantly hypo methylated in relation to healthy/not tumor involved tissue of the larynx with p-value p=0.0407 (p<0.05).

The purpose of this analysis was to define overall methylation status of the promoter region of the MYD88 gene. The results of this analysis showed there was statistically significant difference in overall methylation status between cancers and healthy/not tumor involved tissue in the promoter region of MYD88 gene. The cancer tissue was statistically significantly hypomethylated in relation to healthy/ not tumor involved tissue of the larynx with p-value p=0.0061. In overall difference in the methylation status significant contribution was obtained in 3 CpG islands: CpG2(p=0.0267), CpG7(p=0.0174), and CpG10(p=0.0407) (Figure 1).

Figure 1: The graph (GraphPad Prism 5.0) showing overall methylation status of CpG islands promoter region of MYD88 gene in healthy/ not tumor involved and tumor tissue. Paired t-test; p ≤ 0.05. Results are shown as mean ± SEM. Note: islands in gene promotor region in which we found statistically significant difference in the methylation status between cancer and healthy/ not tumor involved tissue.

Results of analysis of ASC/TMS1 gene methylation status in healthy/not tumor involved and tumor tissue

To accomplish differential methylation analysis of ASC/TMS1 gene in healthy/not tumor involved and tumor tissue, the methylation status of CpG islands of gene promoter region was performed (Table 2).

Table 2: The results of methylation status of promoter region of ASC/TMS1 gene by pyrosequencing of healthy/not tumor involved and tumor tissue.

According to nucleotide sequence of DNA in gene only 1 Region of Interest (ROI) was defined. In this ROI_1 3 CpG islands were detected. The methylation status of CpG1 island in the promoter region of ASC/ TMS1 gene showed there was no statistically significant difference in the methylation status between healthy/not tumor involved and cancer tissue of the larynx according to p-value p=0.0797 (p>0.05). In the CpG2 and CpG3 islands of the promoter region of the ASC/TMS1 gene there was no statistically significant difference between methylation status of the cancer and healthy/not tumor involved tissue of the larynx with p-values p=0.2665 (p>0.05), and p=0.3526 (p>0.05), respectively.

The results of the analysis of the overall methylation status of CpG islands in the promoter region of ASC/TMS1 gene showed no statistically significant difference between cancer and healthy/not tumor involved tissue of the larynx p=0.1562 (p>0.05) (Figure 2).

Figure 2: The graph (GraphPad Prism 5.0) showing overall methylation status of CpG islands promoter region of ASC/TMS1 gene in healthy/not tumor involved and tumor tissue. Paired t-test; p ≤ 0.05. Results are shown as mean ± SEM.

As promoter region methylation is one of the mechanism of gene expression control the purpose of the analysis on the protein level was to investigate if there is a correlation of promoter gene methylation and protein expression.

Results of analysis of MyD88 protein expression in healthy/ not tumor involved and tumor tissue

To accomplish MyD88 protein expression we used Western blot method applied on 21 samples of healthy/not tumor involved and tumor tissue. The results were statistically analyzed and we found statistically significant difference in MyD88 protein expression between healthy/ not tumor involved and tumor tissue of the larynx with p<0.0001 (Figure 3).

Figure 3: The graph (GraphPad Prism 5.0) showing densitometric quantification of protein MYD88 bends. There is statistically significant difference in MyD88 protein expression between healthy/not tumor involved and tumor tissue. The values are shown as mean value ± SEM

Results of analysis of ASC/TMS1 protein expression in healthy/not tumor involved and tumor tissue

ASC/TMS1 protein expression was analyzed by Western blot method applied on 23 samples of healthy/not tumor involved and tumor tissue. The results were statistically analyzed and we found statistically significant difference in MyD88 protein expression between healthy/ not tumor involved and tumor tissue of the larynx with p=0.0157 (Figure 4).

Figure 4: The graph (GraphPad Prism 5.0) showing densitometric quantification of protein ASC/TMS1 bends. There is statistically significant difference in ASC/TMS1 protein expression between healthy/not tumor involved and tumor tissue. The values are shown as mean value ± SEM.

Head and Neck Squamous Cell Carcinoma (HNSCC) which comprise laryngeal carcinoma is frequent cancer with unfortunately increasing rate of incidence [20]. According to our knowledge based on diagnosis and treatment of this cancer we still need development of molecular targets, signaling molecular pathways and reliable diagnostic modality in discovering early disease state. In most European countries according to the data of World Health Organization (WHO) the incidence of laryngeal cancer is decreasing in last two decades [21]. A slight decrease in the overall incidence of HNSCC comprising laryngeal cancer has been detected mostly due to global anti-smoking and anti- alcohol consumption campaign [22]. The relationship between chronic inflammation, innate immunity and carcinogenesis is not new and it is widely recognized but most of the molecular mechanisms involved in this relationship are still not revealed. Oncogenes associated with different molecular patterns and functions led to proinflammatory response and inflammation following infection is associated with carcinogenesis [21]. Chronic inflammation triggers increase the risk of cancer formation and development like infection (Helicobacter pylori for gastric cancer and gastric mucosal lymphoma, Human papilloma virus for cervical and oropharyngeal cancer, Hepatitis B/C virus for hepatocellular cancer), immune deregulation and autoimmune diseases and unknown origin infections [23]. The key mechanisms include transcription factors activation by the activation of the receptors of innate immune system resulting in proinflammatory cytokines secretion. Accordingly, we can say that molecular pathways related to cancer and inflammation are crucial factors in carcinogenesis and genetic instability of important molecules in this process are related to cancer development. It is the reason why molecular pathways related to cancer and inflammation present the aim of research of new strategies for cancer treatment and prevention. The innate immunity system is very important part of immune system and comprises mechanisms to defend organism from dangerous signals like pathogenic microbes or cellular stress signals. The innate immunity response is based on activation of an array of germline-encoded Pattern-Recognition Receptors (PRRs) and by ligand activation of those receptors the cells which expressed receptors are activated. It is well known that innate immunity receptors play a role in cancerogenesis and metastatic cancer as chronic inflammation is induced by innate immunity system and stimulates occurrence and development of cancer [24]. The important role in activation and regulation of activation of innate immunity receptors have adaptor molecules of signal pathways. In our study we investigated the correlation of methylation status and expression of MYD88 gene and ASC/TMS1 gene, adaptor molecules, in TLR and NLR signal pathways and we wanted to search if the methylation status of this genes can be used as potential prognostic biomarker. We hypothesized that changed methylation status of promoter regions of genes involved in activation and regulation of the inflammation, in healthy/not tumor involved and cancer tissue of the larynx, can be related with occurrence and progression of laryngeal cancer. We think that results of our research represent important contribution in investigation of adaptor molecules methylation status as yet in the literature there is no data considering MYD88 gene methylation status in cancer. Also, methodology used in this research is innovative as we used in our analysis of the methylation status the pyrosequencing method which is the “golden standard” method in methylation status determination. All results of ASC/TMS1 gene methylation status in recent literature were defined only by methylation-specific PCR.

The results of this study confirmed that there is significant difference in the methylation status of the promoter region of CpG islands of MYD88 gene between healthy/not tumor involved and cancer tissue of the larynx. Precisely, the results showed that the cancer tissue of the larynx is statistically significantly hypomethylated according to the healthy/not tumor involved tissue. Moreover, results of this research indicated that the overall rate of methylation status of the promoter region of MYD88 gene is statistically significantly reduced in cancer tissue according to healthy/not tumor involved tissue. Furthermore, MyD88 protein expression level, explored to define the correlation between methylation status and protein expression, strongly confirmed the correlation between methylation and expression. According to the results, reduced methylation of the promoter region of MYD88 gene in DNA isolated from cancer tissue corresponds to higher expression of this protein in cancer tissue as opposite to healthy/not tumor involved tissue higher methylation corresponds to reduced expression of MyD88 protein. In recent literature only Lu et al. studied the expression of MyD88 adaptor molecule and clinical impact of this protein expression to laryngeal cancer. According to the author, there is 68.6% of positive expression of MyD88 protein in laryngeal cancer tissue which is significantly higher in relation to healthy surrounding tissue where expression is 11.8%. Positive expression correlates with stage of the disease and occurrence of lymph node metastasis which is inversely proportional to 5 year survival rate. MyD88 can be important factor in development of laryngeal cancer as in future target cancer therapy may improve prognosis of patients with this cancer [25]. Rakoff-Nahoum, et al. showed that adaptor molecule MyD88 has a critical role in spontaneous tumor development in mice with heterozygous mutation in gene APC (Adenomatous Polyposis Coli). Adaptor molecule MyD88- dependent signaling pathway controls expression of main modifying genes in intestinal tumorigenesis and has a crucial role in both spontaneous and carcinogen-induced tumors [26]. Similar results were shown by Naugler, et al. on the model of Hepatocellular Carcinoma (HCC) in mouse. The author showed that Diethylnitrosamine (DEN) induced Hepatocellular Carcinoma (HCC) is associated with increased serum Interleukin-6 (IL-6) concentration whose production is largely dependent on MyD88 signaling pathway [27]. Chefetz, et al. demonstrated that during surgical debunking or chemotherapy in ovarian cancer, cellular debris released during the process of injury and cell death may activate the TLR2-MyD88-NFκB pathway in a specific cancer cell population, the EOC stem cells, leading to tumor repair and hence disease recurrence [28]. Higgins, et al. analyzed TLR-driven inflammation in a mouse model for stage IV breast cancer. The author found that High Mobility Group Box 1 protein (HMGB1) was produced by 4T1 in a Myd88-dependent manner and that neutralizing extracellular HMGB1 resulted in 50% decrease in growth of the tumor cells [29]. Unlike other authors, Kinowaki, et al. reported that tumor progression is associated with reduced expression of MyD88 in Hepatocellular Carcinoma (HCC) [30]. Pyrosequencing results with the aim of determining the methylation status of promoter region of MYD88 gene showed the difference in overall level of methylation status between healthy/not tumor involved tissue and cancer tissue. The overall difference level is statistically significant and three CpG islands highly contribute to this difference; CpG2, CpG7, and CpG10. The other tested CpG islands did not expressed the difference in the overall methylation level between healthy/not tumor involved and cancer tissue. Our results are significant as for the first time according to the literature the methylation status of promoter region of MYD88 gene between healthy/not tumor involved and cancer tissue was analyzed. The result revealed statistically significant difference in the methylation status, MYD88 gene is hypomethylated in cancer tissue, and the results correlates with protein expression. Our results also showed there is a pattern of methylation of promoter region of MYD88 gene as only CpG2, CpG7, and CpG10 islands contributed to the result. If there is tissue specific methylation status of CpG islands for the same gene, than, in the future, according to this model, we can be able to differentiate second primary lung cancer from solitary pulmonary metastasis in laryngeal cancer. It can be a diagnostic problem as patients suffering from laryngeal cancer have a great risk to develop primary lung cancer due to extensive tobacco smoking, or to develop solitary pulmonary metastasis which can sometimes comprise difficulties in obtaining satisfactory biopsy. The results of the methylation status of promotor region of the ASC/TMS1 gene showed no statistically significant difference in the methylation status of different CpG islands between healthy/not tumor involved and cancer tissue. In addition, there is no significant difference in overall methylation status between healthy/not tumor involved and cancer tissue. However, according to our results there is significant difference in expression of ASC/TMS1 gene between healthy/not tumor involved and cancer tissue, expression of protein ASC is significantly higher in cancer tissue. Adaptor molecule ASC/TMS1 plays important role in proinflammatory signaling pathway and cancer related inflammation. Many authors analyzed ASC/TMS1 gene expression in diver’s cancers but the methylation status of this gene with correlation of the expression between healthy/not tumor involved and laryngeal cancer tissue was not yet published. Salaminen, et al. examined the mechanisms involved in the epigenetic regulation of ASC/TMS1 as well as their significance in the coordination of apoptosis and inflammasomes functions [31]. Hong, et al. demonstrated that restoration of ASC expression in human colorectal cancer DLD-1 cells, in which ASC/TMS1 gene is silenced by aberrant methylation, potentiated cell death mediated by DNA damaging agent [32]. According to Virmani, et al. methylation of ASC/TMS1 gene correlated inversely with expression in the pathogenesis of small cell and non- small lung and breast cancers [33]. Xiaofang, et al. analyzed methylation Diagn Pathol Open, an open access journal status of tumor suppressor genes, including ASC/TMS1, in cholangiocarcinoma. The authors revealed that the methylation of DAPK, p14ARF, and ASC/TMS in cholangiocarcinoma is a common event. Furthermore, p53 mutation combined with DAPK, p14ARF, and/or ASC methylation correlates with malignancy and poor prognosis [34]. Unlike former authors Tamandani, et al. described the methylation status of TMS1/ASC and CASP8 genes in cervical cancer. In this study the authors concluded that the frequency of TMS1/ASC and CASP8 genes methylation in cervical cancer is rare (<6%), and plays no critical role in development of cervical cancer [35]. Mirza, et al. observed significant correlation between promoter methylation of ASC/TMS1 gene and loss of protein expression in breast cancer confirming that promoter methylation is an important mechanism for transcriptional silencing of these genes in breast cancer [36]. Deswaerte, et al. revealed a protumorigenic role in gastric cancer for the key inflammasomes adaptor ASC/TMS1 and its effector cytokine IL18. Genetic ablation of ASC/TMS1 in the spontaneous mouse model of intestinal-type gastric cancer suppressed tumorigenesis by augmenting caspase-8-like apoptosis in the gastric epithelium, independently from effects on myeloid cells and mucosal inflammation. The authors concluded that inflammasomes activation that elevates IL18 helps drive gastric cancer by protecting cancer cells against apoptosis, with potential implications for new therapeutic strategies in this setting [37]. Machida, et al. studied proapoptotic ASC/TMS1 gene in lung cancer and used the findings to develop a sputum marker. The authors concluded that hypermethylation of ASC/TMS1 gene is a potential marker for late-stage lung cancer and, in sputum, could predict prognosis in patients resected for early-stage disease [38].

Results of the analysis of the methylation status of ASC/TMS1 gene in our study are different related to results from published articles. According to our results there is no statistically significant difference in the methylation status between healthy/not tumor involved and cancer tissue. This can be explained by the fact that in this study the methylation status was determined using pyrosequencing method which is considerably more precise than commonly used methods, like methylation-specific PCR.

In addition, in this study there is no correlation between methylation status of ASC/TMS1 gene and protein expression which can indicate that protein expression undergoes alternative post-transcriptional mechanisms of regulation.

The main contribution of the present study is in determination of the methylation status of MYD88 gene and ASC/TMS1 gene by pyrosequencing method which is still considered to be the "golden standard" in methylation status analysis. Furthermore, we found statistically significant difference in the methylation status of MYD88 gene between healthy/not tumor involved and laryngeal cancer tissue. We also showed that regarding gene/protein MYD88 there is a positive correlation between methylation status and gene expression- hypermethylation correlates with reduced protein expression. It is worth mentioning that regarding gene/protein ASC/TMS1 there is no positive correlation of methylation status and protein expression. In fact, our results showed there is no statistically significant difference in gene methylation status but there is significantly higher protein expression in laryngeal cancer tissue. The pattern of methylation which comprises the tissue specific methylation status indicates potential biomarkers in differential diagnosis of Head and Neck and lung squamous cell carcinoma.

Ethics approval and consent to participate

This study was approved by Ethics Committee of University Hospital Center Zagreb and all procedures followed were in the accordance with the ethical standards of the Committee. All patients signed informed consent prior to a surgical intervention.

Consent for publication

All participants in this study signed informed consent to participate in the study and to publish their data.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

No funding was obtained for the study.

Authors' contributions

All persons designated as authors have participated sufficiently in the work to take public responsibility for the content of the manuscript. Associate professor Lana Kovač Bilić and professor Mario Bilić are mainly responsable for conception and design of this study. They have both participated in all surgical procedures and literature examination. All analysis and data interpretation were done by Jelena Knežević, PhD and biologist Maja Šutić from Department for Molecular Medicine, Laboratory for Advanced Genomics Ruđer Bošković Institute. Important intellectual content and final approval of the version was done by associate professor Krsto Dawidowsky and professor Srećko Branica.

Acknowledgement

Not applicable

Conflict of interest

All authors declare that they have no conflict of interest.

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