Oral Oncology xxx (2014) xxx–xxx
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Review
Methylation as a biomarker for head and neck cancer L.M.R.B. Arantes a,b, A.C. de Carvalho b, M.E. Melendez b, A.L. Carvalho b, E.M. Goloni-Bertollo a,⇑ a b
Department of Molecular Biology, São José do Rio Preto Medical School (FAMERP), São José do Rio Preto – SP, Brazil Center for Research in Molecular Oncology, Barretos Cancer Hospital – Pio XII, Barretos – SP, Brazil
a r t i c l e
i n f o
Article history: Received 26 November 2013 Received in revised form 13 February 2014 Accepted 20 February 2014 Available online xxxx Keywords: Head and neck cancer Biomarkers DNA methylation Serum Plasma Exfoliated cells Saliva
s u m m a r y Head and neck cancer is a collective term that describes malignant tumors of the oral cavity, pharynx, and larynx characterized by high incidence and mortality rates. Although most HNSCC originate from the mucosal surface of the upper aerodigestive tract, where they can be easily detected during a routine clinical examination. Often the definitive diagnosis is delayed because of the difficulty in differentiating from other similar lesions. Activation of proto-oncogenes and inactivation of tumor suppressor genes are the major molecular alterations involved in carcinogenesis. In addition, epigenetic changes can alter the expression of critical genes important in the development of a variety of cancers. The detection of aberrant gene promoter methylation as a tool for the detection of tumors or its use as prognostic marker have been described for many different cancers including HNSCC. The search for biomarkers has as its main aim the evaluation and measurement of the status of normal and pathological biological processes as well as pharmacological responses to certain treatments. The tracking of these biomarkers is an important part for the identification of individuals in the early stages of head and neck cancer for its diagnostic and prognostic relevance reflecting in high survival rates, better quality of life and less cost to the healthcare system. Therefore, assuming that cancer results from genetic and epigenetic changes, analyzes based on gene methylation profile in combination with the pathological diagnosis would be useful in predicting the behavior of these head and neck tumors. Ó 2014 Published by Elsevier Ltd.
Introduction Head and neck cancer is a collective term defined by topographical and anatomical bases to describe malignant tumors of the upper aerodigestive tract. It is a disease with high incidence and mortality affecting mainly the oral cavity (lips, hard palate, tongue, gums and floor of mouth), pharynx (naso-, oro- and hypopharynx) and larynx. It is considered to be the fifth most common cancer site worldwide, and is associated with low survival and high mortality rates, when diagnosed in advanced stages. The World Health Organization (WHO) estimated for 2012, approximately 685,000 new cases, with the oral cavity as the most frequent site with 300,200 cases, followed by the pharynx with 228,800 and the larynx with 156,800 [1]. The WHO defines as pre-neoplastic lesions (or precancerous) tissue changes that may undergo neoplastic transformation more frequently than normal tissue, but can also remain stable and even undergo regression, especially if the irritant factor is removed [2]. ⇑ Corresponding author. Address: UPGEM, FAMERP (bloco U6), Avenida Brigadeiro Faria Lima, no. 5416, São José do Rio Preto – SP, CEP: 15.090-000, Brazil. Tel.: +55 17 3201 5720; fax: +55 17 3201 5708. E-mail address: [email protected] (E.M. Goloni-Bertollo).
The main histologic type is the squamous cell carcinoma (SCC) that develops mainly from a field cancerization and corresponds to around 90% of cases [3]. The factors promoting this tumor type include environmental exposure to tobacco and alcohol. But also, viral infections, especially by Epstein–Barr Virus and Human Papilloma Virus subtypes 16 and 18, and deficiencies or imbalances of vitamins and micronutrients, such as folic acid, vitamins A, C and E, zinc and selenium are related to carcinogenesis of SCC [4–6]. A careful analysis of clinical signs and symptoms obtained by history and physical examination, and their appropriate interpretation is essential for the diagnosis of any disease. Patients with early stage HNSCC have vague symptoms and minimal physical changes. The physical presentation varies with the primary site involved. Patients with tumors in the oral cavity usually present ulcers that do not heal and pain, while the oropharyngeal involvement results in persistent sensitivity, chronic dysphagia or odynophagia for more than 6 weeks [7]. Despite the different strategies used in the treatment of head and neck cancer, the overall survival rate is around 50%, [8] and the main reason for treatment failure is the frequent development of loco-regional recurrences, which affects around 30% of HNSCC patients [9]. The theory of field cancerization, by Slaughter et al. [10] proposes
http://dx.doi.org/10.1016/j.oraloncology.2014.02.015 1368-8375/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Arantes LMRB et al. Methylation as a biomarker for head and neck cancer. Oral Oncol (2014), http://dx.doi.org/10.1016/ j.oraloncology.2014.02.015
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that, although the epithelium has a normal appearance, there may be changes in the mucosa. These altered mucosa is frequently found in surgical margins after the tumor is resected, remaining in the patient and contributing to the high frequency of local recurrence and second primary tumors development in patients with HNSCC. Although most of HNSCC originate from the mucosal surface of the oral cavity, where they can be easily detected during a routine clinical examination, the definitive diagnosis will not happen quickly, due to difficulties in differentiating from other similar lesions. Thus, early detection of this tumor type is of paramount importance, requiring highly sensitive and specific tests [11].
Epigenetic changes: DNA methylation Cancer cells have unlimited replicative potential, self-sufficiency in growth factors, insensitivity to anti-growth signals and apoptosis, capacity of invasion, metastasis and angiogenesis. The process of carcinogenesis is comprised of multiple steps in which several genetic (punctual mutations, loss of heterozygosity, deletions, insertions, aneuploidy, gene amplification, among others) and epigenetic changes are progressively accumulated [12,13]. Epigenetic changes are inheritable and reversible, affecting the spatial conformation of DNA and its transcriptional activity, thus ensuring the maintenance of stability and integrity of the DNA, leading to changes in chromatin structure. This process leads to changes in the phenotype without changing the sequence of DNA bases, allowing the generation of diverse forms of expression and resulting in different phenotypes. Epigenetic changes occur during development of the organism, and are reproduced during DNA replication [14,15]. There are several known epigenetic mechanisms, which include DNA methylation (addition of methyl groups – CH3), changes in chromatin conformation, histone modification and post transcriptional modifications that may influence gene expression. It is important to remember that the interaction between epigenetic factors with the observed result is always a result of the sum of its interactions with the various mechanisms of positive and negative feedback [15]. One of the main epigenetic changes that occur in mammals is DNA methylation, which is the main focus of this report. This is a biological process consisting in adding a methyl group (CH3) on the carbon 5 of nitrogenous bases cytosines (C) located 5’ of guanines (G). The primary targets of this process are CpG islands, regions rich in ‘‘CG’’ dinucleotides mainly present in the promoter region of certain genes. The presence of aberrant methylation in the promoter region of genes is associated with the suppression of gene expression [14–17]. In normal cells, methylation is an important event in the control of gene expression occurring at predetermined sites and contributing to mechanisms such as the inactivation of one X chromosome in females and the mechanism of genomic imprinting in which there is the inactivation of specific alleles according to parental inheritance, leading to a monoallelic expression [15]. This process involves an enzymatic reaction catalyzed by a family of enzymes named DNA methyltransferases (DNMTs). Three subtypes of DNMT have been identified: DNMT1, DNMT3A and DNMT3B. While DNMT1 is responsible for maintaining the methylation pattern in the daughter cells during mitosis or meiosis, DNMT3A and DNMT3B are involved in the de novo methylation process, during genomic imprinting. During embryogenesis and depending on the gender of the newly formed germ cells, de novo methylation occurs in paternal or maternal loci. The possible mechanisms which initiate the de novo methylation are: DNA target sequences, interference RNA, chromatin changes induced by histones and protein interactions [18–20].
DNA methylation and cancer The pattern of DNA methylation is cell-specific, and determined during the embryonic development of organisms. Changes in the methylation profile have been found in all types of cancer, in developmental disorders and in cellular aging. Several methods have been developed to determine the rate of DNA methylation, some provide information about the DNA methylation profile, from the methylation status of specific genes to the methylation content of the whole genome, known as global methylation [16,21]. Activation of proto-oncogenes and inactivation of tumor suppressor genes are the major genetic alterations involved in carcinogenesis. In addition, epigenetic changes (i.e., altered patterns of gene expression that do not affect the primary sequence of the DNA) can alter the expression of critical genes important in the development of a variety of cancers [22]. Studies show that cancer cells are associated with global DNA hypomethylation accompanied by the hypermethylation of CpG islands in the promoter region of genes involved in DNA damage repair, detoxification, cell cycle regulation and apoptosis [23,24]. Increased methylation in the promoter region of a tumor suppressor gene can lead to a progressive reduction of its expression, resulting in its silencing and the selection of cells with proliferative advantage. Analyzes carried out on DNA methylation patterns show that this process can be used as biomarker for early diagnosis as well as for classification, prognosis, and therapy of human cancers. The detection of aberrant gene promoter methylation has been considered a potential molecular marker for several tumor types [25–27]. It is noteworthy that the methylation profile of gene promoters is different for each type of tumor, allowing the identification of patterns of tumor-specific hypermethylation [28]. The evaluation of the methylation profile as a tool for the detection of tumors or their use as prognostic factors has been described for many different cancers such as bladder cancer, [29] colorectal, [30] lung, [31] and HNSCC [32]. Gene-specific DNA methylation and head and neck cancer Tumor suppressor genes CDKN2A/p16, CDH1, DAPK and MGMT were described as hypermethylated in larynx and hypopharynx [33] and genes AIM1, APC, CDH1 and UCHL1 in nasopharyngeal carcinoma [34]. Several analyzes of genes involved in oral carcinogenesis, indicated a higher frequency of hypermethylation at the promoter region of the gene CDKN2A/p16 in primary HNSCC [35–37]. Hypermethylation in the promoter region of SEMA3B was found in oral squamous cell carcinoma tissues, and a significant difference in the frequency of methylation of this gene was observed between oral squamous cell carcinoma and non-cancerous tissues [38]. Hwang et al. [39] reported that the expression of the GLT8D1 and C6orf136 genes, which methylation status may be regulated by the aberrant upregulation of the FOXM1 gene, were differentially expressed probably due to their promoter methylation, on independent HNSCC patient cohorts from Norway and the United Kingdom. Methylation in precursor lesions In head and neck tumors, as well as in other tumors, methylation is a frequent event in the early stages of carcinogenesis and can be detected in precursor lesions. Schussel et al. [40] demonstrated that HOXA9, EDNRB, and DCC methylation was associated with premalignant or malignant disease in saliva of a high-risk population, comparing well with
Please cite this article in press as: Arantes LMRB et al. Methylation as a biomarker for head and neck cancer. Oral Oncol (2014), http://dx.doi.org/10.1016/ j.oraloncology.2014.02.015
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examinations of an expert clinician in clinical risk classification of oral lesions. Methylation in advanced disease Hypermethylation appears to correlate with the severity of the disease and the potential to metastasize [24,41,42]. A significant correlation between the methylation profile of specific genes and clinical aspects of HNSCC patients has been shown. For instance, aberrant methylation of DAPK is correlated with advanced stage and positive lymph nodes at diagnosis [43]. The hypermethylation of DCC in squamous cell carcinoma of the oral cavity is correlated with bone invasion and worse overall survival, while hypermethylation of MINT31 was described as a predictor of poor prognosis [44]. It was also observed that hypermethylation of both galanin and galanin receptor 2 (GALR2) genes was statistically correlated with a decrease in disease-free survival in HNSCC patients [40,45,46]. Methylation and HNSCC biological features The literature also correlates the hypermethylation profile of genes DAPK, DCC and MINT31 with histological and biological features of oral carcinomas. An evaluation of the hypermethylation present in the promoter region of nine genes in oral cancer showed a strong link between clinic-pathological aspects and the ‘‘status’’ of methylation of at least six of these genes. This statement could be illustrated by the strong correlation observed between the consumption of tobacco and/or alcohol and the presence of hypermethylation in the promoter region of the tumor suppressor gene CDKN2A/p14 and CDKN2A/p16 [47]. Recently, Weiss et al. [48] showed that the methylation frequency of TCF21 gene is significantly higher in HNSCC than in benign tonsil controls, in tumors of patients without nicotine abuse, HPV-positive tumors and in tumors exhibiting over-expression of CDKN2A/p16. Pierini et al. [49] analyzed the promoter methylation status of CDKN2A/p16, MGMT, MLH1, and DAPK genes, using methylationsensitive high resolution melting in 100 patients with laryngeal squamous cell carcinoma, finding that the prevalence of promoter methylation was 60.8%, 47.4%, 46.4% and 42.3%, respectively. Moreover, authors found significantly increased methylation of CDKN2A/p16 in heavy smokers and epigenetic inactivation of the same gene and MLH1 were associated with lymph node involvement. Finally, the authors described that an inverse correlation was present between MLH1 methylation and alcohol consumption. Additional tissue source of samples for DNA methylation detection Serum/plasma Several studies evaluating the presence of hypermethylated DNA in body fluids have been performed [35,43,50,51]. In serum of patients who developed tumors, the presence of circulating DNA from tumor cells was identified. The mechanism of serum DNA release has not been fully clarified. Sidransky et al. [52] suggested that it might occur by two ways: the first one suggests that the circulating DNA is derived from apoptotic neoplastic corpuscles and the other that this DNA originates from viable tumor cells, not capable of generating metastases, present in the bloodstream. The analysis of the serum of patients with HNSCC is a promising method, since it is less invasive and can present a considerable prognostic value. Sanchez-Cespedes et al. [43] examined the methylation profile in a panel of genes in serum samples from
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HNSCC patients and demonstrated that the analysis of the hypermethylation profile in serum can be a very effective method for monitoring patients. CDH1, CDKN2B/p15, CDKN2A/p16 and DAPK were described as being hypermethylated in plasma samples of patients with carcinoma of the nasopharynx, and even after treatment, the aberrant methylation could still be detected, demonstrating that this analysis could be used as a serological tumor marker for early detection of locoregional recurrence [53]. Saliva Rosas et al. [35], demonstrated for the first time the possibility to detect hypermethylation in saliva. This study evaluated the presence of hypermethylation in the promoter region of genes involved in tumor suppression, tumor metastasis and DNA repair such as CDKN2A/p16, MGMT and DAPK. Paired tumor and saliva samples of HNSCC patients were evaluated in order to verify whether the changes found in tumors could also be detected in saliva, since this body fluid contains cells from the oral mucosa and pharynx. The hypermethylation rate was of 56% for HNSCC, and in 55% of these hypermethylated samples it was also possible to detect hypermethylation in paired tumor and saliva. Likewise, Righini et al. [54], evaluated paired tumor and saliva samples collected at diagnosis and identified a panel of 6 genes (CDH1, CDKN2A/p16, DAPK, MGMT, RASSF1A and TIMP3) with frequencies of hypermethylation of 82% and 78%, respectively. Carvalho et al. [55], were able to confirm an elevated rate of promoter hypermethylation detected in HNSCC patient salivary rinses by using a panel of gene promoters previously described as methylated in HNSCC but not in control subjects, by the same group [8]. In addition, detection of hypermethylation in pretreatment saliva DNA was associated with local recurrence [55]. Salivary rinses collected from HNSCC patients were also evaluated by Rettori et al. [56] that found a significant association between TIMP3 methylation in HNSCC samples collected 6 months after the last curative treatment and a lower local recurrence-free survival. The multivariate analysis confirmed for the first time, TIMP3 promoter hypermethylation in post-treatment salivary rinse as an independent prognostic marker for local recurrence-free survival in patients with HNSCC. These findings justify the use of DNA hypermethylation detection in saliva as a tool for identifying and monitoring subgroups of HNSCC patients subgroups with high risk of developing local recurrence. Exfoliated cells Exfoliating techniques have the advantage of being minimally invasive and of not requiring local anesthetic. The use of an exfoliating brush allows sampling of the full thickness of stratified squamous epithelium of the oral mucosa. In light of these considerations, biomarkers based on the methylation profile evaluation in exfoliated mucosa cell samples could represent a powerful class of minimally invasive markers for HNSCC detection [57]. Longo et al. [57] demonstrated that exfoliated tumor cells collected from patients with HNSCC could be used to access their methylation profile. In this work, authors showed that CCNA1 (60.4%), DCC (54.2%), and TIMP3 (35.4%) were frequently methylated in the 96 HNSCC patient cohort. Moreover, their data provided evidence that hypermethylation of DCC could be useful as a prognostic indicator for this malignancy. Surgical margins Local recurrences and/or multiple primary tumors develop despite an apparently complete excision and histopathologically
Please cite this article in press as: Arantes LMRB et al. Methylation as a biomarker for head and neck cancer. Oral Oncol (2014), http://dx.doi.org/10.1016/ j.oraloncology.2014.02.015
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Table 1 Statistically significant genes associated with diagnosis and/or prognosis for HNSCC classified by their clinical use and tissue source. Gene
Function
Clinical use
Source
References
ALDH3A1 CCNA1
Cell detoxification Cell cycle control
Prognostic Prognostic
CDH1 CDKN2A/p16
Adhesion glycoprotein Receptor of sonic hedgehog
CDKN2B/p15 DAPK
Inhibitor of CDK4 kinase Programmed cell death
Prognostic Diagnostic Prognostic Diagnostic Diagnostic Prognostic
DCC
Receptor for netrin required for axon guidance
EDNRB
G protein-coupled receptor
Prognostic Diagnostic Prognostic Diagnostic
ERCC1 ESR1 FANCC FHIT GALR1 GALR2 HIC1 HOTAIR KIF1A LKB1 MGMT MLH1 PTCH1 RARb2 RASSF4 RASSF5 RUNX3 SEMA3B SPARC TAP1 TCF21 TIMP3
Nuclear hormone receptor DNA repair protein Purine metabolismo Receptor for the hormone galanin Receptor for the hormone galanin and for GALP Unknown Transcriptional repressor Pattern formation in the embryo Protein kinase involved in apoptosis and DNA damage response DNA repair protein DNA mismatch repair system RNA binding Retinoic acid receptor Undetermined Regulation of lymphocyte adhesion and suppression of cell growth Transcription factor Axonal guidance during neuronal development Matrix-associated protein Membrane-associated pump Transcription factor Inhibitor of the matrix metalloproteinases T-Cell antigen receptor, recognition of foreign antigens
Prognostic Prognostic Diagnostic and prognostic Prognostic Prognostic Prognostic Prognostic Prognostic Diagnostic Prognostic Prognostic Prognostic Diagnostic and prognostic Prognostic Prognostic Prognostic Prognostic Diagnostic Diagnostic and prognostic Prognostic Diagnostic Prognostic
TRG
Cell detoxification
Prognostic
Tissue Surgical margin Tissue Tissue Tissue Surgical margin Plasma Plasma Tissue and serum Tissue Surgical margin Surgical margin Saliva Exfoliated Saliva Saliva and tissue Saliva Tissue Tissue Tissue Tissue Tissue Tissue Tissue Saliva and tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Saliva Tissue Tissue
[63] [59] [64] [41,65,66] [32,49,59], [67,68] [69] [70] [70] [43] [32] [58] [59] [40] [57] [40,71] [60] [72] [73] [74] [75] [76] [45] [73] [77] [78] [79] [80] [49] [74] [81] [42] [42] [82] [38] [83] [63] [48] [56,84] [41,64] [42]
tumor-free surgical margins. This raised the question of molecular alterations existence in negative surgical margins, which may not be detectable by conventional microscopic histopathologic analysis, but could be detected with molecular analyzes for genetic alterations associated with carcinogenesis [58]. The prognosis of patients with non-informative tumors or positive surgical margins justify the monitoring of these cases, with recommendation for adjuvant therapy, even in the absence of other risk factors such as advanced stage, perineural invasion and perivascular invasion. However, the assessment of the hypermethylation profile of the surgical margins can serve as an additional strategy for more sensitive and appropriate treatment, which can minimize morbidity [59]. Tan et al. [59] proposed that the presence of hypermethylated promoters in surgical margins of HNSCC can predict local recurrences and disease-specific deaths, by performing a panel of three genes (CDKN2A/p16, CCNA1 and DCC). Methylation analyzes of their resection margins correctly predicted all the recurrences in their cohort of patients. Also, the presence of DAPK promoter hypermethylation detected in histologically negative surgical margins of oral squamous cell carcinoma patients was significantly associated with the decrease in overall survival, showing the feasibility of this molecular marker for prognostic [58].
early disease detection, treatment selection and prognostication, especially in HNSCC [8,60–62]. A systematic search in the PUBMED database using the terms: methylation AND head neck was performed in order to identify studies reporting genes in which the detection of hypermethylation on their promoter region showed a statistical significant association for their use as biomarker for diagnosis and/or prognosis. Table 1 summarizes these results for these genes classified by their clinical use and tissue source.
Conclusion The search for biomarkers has as its main aim to evaluate and measure the status of normal and pathological biological processes as well as pharmacological responses to certain treatments. The tracking of these biomarkers is an important step for the identification of individuals in the early stages of head and neck cancer for its diagnostic and prognostic relevance. Therefore, assuming that cancer results from genetic and epigenetic changes, analyzes based on the methylation profile in combination with the pathological diagnosis would be useful in predicting the behavior of these head and neck cancers.
Methylation as biomarkers Conflict of interest statement Publications have shown that DNA methylation is an early event and new efforts are focused on identifying markers of
None declared.
Please cite this article in press as: Arantes LMRB et al. Methylation as a biomarker for head and neck cancer. Oral Oncol (2014), http://dx.doi.org/10.1016/ j.oraloncology.2014.02.015
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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Review
Methylation as a biomarker for head and neck cancer L.M.R.B. Arantes a,b, A.C. de Carvalho b, M.E. Melendez b, A.L. Carvalho b, E.M. Goloni-Bertollo a,⇑ a b
Department of Molecular Biology, São José do Rio Preto Medical School (FAMERP), São José do Rio Preto – SP, Brazil Center for Research in Molecular Oncology, Barretos Cancer Hospital – Pio XII, Barretos – SP, Brazil
a r t i c l e
i n f o
Article history: Received 26 November 2013 Received in revised form 13 February 2014 Accepted 20 February 2014 Available online xxxx Keywords: Head and neck cancer Biomarkers DNA methylation Serum Plasma Exfoliated cells Saliva
s u m m a r y Head and neck cancer is a collective term that describes malignant tumors of the oral cavity, pharynx, and larynx characterized by high incidence and mortality rates. Although most HNSCC originate from the mucosal surface of the upper aerodigestive tract, where they can be easily detected during a routine clinical examination. Often the definitive diagnosis is delayed because of the difficulty in differentiating from other similar lesions. Activation of proto-oncogenes and inactivation of tumor suppressor genes are the major molecular alterations involved in carcinogenesis. In addition, epigenetic changes can alter the expression of critical genes important in the development of a variety of cancers. The detection of aberrant gene promoter methylation as a tool for the detection of tumors or its use as prognostic marker have been described for many different cancers including HNSCC. The search for biomarkers has as its main aim the evaluation and measurement of the status of normal and pathological biological processes as well as pharmacological responses to certain treatments. The tracking of these biomarkers is an important part for the identification of individuals in the early stages of head and neck cancer for its diagnostic and prognostic relevance reflecting in high survival rates, better quality of life and less cost to the healthcare system. Therefore, assuming that cancer results from genetic and epigenetic changes, analyzes based on gene methylation profile in combination with the pathological diagnosis would be useful in predicting the behavior of these head and neck tumors. Ó 2014 Published by Elsevier Ltd.
Introduction Head and neck cancer is a collective term defined by topographical and anatomical bases to describe malignant tumors of the upper aerodigestive tract. It is a disease with high incidence and mortality affecting mainly the oral cavity (lips, hard palate, tongue, gums and floor of mouth), pharynx (naso-, oro- and hypopharynx) and larynx. It is considered to be the fifth most common cancer site worldwide, and is associated with low survival and high mortality rates, when diagnosed in advanced stages. The World Health Organization (WHO) estimated for 2012, approximately 685,000 new cases, with the oral cavity as the most frequent site with 300,200 cases, followed by the pharynx with 228,800 and the larynx with 156,800 [1]. The WHO defines as pre-neoplastic lesions (or precancerous) tissue changes that may undergo neoplastic transformation more frequently than normal tissue, but can also remain stable and even undergo regression, especially if the irritant factor is removed [2]. ⇑ Corresponding author. Address: UPGEM, FAMERP (bloco U6), Avenida Brigadeiro Faria Lima, no. 5416, São José do Rio Preto – SP, CEP: 15.090-000, Brazil. Tel.: +55 17 3201 5720; fax: +55 17 3201 5708. E-mail address: [email protected] (E.M. Goloni-Bertollo).
The main histologic type is the squamous cell carcinoma (SCC) that develops mainly from a field cancerization and corresponds to around 90% of cases [3]. The factors promoting this tumor type include environmental exposure to tobacco and alcohol. But also, viral infections, especially by Epstein–Barr Virus and Human Papilloma Virus subtypes 16 and 18, and deficiencies or imbalances of vitamins and micronutrients, such as folic acid, vitamins A, C and E, zinc and selenium are related to carcinogenesis of SCC [4–6]. A careful analysis of clinical signs and symptoms obtained by history and physical examination, and their appropriate interpretation is essential for the diagnosis of any disease. Patients with early stage HNSCC have vague symptoms and minimal physical changes. The physical presentation varies with the primary site involved. Patients with tumors in the oral cavity usually present ulcers that do not heal and pain, while the oropharyngeal involvement results in persistent sensitivity, chronic dysphagia or odynophagia for more than 6 weeks [7]. Despite the different strategies used in the treatment of head and neck cancer, the overall survival rate is around 50%, [8] and the main reason for treatment failure is the frequent development of loco-regional recurrences, which affects around 30% of HNSCC patients [9]. The theory of field cancerization, by Slaughter et al. [10] proposes
http://dx.doi.org/10.1016/j.oraloncology.2014.02.015 1368-8375/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Arantes LMRB et al. Methylation as a biomarker for head and neck cancer. Oral Oncol (2014), http://dx.doi.org/10.1016/ j.oraloncology.2014.02.015
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that, although the epithelium has a normal appearance, there may be changes in the mucosa. These altered mucosa is frequently found in surgical margins after the tumor is resected, remaining in the patient and contributing to the high frequency of local recurrence and second primary tumors development in patients with HNSCC. Although most of HNSCC originate from the mucosal surface of the oral cavity, where they can be easily detected during a routine clinical examination, the definitive diagnosis will not happen quickly, due to difficulties in differentiating from other similar lesions. Thus, early detection of this tumor type is of paramount importance, requiring highly sensitive and specific tests [11].
Epigenetic changes: DNA methylation Cancer cells have unlimited replicative potential, self-sufficiency in growth factors, insensitivity to anti-growth signals and apoptosis, capacity of invasion, metastasis and angiogenesis. The process of carcinogenesis is comprised of multiple steps in which several genetic (punctual mutations, loss of heterozygosity, deletions, insertions, aneuploidy, gene amplification, among others) and epigenetic changes are progressively accumulated [12,13]. Epigenetic changes are inheritable and reversible, affecting the spatial conformation of DNA and its transcriptional activity, thus ensuring the maintenance of stability and integrity of the DNA, leading to changes in chromatin structure. This process leads to changes in the phenotype without changing the sequence of DNA bases, allowing the generation of diverse forms of expression and resulting in different phenotypes. Epigenetic changes occur during development of the organism, and are reproduced during DNA replication [14,15]. There are several known epigenetic mechanisms, which include DNA methylation (addition of methyl groups – CH3), changes in chromatin conformation, histone modification and post transcriptional modifications that may influence gene expression. It is important to remember that the interaction between epigenetic factors with the observed result is always a result of the sum of its interactions with the various mechanisms of positive and negative feedback [15]. One of the main epigenetic changes that occur in mammals is DNA methylation, which is the main focus of this report. This is a biological process consisting in adding a methyl group (CH3) on the carbon 5 of nitrogenous bases cytosines (C) located 5’ of guanines (G). The primary targets of this process are CpG islands, regions rich in ‘‘CG’’ dinucleotides mainly present in the promoter region of certain genes. The presence of aberrant methylation in the promoter region of genes is associated with the suppression of gene expression [14–17]. In normal cells, methylation is an important event in the control of gene expression occurring at predetermined sites and contributing to mechanisms such as the inactivation of one X chromosome in females and the mechanism of genomic imprinting in which there is the inactivation of specific alleles according to parental inheritance, leading to a monoallelic expression [15]. This process involves an enzymatic reaction catalyzed by a family of enzymes named DNA methyltransferases (DNMTs). Three subtypes of DNMT have been identified: DNMT1, DNMT3A and DNMT3B. While DNMT1 is responsible for maintaining the methylation pattern in the daughter cells during mitosis or meiosis, DNMT3A and DNMT3B are involved in the de novo methylation process, during genomic imprinting. During embryogenesis and depending on the gender of the newly formed germ cells, de novo methylation occurs in paternal or maternal loci. The possible mechanisms which initiate the de novo methylation are: DNA target sequences, interference RNA, chromatin changes induced by histones and protein interactions [18–20].
DNA methylation and cancer The pattern of DNA methylation is cell-specific, and determined during the embryonic development of organisms. Changes in the methylation profile have been found in all types of cancer, in developmental disorders and in cellular aging. Several methods have been developed to determine the rate of DNA methylation, some provide information about the DNA methylation profile, from the methylation status of specific genes to the methylation content of the whole genome, known as global methylation [16,21]. Activation of proto-oncogenes and inactivation of tumor suppressor genes are the major genetic alterations involved in carcinogenesis. In addition, epigenetic changes (i.e., altered patterns of gene expression that do not affect the primary sequence of the DNA) can alter the expression of critical genes important in the development of a variety of cancers [22]. Studies show that cancer cells are associated with global DNA hypomethylation accompanied by the hypermethylation of CpG islands in the promoter region of genes involved in DNA damage repair, detoxification, cell cycle regulation and apoptosis [23,24]. Increased methylation in the promoter region of a tumor suppressor gene can lead to a progressive reduction of its expression, resulting in its silencing and the selection of cells with proliferative advantage. Analyzes carried out on DNA methylation patterns show that this process can be used as biomarker for early diagnosis as well as for classification, prognosis, and therapy of human cancers. The detection of aberrant gene promoter methylation has been considered a potential molecular marker for several tumor types [25–27]. It is noteworthy that the methylation profile of gene promoters is different for each type of tumor, allowing the identification of patterns of tumor-specific hypermethylation [28]. The evaluation of the methylation profile as a tool for the detection of tumors or their use as prognostic factors has been described for many different cancers such as bladder cancer, [29] colorectal, [30] lung, [31] and HNSCC [32]. Gene-specific DNA methylation and head and neck cancer Tumor suppressor genes CDKN2A/p16, CDH1, DAPK and MGMT were described as hypermethylated in larynx and hypopharynx [33] and genes AIM1, APC, CDH1 and UCHL1 in nasopharyngeal carcinoma [34]. Several analyzes of genes involved in oral carcinogenesis, indicated a higher frequency of hypermethylation at the promoter region of the gene CDKN2A/p16 in primary HNSCC [35–37]. Hypermethylation in the promoter region of SEMA3B was found in oral squamous cell carcinoma tissues, and a significant difference in the frequency of methylation of this gene was observed between oral squamous cell carcinoma and non-cancerous tissues [38]. Hwang et al. [39] reported that the expression of the GLT8D1 and C6orf136 genes, which methylation status may be regulated by the aberrant upregulation of the FOXM1 gene, were differentially expressed probably due to their promoter methylation, on independent HNSCC patient cohorts from Norway and the United Kingdom. Methylation in precursor lesions In head and neck tumors, as well as in other tumors, methylation is a frequent event in the early stages of carcinogenesis and can be detected in precursor lesions. Schussel et al. [40] demonstrated that HOXA9, EDNRB, and DCC methylation was associated with premalignant or malignant disease in saliva of a high-risk population, comparing well with
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examinations of an expert clinician in clinical risk classification of oral lesions. Methylation in advanced disease Hypermethylation appears to correlate with the severity of the disease and the potential to metastasize [24,41,42]. A significant correlation between the methylation profile of specific genes and clinical aspects of HNSCC patients has been shown. For instance, aberrant methylation of DAPK is correlated with advanced stage and positive lymph nodes at diagnosis [43]. The hypermethylation of DCC in squamous cell carcinoma of the oral cavity is correlated with bone invasion and worse overall survival, while hypermethylation of MINT31 was described as a predictor of poor prognosis [44]. It was also observed that hypermethylation of both galanin and galanin receptor 2 (GALR2) genes was statistically correlated with a decrease in disease-free survival in HNSCC patients [40,45,46]. Methylation and HNSCC biological features The literature also correlates the hypermethylation profile of genes DAPK, DCC and MINT31 with histological and biological features of oral carcinomas. An evaluation of the hypermethylation present in the promoter region of nine genes in oral cancer showed a strong link between clinic-pathological aspects and the ‘‘status’’ of methylation of at least six of these genes. This statement could be illustrated by the strong correlation observed between the consumption of tobacco and/or alcohol and the presence of hypermethylation in the promoter region of the tumor suppressor gene CDKN2A/p14 and CDKN2A/p16 [47]. Recently, Weiss et al. [48] showed that the methylation frequency of TCF21 gene is significantly higher in HNSCC than in benign tonsil controls, in tumors of patients without nicotine abuse, HPV-positive tumors and in tumors exhibiting over-expression of CDKN2A/p16. Pierini et al. [49] analyzed the promoter methylation status of CDKN2A/p16, MGMT, MLH1, and DAPK genes, using methylationsensitive high resolution melting in 100 patients with laryngeal squamous cell carcinoma, finding that the prevalence of promoter methylation was 60.8%, 47.4%, 46.4% and 42.3%, respectively. Moreover, authors found significantly increased methylation of CDKN2A/p16 in heavy smokers and epigenetic inactivation of the same gene and MLH1 were associated with lymph node involvement. Finally, the authors described that an inverse correlation was present between MLH1 methylation and alcohol consumption. Additional tissue source of samples for DNA methylation detection Serum/plasma Several studies evaluating the presence of hypermethylated DNA in body fluids have been performed [35,43,50,51]. In serum of patients who developed tumors, the presence of circulating DNA from tumor cells was identified. The mechanism of serum DNA release has not been fully clarified. Sidransky et al. [52] suggested that it might occur by two ways: the first one suggests that the circulating DNA is derived from apoptotic neoplastic corpuscles and the other that this DNA originates from viable tumor cells, not capable of generating metastases, present in the bloodstream. The analysis of the serum of patients with HNSCC is a promising method, since it is less invasive and can present a considerable prognostic value. Sanchez-Cespedes et al. [43] examined the methylation profile in a panel of genes in serum samples from
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HNSCC patients and demonstrated that the analysis of the hypermethylation profile in serum can be a very effective method for monitoring patients. CDH1, CDKN2B/p15, CDKN2A/p16 and DAPK were described as being hypermethylated in plasma samples of patients with carcinoma of the nasopharynx, and even after treatment, the aberrant methylation could still be detected, demonstrating that this analysis could be used as a serological tumor marker for early detection of locoregional recurrence [53]. Saliva Rosas et al. [35], demonstrated for the first time the possibility to detect hypermethylation in saliva. This study evaluated the presence of hypermethylation in the promoter region of genes involved in tumor suppression, tumor metastasis and DNA repair such as CDKN2A/p16, MGMT and DAPK. Paired tumor and saliva samples of HNSCC patients were evaluated in order to verify whether the changes found in tumors could also be detected in saliva, since this body fluid contains cells from the oral mucosa and pharynx. The hypermethylation rate was of 56% for HNSCC, and in 55% of these hypermethylated samples it was also possible to detect hypermethylation in paired tumor and saliva. Likewise, Righini et al. [54], evaluated paired tumor and saliva samples collected at diagnosis and identified a panel of 6 genes (CDH1, CDKN2A/p16, DAPK, MGMT, RASSF1A and TIMP3) with frequencies of hypermethylation of 82% and 78%, respectively. Carvalho et al. [55], were able to confirm an elevated rate of promoter hypermethylation detected in HNSCC patient salivary rinses by using a panel of gene promoters previously described as methylated in HNSCC but not in control subjects, by the same group [8]. In addition, detection of hypermethylation in pretreatment saliva DNA was associated with local recurrence [55]. Salivary rinses collected from HNSCC patients were also evaluated by Rettori et al. [56] that found a significant association between TIMP3 methylation in HNSCC samples collected 6 months after the last curative treatment and a lower local recurrence-free survival. The multivariate analysis confirmed for the first time, TIMP3 promoter hypermethylation in post-treatment salivary rinse as an independent prognostic marker for local recurrence-free survival in patients with HNSCC. These findings justify the use of DNA hypermethylation detection in saliva as a tool for identifying and monitoring subgroups of HNSCC patients subgroups with high risk of developing local recurrence. Exfoliated cells Exfoliating techniques have the advantage of being minimally invasive and of not requiring local anesthetic. The use of an exfoliating brush allows sampling of the full thickness of stratified squamous epithelium of the oral mucosa. In light of these considerations, biomarkers based on the methylation profile evaluation in exfoliated mucosa cell samples could represent a powerful class of minimally invasive markers for HNSCC detection [57]. Longo et al. [57] demonstrated that exfoliated tumor cells collected from patients with HNSCC could be used to access their methylation profile. In this work, authors showed that CCNA1 (60.4%), DCC (54.2%), and TIMP3 (35.4%) were frequently methylated in the 96 HNSCC patient cohort. Moreover, their data provided evidence that hypermethylation of DCC could be useful as a prognostic indicator for this malignancy. Surgical margins Local recurrences and/or multiple primary tumors develop despite an apparently complete excision and histopathologically
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Table 1 Statistically significant genes associated with diagnosis and/or prognosis for HNSCC classified by their clinical use and tissue source. Gene
Function
Clinical use
Source
References
ALDH3A1 CCNA1
Cell detoxification Cell cycle control
Prognostic Prognostic
CDH1 CDKN2A/p16
Adhesion glycoprotein Receptor of sonic hedgehog
CDKN2B/p15 DAPK
Inhibitor of CDK4 kinase Programmed cell death
Prognostic Diagnostic Prognostic Diagnostic Diagnostic Prognostic
DCC
Receptor for netrin required for axon guidance
EDNRB
G protein-coupled receptor
Prognostic Diagnostic Prognostic Diagnostic
ERCC1 ESR1 FANCC FHIT GALR1 GALR2 HIC1 HOTAIR KIF1A LKB1 MGMT MLH1 PTCH1 RARb2 RASSF4 RASSF5 RUNX3 SEMA3B SPARC TAP1 TCF21 TIMP3
Nuclear hormone receptor DNA repair protein Purine metabolismo Receptor for the hormone galanin Receptor for the hormone galanin and for GALP Unknown Transcriptional repressor Pattern formation in the embryo Protein kinase involved in apoptosis and DNA damage response DNA repair protein DNA mismatch repair system RNA binding Retinoic acid receptor Undetermined Regulation of lymphocyte adhesion and suppression of cell growth Transcription factor Axonal guidance during neuronal development Matrix-associated protein Membrane-associated pump Transcription factor Inhibitor of the matrix metalloproteinases T-Cell antigen receptor, recognition of foreign antigens
Prognostic Prognostic Diagnostic and prognostic Prognostic Prognostic Prognostic Prognostic Prognostic Diagnostic Prognostic Prognostic Prognostic Diagnostic and prognostic Prognostic Prognostic Prognostic Prognostic Diagnostic Diagnostic and prognostic Prognostic Diagnostic Prognostic
TRG
Cell detoxification
Prognostic
Tissue Surgical margin Tissue Tissue Tissue Surgical margin Plasma Plasma Tissue and serum Tissue Surgical margin Surgical margin Saliva Exfoliated Saliva Saliva and tissue Saliva Tissue Tissue Tissue Tissue Tissue Tissue Tissue Saliva and tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Tissue Saliva Tissue Tissue
[63] [59] [64] [41,65,66] [32,49,59], [67,68] [69] [70] [70] [43] [32] [58] [59] [40] [57] [40,71] [60] [72] [73] [74] [75] [76] [45] [73] [77] [78] [79] [80] [49] [74] [81] [42] [42] [82] [38] [83] [63] [48] [56,84] [41,64] [42]
tumor-free surgical margins. This raised the question of molecular alterations existence in negative surgical margins, which may not be detectable by conventional microscopic histopathologic analysis, but could be detected with molecular analyzes for genetic alterations associated with carcinogenesis [58]. The prognosis of patients with non-informative tumors or positive surgical margins justify the monitoring of these cases, with recommendation for adjuvant therapy, even in the absence of other risk factors such as advanced stage, perineural invasion and perivascular invasion. However, the assessment of the hypermethylation profile of the surgical margins can serve as an additional strategy for more sensitive and appropriate treatment, which can minimize morbidity [59]. Tan et al. [59] proposed that the presence of hypermethylated promoters in surgical margins of HNSCC can predict local recurrences and disease-specific deaths, by performing a panel of three genes (CDKN2A/p16, CCNA1 and DCC). Methylation analyzes of their resection margins correctly predicted all the recurrences in their cohort of patients. Also, the presence of DAPK promoter hypermethylation detected in histologically negative surgical margins of oral squamous cell carcinoma patients was significantly associated with the decrease in overall survival, showing the feasibility of this molecular marker for prognostic [58].
early disease detection, treatment selection and prognostication, especially in HNSCC [8,60–62]. A systematic search in the PUBMED database using the terms: methylation AND head neck was performed in order to identify studies reporting genes in which the detection of hypermethylation on their promoter region showed a statistical significant association for their use as biomarker for diagnosis and/or prognosis. Table 1 summarizes these results for these genes classified by their clinical use and tissue source.
Conclusion The search for biomarkers has as its main aim to evaluate and measure the status of normal and pathological biological processes as well as pharmacological responses to certain treatments. The tracking of these biomarkers is an important step for the identification of individuals in the early stages of head and neck cancer for its diagnostic and prognostic relevance. Therefore, assuming that cancer results from genetic and epigenetic changes, analyzes based on the methylation profile in combination with the pathological diagnosis would be useful in predicting the behavior of these head and neck cancers.
Methylation as biomarkers Conflict of interest statement Publications have shown that DNA methylation is an early event and new efforts are focused on identifying markers of
None declared.
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