**1. Introduction**

176 Otolaryngology

Kujath M, Kerr P, Myers C, Bammeke F, Lambert P, Cooke A, Sutherland D. Functional

Mahler V, Boysen M, Brøndbo K. Radiotherapy or CO(2) laser surgery as treatment of T1a glottic carcinoma? Eur Arch Otorhinolaryngol. 2010 May;267(5):743-50. Motta G, Esposito E, Motta S, Tartaro G, Testa D. CO(2) laser surgery in the treatment of

Osborn HA, Hu A, Venkatesan V, Nichols A, Franklin JH, Yoo JH et al. Comparison of

Peeters AJ, van Gogh CD, Goor KM, Verdonck-de Leeuw IM, Langendijk JA, Mahieu HF.

Phillips TJ, Sader C, Brown T, Bullock M, Wilke D, Trites JR et al. Transoral laser

Rucci L, Romagnoli P, Scala J. CO(2) laser therapy in Tis and T1 glottic cancer: indications

Schrijvers ML, van Riel EL, Langendijk JA, Dikkers FG, Schuuring E, van der Wal JE et al.

Shah JP, Karnell LH, Hoffman HT, Ariyan S, Brown GS, Fee WE et al. Patterns of care for

Silver CE, Beitler JJ, Shaha AR, Rinaldo A, Ferlito A. Current trends in initial management of

Sjögren EV, Wiggenraad RG, Le Cessie S, Snijder S, Pomp J, Baatenburg de Jong RJ.

Spielmann PM, Majumdar S, Morton RP. Quality of life and functional outcomes in the

Steiner W. Results of curative laser microsurgery of laryngeal carcinomas. Am J Otolaryngol

Stoeckli SJ, Guidicelli M, Schneider A, Huber A, Schmid S. Quality of life after treatment for early laryngeal carcinoma. Eur Arch Otorhinolaryngol. 2001 Feb;258(2):96-9. Strong M, Jako G. Laser surgery in the larynx: early clinical experience with continuous CO2

[SEER] Surveillance Epidemiology and End Results. 2011. Cancer of the Larynx. Available from: http://seer.cancer.gov/statfacts/html/laryn.html. Accessed 2011 Sep 26. Tateya I, Hirano S, Kitamura M, Kada S, Ishikawa S, Kanda T et al. Management and pitfalls of stage I/II glottic cancer. J.Acta Otolaryngol Suppl. 2010 Nov;(563):62-7. Webb 2003. The physical basis of IMRT and inverse planning. Br J Radiol. 2003

glottis cancer. Head Neck. 2005 Jul;27(7):566-73; discussion 573-4.

carcinoma. J Otolaryngol Head Neck Surg. 2011 Jun 1;40(3):200-4.

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and results. Head Neck. 2010 Mar;32(3):392-8.

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laser. Ann Otol Rhinol Laryngol 1972;81:791–8.

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outcomes and laryngectomy-free survival after transoral CO2 laser microsurgery for stage 1 and 2 glottic carcinoma. J Otolaryngol Head Neck Surg. 2011 Feb; Suppl

endoscopic laser resection versus radiation therapy for the treatment of early glottic

Health status and voice outcome after treatment for T1a glottic carcinoma. Eur

microsurgery versus radiation therapy for early glottic cancer in Canada: cost

Higher laryngeal preservation rate after CO2 laser surgery compared with radiotherapy in T1a glottic laryngeal carcinoma. Head Neck. 2009 Jun;31(6):759-64.

cancer of the larynx in the United States. Arch Otolaryngol Head Neck Surg

laryngeal cancer: the declining use of open surgery. Eur Arch Otorhinolaryngol.

Outcome of radiotherapy in T1 glottic carcinoma: a population-based study. Eur

management of early glottic carcinoma: a systematic review of studies comparing radiotherapy and transoral laser microsurgery. Clin Otolaryngol. 2010 In addition to the genetic information required to establish an organism, recent decades have unveiled a previously unknown type of chromatin modification, known as epigenetic, which is defined as heritable DNA changes that are not encoded in the sequence itself. Unlike genetic modifications, the epigenetic ones are reversible, and increasingly appear to serve fundamental roles in cell differentiation and development.

It is increasingly evident that genetics alone cannot explain the complexity of phenotypes in the living world. Heritable phenotypic characteristics that are not caused by DNA sequence alterations represent the object of epigenetics and include potentially reversible changes such as histone modifications, DNA methylation, and imprinting. At the interface between epigenetics and genomics, a new discipline that is emerging, epigenomics, promises to profoundly change the way we envision phenomena in the biological and medical sciences. Epigenetic modifications can provide an astronomic number of distinct signatures, with huge diagnostic and prognostic value, but it is essential to consider all the different sources of information.

Epigenomics-based diagnostic tools for early cancer detection represent an exciting development. Tumors shed their DNA into the blood, and epigenetic changes that occur early during tumorigenesis, sometimes even in premalignant lesions, can provide valuable biomarkers. Previous research at Epigenomics identified Septin 9 as a single gene in which DNA methylation changes occur very early in colorectal cancer development and are present in the vast majority of tumors of all stages. In most tissues, CpG (cytosine 5 phosphorylated guanine) islands around transcription start sites are largely unmethylated, but their methylation has been described in many tumors and can serve as potential biomarkers. One of the advantages of using epigenomic biomarkers is that, in most cases, DNA methylation changes precede clinical symptoms.

Stephen B. Baylin, M.D., professor of cancer research and deputy director of The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University and colleagues recently presented a molecular model to explain how DNA methylation causes gene silencing in mammalian cells. They used the GATA-4 gene as a model to investigate how polycomb protein complexes and DNA methylation maintain the chromatin in its silent state. They found that polycomb protein occupancy at genomic regions enriched in trimethylated

Epigenetics in Head and Neck Squamous Cell Carcinoma 179

Carcinogenesis in human OSCC is a multistep phenomenon (Scully et al, 2000), which is usually associated with or preceded by potentially malignant oral disorders. A series of genetic hits are required for a cell to progress through dysplasia, carcinoma in situ, invasion, and metastasis. Crucial genetic events that trigger carcinogenesis include the activation of oncogenes and inactivation of tumor suppressor genes. The current understanding in the molecular pathogenesis of oral cancer suggests that both the genetic and epigenetic alterations are implicated in this multistep process as they are known to complement each other during successive stages of oral carcinogenesis. A change in gene expression profiles is evident in cancer cells at the epigenetic level via transcriptional inactivation owing to DNA methylation (Jones,2003). Among the various epigenetic alterations that lead to altered gene expression, the most important are believed to be DNA methylation and chromatin remodeling by histone modification (Baylin & Herman, 2000). DNA methylation markers stand out for their potential to provide a unique combination of specificity, sensitivity, high information content, and applicability to a wide variety of clinical specimens. Methylation markers are particularly suited for situations where sensitive detection is necessary, such as

While the effects of genotoxic agents such as tobacco smoke and alcohol are the most important risk factors for the development of oral cancers, the interaction of epigenetic factors and genotoxic agents may synergistically increase the risk of oral carcinogenesis. It may be argued that an epigenetic disruption in progenitor cells might be a common event in human cancer and the epigenome is a logical target for early events in carcinogenesis. The presence of methylated CpG islands in the promoter region of human genes can suppress their expression because of the presence of 5-methylcytosine, which interferes with the binding of transcription factors or other DNA-binding proteins repressing transcription activity. Inactivation of some tumor suppressor genes, such as p16, is an initial event in head and neck cancers (Von Zeidler et al, 2004), which is frequent during early oral carcinogenesis and more so in the later stages (Lopez et al, 2003). Although many important genes and gene products have been identified through DNA methylation changes, no single unifying pathway has been identified that accounts for all OSCC. Aberrant promoter hypermethylation of tumor-associated gene leads to their inactivation, as it may inactivate one or both the alleles of the proven tumor suppressor genes in sporadic cancers and can

when tumor DNA is either scarce or contaminated by excess normal DNA.

potentially act as a second hit during the development of hereditary cancer.

Cell cycle regulation, which is coordinated by cyclin-dependent kinases, its binding partners, and the inhibitory molecules such as p16, pRb, and p15 have been widely studied in oral cancer (Chang et al, 2004; Nakahara et al, 2001; Lee et al, 2004; Soni et al, 2005). Cellular signal transduction involves the conversion of one signal or stimulus (mechanical or chemical) to another. The transduction process is usually performed by enzymes in association with second messengers (Oshiro et al, 2005; Mhaweck, 2005; Nakajima et al, 2003; Gasco et al, 2002). Wnt signaling pathway is a network of proteins best known for their role in embryogenesis and cancer. E-cadherin, Adenomatosis polyposis coli APC, ß-catenin, CDH3, SFRP family, WIF1, and DKK3 are some of the genes of Wnt pathway, which are epigenetically silenced in cancer. Wnt pathway is also involved in calcium-dependent cell adhesion through the interaction of E-cadherin and ß-catenin (Nakayama et al, 2001; Uesugi et al, 2005; Gao et al, 2005). One of the hallmarks of cancer is the down-regulation of genes involved in DNA repair pathways. O-6-methylguanine-DNA methyltransferase (MGMT), MutL homolog 1 (MLH1), and fragile histidine triad (FHIT) are most widely studied in oral

histone H3 lysine 27 marks establishes long-range interactions by chromatin looping. This finding promises to significantly improve our understanding of higher order chromatin organization and gene silencing both in stem cells and in cancer cells, which share intriguing similarities with respect to chromatin organization.

In addition to the work on acetylation and methylation in the context of cancer epigenetics another interest focuses on the involvement of heat shock proteins (Hsp) as molecular chaperones in cancer. Cancer cell metabolism creates a considerable amount of stress, and one of the main categories, known as proteotoxic stress, is mediated by the misfolded or unfolded protein response. Heat shock proteins are essential in maintaining the correctly folded conformation and activity of oncoproteins, and thus allow cancer cells to survive the stress response. The use of molecular chaperones as therapeutic targets for malignant tumors emerges as an exciting idea, and several Hsp90 inhibitors are currently being investigated as potential anticancer agents.

 "Our individual life history is inscribed in our epigenome," states Toshikazu Ushijima, Ph.D., chief of the carcinogenesis division at the National Cancer Center Research Institute, Tokyo. Dr. Ushijima and collaborators screened genes that were silenced in esophageal squamous cell carcinomas and demonstrated that methylation levels in five promoters are significantly correlated with the duration of tobacco smoking, indicating that chronic smoking induces methylation changes in many of these genes. This finding supports the idea that smoking induces an epigenetic field of cancerization, a term that was previously described for breast, colon, liver, and stomach cancers, and is used to denote epigenetic modifications that occur during the early stages of carcinogenesis. The use of molecular chaperones as therapeutic targets for malignant tumors emerges as an exciting idea, and several Hsp90 inhibitors are currently being investigated as potential anticancer agents.

Increasingly, new revelations about epigenetic modifications promise to transform all facets of cancer biology and to provide prophylactic, diagnostic, and therapeutic benefits. Epigenetic modifications could, in addition, become one of the missing links between infectious diseases and cancer. The ability of certain viruses, bacteria, parasites, and protozoa to cause malignant transformation represents one of the most fascinating topics in life sciences. This connection was regularly re-discovered throughout the past century, it repeatedly fell into oblivion and, historically, demonstrating causality often proved challenging. It is currently estimated that approximately 20% of all cancers worldwide are linked to pathogens, and the involvement of epigenetic changes in shaping this connection could soon lead to new chapters in cancer biology, establishing links that we never would have thought could exist.

#### **2. DNA methylation and gene dysregulation in oral carcinogenesis**

Oral squamous cell carcinoma (OSCC) encompasses all the malignancies that originate in the oral tissues, which include cancer of the lip, tongue, gingiva, floor of the mouth, buccal mucosa, palate, and the retromolar trigone. It is the largest group of cancers that fall into the head and neck squamous cell cancer (HNSCC) category, making it the 6th most common cancer in the world (Silverman, 1998).

 Oral cancer begins as a focal clonal overgrowth of altered progenitor cells near the basement membrane, expanding upward and laterally, replacing the normal epithelium.

histone H3 lysine 27 marks establishes long-range interactions by chromatin looping. This finding promises to significantly improve our understanding of higher order chromatin organization and gene silencing both in stem cells and in cancer cells, which share intriguing

In addition to the work on acetylation and methylation in the context of cancer epigenetics another interest focuses on the involvement of heat shock proteins (Hsp) as molecular chaperones in cancer. Cancer cell metabolism creates a considerable amount of stress, and one of the main categories, known as proteotoxic stress, is mediated by the misfolded or unfolded protein response. Heat shock proteins are essential in maintaining the correctly folded conformation and activity of oncoproteins, and thus allow cancer cells to survive the stress response. The use of molecular chaperones as therapeutic targets for malignant tumors emerges as an exciting idea, and several Hsp90 inhibitors are currently being

 "Our individual life history is inscribed in our epigenome," states Toshikazu Ushijima, Ph.D., chief of the carcinogenesis division at the National Cancer Center Research Institute, Tokyo. Dr. Ushijima and collaborators screened genes that were silenced in esophageal squamous cell carcinomas and demonstrated that methylation levels in five promoters are significantly correlated with the duration of tobacco smoking, indicating that chronic smoking induces methylation changes in many of these genes. This finding supports the idea that smoking induces an epigenetic field of cancerization, a term that was previously described for breast, colon, liver, and stomach cancers, and is used to denote epigenetic modifications that occur during the early stages of carcinogenesis. The use of molecular chaperones as therapeutic targets for malignant tumors emerges as an exciting idea, and several Hsp90 inhibitors are currently being investigated as potential anticancer agents.

Increasingly, new revelations about epigenetic modifications promise to transform all facets of cancer biology and to provide prophylactic, diagnostic, and therapeutic benefits. Epigenetic modifications could, in addition, become one of the missing links between infectious diseases and cancer. The ability of certain viruses, bacteria, parasites, and protozoa to cause malignant transformation represents one of the most fascinating topics in life sciences. This connection was regularly re-discovered throughout the past century, it repeatedly fell into oblivion and, historically, demonstrating causality often proved challenging. It is currently estimated that approximately 20% of all cancers worldwide are linked to pathogens, and the involvement of epigenetic changes in shaping this connection could soon lead to new chapters in cancer biology, establishing links that we never would

**2. DNA methylation and gene dysregulation in oral carcinogenesis** 

Oral squamous cell carcinoma (OSCC) encompasses all the malignancies that originate in the oral tissues, which include cancer of the lip, tongue, gingiva, floor of the mouth, buccal mucosa, palate, and the retromolar trigone. It is the largest group of cancers that fall into the head and neck squamous cell cancer (HNSCC) category, making it the 6th most common

 Oral cancer begins as a focal clonal overgrowth of altered progenitor cells near the basement membrane, expanding upward and laterally, replacing the normal epithelium.

similarities with respect to chromatin organization.

investigated as potential anticancer agents.

have thought could exist.

cancer in the world (Silverman, 1998).

Carcinogenesis in human OSCC is a multistep phenomenon (Scully et al, 2000), which is usually associated with or preceded by potentially malignant oral disorders. A series of genetic hits are required for a cell to progress through dysplasia, carcinoma in situ, invasion, and metastasis. Crucial genetic events that trigger carcinogenesis include the activation of oncogenes and inactivation of tumor suppressor genes. The current understanding in the molecular pathogenesis of oral cancer suggests that both the genetic and epigenetic alterations are implicated in this multistep process as they are known to complement each other during successive stages of oral carcinogenesis. A change in gene expression profiles is evident in cancer cells at the epigenetic level via transcriptional inactivation owing to DNA methylation (Jones,2003). Among the various epigenetic alterations that lead to altered gene expression, the most important are believed to be DNA methylation and chromatin remodeling by histone modification (Baylin & Herman, 2000). DNA methylation markers stand out for their potential to provide a unique combination of specificity, sensitivity, high information content, and applicability to a wide variety of clinical specimens. Methylation markers are particularly suited for situations where sensitive detection is necessary, such as when tumor DNA is either scarce or contaminated by excess normal DNA.

While the effects of genotoxic agents such as tobacco smoke and alcohol are the most important risk factors for the development of oral cancers, the interaction of epigenetic factors and genotoxic agents may synergistically increase the risk of oral carcinogenesis. It may be argued that an epigenetic disruption in progenitor cells might be a common event in human cancer and the epigenome is a logical target for early events in carcinogenesis. The presence of methylated CpG islands in the promoter region of human genes can suppress their expression because of the presence of 5-methylcytosine, which interferes with the binding of transcription factors or other DNA-binding proteins repressing transcription activity. Inactivation of some tumor suppressor genes, such as p16, is an initial event in head and neck cancers (Von Zeidler et al, 2004), which is frequent during early oral carcinogenesis and more so in the later stages (Lopez et al, 2003). Although many important genes and gene products have been identified through DNA methylation changes, no single unifying pathway has been identified that accounts for all OSCC. Aberrant promoter hypermethylation of tumor-associated gene leads to their inactivation, as it may inactivate one or both the alleles of the proven tumor suppressor genes in sporadic cancers and can potentially act as a second hit during the development of hereditary cancer.

Cell cycle regulation, which is coordinated by cyclin-dependent kinases, its binding partners, and the inhibitory molecules such as p16, pRb, and p15 have been widely studied in oral cancer (Chang et al, 2004; Nakahara et al, 2001; Lee et al, 2004; Soni et al, 2005). Cellular signal transduction involves the conversion of one signal or stimulus (mechanical or chemical) to another. The transduction process is usually performed by enzymes in association with second messengers (Oshiro et al, 2005; Mhaweck, 2005; Nakajima et al, 2003; Gasco et al, 2002). Wnt signaling pathway is a network of proteins best known for their role in embryogenesis and cancer. E-cadherin, Adenomatosis polyposis coli APC, ß-catenin, CDH3, SFRP family, WIF1, and DKK3 are some of the genes of Wnt pathway, which are epigenetically silenced in cancer. Wnt pathway is also involved in calcium-dependent cell adhesion through the interaction of E-cadherin and ß-catenin (Nakayama et al, 2001; Uesugi et al, 2005; Gao et al, 2005). One of the hallmarks of cancer is the down-regulation of genes involved in DNA repair pathways. O-6-methylguanine-DNA methyltransferase (MGMT), MutL homolog 1 (MLH1), and fragile histidine triad (FHIT) are most widely studied in oral

Epigenetics in Head and Neck Squamous Cell Carcinoma 181

acute infectious disease is sometimes controlled, while on other occasions it progresses to malignant tumors, is still a mystery, and the mechanisms of how pathogens may accomplish this are still elusive but, for increasing numbers of pathogens, epigenetic modifications have

Three of the most extensively studied oncogenic viruses, the hepatitis B virus (HBV), human papilloma viruses (HPV), and the Epstein-Barr virus (EBV) are, at the same time, major public health concerns. HBV infects over 2 billion people worldwide, and causes chronic infection in approximately 350 million of them (Fernandez & Esteller, 2010). Human papillomavirus (HPV) is thought to be the most frequent infectious agent that causes cancer. Specific human papillomavirus serotypes were causally linked to more than 70% of cervical cancers, to most anal cancers, and to respiratory papillomatosis. In addition, this virus emerges as an increasingly important causal agent of head and neck cancers, particularly in

To dissect the link between inflammation and cancer, Iliopoulos et al (Illiopoulos 2009, 2010) fused the estrogen receptor ligand-binding domain to the Src kinase oncoprotein to create a tamoxifen-inducible fusion protein. By using this system, the authors found that a transient inflammatory signal generated by an induction as short as 5 minutes triggered, in an NF-\_B (transcription factor)–dependent manner, an epigenetic switch similar to the one that controls tissue differentiation during development. This switch established a positive feedback loop, and as a result, nontransformed human mammary epithelial cells were becoming transformed, as revealed 24 to 36 hours later. The transformed state was propagated for many generations in the absence of the initiating inflammatory signal. Subsequently, the authors found several microRNAs that are differentially regulated as part of this positive feedback loop. The 2 most up-regulated ones, miR-21 and miR-181b-1, are

Infection with HPV, notably with high-risk HPV types as HPV-16 and 18, is a necessary step in the etiology of anogenital cancers, specifically carcinoma of the cervix uteri. DNA diagnosis confirmed the presence and transcription of HPV genomes in all cervical carcinomas, and the study of HPV oncoproteins catalyzed a variety of molecular mechanisms that convert normal into malignant cells (Munoz et al, 2003). In case of head and neck squamous cell carcinomas (HNSCC), approximately 80% of all HNSCC do not contain HPV genomes, and must therefore originate from HPV independent etiological processes, likely including mutational events triggered by tobacco and alcohol consumption. Among head and neck sites, squamous cell carcinomas of the tonsils have the strongest statistical support for an HPV dependent etiology (Syrjianen, 2005). There is less strength and consistency for a linkage between HPV infection and carcinogenesis at sites of the oral mucosa such as tongue, palate, floor of mouth and gingiva. Analysis of some HPV containing oral carcinomas revealed recombination between HPV genomes and cellular DNA as well as HPV oncogene expression (Ragin et al, 2004), properties that are generally viewed as support of carcinogenic processes under the influence of HPV. On the basis of these observations one must conclude that infection with high-risk HPV plays an etiological role in at least a proportion of malignancies of the oral cavity (Balderas-Loaeza et al, 2007). With the recent reported increase in young HNSCC cases many of them nonsmokers, the question arises as to whether these cases have the same genetic alterations as seen in the classic progression model. Toner and O'Regan (2009) studied HNSCC in young adults using

younger patients, and has also been associated with breast cancer (Westra, 2009).

been implicated (Paschos & Alladay, 2010).

key regulators of tumor-suppressor genes (Stein, 2011).

cancer. Frequent inactivation of some of these repair genes in cancer has been reported to be due to promoter hypermethylation (Mikami et al, 2007; Murakami et al, 2004; Rohatgi et al, 2005; Kato et al, 2006; Rodriguez et al, 2007; Kim et al, 2004; Paradiso et al, 2004; Baer-Desurmont et al, 2007).

Failure of cells to undergo apoptosis is a common and frequent event in carcinogenesis. Resistance to apoptosis of cancer cells has great clinical significance as these cells are resistant to chemo-and radiotherapies. Thus, identifying the checkpoints of apoptosis in cancer may offer newer therapeutic modalities for the treatment of cancer. The genes, which are involved in the apoptosis, such as Death-associated protein kinase 1, p73, and RASSF2, have shown to be down-regulated in oral cancer because of promoter hypermethylation (Hasegawa et al, 2002). In head and neck cancers, a statistically significant correlation was detected between the presence of DAP kinase gene promoter hypermethylation, lymph node involvement, and advanced disease stage. Tobacco smoking was slated to play an important role in the occurrence of promoter methylation and in delineating the precise pathway that eventually resulted in a tumorigenic phenotype (Araki et al, 2002). Some of the molecular changes characteristic of early oral cancer development have been identified in immortal oral dysplasia cultures and are associated with the loss of the expression of RARbeta and the cell cycle inhibitor p16INK4A. RAR-beta and or p16 reexpression could be reinduced by treatment with 5-aza-2¢-deoxycytidine in some immortal dysplasias, and the possibility of reversing the immortal phenotype of some dysplasias by 5-aza-2¢ deoxycytidine was considered to be of clinical usefulness (McGregor et al, 2002).

The ability to determine methylation states in primary tumors, saliva, and serum may have a potential clinical application in creating methylation gene panels for cancer screening. Gene methylation in saliva is a promising biomarker for the follow-up and early detection of still curable relapses of patients with HNSCC (Righini et al, 2007). Sanchez-Cespedes et al. (2000) studied the promoter hypermethylation patterns of the p16, MGMT, and DAP-K genes in tumor DNA of head and neck primary tumors and paired saliva samples to test whether the cells with tumor-specific aberrant DNA methylation might be found in the saliva of affected patients. Promoter methylation in saliva DNA was found in all tumor stages and more frequently in tumors located in the oral cavity. Moreover, none of the saliva from patients with methylation-negative tumors displayed methylation of any marker. This assay allowed sensitive and accurate detection of tumor DNA in saliva and may be potentially useful for detecting and monitoring recurrence in patients with head and neck cancers.

#### **3. Human papillomavirus-16 DNA methylation patterns**

This year marks a century since Francis Peyton Rous' seminal discovery that laid the foundations of a new field at the crossroads between microorganisms and human cancer. In 1911, Rous showed that cell-free filtrates from birds with sarcoma were able to cause tumors in healthy birds. The idea that viruses are causally linked to cancer was viewed at the time with skepticism, and fell into oblivion for decades, but re-emerged later. Fifty-five years after his groundbreaking discovery, Rous received the 1966 Nobel Prize in Physiology or Medicine.

A century after this major discovery, over 20% of the cancers have been causally linked to human pathogens, including viruses, bacteria, and parasites (Zur Hausen, 2009). Why an

cancer. Frequent inactivation of some of these repair genes in cancer has been reported to be due to promoter hypermethylation (Mikami et al, 2007; Murakami et al, 2004; Rohatgi et al, 2005; Kato et al, 2006; Rodriguez et al, 2007; Kim et al, 2004; Paradiso et al, 2004; Baer-

Failure of cells to undergo apoptosis is a common and frequent event in carcinogenesis. Resistance to apoptosis of cancer cells has great clinical significance as these cells are resistant to chemo-and radiotherapies. Thus, identifying the checkpoints of apoptosis in cancer may offer newer therapeutic modalities for the treatment of cancer. The genes, which are involved in the apoptosis, such as Death-associated protein kinase 1, p73, and RASSF2, have shown to be down-regulated in oral cancer because of promoter hypermethylation (Hasegawa et al, 2002). In head and neck cancers, a statistically significant correlation was detected between the presence of DAP kinase gene promoter hypermethylation, lymph node involvement, and advanced disease stage. Tobacco smoking was slated to play an important role in the occurrence of promoter methylation and in delineating the precise pathway that eventually resulted in a tumorigenic phenotype (Araki et al, 2002). Some of the molecular changes characteristic of early oral cancer development have been identified in immortal oral dysplasia cultures and are associated with the loss of the expression of RARbeta and the cell cycle inhibitor p16INK4A. RAR-beta and or p16 reexpression could be reinduced by treatment with 5-aza-2¢-deoxycytidine in some immortal dysplasias, and the possibility of reversing the immortal phenotype of some dysplasias by 5-aza-2¢-

deoxycytidine was considered to be of clinical usefulness (McGregor et al, 2002).

**3. Human papillomavirus-16 DNA methylation patterns** 

The ability to determine methylation states in primary tumors, saliva, and serum may have a potential clinical application in creating methylation gene panels for cancer screening. Gene methylation in saliva is a promising biomarker for the follow-up and early detection of still curable relapses of patients with HNSCC (Righini et al, 2007). Sanchez-Cespedes et al. (2000) studied the promoter hypermethylation patterns of the p16, MGMT, and DAP-K genes in tumor DNA of head and neck primary tumors and paired saliva samples to test whether the cells with tumor-specific aberrant DNA methylation might be found in the saliva of affected patients. Promoter methylation in saliva DNA was found in all tumor stages and more frequently in tumors located in the oral cavity. Moreover, none of the saliva from patients with methylation-negative tumors displayed methylation of any marker. This assay allowed sensitive and accurate detection of tumor DNA in saliva and may be potentially useful for detecting and monitoring recurrence in patients with head and neck

This year marks a century since Francis Peyton Rous' seminal discovery that laid the foundations of a new field at the crossroads between microorganisms and human cancer. In 1911, Rous showed that cell-free filtrates from birds with sarcoma were able to cause tumors in healthy birds. The idea that viruses are causally linked to cancer was viewed at the time with skepticism, and fell into oblivion for decades, but re-emerged later. Fifty-five years after his groundbreaking discovery, Rous received the 1966 Nobel Prize in Physiology or

A century after this major discovery, over 20% of the cancers have been causally linked to human pathogens, including viruses, bacteria, and parasites (Zur Hausen, 2009). Why an

Desurmont et al, 2007).

cancers.

Medicine.

acute infectious disease is sometimes controlled, while on other occasions it progresses to malignant tumors, is still a mystery, and the mechanisms of how pathogens may accomplish this are still elusive but, for increasing numbers of pathogens, epigenetic modifications have been implicated (Paschos & Alladay, 2010).

Three of the most extensively studied oncogenic viruses, the hepatitis B virus (HBV), human papilloma viruses (HPV), and the Epstein-Barr virus (EBV) are, at the same time, major public health concerns. HBV infects over 2 billion people worldwide, and causes chronic infection in approximately 350 million of them (Fernandez & Esteller, 2010). Human papillomavirus (HPV) is thought to be the most frequent infectious agent that causes cancer. Specific human papillomavirus serotypes were causally linked to more than 70% of cervical cancers, to most anal cancers, and to respiratory papillomatosis. In addition, this virus emerges as an increasingly important causal agent of head and neck cancers, particularly in younger patients, and has also been associated with breast cancer (Westra, 2009).

To dissect the link between inflammation and cancer, Iliopoulos et al (Illiopoulos 2009, 2010) fused the estrogen receptor ligand-binding domain to the Src kinase oncoprotein to create a tamoxifen-inducible fusion protein. By using this system, the authors found that a transient inflammatory signal generated by an induction as short as 5 minutes triggered, in an NF-\_B (transcription factor)–dependent manner, an epigenetic switch similar to the one that controls tissue differentiation during development. This switch established a positive feedback loop, and as a result, nontransformed human mammary epithelial cells were becoming transformed, as revealed 24 to 36 hours later. The transformed state was propagated for many generations in the absence of the initiating inflammatory signal. Subsequently, the authors found several microRNAs that are differentially regulated as part of this positive feedback loop. The 2 most up-regulated ones, miR-21 and miR-181b-1, are key regulators of tumor-suppressor genes (Stein, 2011).

Infection with HPV, notably with high-risk HPV types as HPV-16 and 18, is a necessary step in the etiology of anogenital cancers, specifically carcinoma of the cervix uteri. DNA diagnosis confirmed the presence and transcription of HPV genomes in all cervical carcinomas, and the study of HPV oncoproteins catalyzed a variety of molecular mechanisms that convert normal into malignant cells (Munoz et al, 2003). In case of head and neck squamous cell carcinomas (HNSCC), approximately 80% of all HNSCC do not contain HPV genomes, and must therefore originate from HPV independent etiological processes, likely including mutational events triggered by tobacco and alcohol consumption. Among head and neck sites, squamous cell carcinomas of the tonsils have the strongest statistical support for an HPV dependent etiology (Syrjianen, 2005). There is less strength and consistency for a linkage between HPV infection and carcinogenesis at sites of the oral mucosa such as tongue, palate, floor of mouth and gingiva. Analysis of some HPV containing oral carcinomas revealed recombination between HPV genomes and cellular DNA as well as HPV oncogene expression (Ragin et al, 2004), properties that are generally viewed as support of carcinogenic processes under the influence of HPV. On the basis of these observations one must conclude that infection with high-risk HPV plays an etiological role in at least a proportion of malignancies of the oral cavity (Balderas-Loaeza et al, 2007).

With the recent reported increase in young HNSCC cases many of them nonsmokers, the question arises as to whether these cases have the same genetic alterations as seen in the classic progression model. Toner and O'Regan (2009) studied HNSCC in young adults using

Epigenetics in Head and Neck Squamous Cell Carcinoma 183

obtained from microdissected areas of normal and papilloma areas and examined using a panel of 41 gene probes, designed to interrogate 35 unique genes for aberrant methylation status (22 genes) using the methylation-specific multiplex-ligation-specific polymerase assay. Methylation-specific PCR was employed to confirm aberrant methylation detected by the methylation-specific multiplex-ligation-specific polymerase assay. All seven cases indicated at least one epigenetic event of aberrant promoter hypermethylation. The *CDKN2B* gene was a consistent target of aberrant methylation in six of seven cases. Methylation-specific PCR confirmed hypermethylation of *CDKN2B*. Recurrent biopsies from two inverted papilloma cases had common epigenetic events. Promoter hypermethylation of *CDKN2B* was a consistent epigenetic event. Common epigenetic alterations in recurrent biopsies underscore a monoclonal origin for these lesions. Epigenetic events contribute to the underlying pathogenesis of benign inverted and exophytic papillomas. As a consistent target of aberrant promoter hypermethylation, *CDKN2B* may serve as an important

Nasopharyngeal carcinoma (NPC) is rare in most part of the world but prevalent in southern China, including Guangdong and Hong Kong, and Southeast Asia, with an incidence rate of 20 to 30 per 100 000 people/year (Li et al, 2011). The unique ethnic and geographic distribution of NPC indicates its unusual etiology. Three major etiologic factors, genetic, environmental, and viral factors – Epstein-Barr virus (EBV), have been identified to lead to multiple genetic and epigenetic alterations during NPC pathogenesis by either acting

EBV is a prototype of gamma herpes virus which infects >90% of the world adult population (Liebowitz, 1994). Humans are the only natural host for EBV. Primary infection with EBV normally occurs in early childhood and is usually asymptomatic in most underdeveloped countries. But when the exposure to EBV is delayed until adolescence it occasionally presents as mononucleosis. Long-term EBV coexists with most human hosts without overt serious consequences. However, in some individuals, the virus is implicated in the development of malignancy. EBV has a strong tropism for human lymphocytes and for epithelium of the upper respiratory tract (Young & Rickinson, 2004). EBV was the first human virus identified to be associated with human lymphomas as well as epithelial tumors, such as post-transplant lymphoma, AIDS-associated lymphomas, Burkitt lymphoma, Hodgkin's disease, T-cell lymphoma, nasopharyngeal carcinoma, parotid gland

Tumorigenesis of nasopharyngeal carcinoma is a multistep process. EBV may play an important role in the progression of NPC, involving activation of oncogenes and/or the inactivation of tumor suppressor genes (TSGs). Early genetic changes may predispose the

Nasopharyngeal carcinoma also distinguishes itself from other tumors by the number of genes targeted for silencing by promoter methylation. The key tumor suppressor genes like p53 or Rb which are found to be mutated in 50% of all the tumors were rarely found to be mutated in nasopharyngeal carcinoma (Wensing & Farrel, 2000). On the contrary, hypermethylation of known or candidate tumor suppressor genes involved in various

epigenetic biomarker for gene reactivation studies (Stephen et al, 2007).

**4. Epigenetic changes in nasopharyngeal carcinoma** 

carcinoma, and gastric carcinoma. (Murray & Young, 2002)

epithelial cells to EBV infection or persistent maintenance of latent cycle.

alone or in synergy.

array comparative genomic hybridization on a cohort of predominantly nonsmoking young adults and compared them with a cohort of mostly smoking older adults. Results from this study showed that when stratified by age the young cohort do not have the genetic alterations that are seen so consistently in older HNSCC. In fact, the mean number of aberrations in the young nonsmokers was less than 50% of that in the older smokers. Molecular alterations at the p53 gene have been documented as being the most frequent genetic alteration observed in carcinomas and has been found to be altered in over 70% of HNSCC (Gillison et al, 2000). It has been shown that p53 sequence alteration decreased in the setting of HPV infection, since there is an alternative means of p53 silencing with the production of E6 (Mork et al, 2001). Overexpression of p16INK4 has also been reported in head and neck cancers, and it is believed that HPV infection, via inactivation of retinoblastoma gene, accounts for these high levels of p16INK4 expression [Mork et al, 2001]. Toner and O'Regan (2009) found that p16 methylation is a more common event in those younger than 40 years in contrast to p16 deletions, which are more common in those older than 40 years. Consequently, it appears that specific modes of inactivation of p16 in HNSCC are related to specific patient risk profiles. After detailed study of p16 mRNA expression and p16 immunohistochemistry in this cohort, it was clear that all HPV mRNA positive cases showed p16 overexpression. In relation to copy gain and loss, previous studies have found only occasional chromosomal loss in HPV16 positive cases, suggesting that HPV16 infection is an early event in HNSCC development.

Inverted papillomas are benign, rare sinonasal lesions well known for their local recurrence, invasiveness and predisposition for malignant transformation. Recurrence rates for inverted papillomas range from 6 to 33% and malignant transformation occurs in 7–10% of cases (Batsakis, 2001). Endophytic sinonasal papillomas, comprising inverted papillomas and cylindrical cell papillomas according to the World Health Organization classification, show malignant progression in up to 25% of cases (Gujrathi et al, 2003).

The exact nature and biological evolution of sinonasal papillomas is not well known. The suggested hypothesis includes viral infections, chronic inflammation, and proliferation of nasal polyps, allergy, and environmental carcinogens. In view of its epitheliotropic nature and increasing evidence, Human Papilloma Virus (types 6 and 11) is postulated to be the most probable aetiological factor. Causative factor form alignant transformation in a papilloma is still unknown. It has been seen that HPV 16 and 18 are more carcinogenic than HPV 6 and 11. Over-expression of p53 & reduced expression of CD44 has been seen in carcinomas associated with inverted and exophytic papillomas. Overexpression of p53 may serve as a marker for malignant transformation of inverted papilloma (el-Deiry, 1998). Induction of p21waf1/cip1 is associated with terminal differentiation, senescence, and apoptosis in several tissues (Yook & Kim, 1998). Expression of p21waf1/cip1 has been detected in head and neck cancers, in particular oral SCCs and its precursors (Ng et al, 1999).

Epigenetic alterations of promoter hypermethylation have not been previously reported in sinonasal papillomas. Stephen et al. (2007) investigated whether epigenetic events of aberrant promoter hypermethylation in genes known to be involved in squamous head and neck cancer underlie the pathogenesis of sinonasal papillomas. Ten formalin-fixed paraffin DNA samples from three inverted papilloma cases, two exophytic (everted) papilloma cases, and two cases with inverted and exophytic components were studied. DNA was

array comparative genomic hybridization on a cohort of predominantly nonsmoking young adults and compared them with a cohort of mostly smoking older adults. Results from this study showed that when stratified by age the young cohort do not have the genetic alterations that are seen so consistently in older HNSCC. In fact, the mean number of aberrations in the young nonsmokers was less than 50% of that in the older smokers. Molecular alterations at the p53 gene have been documented as being the most frequent genetic alteration observed in carcinomas and has been found to be altered in over 70% of HNSCC (Gillison et al, 2000). It has been shown that p53 sequence alteration decreased in the setting of HPV infection, since there is an alternative means of p53 silencing with the production of E6 (Mork et al, 2001). Overexpression of p16INK4 has also been reported in head and neck cancers, and it is believed that HPV infection, via inactivation of retinoblastoma gene, accounts for these high levels of p16INK4 expression [Mork et al, 2001]. Toner and O'Regan (2009) found that p16 methylation is a more common event in those younger than 40 years in contrast to p16 deletions, which are more common in those older than 40 years. Consequently, it appears that specific modes of inactivation of p16 in HNSCC are related to specific patient risk profiles. After detailed study of p16 mRNA expression and p16 immunohistochemistry in this cohort, it was clear that all HPV mRNA positive cases showed p16 overexpression. In relation to copy gain and loss, previous studies have found only occasional chromosomal loss in HPV16 positive cases, suggesting that HPV16

Inverted papillomas are benign, rare sinonasal lesions well known for their local recurrence, invasiveness and predisposition for malignant transformation. Recurrence rates for inverted papillomas range from 6 to 33% and malignant transformation occurs in 7–10% of cases (Batsakis, 2001). Endophytic sinonasal papillomas, comprising inverted papillomas and cylindrical cell papillomas according to the World Health Organization classification, show

The exact nature and biological evolution of sinonasal papillomas is not well known. The suggested hypothesis includes viral infections, chronic inflammation, and proliferation of nasal polyps, allergy, and environmental carcinogens. In view of its epitheliotropic nature and increasing evidence, Human Papilloma Virus (types 6 and 11) is postulated to be the most probable aetiological factor. Causative factor form alignant transformation in a papilloma is still unknown. It has been seen that HPV 16 and 18 are more carcinogenic than HPV 6 and 11. Over-expression of p53 & reduced expression of CD44 has been seen in carcinomas associated with inverted and exophytic papillomas. Overexpression of p53 may serve as a marker for malignant transformation of inverted papilloma (el-Deiry, 1998). Induction of p21waf1/cip1 is associated with terminal differentiation, senescence, and apoptosis in several tissues (Yook & Kim, 1998). Expression of p21waf1/cip1 has been detected in head and neck cancers, in particular oral SCCs and its precursors (Ng et al,

Epigenetic alterations of promoter hypermethylation have not been previously reported in sinonasal papillomas. Stephen et al. (2007) investigated whether epigenetic events of aberrant promoter hypermethylation in genes known to be involved in squamous head and neck cancer underlie the pathogenesis of sinonasal papillomas. Ten formalin-fixed paraffin DNA samples from three inverted papilloma cases, two exophytic (everted) papilloma cases, and two cases with inverted and exophytic components were studied. DNA was

infection is an early event in HNSCC development.

1999).

malignant progression in up to 25% of cases (Gujrathi et al, 2003).

obtained from microdissected areas of normal and papilloma areas and examined using a panel of 41 gene probes, designed to interrogate 35 unique genes for aberrant methylation status (22 genes) using the methylation-specific multiplex-ligation-specific polymerase assay. Methylation-specific PCR was employed to confirm aberrant methylation detected by the methylation-specific multiplex-ligation-specific polymerase assay. All seven cases indicated at least one epigenetic event of aberrant promoter hypermethylation. The *CDKN2B* gene was a consistent target of aberrant methylation in six of seven cases. Methylation-specific PCR confirmed hypermethylation of *CDKN2B*. Recurrent biopsies from two inverted papilloma cases had common epigenetic events. Promoter hypermethylation of *CDKN2B* was a consistent epigenetic event. Common epigenetic alterations in recurrent biopsies underscore a monoclonal origin for these lesions. Epigenetic events contribute to the underlying pathogenesis of benign inverted and exophytic papillomas. As a consistent target of aberrant promoter hypermethylation, *CDKN2B* may serve as an important epigenetic biomarker for gene reactivation studies (Stephen et al, 2007).
