Biomarkers in Cancer Detection and Monitoring of Therapeutics: Volume 2: Diagnostic and Therapeutic Applications

Molecular Biomarkers in Cancer Detection and Monitoring of Therapeutics: Volume Two: Diagnostic and Therapeutic Applications discusses how molecular biomarkers are used to determine predisposition, facilitate detection, improve treatment and offer prevention guidelines for different cancers. It focuses on novel diagnostic techniques based on molecular biomarkers and their impact on treatment, covering different cancer types such as tumors in the nervous system, head and neck, oral and GI tractor, lung, breast, gastric system, leukemia and urogenital tract cancers. For each type, the book discusses the best diagnostic techniques and therapeutic approaches, thus helping readers easily identify the best solution for each case.

This is a valuable resource for cancer researchers, oncologists, graduate students and other members of the biomedical field who are interested in the potential of biomarkers in cancer research.

• Provides up-to-date information on current molecular biomarkers research for cancer
• Presents new diagnostic techniques using biomarkers that can leverage quicker results that can be essential for patient’s survivorship
• Discusses several novel therapeutics applied to specific cancer types, such as head and neck, oral, GI tract, breast, lung and hepatocellular carcinomas

• Language: English
• Publisher: Academic Press
• Release date: Nov 16, 2023
• ISBN: 9780323951159

Book preview

Chapter One Oral squamous cell carcinoma

Anubha Gulatia; R.C. Sobtib    a H S Judge Institute of Dental Sciences, Panjab University, Chandigarh, India

b Department of Biotechnology, Panjab University, Chandigarh, India

Abstract

Introduction—The world over is afflicted by oral cancer, which is an aggressive tumor with poor response to chemotherapy and resistance to most standards of care therapies. 90% of these are reported as oral squamous cell carcinoma, the sixth most common malignancy in the world.

Etiology—The higher risk of developing oral squamous cell carcinoma in the developing nations is attributable to self-neglect and lack of advertence to the consequences of exposure to avoidable risk factors such as tobacco and alcohol. There is also an interplay of genetic factors and epigenetic modifications.

Potentially malignant disorders are precursor lesions of oral squamous cell carcinoma with a moderate to high risk of transformation into malignancy of many of these disorders. A bidirectional role of autophagy is also noted in oral squamous cell carcinoma. The tumor microenvironment influences the proliferation of cancer cells and progression of this malignancy including its metastasis to distant locations.

Diagnosis—Salivaomics and liquid biopsy are upcoming fields for early and effective diagnosis of oral squamous cell carcinoma which can improve the prognosis of this malignancy that is associated with considerable morbidity and mortality.

Treatment—in addition to the conventional therapeutic modalities of surgery, radiotherapy and chemotherapy, the ongoing preclinical and clinical trials of immunotherapy targeting the tumor microenvironment and cancer stem cells are paving a promising way for treating this malignancy.

Keywords

Oral squamous cell carcinoma; Oral cavity squamous cell carcinoma; Squamous cell carcinoma of the mouth

1 Oral cancer

Head and neck cancer is a broad term used to describe a variety of neoplasms occurring in different anatomical structures including oral cavity, oropharynx and the larynx (Hussein et al., 2017). It is typically regarded as a disease of the elderly and is predominantly seen in men in their sixth and seventh decades after many years of tobacco and alcohol abuse (Llewellyn et al., 2004). However, the incidence of patients being diagnosed with head and neck squamous cell carcinoma at a younger age (<40–45 years old) has been on the rise worldwide over the last 3 decades (Majchrzak et al., 2014). This new trend of high incidence among young patients was primarily observed in oropharyngeal cancer involving the areas of the base of the tongue, tonsil and oropharynx and cancers affecting the body of the tongue (Shoboski et al., 2005). In their systematic review, Hussein et al. found that oral tongue was the most common site of occurrence of oral squamous cell carcinoma among the younger age group, especially in females (Hussein et al., 2017).

The world over is afflicted by oral cancer which is an aggressive tumor with poor response to chemotherapy and resistance to most standards of care therapies (Chi et al., 2015). The annual report of new cases of oral cancer is about 405,000 cases. The prevalence of this disease varies widely in different parts of the world or even within the minorities or subpopulations of the same countries. Sri Lanka, India, Pakistan, Bangladesh, Hungary and France attribute to the main burden of oral cancer. Head and neck cancer is known to be the sixth most common malignancy in the world; its occurrence in South-central Asia is much more common with it being the third most common type of cancer here (Warnakulasuriya, 2009).

This complex disease reveals a marked variation in the trends and patterns of mortality between countries and across specific cancer types that are linked to differences in changing lifestyles and in local exposures to known or presumptive determinants, as well as an altering environment. The recent few decades have shown the evolution of cancer as the first or second leading cause of premature death (i.e., at ages 30–69 years) in most countries (WHO, 2018). Specifically, cancer is currently the leading cause of premature death in most of the countries with high or very high HDI. This epidemiological transition based on Omran's theory (Omran, 1971) is owed to dramatic decline in mortality from infectious diseases due to better sanitation and the development of vaccines and antibiotics and the improving primary and secondary prevention for cardiovascular diseases. The differences in levels of medical practice and health infrastructure also influence the diverging patterns and trends in cancer mortality (World Cancer Report, 2020).

2 Oral squamous cell carcinoma

Squamous cell carcinoma represents the most common form of head and neck cancer—representing around 90% of all head and neck malignancies (Warnakulasuriya, 2009). Squamous cell carcinomas can originate from any part of the lining of the upper aerodigestive tract—nasopharynx, lip vermilion, oral cavity, oropharynx, and larynx (Hussein et al., 2017). Of all these anatomic locations, squamous cell carcinomas most commonly arise within the oral cavity. Recently, oral squamous cell carcinoma (OSCC) has emerged as the sixth most common malignancy in the world (as of 2016) (Seigel et al., 2016).

In the 2017 WHO classification of tumors, oral squamous cell carcinoma was defined as the carcinoma with squamous differentiation arising from the mucosal epithelium (WHO, 2017). The 21st century has evidenced a rise in the morbidity and mortality associated with oral squamous cell carcinoma especially in younger patients. Over 300,000 new cases are reported each year contributing to the global impact of this malignancy. Despite sizable advances in diagnosis and management, 50% of patients suffering from oral squamous cell carcinoma die within 5 years. Even with the relatively easy visualization of the tumor in the clinic and/or successful treatment intervention, the long-term prognosis is compromised due to late initial presentation of advanced tumors and widespread, multifocal presentation throughout the upper aerodigestive tract (Thomson, 2018).

Early diagnosis of oral squamous cell carcinoma plays a crucial role in saving a patient's life as well as minimizing the negative impact on quality of life linked to invasive surgical intervention. A vast array of diagnostic tools are available for screening and visual devices that can substantively improve the ability of the clinician to characterize any suspicious lesion. Additionally, with the advent of salivary biomarkers and liquid biopsy, recognition of the risk of malignant transformation has been made easier. Although surgical biopsy and histology are still considered the mainstay of diagnosis, auxiliary methods should be considered during objective clinical examination. The scientific community is constantly devising preventive measures and screening methods for early detection of oral cancer with the aim of reducing the diagnostic delay to help save the patient's life (Abati et al., 2020).

3 Epidemiology

One of the key variables in the occurrence of oral cancer is geographic location with low socioeconomic status group in developing countries being at a higher risk of developing oral squamous cell carcinoma attributable to their self-neglect and lack of advertence to the consequences of exposure to the avoidable risk factors such as tobacco and alcohol (Conway et al., 2018). A diversity in reporting of oral cancer in epidemiological studies is related to the several anatomical subsites of its presentation (Conway et al., 2006). There is a lack of consensus regarding the sites that can be included in cancers of the oral cavity and oropharynx which has been detrimental for the understanding and comparison of the actual disease prevalence across the globe. Conway et al. proposed a compromise way of describing oral squamous cell carcinoma and oropharyngeal cancers distinctly in concurrence with the International Classification of Diseases for Oncology (ICD-O) coding. According to them, the oral cavity cancer comprises of the involvement of the labial mucosa, dorsal surface of the tongue, gingiva, hard and soft palate, buccal mucosa, floor of the mouth and other unspecified parts of the mouth, while oropharyngeal cancer is cancer involving base of the tongue, tonsils including lingual tonsil, anterior surface of the epiglottis, lateral wall of the oropharynx and pharynx unspecified involving Waldeyer's ring/overlapping sites of oral cavity and pharynx (Conway et al., 2018).

The incidence rate, mortality rate and survival rate within the specific population help gauge the impact of oral cancer. Globally there has been a substantial rise in incidence rates of cancer owing to the development of advanced technologies in cancer detection even in the subclinical stages. GLOBOCAN 2018 by the International Association of Cancer Registries (IACR) provides the global picture of different cancers, including oral cancer. Approximately 354,864 newly diagnosed cases of lip and oral cavity cancer and around 92,8887 new cases of oropharynx cancer were reported accounting for about 2% and 0.5% of all the malignancies. In 2018, 4.0 per 100,000 was the projected age-standardized incidence rate of lip and oral cavity cancer worldwide. Similarly, a mortality rate of 177,384 was reported across the globe for oral cancer in 2018, with an age-standardized mortality rate of 2.0 per 100,000 individuals (Bray et al., 2018) (Fig. 1).

Fig. 1

Fig. 1 Global oral cancer rates.

The global age-standardized rates (ASR) of incidence of oral cancer are consistently higher in males (5.8 per 100,000) as compared to females (2.3 per 100,000). Some studies show an inverse trend with greater incidence in women. This change in trend could be ascribed to increasing habits of tobacco use, especially the smoking form, among women as compared to the traditional days (Jemal et al., 2018).

The highest burden of oral cancer is linked to cultural practices like tobacco chewing, alcohol consumption, and extreme use of betel quid in Asia. According to GLOBOCAN 2018, oral cancer is the eleventh most common cancer in Asia, with a projected 227,906 newly diagnosed cases. The Asian population presented with a mortality rate and a 5-year prevalence of the disease of 129,939 and 536,185, respectively (Bray et al., 2018).

Differing population habits, life expectancies, preventive education, and the quality of medical records in various countries (poverty, illiteracy, advanced stage at presentation, lack of access to health care, and poor treatment infrastructure) are major contributors for the differences in oral cancer between the developing world and the Western world (Inchingolo et al., 2020). However, as the rural populations are not well represented in the cancer registries so the GLOBOCAN data may not be reflective of the true incidence of oral squamous cell carcinoma (Fig. 2).

Fig. 2

Fig. 2 5-Year survival in oral squamous cell carcinoma after diagnosis.

4 Etiology

Tobacco smoking which was once prevalent mostly among men in high-income countries is now much more prevalent among women in many countries. Asia, Africa, and South America account for the highest tobacco use (World Cancer Report, 2020).

1.Tobacco

Tobacco which is available in many forms has a long-established cause relationship with multiple types of cancer and other major noncommunicable diseases. Cigarette smoking has been found to result in at least 20 different types of cancer (US Department of Health and Human Services, 2014). An estimated 1.3 billion people use tobacco products worldwide (WHO, 2019). Together, cigarettes and other tobacco products are estimated to cause 2.4 million tobacco-related cancer deaths worldwide every year (Stanaway et al., 2017).

Tobacco is used in the following forms:

Smoked (Fig. 3)

Fig. 3 Forms of smoked/combustible tobacco.

Smokeless (Fig. 4)

Fig. 4 Forms of smokeless tobacco products across the world.

E-cigarettes

Heated tobacco products

•E-cigarettes and other electronic nicotine delivery systems (ENDS)—An aerosol is produced by heating a nicotine-containing solution.

•Heated tobacco products—tobacco sticks are heated to produce an aerosol (World Cancer Report, 2020).

Impact of tobacco products

The most predominant form of tobacco consumption worldwide is in the smoked form as cigarettes, manufactured or hand-rolled ones. The carcinogenicity of smoked and smokeless tobacco products (US Department of health and human services, 2014) includes enzymatic and nonenzymatic transformations of nitrosamines, benzopyrenes, and aromatic amines into molecules covalently bound to various regions of DNA. This results in various mutations and the formation of DNA adducts. High oxidative stress is also generated by tobacco smoke due to its high concentration of free radicals (oxygen and nitrogen species) which deplete the enzymatic and nonenzymatic cellular antioxidants causing cell damage which in turn leads to cancer (Choudhari et al., 2013). Tobacco also impacts nonmalignant diseases, and the harmful effects of second-hand smoke have been widely studied (US Department of health and human services, 2014).

Smoke from cigarettes comprises more than 8000 compounds, which include over 70 carcinogens, including tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, and aromatic amines. Over the past 5 years, cigarette smoking has been linked to altered patterns of circulating inflammatory markers, altered DNA methylation patterns, altered airway gene expression patterns, an altered oral microbiome, specific mutational signatures, and Y chromosome loss. Almost all the carcinogens found in cigarette smoke are also produced by other combustible products, including bidis, cigars, and pipes.

The levels of specific carcinogens vary across the different products, but smokeless tobacco has been shown to contain at least 30 carcinogens and release high levels of tobacco-specific nitrosamines (World Cancer Report, 2020).

ENDS have emerged only during the past decade. Contents of ENDS products are nicotine, propylene glycol, glycerine, and flavors mixed in a solution, which is then evaporated to produce an aerosol. They are available in fruit and caramel flavors. Laboratory studies have shown that ENDS devices generally heat to a lower temperature and have lower levels of most carcinogens than combusted cigarettes (Shahab et al., 2017).

Patterns and trends in tobacco use

The most common pattern of smoked tobacco use is daily smoking. In 2015, an estimated 1.3 billion people worldwide used tobacco products and 1.1 billion people smoked, of which more than 80% smoked daily. The prevalence of smoking is higher in men than in women. About 25% of men in the world are daily smokers, compared with about 5% of women. Geographical patterns of smoking prevalence also differ by sex. Among men, the prevalence of daily smoking is highest in central and eastern Europe and South-East Asia; among women, the prevalence is highest in selected countries in eastern and western Europe.

The prevalence of daily smoking in both men and women has declined from 1990 to 2015. The Global Burden of Disease collaboration surveyed 195 countries and found estimated reductions in smoking of 28% in men and 34% in women since 1990. Other studies report similar reductions in smoking prevalence. The reductions were largest in high-income countries and in Latin America with the largest reduction in smoking prevalence occurring in Brazil, where the prevalence dropped by more than 50%. Pakistan, Panama, and India have implemented numerous tobacco controls that have led to large declines in daily smoking prevalence since 2005.

Nevertheless, the worldwide prevalence of tobacco use remains high. Low- and middle-income countries are home to about 80% of the world's smokers with more than 50% of the world's male smokers living in three countries: China, India, and Indonesia. Due to the size and the growth of populations and the aging of long-term, continuing smokers, the disease burden from tobacco use continues to increase rapidly in low- and middle-income countries despite a decrease in smoking prevalence.

Use of smokeless tobacco products

WHO has estimated that worldwide there are more than 367 million smokeless tobacco users aged 15 years or older with more men (237 million) than women (129 million). Smokeless tobacco accounts for an estimated more than 101,000 cancer deaths per year. Comparable results of 76,000 cancer deaths per year from the use of smokeless tobacco have been reported by the Global Burden of Disease project. An estimated 82% of users (301 million users) are in the South-East Asia Region with an estimated 87% of cancer deaths from smokeless tobacco occurring in this region.

In much of the world, 13- to 15-year-old children use smokeless tobacco. The highest prevalence in this age group is in the South-East Asia Region (7.3% overall; 9.5% in boys and 4.8% in girls), which accounts for almost 60% of smokeless tobacco use in this age group worldwide (World Cancer Report, 2020).

2Alcohol

Alcoholic beverages contain numerous carcinogenic compounds, but most of the risk relationship between alcohol consumption and the development of cancer is due to ethanol. Alcohol-induced carcinogenesis is not fully understood. However, the postulated main pathophysiological carcinogenic mechanisms of ethanol include its breakdown into the carcinogenic metabolite acetaldehyde, its one-carbon metabolism pathway inhibition, and DNA methylation (especially in people with lower intake of dietary folate), and its effect on elevation of serum levels of endogenous estrogens. Ethanol is also said to increase the risk of cancer via the production of reactive oxygen species and polar metabolites; through the conversion of procarcinogens in the metabolic pathway of ethanol; by peroxidation of lipids; by prostaglandin production; by alteration of the insulin-like growth factor 1 pathway; and by acting as a solvent for cellular penetration of environmental carcinogens (e.g., tobacco) (Pflaum et al., 2016). There is a site-specific difference in the biological pathways involved, and the relative contributions of these pathways to carcinogenesis. There are dose–response relationships, with almost linear gradients of relative risks and no apparent lower risk threshold. The level of lifetime exposure to alcohol is said to impact risk relationships. Alcohol was responsible for 26.4% of all cancers of the lip and oral cavity, 30.5% of all other pharyngeal cancers (excluding nasopharyngeal cancers), 21.6% of all laryngeal cancers, and 16.9% of all esophageal cancers (Rehm et al., 2017). These findings reflect the stronger associations—i.e., the higher gradients of the dose–response curves—between levels of alcohol consumption and cancers of the upper aero-digestive tract compared with cancers of the colorectum, liver, and breast (World Cancer Report, 2020).

3.Human papillomavirus

Human papillomavirus (HPV) has a strong affinity for the epithelium and infects the basal epithelial cells of stratified squamous epithelium in both skin and mucous membranes. It causes lesions that include common warts and invasive neoplasia. Based on their potential oncogenic activity, HPV subtypes have been divided into high-risk and low-risk groups. The high-risk Human papilloma viruses are associated with the development of cancer and are called oncogenic viral types and belong to 16, 18, 31, 33, 35, 45, 51, 52, 56, 58, 59, 68, 73, and 82 subtypes of which types 16 and 18 have been particularly associated with cervical carcinoma, anogenital neoplasia, and oropharyngeal lesions (Bouvard et al., 2009; Bernard et al., 2010). Benign epithelial lesions result from infection with the low-risk HPV, such as types 6, 11, 42, 43, and 44. The early region of the HPV genome encodes 7 proteins of which E6 induces DNA synthesis, prevents cell differentiation, and interacts with tumor suppressors and DNA repair factors and E7 protein that induces cell proliferation by interacting with negative regulators of cell cycle proteins and tumor suppressors are considered oncoproteins which result in carcinogenesis. E6 is a transforming protein that functions to promote p53 degradation which is a tumor suppressor protein; E7, on the other hand, is a transforming protein that has an affinity for the retinoblastoma protein (Valencia et al., 2008). Oral sexual practices have been associated with HPV transmission to the oral mucosa in both young and adult populations, because this sexual behavior is associated with a greater number of partners, especially in men. Sharing of smoking devices, lipstick, or toothbrushes has been reported as an alternate route of transmission that needs further validation. Occasionally, HPV can be transmitted to the oral mucosa by vertical transmission from mother to son.

HPV 16 and 6 are the most persistent viruses associated with HPV infection of the oral mucosa. Other factors linked to the persistence of HPV infection include smoking, patients over the age of 40 years, HIV patients treated with highly active antiretroviral therapy (HAART), and a CD4 count of 500 (Lafaurie et al., 2018).

HPV infects the basal cells by entering through wounds in the epithelial layer. The virus then resides in the nucleus of the infected cell where 1000 virus particles are produced per cell and maintain a high number of copies in squamous cells following which the viral progenies are subsequently released into the microenvironment. HPV protein E7 degrades all members of the Rb family by binding to them, thus resulting in the release and activation of the transcription factor E2F and deregulation of the G1/S checkpoint; E7 of high-risk HPV bind with higher affinity (Gage et al., 1990). A p53-dependent inhibition of cell growth and apoptosis occurs from the interaction of E7 with Rb. In addition, E6 proteins target the p53 tumor suppressor that leads to degradation, prevention of the inhibition of growth in differentiated and undifferentiated cells (Cooper et al., 2003). Cancer develops many years later when the immune system fails to clear persistent HPV infections. Two forms of HPV viral genome persist in the infected cells—the stable/episomal form that is responsible for latent disease and the integrated form present within the host DNA. A growth advantage is conferred to the cell through viral integration, which contrasts with harboring copies of viral episomal DNA. An important step in carcinogenesis associated with HPV is the coexistence of episomes with integrated copies (Kadaja et al., 2009; Lafaurie et al., 2018).

Oncogenic HPVs are responsible for approximately 60% of oropharyngeal cancer in North America; 36%–45% in Asia, Oceania, and Europe; and 15% in South and Central America. An increase in HPV-associated oropharyngeal cancers from 16% in the 1980s to the current 60% has been reported in the United States with increases in Europe mirroring this trend. This dramatic rise in HPV-related oropharyngeal cancer points to the emergence of a cancer epidemic. Of all oncogenic forms, HPV-16 is responsible for 90% of HPV-related oropharyngeal cancer (Chi et al., 2015). A subgroup analysis of oral potentially malignant disorders revealed an HPV association for oral leukoplakia (OR: 4.03, 95% CI: 2.34–6.92), oral lichen planus (OR: 5.12, 95% CI: 2.40–10.93), and epithelial dysplasia (OR: 5.10, 95% CI: 2.03–12.80) (Lafaurie et al., 2018).

4Other microbes/microbial infections

Syphilis—It is a systemic bacterial infection caused by Treponema pallidum. The incubation period of the infection is usually 21–30 days after contact, although it can vary from 10 to 90 days, depending on the number and virulence of the host to the causative agent. One of the clinical presentations of syphilis in the oral cavity is Syphilitic leukoplakia which involves the dorsum of the tongue and presents as a homogenous white patch. Both clinically- and serology-based studies have suggested an increased prevalence of syphilis in patient groups with squamous cell carcinoma of the tongue (up to 60% in one study), the association is stronger in males than females. A relatively recent study of 16,420 people with syphilis, resident in the United States, found a significantly raised frequency of cancer of the tongue (and Kaposi's sarcoma) in males (Leão et al., 2006).

Candida albicans—It is the most common opportunistic organism residing in the oral cavity as a normal commensal. A complex process involving factors related to yeast cells, host cells, and environmental factors exists which contributes to the virulence of the organism with a predisposition to infection, causing several clinical manifestations.

Candida infection was first recognized and introduced as candidal leukoplakia by Jepsen and Winther. They mentioned the adherent white patch which is infected by Candida. This tends to undergo malignant transformation (Jepsen and Winther, 1965). In his study, Cawson found that 6 out of 10 tissue biopsies initially diagnosed as chronic hyperplastic candidiasis underwent a malignant transformation to oral squamous cell carcinoma (Cawson, 1966). McCullough et al. postulated that the progression of chronic hyperplastic candidiasis to dysplasia is advanced by Candida albicans. It is due to the higher nitrosation potential of certain species which elaborate nitrosamine compounds which play a role in the initiation of carcinogenesis (McCullough et al., 2002).

5Chronic irritation

The constant action of a deleterious agent in the oral cavity (defective restorations, broken tooth, constant biting of the oral mucosa, or ill-fitting dentures with sharp or retentive edges) results in chronic mechanical irritation which can also be another cause of oral cancer. These agents maintain a chronic state of inflammation that invokes epigenetic transformation of these affected cells (Piemonte et al., 2018).

6.Oral cancer can be caused by genetic factors, epigenetic modifications (such as histones modifications; nucleosome integrity, DNA methylation and expression of noncoding RNAs (ncRNAs) (World Cancer Report, 2020).

Genetic alterations—Studies have revealed that oral carcinoma is not genetically stable as there is an accumulation of genetic variations in proto-oncogenes and tumor suppressor genes that leads to the development of oral squamous cell carcinoma through a multistep process (Califano et al., 1996). Two important observations were made in the Cancer Genome Atlas (TCGA) project (2006) while analyzing 10,000 samples from 20 different types of tumors.

(1)Genetic variations exist in tumors with an equivalent origin, and

(2)Similar patterns of genomic variations are shown by tumors with different origins.

Tumor cells acquire genetic instability by defects in segregation of chromosomes, copy number alterations, loss of heterozygosity, telomere stabilities, regulation of cell-cycle checkpoints, Notch signaling pathway, and DNA damage repairs (Ali et al., 2017; World Cancer Report, 2020).

Epigenetic modifications—Epigenetic modifications include hypermethylation within the promoter region of genes, posttranslational histone modifications, and posttranscriptional regulation by microRNAs. Epigenetic regulation occurs early in the process of oral carcinogenesis. The concept of genetic control of cancer has paved the way for a more comprehensive picture where DNA methylation, (Sharma et al., 2010) modifications of histones and nucleosome positioning are now considered to play an important role. Also, the expression of noncoding RNAs (ncRNAs), especially microRNAs (miRNAs), could also influence the epigenetic mechanisms (Irimie et al., 2018). The basic characterization of the epigenetic concept states that these mechanisms are reversible changes that are not associated with modifications within the structure of DNA and may be inherited and preserved for multiple generations (Sharma et al., 2010).

7.Others

A variety of suspected risk factors such as poor oral hygiene, occupational exposure, and malnutrition as well as low fruit and vegetable diets, have been proposed for the development of oral cancer (WHO, 2017; World Cancer Report, 2020).

5 Precursor lesions

WHO termed the pathologies that are associated with an increased risk of transforming into cancer as potentially malignant disorders. These include leukoplakia, erythroplakia, oral submucous fibrosis, oral lichen planus, palatal changes due to reverse smoking, discoid lupus erythematosus, actinic cheilosis, epidermolysis bullosa, and dyskeratosis congenita (Warnakulasuriya et al., 2007). All these arise either from genetic aberrations, immune disorders, or exposure to exogenous agents like tobacco. Some may arise as a rare inherited disease.

Ganesh et al. grouped these disorders according to their etiology as

a.Genetically acquired disorders: leukoplakia, erythroplakia, actinic cheilitis.

b.Tobacco-induced disorders: oral submucous fibrosis, palatal keratosis associated with reverse smoking.

c.Immune-mediated disorders: oral lichen planus, discoid lupus erythematosus.

d.Genetically inherited disorders: dyskeratosis congenita, epidermolysis bullosa (Ganesh et al., 2018).

5.1 Genetically acquired potentially malignant disorders

Leukoplakia—It is a diagnosis of exclusion. Leukoplakia has been defined as, white plaque of questionable risk having excluded (other) known diseases by WHO (WHO, 2017). Two clinical forms exist: homogeneous and nonhomogeneous. A uniform pattern of the white lesion is exhibited in homogenous leukoplakia. In the nonhomogeneous forms, a speckled (mixture of red and white with a predominance of white), nodular (red or white polypoid outgrowths), or verrucous (wrinkled or corrugated) appearance is seen at the time of presentation. Etiologic factors include the use of tobacco, alcohol, betel quid, and genetic abnormalities. Leukoplakia arising from the latter are termed Idiopathic leukoplakia.

Middle-aged or older men are most affected by leukoplakia. A rare form of nonhomogeneous leukoplakia is proliferative verrucous leukoplakia. It is most frequent on the gingiva and buccal mucosa and rapidly progresses to involve surrounding areas, both contiguous and noncontiguous (Warnakulasuriya et al., 2007).

The malignant transformation rate of leukoplakia has been reported to range between 0.13% and 34% in different studies. Sixty-one percent of patients with proliferative verrucous leukoplakia transform into malignancy up to 7 years postdiagnosis (Warnakulasuriya and Ariyawardana, 2016).

Hyperorthokeratosis or hyperparakeratosis with acanthosis is the typical histological presentation. Different degrees of dysplasia are also reported. Diagnosis of leukoplakia requires correlation between clinical presentation and histopathological findings.

Erythroplakia—WHO defined it as, a fiery red patch that cannot be characterized clinically or pathologically as any definable disease (WHO, 2017). This too is a diagnosis of exclusion like leukoplakia. It is strongly associated with the use of tobacco and alcohol. A mean global prevalence of 0.1% (range: 0.01%–0.21%) has been reported (Villa et al., 2011). Males in the 6th to 8th decades are most frequently affected by erythroplakia (WHO, 2017). A malignant transformation rate of 51% has been reported (Shafers and Waldron, 1975) with a range of 14%–50% reported in the literature (WHO, 2017). Thus, early diagnosis and immediate treatment are desired.

Histopathology reveals mild to moderate dysplasia in about 9% of cases of erythroplakia and carcinoma in situ in 40% cases. At times frank invasive carcinoma may be the histopathologic picture of a case clinically diagnosed as erythroplakia (Shafers and Waldron, 1975).

Actinic cheilitis—It is characterized by mottled lips with atrophic or erosive areas along with scaly, rough, flaky, keratotic patches on the exposed portion of lips. Wrinkling of the vermilion border has also been reported. The lower lip is more commonly affected but both lips may be involved in patients with bimaxillary protrusion. Labial mucosa of the lower lip may also be involved when the lip is everted due to exposure to UV radiations of the sun (Schwartz et al., 2008).

Pathogenesis involves UVA- and UVB-induced damage to collagen resulting in the aging of skin, breakdown of vitamin A and release of hydroxyl and oxygen radicals due to ionization which damage the DNA (Matsumura and Ananthaswamy, 2004).

There is a high risk of transformation of actinic cheilitis of the lower lip into squamous cell carcinoma of the lip. A malignant transformation rate of 6% to 10% has been reported. A study by Kwon et al. revealed a higher risk of malignant transformation of these lesions compared to other parts of the lip (Kwon et al., 2011).

Histopathologic presentation is in the form of atrophy or hyperplasia of the epithelium along with drop-shaped rete ridges, cytological atypia, and keratinization in the vermilion border. The underlying connective tissue exhibits UV radiation-induced basophilic degeneration (Warnakulasuriya et al., 2007).

5.2 Tobacco-induced potentially malignant disorders

Oral submucous fibrosis—It is a chronic disease involving the oral mucosa, pharynx, and esophagus at times. The patient presents with rigidity of the oral mucosa brought upon by fibroelastic changes in the juxta-epithelial connective tissue which leads to progressive trismus.

Etiologic factors include nutritional deficiencies, consumption of chillies, tobacco, areca nut, collagen disorder and genetic susceptibility (Murti et al., 1995). 20- to 40-year-old Indians are commonly affected by this condition. These patients are more likely to undergo malignant transformation than healthy adults. The transformation is generally reported in the 5th decade with an almost 32 times higher incidence in males. This malignancy is more invasive and with a higher metastatic potential than oral squamous cell carcinoma arising elsewhere. A 2% to 8% malignant transformation risk exists for patients with oral submucous fibrosis (Ray et al., 2016).

Atrophic epithelium and juxta-epithelial hyalinization along with collagen of differing densities are seen on histopathological examination (Warnakulasuriya et al., 2007).

Palatal keratosis associated with reverse smoking—A practice of keeping the lit end of a rolled tobacco leaf in the mouth (reverse of the conventional form of smoking) has been reported in people from India and Philippines (Asia), Columbia, Venezuela, Panama, and the Caribbean islands (South America) as well as Sardinia (Europe). This is termed reverse smoking and is associated with palatal and tongue changes of which the palatal changes have a malignant transformation potential ascribed to them (Ramesh et al., 2014; Ganesh et al., 2018).

Most patients are women belonging to the lower socioeconomic status. Clinical presentation on the palate ranges from keratosis, excrescences, white patches, ulcerations to even frank malignancy. 83% of these lesions present with some form of epithelial dysplasia while 13% exhibit oral squamous cell carcinoma (Gómez et al., 2008).

The histopathology reveals atypia of the epithelium, papules with umbilication at the ductal orifices resulting from hyperplastic changes in the mucous salivary gland, and microinvasive cancer (Warnakulasuriya et al., 2007).

5.3 Immune-mediated potentially malignant disorders

Oral lichen planus—It is a chronic immune-mediated disease that affects the skin and mucous membranes. Six clinical forms exist: reticular, papular, plaque, erosive, bullous, and atrophic with Wickham's Striae (lacy white network of fine lines) being its hallmark feature. A viral etiology has been proposed, citing an association with Epstein–Barr virus, hepatitis C and even human papilloma virus (Warnakulasuriya et al., 2007).

Approximately 0.5%–2.6% of prevalence has been reported. Middle-aged females are most afflicted with lichen planus. Controversy exists regarding the premalignant nature of this lesion. Approximately 0.4%–3.7% malignant transformation rate risk exists for oral lichen planus (Epstein et al., 2003).

A histopathological diagnosis is made based on the findings of hyperkeratosis with saw-tooth rete ridges, basement membrane degeneration, along with basal cell degeneration and intraepithelial T-cell migration. The underlying connective tissue shows a band lymphocytic infiltrate subepithelially (Warnakulasuriya et al., 2007; Epstein et al., 2003).

Discoid lupus erythematosus—It is a chronic immunological disorder that results in scarring of the involved mucocutaneous region (Warnakulasuriya et al., 2007). These patients present characteristic keratinized plaques exhibiting elevated borders, white radiating striae, and telangiectasia (Lourenço et al., 2006). Females are more commonly affected, and this disorder rarely transforms to malignancy unless it displays epithelial dysplasia (high-risk dysplastic lesions are at 19.25 times higher risk of transformation) or there is prolonged UV exposure (Liu et al., 2011).

Histopathological features include atrophic epithelium with hyperkeratosis, inflammatory cell infiltrate within the underlying connective tissue along with edema. PAS staining reveals thick (patchy or continuous) deposits along the basal lamina (Lourenço et al., 2006).

5.4 Genetically inherited potentially malignant disorders

Dyskeratosis congenita—It is a very rare inherited disease which is alternatively termed Cole–Engman syndrome or Zinsser–Cole–Engman syndrome. Patients present with a classic triad of reticular pigmentation of the skin, dystrophy of the nails, and oral leukoplakia (87% cases) (Auluck, 2007; Bongiorno et al., 2017). The most common form of inheritance is an X-linked recessive trait where the affected males are usually between the ages of 5 and 13 (Bongiorno et al., 2017). Mutation of DKC1 (dyskerin pseudouridine synthase 1) gene is seen on Xq28 locus (Kirwan and Dokal, 2008).

Oral manifestations include hypodontia with remaining teeth exhibiting short, blunted roots and hypocalcification. Gingival recession and inflammation, gingival bleeding, bone loss, extensive caries, leukoplakia, and lichen planus are other presentations (Auluck, 2007). This disease is associated with an increased malignant transformation potential (Bongiorno et al., 2017).

Epidermolysis bullosa—It is a rare inherited disease that presents as blistering of skin and mucous membranes. Four major forms exist: simplex (intraepidermal), junction, dystrophic (dermolytic), and mixed (Kindler syndrome) which are associated with different mutations.

(Fine et al., 2008; Ganesh et al., 2018).

Oral blistering which heals with scarring, enamel defects, and microstomia are common oral manifestations (Wright, 2011). The junctional form has a 25% malignant transformation risk (Yuen and Jonkman, 2011). In addition, these patients show a higher risk of developing basal cell carcinoma or malignant melanoma (Fine and Mellerio, 2009) (Fig. 5).

Fig. 5

Fig. 5 Clinical images: (A) Oral leukoplakia. (B) Erythroleukoplakia. (C) Erythroplakia. (D) Oral submucous fibrosis.

6 Oral epithelial dysplasia

WHO in 2017 defined dysplasia as a spectrum of architectural and cytological epithelial changes caused by accumulation of genetic changes, associated with an increased risk of progression to squamous carcinoma. In dysplasia the epithelial cell undergoes abnormal proliferation, maturation, and differentiation. The epithelium may be atrophic or show acanthosis and may or may not be keratinized.

WHO listed the dysplastic features of oral epithelium as architectural changes and cytological changes. Irregular stratification of the epithelium, loss of basal cell polarity, drop-shaped rete ridges, increased mitosis or presence of abnormally superficial mitotic figures, individual cell keratinization, keratin pearl formation, and loss of cellular cohesion make up the architectural dysplastic changes. The cytological abnormalities in dysplasia include altered cell size or shape, altered nuclear size or shape, increased nuclear:cytoplasmic ratio, presence of atypical mitotic figures, increase in number or size of nucleoli, and hyperchromasia of cells. The grading of epithelial dysplasia is then done based on the third affected with the involvement of the basal one-third of epithelium is allotted a mild grade, involvement up to the middle third is termed moderate dysplasia and in severe dysplasia epithelial involvement up to the upper third is noted (WHO, 2017). In the 1997 WHO classification, Pindborg et al., defined carcinoma in situ as a lesion in which the full thickness, or almost the full thickness, of squamous epithelium shows the cellular features of carcinoma without stromal invasion (Pindborg et al., 1997) (Fig. 6).

Fig. 6

Fig. 6 Photomicrographs (A) Mild dysplasia (4×, H&E), (B) moderate dysplasia (10×, H&E), (C) severe dysplasia (10×, H&E) (D) carcinoma in situ (4×, H&E).

In 2017, WHO pointed out that architectural and cytological atypia alone can form the basis for grading dysplasia. Marked atypia in the basal third can be used to give a diagnosis of severe dysplasia. Dysplasia grading, however, has a lower predictive value in cases of high malignant transformation. In HPV oral potentially malignant disorders, there is epithelial hyperplasia with marked karyorrhexis and apoptosis. Presence of these features warrants an assignment of severe grade of dysplasia (WHO, 2017).

Risk factors for malignant transformation of oral potentially malignant disorders include old age, large size of lesion, long duration of disease, presence of epithelial dysplasia, erythroplakia or speckled leukoplakia presentation, female gender, multifocal lesion, involvement of the tongue, and occurrence in nonsmokers. Of these, the presence of epithelial dysplasia is said to be the best predictor for malignant change (Reibel, 2003).

7 Pathogenesis

The normal oral epithelium undergoes transformation into first a premalignant and then a malignant tissue as a part of a complex, multistep process that is affected by multiple factors. There is an accumulation of genetic alterations that result in disruption of the normal functioning of oncogenes and tumor suppressor genes. In its earliest phase, the cell cycle is disrupted by dysregulation, increased proliferation and alterations in differentiation, DNA repair, apoptosis and cellular immunity (Thomson, 2012).

Any genetic, phenotypic and/or functional alterations acquired in oral stem cells result in loss of a cell cycle regulatory mechanism and causes abnormal cell proliferation. These seem to be the core mechanisms that drive the process of carcinogenesis (Thomson, 2018).

Several genes are involved in the pathogenesis of oral cancers via four major groups: regulatory genes involved in response to DNA damage, genes controlling the cell cycle, those that control growth inhibition and apoptosis, as well as the genes involved in signal transduction and cell–cell collaboration. In their systematic review, Khattak et al. concluded that tumor protein 53 (TP53) is the protein with the highest degree (maximum number of neighbor proteins for interactions), TSPO is the protein with the largest betweenness centrality (BC) value, and EGFR is the protein with the highest closeness centrality (CC) values. However, TP53 has a key position in the network due to its degree, BC, and CC values, thus indicating that TP53 is centrally localized in the network and plays a significant role in the protein–protein interaction in oral cancer. Based on these findings, the authors suggested that in pathogenesis of oral cancer, variation was carried out via an integrated network of protein-to-protein interaction that centered around TP53. Through its functions of regulating cell division and restriction of uncontrolled growth and division of cells, TP53 executes the function of at least one of the gene groups in oral cancer (Khattak et al., 2021).

Progression of dysplasia to carcinoma was initially explained as follows: there was a sequential progression from one stage to the other via a series of mutations and chromosomal changes that occurred until several genetic changes that were required for the development of cancer had accumulated (Califano et al., 1996) (Fig. 7).

Fig. 7

Fig. 7 Sequential progression to carcinoma.

In the Progressive mutation or selective sweep model, it was suggested that clones of cells that undergo dysplastic changes have a growth advantage over the normal cells. Through continuous growth, these dysplastic cells replace the normal cells of epithelium. Exposure of amplifying cells (parabasal cells that have the capability of undergoing mitotic divisions) to carcinogens which are modulated through factors such as stem cell quiescence, metabolic insults, telomere loss, error in DNA replication, epigenetic changes and/or inflammation leads to the daughter cells receiving the genetic effects of these carcinogens. The mature cells that have exited the cell cycle, however, cannot pass on these genetic changes to any other cell. Clones of cells that are actively dividing and have undergone genetic changes start taking over the entire epithelium due to their growth advantage and proliferation. A repeated exposure to carcinogens would further accumulate more mutations in these altered surviving clones. These clones lead to the production of a plethora of altered stem cells resulting from clonal divergence and selection. They, however, share the same clonal origin. It is understood that with each increasing growth advantage, these altered cells start becoming closer to a carcinoma and ultimately take over the entire epithelium (Mohan and Jagannathan, 2014).

An alternate hypothesis has been proposed by Cross et al., where evolutionary stasis is seen in dysplastic or precursor lesions. They are genetically stable and remain unchanged for long periods. Dysplasia is more common than cancer, so only a minority of dysplastic lesions undergo malignant transformation. Most of the mutations found in cancer also exist in dysplastic lesions (Cross et al., 2016). It is now thought that carcinoma arises because of chromothripsis and chromoplexy, which are catastrophic events where sudden chromosomal rearrangements are seen in the cells. Oral cancers are molecularly diverse with few driver genes. Carcinoma evolves in dysplasia and not due to progression of dysplasia. Thus, no predictive pathway exists for cancer (Makarev et al., 2017).

Punctuated equilibrium theory (based on other body sites but fits the oral cavity) states that small clones of altered cells are formed within the actively dividing cell populations that are exposed to carcinogens. Their genetic pathways are inhibited. Some of these clones are unable to survive in a solid population and require intermingling with normal cells as they require signaling and obtain their nutrients from these normal cells. Ultimately, genetically abnormal clones of cells are found in the altered epithelium (Gould and Eldredge, 1993).

Henry Wood et al. stated that there was a rare mutation in the known cancer genes found only in the dysplasia samples. This suggests that much of the evolution of dysplasia is related to accumulation of passenger events. It is probable that these gene mutations in cancer were likely to be shared. Dysplasia seems to develop through accumulation of random nonsignificant mutations in cells. A separate unconnected event leads to the development of cancer in these dysplastic lesions. TP53 mutations, however, were almost always common to both lesions, presaging that, where displayed, they were essentially early events in the development of squamous cell carcinoma in the head and neck region. Thus, making p53 the most promising marker of field cancerization as there is a strong positive correlation with tumor progression from a benign to a malignant state (Wood et al., 2017).

7.1 Autophagy

Autophagy is natural cell-housekeeping which functions to remove damaged or senescent cell proteins and/or organelles. Cellular components are degraded by sequestration in vesicles (autophagosomes) which eventually fuse with lysosomes. At low pH in these organelles, now termed autolysosomes, the hydrolytic enzymes degrade the sequestered material (Feng et al., 2014; Yang and Klionsky, 2020). By virtue of this survival-promoting pathway, autophagy prevents the buildup of toxic cellular waste products and provides substrates required for sustenance of metabolism during starvation (Alexandra et al., 2020). Stress up-regulates autophagy for coping with the damage in the cell. Autophagy-related proteins (ATGs) participate in autophagy (Feng et al., 2014) (Fig. 8).

Fig. 8

Fig. 8 Autophagy cascade.

ATG1 serves as an on/off switch for autophagy (Peña-Oyarzún et al., 2020). ATG1 also termed ULK1 (Unc-51-like autophagy activating kinase) is activated by AMPK (AMP-activated protein kinase) and inactivated by mTOR (mechanism target of rapamycin). The nutritional status of a cell is kept under check by AMPK and mTOR. mTOR is elevated in normal nutritional conditions where it suppresses autophagy. On the other hand, starvation leads to activation of AMPK which in turn promotes autophagy (Li et al., 2013; Meijer et al., 2015).

Inflammatory pathways like transcription factor nuclear factor -Kappa B (NF-κB) also regulate the process of autophagy. NF-κB is translocated to the nucleus on the degradation of its inhibitors. This results in an increase in the expression of genes that control inflammation, the proliferation of cells, cell survival, an epithelial–mesenchymal transition which in turn leads to invasion, angiogenesis as well as metastasis (Liu et al., 2017). Thus, activation of NF-κB leads to increased autophagy via ATG5 and LC3 expression (Copetti et al., 2009). Another effect of NF-κB is to promote mTOR expression through which autophagy is repressed (Lee et al., 2007).

For cancers to progress, autophagy modulation is to be carried out. Autophagy not only results in tumor formation due to its inhibition in normal cells but is also increased in frank tumors such that their growth is facilitated by enabling the tumor cells to survive microenvironmental stress. These cancer cells then exhibit an increase in both growth and aggressiveness (Singh et al., 2018). Mechanisms that include suppression of the P53 tumor suppressor protein by autophagy promote cancer development. Autophagy also helps to maintain the metabolic function of mitochondria. Genetics, tumor microenvironment, type of tumor, and its stage of development influence autophagy which in turn affects the process of carcinogenesis (Sakakura et al., 2015).

In oral squamous cell carcinoma, impaired autophagy is linked to a poor prognosis. Advanced cases of oral squamous cell carcinoma show high levels of SQSTM1/p62. As a result, there is absence of fusion of lysosomes in the tumor cells with autophagosomes (Liu et al., 2014). Immune infiltration of T lymphocytes and tumor associated macrophages along with the accumulation of SQSTM1 and LC3 portray inhibition of autophagy during these advanced stages. These help to establish a tumor immune niche (Sakakura et al., 2015). Downregulation of autophagy through activation of AMPK results in the transformation of normal fibroblasts to tumor-associated fibroblasts that release chemokines which promote epithelial–mesenchymal transition in cancer cells (Zhang et al., 2019) (Fig.