Molecular basis of

Oral Potentially Malignant Disorders

Presented by:Ujwal Gautam

BDS 2009(4th year)BPKIHS

Contents

• Malignant Transformation• Oral Epithelial Dysplasia • Multistep Carcinogenesis• Field Cancerization• Molecular diagnosis• Genes playıng role ın malignancy• Role of growth factors• Telomere and Cancer• Role of Alcohol• Role of Viruses• Chronic Hyperplastic Candidiasis• Graft Versus Host Disease• Systemic Lupus Erythematosus

multifactorial process

Genetic predisposition, immunodeficiency, diet and viral infections, e.g. HPV (human papillomavirus) and HHV (human herpes virus)

Malignant Transformation

NORMAL tissue

CANCEROUS lesion

PREMALIGNANCY

Histological connotationat tissue level

DYSPLASIA

Oral precancerous lesions are usually histologically classified by

the presence or absence of

Oral Epithelial Dysplasia (OED)

the cellular atypia or dysplasia is similar to that seen in squamous cell carcinoma;

there is no evidence of invasion into underlying stroma (the diagnosis would then change to carcinoma)

the epithelium with the greatest proportion of atypical cells has the greatest risk of being or becoming a carcinoma;

the epithelium with the most extreme atypia of cells has the greatest risk of being or becoming a carcinoma;

the final grading or diagnosis should be based on the most severely involved area of change, even if that area includes no more than a few rete processes

oral precancer grading criteria

The malignant potential of oral precancerous lesions is assessed by histopathology and mainly based on the presence and degree of dysplasia in biopsy material.

Not exclusive to carcinogenesis, as they may be seen in reactive epithelium or epithelium influenced by a variety of systemic alterations.

Dysplastic features

o Cells high in the epithelium have the same immature appearance as those in the basal layers.

o due to an apparent inability to properly differentiate and mature from basal cells to prickle cells to flattened keratinocytes.

o This feature is especially pronounced in severe epithelial dysplasia and carcinoma in situ.

o Drop-shaped rete processes, regardless of their size, especially if secondary projections or nodules are seen to arise from the basal layer and branch at in different angles into the lamina propria and connective tissue papillae are suggestive of poor prognosis.

o Enlarged, tripolar or star-shaped mitotic figures (abnormal mitoses), however, are much more indicative of precancerous changes.

o Abnormal mitosis may also be defined as mitotic figures found in unusual locations above the basal cell layer.

o much more commonly seen in oral carcinomas than in oral premalignancies.

o dyskeratosis may be represented by individually keratinized cells or by tight concentric rings of flattened keratinocytes (epithelial pearls).

o loss of cellular cohesiveness (acantholysis) are major signs of poorly differentiated carcinoma but are extremely rare in the epithelial dysplasia of oral precancer.

o When present, these features must be distinguished from intercellular edema, intraepithelial inflammatory cells and degenerating cells with pyknotic nuclei and vacuolated cytoplasm

o Basal cell hyperplasia is of major importance to the diagnosis as well as to the grading of dysplasia

– Denotes the accumulation of genetic and/or epigenetic changes resulting in the progression from a normal cell into a cancer cell.

– changes may appear in less important regions of the genome and are therefore a mere reflection of the genetic instability of the tumors, but some of these alterations specifically occur in genes that play a crucial role in signalling pathways that regulate DNA maintenance, cell cycle or other important physiological processes.

Multistep Carcinogenesis

Genetic progression model for OSCC

A model for field cancerization

Field Cancerization

Molecular diagnosis

The hypothesis that oral cancers are preceded by precancerous fields of genetically altered cells, suggests that screening approaches for precancerous changes might become an option with molecular tools detecting genetic alterations

As the fields are characterized by genetic changes, the markers of choice include;

I. Numerical chromosomal alterations

II. Allelic imbalance analysis by microsatellitesChromosomal changes in (pre)cancer cells can be determined by allele-specific markers such as microsatellites. The loss of a given marker is generally considered a hallmark of the loss of a specific chromosomal locus harboring a tumor suppressor gene, or an unbalanced amplification.

III. DNA content: Abnormal nuclear DNA content (aneuploidy) is an indicator of chromosomal aberrations and is associated with malignant and premalignant lesions

Molecular diagnosis

23

• Oncogenes • Tumor suppressor genes • DNA repair genes

Genes playıng role ın malignancy

24

What are the genes responsible for tumorigeniccell growth?

Normal

Cancer

Proto-oncogenes Cell growthand

proliferationTumor suppressor genes

+

-

Mutated or “activated”oncogenes Malignant

transformationLoss or mutation of

Tumor suppressor genes

++

25

ONCOGENES

• Oncogenes are mutated forms of cellular proto-oncogenes.

• Proto-oncogenes code for cellular proteins which regulate normal cell growth and differentiation.

p21 ras oncoprotein has been observed in precancers

Genes playıng role ın malignancy

26

TUMOR SUPPRESSOR GENES

• Normal function inhibit cell proliferation• Absence/inactivation of inhibitor cancer• Both gene copies must be defective for loss of function

p53 gene Rb gene p16INK4A FHIT

Genes playıng role ın malignancy

27

These are genes that ensure each strand of genetic information is accurately copied during cell division of the cell cycle.

Mutations in DNA repair genes lead to an increase in the frequency of mutations in other genes, such as proto-oncogenes and tumor suppressor genes.

Eg. Defect in the genes encoding proteins involved in nucleotide-excision repair of damaged DNA causes Xeroderma pigmentosum.

DNA REPAIR GENES

Genes playıng role ın malignancy

28

Importance of DNA repairGenes playıng role ın malignancy

Role of growth factors

Growth factors necessary for normal growth and development may also play a role in oral cancer and precancer.

Fibroblast grown factors (FGF) – widely distributed in normal and neoplastic tissues– involved in angiogenesis and wound healing, and have also been shown

to be mitogenic for keratinocytes. – Almost all oral carcinomas are immunoreactive to FGF and oral

carcinoma cells in culture are capable of expressing FGF. – Biopsy samples of oral dysplastic lesions have also demonstrated

positive focal staining which becomes stronger with increasing immaturity or severity of the dysplastic cells.

Epidermal growth factor receptor (EGFR)– protein of the proto-oncogene c-erb– Its expression has been correlated somewhat with an increased rate of

recurrence in some cancers of the head and neck region– Role in oral precancers has not yet been proven

Proliferation markers– measure of its biological potential, proliferative activity, can be measured by

immunohistochemical demonstration of proliferation markers such as proliferating cell nuclear antigen (PCNA) or the nonhistone nuclear protein, Ki67 (MIB 1)

– Local recurrence has been associated with a decreased PCNA expression or increased Ki67 expression in head and neck cancers

– evaluation of these events in oral precancers has not yet been reported.

Telomere and Cancer

Telomere and Cancer

carcinogens contained in tobacco products are responsible for cancers.

More than 60 carcinogens are in cigarette smoke and at least 16 in unburned tobacco. Among these, tobacco-specific nitrosamines (such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N -′nitrosonornicotine (NNN)), polycyclic aromatic hydrocarbons (such as benzo[a]pyrene) and aromatic amines (such as 4-aminobiphenyl) seem to have an important role as causes of cancer.

Role of Tobacco

• PAH,NNK and NNN are the most likely causes of oral cancer in smokers.• NNK and NNN, perhaps together with enhancing agents, are the carcinogens most

likely to cause oral cancer in smokeless-tobacco users.

ORALCANCER

Role of Tobacco

– characterized by a intracellular vacuolization or "edema" of superficial layers, perhaps interspersed with streaks of parakeratinized cells.

– most likely results from a low-grade chemical burn from the alkaline tobacco used. It has been referred to as surface etching and has no implications one way or another relative to the risk of malignant transformation

Smokeless tobacco keratosis

Role of Tobacco

– benign hyperkeratosis and acanthosis to hyperkeratosis accompanied with graded epithelial dysplasia or invasive squamous cell carcinoma.

– Cigarette smoke causes oxidative damage, probably because it contains free radicals such as nitric oxide and mixtures of hydroquinones, semiquinones and quinones, which can induce redox cycling

Reverse Smoker’s Palate

Role of Tobacco

–an important risk factor for the development of OSCC–Metabolism of ethanol results in the generation of toxic compounds

such as acetaldehyde, hydroxyethyl radicals, ethoxy radicals, and hydroxy radicals –Acetaldehyde is highly toxic, mutagenic, and carcinogenic – In the mouth the conversion of alcohol to acetaldehyde can be

catalyzed by alcohol dehydrogenase (ADH) enzymes from the epithelium and also from oral microorganisms.–Acetaldehyde in the oral cavity can also come from tobacco smoking.

Tobacco smoke contains toxic aldehydes, and the combined use of tobacco and alcohol has a synergistic effect on the risk of developing oral cancer

Role of Alcohol

Actinic Cheilitis and Role of UV radiation

Result of clonal expansion of UVB-induced transformed keratinocytes characterised by molecular and genomic alterations causing genomic instabilityThough additional genetic changes are necessary for full malignant transformation with the potential for invasiveness and metastasis.

UVB from the sun leads to mutations in the epithelial p53 tumour-suppressor gene that result in the dysregulation of its functions. Formation of aberrant covalent bonds between adjacent cytosine bases in epithelial DNA.

Role of Viruses

With latent or chronic infections, the viral agent becomes incorporated into the human, immortalize the host cell, and facilitate malignant transformation;

1. Viral interference with tumor suppressor gene function

2. Viruses may act as vectors for mutated proto-oncogenes (oncogenes).

Overexpression of these oncogenes in viral-infected cells

Pathogenesis:

Oncoproteins, E6 and E7, from these virusesbind with wild-type p53 and pRb proteins and

remove their ability to stimulate DNA repair or apoptosis.

Viral-infected cells may continue to undergo additional mutations induced by other `DNA-damaging' events, including tobacco, alcohol, carcinogens, toxins, ionizing radiation, prolonged ultraviolet light/sun exposure, chemical insult, other viral infections, which may result in the development of a malignant tumor (transformation process).

Role of Viruses

Human herpes virus 4 (HHV-4, Epstein- Barr virus)

– allow immortalization of epithelial cells and B lymphocytes– EBV latent infection is capable of inducing malignant tumor formation in

oncogenic genes.– EBV nuclear antigens (EBNA1, EBNA2, LMP1) are themselves known to be

oncogenic while another EBV nuclear antigen (EBNA-LP) interferes with wild type p53 and pRb function and results in dysregulation of the cell cycle.

Role of Viruses

Human herpes virus 8 (HHV-8, Kaposi's sarcoma- associated herpes virus, KSHV)

– Has oncogenic (transforming) ability via a viral encoded receptor (G-protein coupled receptor), which induces cell proliferation mediated by vascular endothelial growth factor.

– In addition, ras/raf oncogenes are activated by a proliferation cascade initiated by HHV-8.

– Additional oncogenic proliferation factors, prad-1, and bcl-2, expressed by HHV-8 interfere with wild-type p53 and wild-type pRb regulation of cell proliferation.

Role of Viruses

Human herpes virus 6 (HHV-6)

– Tumor necrosis factor-alpha is over expressed and this appears to induce HIV replication which in turn increases HIV tat protein, a protein with oncogenic potential.

– In both immunocompetent and immunosuppressed hosts, HHV-6 produces an oncogenic protein (ORF-1) which binds to wild-type p53 and inhibits regulation of cell proliferation, which allows for the escape of HHV-6 infected host cells from cell growth regulation

Role of Viruses

Human papilloma virus (HPV)

have tropism for squamous cells and their full reproductive life cycle is only supported in these epithelial cells as their viral replication functions are limited to the most terminally differentiated squamous cells or keratinocytes.

viral DNA synthesis occurs within the basal layer of squamous epithelium and the incorporation of HPV genes into the basal cells ensures that a persistent lesion will develop.

Role of Viruses

HPV as an etiologic factor for OSCC

Gene products E5, E6 and E7, are considered to be oncoproteins and are associated with the development of squamous cell carcinoma of the oral cavity.

1. E6 and E7 mediated inhibition of cell cycle regulation tumor suppressor genes

2. E5 mediated stimulation of growth factor activity, which enhances cell proliferation and may influence transformation to a malignant process.

Role of Viruses

HPV Cofactors

Squamous papillomas 6, 11 Alcohol, carcinogenSmoking, HSV, HIV

Verruca vulgaris 1, 2, 4, 7, 57

focal epithelial hyperplasia (Heck's disease)

13, 32

Condyloma acuminatum 2, 6, 11, 16, 18, 31, 33, 35

Oral epithelial dysplasia (koilocytic dysplasia)

16, 18, 31, 33, 35

verrucous carcinoma 2, 6, 11, 16, 18

squamous cell carcinoma 2, 3, 13, 16, 18, 31, 33, 35, 52, 57

Role of Viruses

Herpes simplex virus (HSV)

– binds and inactivates basic fibroblastic growth factor, activating certain oncogenes (c-myc) that results in the activation, amplification and overexpression of pre-existing oncogenes within neoplastic tissue, such as c-erb-B-1 and c-myc.

– HSV participates in malignant transformation as a cofactor in squamous cell carcinoma development.

– Possible reactivation of HSV infection(hit and run effect) by suppression of natural killer lymphocyte activity due to chromosomal aberration/mutations and gene amplification in the presence of ultraviolet light and carcinogens (nitrosamine and other tobacco extracts)

Role of Viruses

• CHC is a form of oral candidiasis that typically presents as an adherent chronic white patch on the commissures of the oral mucosa

• It is characterized by hyphal invasion of the epithelial surface, which usually becomes parakeratinized; the hyphae rarely extend beyond the parakeratin layer.

• Candida albicans is the most common Candida species present in candidal leukoplakia

• Candida infection in oral leukoplakias was originally termed ‘candidal leukoplakia’ but it was rather turned down since Leukoplakia is itself known to be idiopathic.

Chronic Hyperplastic Candidiasis

It has been shown that leukoplakia with candidal infectionhas a higher rate of malignant transformation

than uninfected leukoplakia,with estimates that

15% of non-dysplastic CHC will progress to dysplastic lesionswith 10% of these developing OSCC

Attributes of C. albicans that may influence oral cancer development

– Colonization of the epithelium– Ability to produce carcinogens and initiate carcinogenesis– Ability to promote carcinogenesis in initiated epithelium– Ability to metabolize procarcinogens– Ability to modify the microenvironment and induce chronic

inflammation

Candida might induce OSCC by directly producing carcinogenic compounds like nitrosamines which binds with DNA to form adducts with bases, phosphate residues, and/or hydrogen bonding sites that could cause miscoding or irregularities with DNA replication.Point mutations thus induced may activate specific oncogenes and initiate the development of oral cancer.

C. albicans has been shown to act as a promoter of oral carcinogenesis in rat and mouse OSCC models

In vitro, C. albicans cultures have shown metabolism of ethanol to the carcinogenic acetaldehyde which is highly toxic, mutagenic, and carcinogenic

Production of Carcinogens

C. albicans has been shown to secrete specific proteinases, capable of degrading basement membrane and extracellular matrix. Degradation of laminin-332, a laminin present in the basement membrane associated with oral epithelium, by C. albicans has been described. C. albicans has also been demonstrated to degrade E-cadherin, a transmembrane glycoprotein important in adhesion of adjacent keratinocytes

Mucosal bacterial infection will induce chronic inflammation in the adjacent connective tissue leading to upregulation of cytokines and growth factors, which in turn may influence carcinogenesis

Induction of Chronic Inflammation

The pathogenesis of acute graft versus host disease (GVHD) is multistep process. GVHD occurs in three sequential steps:

conditioning regimen: simultaneous damage and activation of host tissues, amplification of antigen presentation to allogeneic donor T cells.

donor T cell activation by host alloantigens, proliferation and secretion of a variety of cytokines, Type 1 cytokines (interleukin- 2 and interferon-γ) and

effector mechanisms.

Graft Versus Host Disease

characterised by a myriad of immune system aberrations

involves B cells, T cells, and cells of the monocytic lineage, resulting in polyclonal B cell activation, increased numbers of antibody producing cells, hypergammaglobulinaemia, autoantibody production, and immune complex formation

results in primary pathological findings of inflammation, vasculitis, and vasculopathy

Systemic Lupus Erythematosus

APOPTOSIS AND SLE

Defective apoptosis in SLE leads to the prolonged survival of pathogenic lymphocytes

Under normal circumstances, apoptotic cells are engulfed by macrophages in the early phase of apoptotic cell death without inducing inflammation or the immune response. However, the clearance of apoptotic cells by macrophages in patients with SLE is impaired. This is not confined to monocytes and macrophages of the peripheral blood, but also occurs in the germinal centres of lymph nodes.

Multiple Choice Questions

Which of these is associated with significant risk of malignant transformation?

a. HSV 1,3b. HPV 8, 10c. HPV 16, 18d. CVA 16

Multiple Choice Questions

The current “gold standard” for predicting the malignant potential of the precancerous lesions is the

a. presence & degree of dysplasiab. presence of candidal hyphaec. presence of red areas in the lesiond. site of the lesions

Multiple Choice Questions

Which stage of syphilis is a pre-disposing condition to oral cancer?

a. Primary stageb. Secondary stagec. Tertiary staged. Syphilis is not a pre disposing condition to oral cancer

Multiple Choice Questions

The total amount of carcinogens in cigarette smoke adds up to

a. 1-3 mg per cigaretteb. 7-8 ug per cigarettec. 1-2 ng per cigaretted. Recent studies show tobacco smoke contains no carcinogen

Multiple Choice Questions

Which of these is not a Tumor Suppressor gene?

a. p53b. p16INK4Ac. YNWAd. Rb

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