An Insight into Tumor Markers in Oral Cancer: A Review

DOI: 10.21276/sjds.2017.4.12.5 Abstract: Tumour markers are substances that are produced either by the tumour itself or by the body in response to the presence of cancer or certain benign conditions that can aid in the diagnosis of cancer. These markers may be employed to predict primary or secondary tumor risk. Sometimes, non-cancerous conditions can also cause elevation of some tumor markers to be higher than normal. Besides, not every cancer patient may have raised level of a tumor marker. For these reasons, knowledge about cancer biomarkers is essential. This review highlights potential molecular markers relevant to oral neoplasia in terms of their perspective role of in prevention and detection.


INTRODUCTION
Oral cavity cancer is amongst the most prevalent cancers worldwide and incidence rates are higher in men than women. There are an estimated 529,000 new cases of cancers of the oral cavity andpharynx each year, and more than 300,000 deaths. Oral cancers include the main subsites of lip, oral cavity, nasopharynx, and pharynx and have a particularly high burden in South Central Asia due to risk factor exposures [1].
The diagnosis of cancer is based on the analysis of tissue and cytology specimens obtained through several procedures. When a cell becomes cancerous, new antigens unfamiliar to the immune system appear on the cell's surface. The immune system identifies these new antigens, called tumor antigens, as foreign and may be able to contain or destroy the cancerous cells [2].
A tumor marker can be defined as a molecule that indicates the likely presence of cancer or can also be defined as one that provides information about the likely future behavior of an existing cancer (e.g. ability to metastasize or to respond to therapy). The majority of existing tumor markers is mostly useful in making a clinical decision after initial suspicion of cancer or its behavior which has been already raised by more conventional means [2]. [3]  It should be highly sensitive and should have low false negatives.  It should be highly specific and should have low false positive.  It should have high positive and negative predictive value.  100% accuracy in differentiating between healthy individuals and tumor patients.  It should be able to differentiate between neoplastic and non-neoplastic disease and show positive correlation with tumor volume and extent.

Indications of tumour markers[5]
For Screening and Early Detection of Cancer Screening refers to looking for cancer in people who have no symptoms of the disease, while early detection is finding cancer at an early stage. Although tumour markers were first developed to test for cancer in people without symptoms, very few tumour markers have been found to be helpful in this way because most tumour markers have not been shown to detect cancer much earlier than they would have been found otherwise.

Diagnosing Cancer
In most cases, cancer can only be diagnosed by a biopsy and tumour markers are usually not used to diagnose cancer. However tumour markers can help determine if a cancer is likely in some patients. It can also help diagnose the origin of the cancer in patients presenting with advanced widespread disease.

Determining the Prognosis (Outlook) for Certain Cancers
Some newer tumour markers help to assess how aggressive a cancer is likely to be or even how well it might respond to certain drugs.

Determining the Effectiveness of Cancer Treatment
One of the most important uses for tumour markers is to monitor patients being treated for cancer. If the initially raised tumour marker level goes down with treatment, it indicates that the treatment is working and is having a beneficial effect. On the other hand, if the marker level goes up, then the treatment is probably not working and change of treatment should be considered.

Detecting Recurrent Cancer
Markers are also used to detect cancers that recur after initial treatment. Some tumour markers can be useful once treatment has been completed and with no evidence of residual cancer left. [2]  False elevation may occur in non-neoplastic conditions as many tumour markers are proteins, over expressed not only by cancer cells but also by normal tissues.  Early detection difficult, since low levels are seen in normal individuals.  Large volume of cancer needed for significant elevation above normal.  Many tumour markers are not specific to a particular type of cancer.  Tumour marker levels are not elevated in every person.  No simple tests are yet available with sufficient specificity to detect the presence of a cancer.

Types of tumour markers[6]
Intermediate markers-measure cellular and molecular alterations before the onset of the malignant neoplasm.  Diagnostic markers-useful in confirming the malignant lesion established.  Prognostic markers-determine the growth, metastasis, and invasion potential of tumour.
 Tumour-specific markers-specific for a single tumour  Tumour-associated markers-are found with different tumours of the same tissue type [7] Classification of tumour markers Earlier classification given by Neville AM & Cooper EH 2 is considered to be arbitrary with considerable overlap and grouped Tumour markers into Hormones, Oncofetal products, Enzymes/ isoenzymes and other macromolecules. Even though broader classification was proposed in later years there is no single universally acceptable classification of Tumour markers to date [8]

Neville AM & Cooper EH classification[8]  Hormones
Markers to predict response to therapy  Oestrogen and progesterone receptors  Androgen receptors  Steroid-regulated proteins-Cathepsin D and pS2  c-erbB-2 Gene Markers to monitor drug resistance  P-glycoprotein (a transmembrane protein)  c-erbB-2 Growth factors and receptors  Epidermal growth factor receptors, erb-2 oncoprotein, insulin and insulin-like growth factor receptors, transforming growth factor -receptors, fibroblast growth factor receptors and the somatostatin receptors.

Tumour angiogenesis:
 Microvascular density has been found to be an independent marker of prognostic relevance.

Anti-apoptosis genes  bcl-2
Nm23 Anti-metastasis gene  The nm23 gene family was originally identified in a murine melanoma cell line and nm23-HI was found to be transcribed at a 10-fold higher rate in cells of lower metastatic potential.

DNA Repair genes-microsatellite instability (MSI)
 The human genome is punctuated with an enormous number of short repetitive nucleotide sequences known as microsatellites. They are less likely to be associated with lymphatic and distant metastasis and the improved prognosis applies even they are stratified by stage.

Miscellaneous markers
 K-ras and c-myc oncogenes, transforming factors TGF-a, TGG-b, adhesion proteins E-cadherin and CD 44, and the matrix metaloproteases and inhibitors, etc. [7].

Broad classification of tumour markers[8]
Tissue markers of potential and established malignancy [8] Cell surface markers Carbohydrates Cell surface carbohydrates may be important in the interaction of cells with matrix and other cells and therefore have attracted interest as markers for epithelial maturation, differentiation and neoplastic change. There are regional variations in cell surface carbohydrates in normal oral epithelium but malignancy can be associated with synthesis of new carbohydrates, deletion of complex structures, and the accumulation of precursors [9].

ABH blood group antigens
The ABH blood group antigens are cell surface carbohydrates that may change in oral dysplastic lesions and malignant lesions. The expression of antigens A and B is lost and there is extended expression of precursor antigens. Some leukoplakias showing such cell surface carbohydrate changes but no dysplasia on conventional criteria have later developed into carcinomas, suggesting these changes may be of predictive value [9].

Lectins
Ulex europaeus agglutinin 1 (UEA-1) is a lectin that binds to α-L-fucose and is an approximate marker for the blood group antigen H. The lectin Bandeiraea simplicifolia (BSA-1) binds to a-Dgalactose and is specific to blood group antigen B, Peanut agglutinin (PNA) binds to the carbohydrate Dgalactose-P 1-3-N-acetylgalactosamine in normal oral epithelium, UEA stains mainly the stratum spinosum, BSA the upper stratum spinosum, and PNA the stratum basale of normal oral epithelium [9].

Thomsen-Friedenreieh carbohydrates
The Thomsen-Friedenreich antigen (T antigen) is a carbohydrate which is carried on the same protein as the human blood group antigen system MN on erythrocytes. Antigen Tn is a precursor of T in this system. The sialosyl-T form is found on cell surfaces of basal and some parabasal epithelial cells in normal oral epithelium and, in non-secretors of ABH blood group antigens, also in the stratum spinosum. In contrast, in normal epithelium the less mature carbohydrates Tn and sialosyl-Tn are found in the cytoplasm of some spinous but not basal cells. In dysplastic oral epithelium, sialosyl-T expression in basal cells disappears and conversely, sialosyl-Tn expression extends to the basal cells. The changes in these mucin-type carbohydrates increase with increasing severity of dysplasia but are not specific to potentially malignant lesions [9].

Histocompatibility antigens
HLA class 1 antigens continue to be expressed in normal, potentially malignant and malignant oral epithelium -though there is partial loss in a few carcinomas. HLA class antigens are expressed on some oral carcinomas, most frequently in poorly differentiated tumors [9].

Beta -2 microglobulin (β2m)
β2M is 11 KD light chain constituent of HLA antigen. The Beta 2 M is used clinically as a marker of first choice for B-cell leukemia, lymphomas and multiple Myeloma. However, due to its non-specificity its moderate elevation is observed in cases of solid tumors and also in various inflammatory diseases, benign infectious disorders, and primary biliary cirrhosis and in acquired immune deficiency syndrome. It is used routinely for evaluating tumor cell load, disease activity and prognosis. It is also used to monitor efficacy of patient's response to treatment. Elevated levels of Beta 2 -M are also reported in cerebrospinal fluid (CSF), acute lymphobalstic leukemia, Lymphoma and other Lymphoproliferative disorders/diseases. Hence, the determination of β2M in CSF helps in identifying and managing CNS metastases [3]. Normal value-< 2.5 mg/L

Squamous carcinoma antigens
Several squamous carcinoma antigens have been described but these have not been fully studied in potentially malignant oral lesions.  Antigen Ca-1-Ca-1 antigen is a cell surface glycoprotein found on malignant but not normal cells, but is also unfortunately found on nonmalignant lesions and this is therefore highly unreliable in distinguishing malignant lesions.  Antigen (SCC-Ag) TA-4-SCC-Ag-(TA-4) is found on squamous carcinomas and in serum, preceding recurrence.  Antigen SQM1-SQMl is an antigen detectable on oral and various other carcinomas in the head and neck.  Antigen 3H-1-A monoclonal antibody antibody 3H-1 has recently been reported to stain squamous carcinomas but also stains other rapidly dividing squamous cells.  Differentiation antigens-Two tumour associated antibodies -MAb K 984 and MAb K 928recognizing surface antigens dependent on squamous differentiation have been generated using viable cells of squamous cell carcinomas. MAb K 984 reacts with undifferentiated basal cells and is apparently associated with the proliferative fraction of squamous carcinomas; MAb K 928 binds to suprabasal cells [9].

Growth factors and receptors
Epidermal growth factor Epidermal growth factor (EGF) is a polypeptide, mitogenic to epithelial cells, and acting by binding to a cell surface receptor (EGF receptor: EGFR) thereby stimulating a cellular protein kinase which catalyzes the phosphorylation of tyrosine found in various proteins. EGF is found in small amounts in normal oral mucosa, mainly in the upper lamina propria close to the epithelium; the cellular source is unclear but it seems likely to be mesenchymal, possibly fibroblasts. Increased amounts of EGF at this site are seen in dysplastic and malignant oral epithelial lesions but the epithelia do not express EGF [9].

Transforming growth factor α
Transforming growth factor alpha (TGF-α), which is related to EGF, can stimulate epithelial growth by binding to and activating the EGF receptor, and is not expressed in normal epithelium but is expressed in oral squamous cell carcinomas. Messenger RNAs from TGF-α and EGFR have been demonstrated in oral carcinomas. High levels of TGF-α are associated with poor tumor differentiation. TGF-α appears to be derived mainly from tumor-infiltrating eosinophils [9].

Transforming growth factor β
Transforming growth factor, TGF-β, binds to distinct cell surface receptors, may stimulate or inhibit cell proliferation, and may play a role in the growth of carcinomas by modulating an autocrine growth control loop. Carcinoma cell lines appear to express fewer TGF-β receptors. Tumor cell lines have altered responses to both EGF and TGF-β, which appears to give the tumor a growth advantage over normal keratinocytes [9].

Cytokeratins
Cytokeratins constitute the main structural proteins in epithelial cells. At least 19 cytokeratins have been described and they fall into 2 sub-tamilies -acidic (type 1 or A) and basic (type II or B). Normally, each acidic cytokeratin is co-expressed with a specific basic one, as a "keratin pair" [9] Filaggrin Filaggrin is a histidine-rich basic protein found in the granular and cornified layers in normal oral and other epithelium. Filaggrin is responsible for aggregating keratin intermediate filaments in the final and crucial steps of keratinocyte terminal differentiation and orthokeratinization. In oral leukoplakias, filaggrin appears in the stratum corneum and in oral carcinomas it appears in keratin pearls but is less evident in undifferentiated or anaplastic lesions. No differences have been found between leukoplakias with and without dysplasia, though others report that filaggrin expression correlates with the degree of oral epithelial dysplasia [9].

Involucrin
Involucrin is a precursor of the cross-linked envelope present mostly in and above the stratum spinosum where there is a commitment to terminal differentiation of the keratinocyte. Both proliferative and neoplastic oral mucosa show decreased overall staining for involucrin but positive foci scattered throughout the epithelium and especially positive staining of keratinized areas in carcinomas. No differences have been found in involucrin expression in oral leukoplakias with and without dysplasia [9].

Desmosomal proteins
Desmosomal proteins are complex entities, major components of desmosomes. Potentially malignant oral lesions appear not to have been studied but oral squamous carcinomas -as do all carcinomas, mesotheliomas and carcinoid tumors show strong positive reactivity for desmoplakin, and this correlates with the cytokeratin positivity [9].

Intercellular substance antigen
Intercellular substance antigen is totally or partially absent from 92% of oral leukoplakias with dysplasias and 26.3% of leukoplakias without dysplasia. The degree of antigenic loss correlates with the degree of dysplasia and in carcinomas there is 95% loss [9].

Other cytoplasmic antigens
A monoclonal antibody raised to a specific cytoplasmic antigen in oral squamous carcinoma stains basal cells in normal oral mucosa and benign epithelial lesions but homogeneously stains carcinomas, allowing a good assessment of microinvasion [9].

Silver-binding nucleolar organizer regions
In a cancer cell, chromosome disarray with multiple nucleoli appears to result in an increase in AgNORs and higher AgNOR counts suggest poor prognosis for oral cancer. Ag-NORs appear not to have been examined in potentially malignant oral lesions [9].

Oncogenes
Genetic alterations are involved in the deregulation of cell growth and differentiation that leads to cancer. Specific karyotypic abnormalities involving particularly chromosomes 1 and 11, often at 11ql3 have been found in some head and neck carcinomas. Oncogene expression is a widespread phenomenon and thus quantitative analysis of expression is essential, but the enhanced expression or amplification of c-erb-B with c-myc oncogenes may be related to the biological behaviour of oral carcinomas. The bcl-1 locus on chromosome 11 may be highly susceptible to mutation in individuals who smoke tobacco and it may be that mutation and amplification of this and closely related oncogenes such as int-2 and hst-1 are involved in some oral carcinogenesis, acting via production of factors similar to fibroblast growth factor [9] Arachidonic acid products Lipoxygenase metabolites including prostaglandin E2, 5, 12 and 15 hydroxyeicosatetraenoic acids and leukotriene B4 are increased in oral squamous carcinomas but potentially malignant lesions have not been studied [9].

Enzymes
Gamma-glutamyl transpeptidase (GGT) is a membrane-associated enzyme involved in transport of amino-acids; GGT is expressed in non-dysplastic epithelium of high risk patients and in dysplasias and carcinomas of the oral, pharyngeal and laryngeal mucosa. High activity of lactate dehydrogenase and changes in the isoenzyme pattern has been found in oral carcinoma cells but not in adjacent normal oral epithelium. The protease guanidinobenzoatase which is capable of breaking down extracellular matrix is found in cells with active locomotion and in most malignant tumor cells. In oral squamous cell carcinomas, guanidinobenzoatase can be demonstrated in areas of invasion and it has therefore been advocated as a marker of tumor cells [9].

Basement membrane markers
Epithelial basement membranes are complex structures composed of glycoproteins, proteoglycans and collagens. The basal lamina contains principally type IV collagen, laminin and heparin sulphate, Entactin, nidogen and fibronectin are also present. The lamina reticularis contains collagens types I and III. and type VII collagen anchors the basal lamina to the connective tissue. Fibronectin is consistently present in basement membrane and adjacent connective tissue in normal oral mucosa, leukoplakia and carcinomas, while irregular discontinuities and loss of laminin occur in areas of carcinoma in situ and tumor invasion [9].
The basal lamina fragments in malignant neoplasms and, although this may be a consequence of neoplastic invasion, some components such as laminin might actually promote invasion and metastasis. Changes in epithelial basement membranes may help distinguish invasive from in situ neoplasia [9].

Matrix markers
Tenascin is an extracellular glycoprotein found in mesenchyme during embryogenesis and in some normal adult tissues, healing wounds and neoplasms, including oral squamous carcinomas. However, it is absent or reduced in areas where the tumor is infiltrating and it has been proposed that this could be a contributory factor to invasion [9].

CONCLUSION
Tumour markers cannot be construed as primary modalities for the diagnosis of cancer. Their main utility in clinical medicine has been a laboratory test to support the diagnosis. New investigative techniques at the cellular and molecular level show great promise at defining potentially malignant lesions but further prospective, indepth studies are required to determine their practical usefulness.