Promising Future in the Detection of Oral Cancer by Using Advance Screening Technology

How to cite this article: Kharma MY, Alalwani MS, Amer MF. Promising Future in the Detection of Oral Cancer by Using Advance Screening Technology. J Oral Health Craniofac Sci. 2016; 1: 022-033. Oral cancer has a tendency to be detected at late stage which is detrimental to the patients because of its high mortality and morbidity rates (survival rate 15-50% [1]). The incidence of oral cancer worldwide is approximately 3% of all malignancies, thus creating a signi icant worldwide health problem [2]. The most common form of oral cancer is squamous cell carcinoma (SCC), which accounts for 96% of all cancers of the oral cavity [3]. Oral squamous cell carcinoma (OSCC) and oropharyngeal and pharyngeal SCC is the sixth most common malignancy in the world today [4]. Oral cancer has a mean survival rate of ive years, which is much lower than other cancers [5]. Early detection will allow oral cancer to be 90% curable and less cost effective for treatment [6].

Despite numerous advances in therapy, the long-term survival of patients with OSCC associated with classic etiology, has remained modest. Several factors contribute to this poor outcome. First, OSCC is often diagnosed at an advanced stage. The 5-year survival rate of early-stage disease is ∼80% and in late-stage disease is only ∼20% [12]. Second, " ield cancerization" leads to the development of multiple primary tumors with a major impact on survival. Finally primary tumors are the most common cause of treatment failure and death in patients with early-stage disease [13,14]. Therefore, comprehensive treatment plans must include improved forms of both screening and chemo-preventive strategies to improve long-term outcomes.
Cancer screening can be de ined as testing asymptomatic individuals to sort out those who probably have the disease from those who probably do not. It is generally accepted that screening for OSCC and oral premalignant lesions may decrease the devastating morbidity and mortality associated with this disease. In contrast to skin cancer screening where visual examination has sensitivity and speci icity rates of 93% and 98%, respectively [15,16].
The detection of OSCC and premalignant oral lesions by way of the conventional visual and tactile examination (CVTE) has remained problematic for several reasons. First, oral premalignant lesions and some early OSCC can be subtle in clinical appearance and can mimic in lammatory lesions. Second, it is becoming increasingly clear that some precancerous lesions are not readily identi ied during a conventional visual examination using incandescent light [17]. Therefore, adjunctive screening aids (devices or tests) that can improve the diagnostic accuracy of the CVTE are desperately needed. There is also considerable interest in spectroscopy (narrow ield) for identifying oral premalignancy and OSCC [18,19]. Most other OSCC and oral premalignancy screening adjuncts are based on biomarker assessments [20][21][22].
Furthermore, oropharyngeal SCC is often dif icult to observe during a clinical examination because it may originate in hard-to-examine locations .Early detection of a premalignant or cancerous oral lesion promises to improve the survival and the morbidity of patients suffering from these conditions.Early detection of oral cancer needs more than just understanding of the signs and symptoms of disease (Table  3: signs and symptoms). Oral cancer deaths will be reduced only if the disease is recognized earlier, avoiding delays in diagnosis, and treated sooner.  A signi icant proportion of oral squamous cell carcinomas (OSCC) develop from premalignant lesions such as leukoplakia and oral submucous ibrosis. Detection of lesions and selection of biopsy sites include vital tissue staining (with Toluidine blue and exfoliative cytology. Unfortunately, sensitivity of cytological diagnosis in a metaanalysis of 1306 cases from 14 studies showed an average of only 87.4% (ranging from 73.8 to 100%) [23].
Nowadays malignancy is considered as a process caused by the accumulation of multiple genetic alterations, which affect the cell cycle as well as normal cell differentiation. These genetic alterations, which occur during carcinogenesis, can be used as targets for detecting tumor cells in clinical samples [24][25][26]. Molecular analysis can identify a clonal population of cancerous cells. Mutations in the tumor suppressor gene p53 are the most frequent genetic alterations in human cancer and show a variable frequency in oral cancer [27].
The identi ication of tumoral markers, notably cytokeratins in smears from the oral cavity has attracted considerable interest. Cytokeratin expression pro ile provides useful information on cell differentiation status [28] but its potential for early diagnosis of oral cancer is limited [29] However, certain cytokeratins, such as K8 and K19 are useful as indicators of malignancy, particularly if their presence is interpreted in conjunction with other information, such as DNA pro ile [30]. Early detection of oral cancer needs more than just understanding of the signs and symptoms of disease. Oral cancer deaths will be reduced only if the disease is recognized and treated earlier.

SCREENING PROTOCOL
Early detection of oral cancer and related premalignancy requires an appropriate screening and diagnosis protocol. It has been recommended that all adult patients over 18 should be screened annually [31] even if medical and dental histories elicit no risk factors. Known-risk patients should be screened every six months. The strong association between HPV and oral cancer further underscores the need to screen all patients, as there is a likelihood that risk of HPV transmission will not be elicited by a medical history taken in the dental of ice setting. The screening protocol should include medical and dental history, unaided and aided visual examination, and palpation. All oral structures must be thoroughly examined, and any abnormalities should be recorded on a mouth map. If suspicious lesions are found during the screening procedure, the lesion must be biopsied or the patient referred to a specialist for further evaluation.

Biopsy protocol
The two basic biopsy techniques for de initive diagnosis of oral mucosal lesions are incisional biopsy and excisional biopsy. The brush biopsy (CDx) ( Figure 1) is a third type of biopsy that can be used as a preliminary diagnostic tool. The brush biopsy uses a stiff brush to obtain a full-thickness sampling of epithelial cells for examination, in patients with mucosal lesions. The brush biopsy may also be useful as an intermittent preliminary diagnostic technique in patients under observation [32], while brush biopsies are practical, simple to perform, and less invasive than an incisional or excisional biopsy, they are insuf icient to provide a de initive diagnosis. Incisional or excisional biopsy is the standard-of-care for de initive diagnosis. Brush Biopsy is a simple, relatively inexpensive, high sensitive, risk-free method of screening for cancer and serves as an aid to the clinical examination .There are controversies related to the real value of this technique in the early detection of OSCC. The existence of false positives has been pointed out showing high sensitivity (90%) and low speci icity (3%) [33].
Cowpe et al. [34] found that tissues undergoing malignant transformation typically show a reduction cytoplasmic area (CA), before the reduction in nuclear area (NA). Ramaesh et al. [35] used cytomorphometric techniques to assess nuclear diameter (ND) and cytoplasmic diameter (CD) in normal oral mucosa, in dysplastic lesions and in squamous cell carcinomas. They found that CD was highest in normal mucosa, lower in dysplastic lesions, and lowest in SCCs. By contrast, ND was lowest in normal mucosa, higher in dysplastic lesions, and highest in SCCs. These studies suggested that reduced nuclear size and increased cytoplasm size are useful early indicators of malignant transformation, and thus exfoliative cytology is of value for monitoring clinically suspect lesions and for early detection of malignancy [35].
Mucosal lesions that are microscopically con irmed to be benign must still be evaluated regularly for progression. In this regard, the ability to predict risk of the lesion undergoing malignant transformation could help determine the frequency of follow-up and/or earlier intervention.

Toluidine Blue Staining (Figure 2)
Vital staining of the oral mucosa with toluidine blue (as a one per cent rinse or application) has been suggested as a means of surveillance in patients at risk of developing oral cancer, and for those who have had a con irmed neoplasm in other parts of the aero digestive tract. The results and accuracy of these studies have been variable, with differing false positive and false negative rates. This could be partly due to confusion over inclusion of equivocal (pale) staining areas as positive or negative indings. Toluidine blue is most useful in secondary care for delineating the extent of lesions and for surveillance of patients at risk of recurrent disease [36,37]. It is signi icant for clinicians to realize that if a lesion does not stain with toluidine blue, but remains clinically suspicious for two weeks, it should still be biopsied as false negatives can occur. Toluidine blue may also aid monitoring of patients for recurrent oral cancer but is of limited use in areas with postoperative scarring where a reconstructive lap has been placed [38].  The phenomenon of auto luorescence is based on the interaction of various luorescent tissue compounds ( luorophores) that occur naturally in the body. When excited by an appropriate light stimulus, these compounds emit visible luorescent light in the violet to green region of the spectrum. VELscope® is a portable device comprising a light source and a viewing hand piece. The technology is based on the direct visualization of tissue luorescence. Both keratinized and nonkeratinised squamous epithelium with a normal submucosa show a typical homogeneous, pale green luorescence. Sharply circumscribed areas of decreased auto luorescence might indicate areas of mucosal abnormalities that should de initely be monitored or investigated by tissue biopsy.
Microlux/DL is a hand-held device that uses light-emitting diodes (LEDs) as the illumination source. Prior to exposing the mucosa to the light, the patient rinses for 30-60 seconds with 1% acetic acid. Upon illumination, the abnormal tissue will appear white ("aceto-white") igure 3.

ViziLite and ViziLite-Plus
ViziLite is a hand-held device that emits chemiluminescent light. The patient rinses for 30-60 seconds with 1% acetic acid and the ViziLite device is used to illuminate the oral cavity. Abnormal areas will appear white ("aceto-white") igure 4. The light increases both the brightness and the sharpness of lesions Chemiluminescence has been found to signi icantly assist the clinician in identifying white and erythroleukoplakia lesions [39].
Chemiluminescence examination of the oral cavity with the aid of chemiluminescent blue/white light has been suggested in several studies to improve the identi ication of mucosal abnormalities in comparison with normal incandescent light. Following a rinse with one per cent acetic acid for one minute under the chemiluminescence light, the normal mucosa appears blue, whereas abnormal mucosal areas re lect the light and appear more "acetowhite" with brighter, sharper margins.
Despite the fact that more than 100 potential OSCC salivary biomarkers have been reported, there has been no standardization regarding the condition of the subjects from whom the saliva samples are collected (e.g., the timing in regard to prior food and drink intake, or the use of oral hygiene products). Similarly, a uniform method has not been established for how the saliva samples are collected, processed and stored prior to measurement and comparison of the biomarker levels in the groups studied.
The differences in these factors among the different studies raises the question as to whether the levels of the potential OSCC salivary biomarkers reported in any one lab could be compared to the levels of the same biomarker reported in any other lab. In fact, for the reported potential biomarkers which have been investigated by more than one study, a wide variability was found in the levels of both the diseased and the control groups among different labs. Variability in the levels of potential OSCC salivary biomarkers in both non-cancerous individuals and OSCC patients, suggest unknown confounding factors. Without standardization and validation of biomarkers, valuable research resources are being squandered, and the experts in this research ield is needed to discuss this issue and ind a way to establish the standards needed [67]. Salivary biomarkers represent a promising non-invasive approach for oral cancer detection, and an area of strong research interest. However, some issues (These issues include a lack of standardization for saliva sample collection, processing and storage.) need to be resolved in order to establish this approach as a reliable, highly sensitive and speci ic method for clinical use [67].
The interest is based on several advantages of saliva versus other body luids, for example, serum or urine such as straightforward sample collection, suf icient quantities for analysis, and the lower costs of storage and shipping than those for serum or urine [68]. Previous studies have shown that IL-6, IL-8 are post-in lammatory cytokines, play a prominent role in immune host defense responses to infection. These chemokines are found to stimulate angiogenesis, in luence tissue remodeling and take part in the regulation of cell proliferation and differentiation. They are essential mediators of cancer development and powerful activators of apoptotic and anti-apoptotic signaling cascade. Hence, IL-6 and IL-8 have been implicated in early detection of oral pre malignancies and OSCC [69,70].
Screening test for oral cancer using whole saliva samples, which can be easily and noninvasively collected. A proteomic studies performed to assess changes in protein expressions of whole saliva, revealing that some proteins are either expressed or unexpressed speci ically in oral squamous cell carcinoma (OSCC). Two-dimensional electrophoresis (2-DE) and peptide-mass ingerprinting (PMF) employed to identify biomarkers that speci ically change expression in OSCC, the Results showed that ten protein spots were speci ic to the preoperative whole saliva of OSCC patients, but were absent in the whole saliva of healthy individuals and OSCC patients after surgery. Detection of enolase 1 in the saliva was shown to be signi icantly higher for OSCC patients than for healthy individuals. Immunohistochemical staining con irmed that appearance of enolase 1 was signi icantly higher in OSCC tissue than in healthy tissue [70].

I-Dysplasia and Risk Prediction
The conventional wisdom is that the more severe a lesion's dysplasia, the more likely it is that it will undergo malignant transformation (Conversely, the less severe the dysplasia, the less likely that the lesion will undergo malignant transformation). A number of recent studies do not support this presumption. Holmstrup et al. did not ind any degree of dysplasia, or site and delineation of the lesion, as a statistically signi icant predictor of malignancy [71,72]. Scully et al. have stated that while dysplasia can be predictive, that this is not always the case. They also pointed out that variability in determining the diagnosis exists between examiners as well as by the same examiner [73]. Rosin et al. studied biopsies and outcomes of patients with secondary premalignant lesions following treatment for primary oral cancer and found that the initial microscopic classi ication of the leukoplakia at the previously treated site did not predict a secondary oral malignancy. Forty-seven percent of leukoplakia's classi ied as having either no dysplasia or mild dysplasia developed into a secondary oral malignancy. Primary tumor stage, grade, and location were not signi icantly associated with the outcome [74]. In patients with a previous history of oral cancer, recurrences can occur at the initial site or further away and may or may not be related. Adjacent mucosa that appears clinically normal has been found to harbor geneticallyaltered cells with loss of heterozygocity [75].While a number of cancers recur due to incomplete excision of the actual lesion, it has also been suggested that premalignant cells are able to migrate to a different site out with the original lesion's area [76].

II-Chromosomal abnormalities and risk prediction
Lesions that undergo malignant transformation lose cell cycle regulation and genetic changes are evident. Recent microscopic studies have investigated loss of heterozygocity (LOH) in tumor cells and its potential role as a risk predictor for malignant transformation. A number of chromosomal abnormalities are now known to be predictive of high risk of oral cancer. LOH has been found to indicate high risk of transformation or conversion to malignancy. In particular, aberrations in the 3p, 9p, and 17p chromosomal arm sites have been implicated as high-risk predictors, although other chromosomal arm alterations also occur [77][78][79][80]. Rosin et al. examined biopsies and outcomes for patients with secondary premalignant lesions. They found that LOH in multiple chromosome arms, and in particular in 3p and 9p sites, was predictive of a secondary malignancy [81]. Aberrations in multiple arms rather than in single arms are believed to increase that risk.

III-Risk prediction and treatment planning
Given that speci ic chromosomal and genetic alterations have been found to be predictive of the risk of malignancy developing in a premalignant lesion, histopathology assessment of these changes will help to identify those patients most at risk in the future and enable tailored case management. Several investigators have found correlations between the degree of toluidine blue dye uptake and the presence of chromosomal alterations. Zhang et al. monitored premalignant lesions, for on average 44 months, in 100 patients with no prior oral cancer history. Toluidine blue staining was associated with a 600% increased risk of transformation and occurred in 80% of the lesions that later became malignant. The association of stain and risk factors also applied to lesions initially showing no or mild dysplasia [82]. Epstein et al. studied 32 patients with oral lesions who were biopsied following dye retention. They studied lesions with LOH on three chromosome arms (3p, 9p, and 17p). Toluidine blue positive-stained lesions demonstrated LOH more frequently than dye-negative lesions in 3p and 9p, and more dye-positive cases had LOH on multiple chromosome arms, associated with an increased risk of malignancy [83]. Based on these studies, differential uptake of toluidine blue is associated with chromosomal changes and LOH rather than simply malignancy and/or the degree of dysplasia, and may be a useful adjunct in the future for risk prediction.

CONCLUSION
It is clear that early diagnosis of oral cancers signi icantly improves the patient's long-term survival. Early diagnosis also reduces morbidity and can minimize the extent of treatment required. While in other anatomic sites in the body malignancies can be radically excised, the excision of an oral cancer, depending on the site and size of the tumor, can severely compromise the patient's quality of life and in some cases may not even be possible.
The detection of suspicious lesions is increased through routine, regular screening of patients [83]. Biomarkers help in evaluating the preventive measures or therapies and the detection of the earliest stages of oral mucosal malignant transformation [84]. Early detection of suspicious lesions will result in earlier diagnosis, less aggressive treatment, and decreased need for complicated post-treatment management. The primary goal is to diagnose lesions. The secondary goal is to diagnose the malignancy before it metastasizes in order to minimize the morbidity and maximize the patient's quality of life and survival.
Ethical standards: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/ or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. Human and Animal Rights Statement This article does not contain any studies with human participants performed by any of the authors.