Oral Medicine: Advances in Diagnostic Procedures

In the latter part of the 20th century, the computer and molecular biology have facilitated great scientific progress in medicine and dentistry. In dentistry, emerging clinical methods based in molecular biology and digital technology have the potential to improve the early diagnosis of dental caries, periodontal disease, and oral cancer. In addition, saliva shows potential as a convenient substitute for blood in diagnostic testing for systemic and oral diseases. DNA chip technology, a new system that combines these two technologies, has potential diagnostic value in dentistry as well as medicine. For each of the three common oral disease processes, emerging diagnostic procedures are discussed, with an emphasis on their potential utility for the practicing dentist of the 21 century.

In the latter part of the 20th century, both computer science and molecular biology have facilitated great scientific progress in medicine and dentistry. In dentistry, emerging technologies based in molecular biology and digital technology have the potential to improve the early diagnosis of dental caries, periodontal disease, and oral cancer. In dental caries, for example, the dental explorer and bitewing radiograph have always been integral to the caries examination. Soon, they may be replaced by electrical conductivity methods and quantitative laser fluorescence measurements. The scientific principles behind these methods are made clinically relevant through the use of digital technology. Also, the concept of risk assessment in oral diagnosis and treatment planning may have a greater chance of becoming a practical reality in private dental practice, if computer algorithms can be applied to clinical data to quickly and accurately calculate risk. Measurements of disease indicators such as cytokines or DNA analysis may be included in such risk assessments in the future, thus combining digital and molecular technologies. Similarly, digital subtraction radiography in periodontal disease and computer-aided cytology in oral cancer represent new diagnostic procedures that dentists of the 21st century can be expected to use to benefit their patients through early diagnosis and disease intervention. These and other selected diagnostic procedures will be discussed.

Saliva is a potential source of material for diagnostic testing in both systemic disease and oral disease. With new interest in the link between oral and systemic health¹ and recent success using saliva-based molecular diagnostics, it is possible that saliva sampling may become part of the routine examination and assessment of dental patients. In addition to the biologically active proteins and exogenous substances such as drugs that can be found, saliva is also a source of the patient’s DNA. The imminent completion of the Human Genome Project and its progress to date suggest that DNA analysis may become important in risk assessment, disease prevention, and health promotion efforts of all health professionals.² Therefore, this paper will conclude with a brief description of micro-array or "DNA chip" technology. This emerging technology represents the combination of the "state-of the art" of both digital and molecular biology techniques and shows promise as a diagnostic aid in medicine and dentistry in the very near future.

The aim of this paper is to:

* Give an overview of recent progress in oral diagnosis in those diseases most relevant to practicing dentists, and

* Serve as an introduction to the diagnostic procedures that are expected to become part of dental practice in the next century.

Dental Caries

By the time the earliest signs of caries are detectable, the disease is already well-established. ³ Dentists cannot detect the onset of caries. In most Western countries, caries prevalence has declined, although there are certainly portions of the population that demonstrate high caries rates. Caries is harder to detect in populations with low caries prevalence, because lesions are smaller and progress slower. Fluoride may be partially responsible for the declining prevalence and also the difficulty in detecting lesions. Research has shown that caries can be arrested and noncavitated lesions that are confined to the enamel can remineralize. To take advantage of the potential for remineralization, the caries must be detected at an early stage so that aggressive medical treatment can be used at the right time. In addition, dentists need to be able to monitor lesions for small changes indicative of progression or remineralization.³

It takes more time to diagnose early lesions because they are harder to detect and the dentist is more likely to detect lesions in those patients at greatest risk of developing caries. If dentists are to successfully remineralize early lesions in their patients, they must first detect those early lesions. Caries risk assessment must be performed to identify those patients for whom additional diagnostic procedures may be indicated. There are several excellent reviews of risk assessment in dentistry.^4-6

Methods of detection are different for different types of caries.³ The pattern of caries has changed. Occlusal surfaces are proportionately affected more often than smooth surface or approximal surfaces; therefore, recent attention has focused on the early detection of occlusal surface caries. However, methods of detection of approximal, smooth surface, and root caries will also be discussed: direct digital radiography, quantitative light (laser) fluorescence, electrical conductivity measurements, and microbial testing.

Figure 1. The charge-coupled-device sensor for the digital radiography is attached to the computer by a cable. The sensor fits into the film holder for a parallel projection periapical image.

Figure 2. A view of the display screen using a currently available digital radiography software program shows the image enhancement options available. Most commercially available software packages include these options (Photo courtesy of Dr. James Geist)

Wenzel⁷ recently reviewed the use of digital radiography for caries diagnosis. There are two types of digital radiography image systems:

1. Charge-coupled-device-based (Figure 1), and

2. Storage-phosphor-based.

In the charge-coupled-device system, a cable connects the sensor to the computer, and the image is displayed on the monitor (Figure 2). In the storage-phosphor system, the reusable image plate is exposed to X radiation to create a latent image. The plate is exposed to a laser scanner to obtain the stored information. The advantages of digital radiography over conventional film-based radiography are:

* The image can be manipulated to change contrast and density according to the diagnostic task.

* One can avoid the potential errors associated with chemical processing.

* Radiation dose to acquire a diagnostic image can be lowered to 50 percent or less of that needed for conventional film-based radiography. This increases x- ray tube life.

* It takes less time to acquire an image.

* Image storage and communication is easier with digital networking.⁵

Based on the results of the studies she reviewed, Wenzel concluded that digital radiography appears to be at least as accurate as current dental films for the detection of caries. However, currently radiography is of no value for the detection of occlusal caries confined to the enamel. Also, for all radiographic methods, predictive values for the detection of approximal enamel lesions are poor. Radiographic methods are better for the detection of approximal dentinal lesions.⁷

Most digital radiography systems offer image enhancement software as part of a package (Figure 2). Wenzel and colleagues⁸ showed that contrast-enhanced digitized films and charge-coupled-device-based images tended to perform better than unenhanced images within the same systems. Other studies have not shown any improvement with task-dependent algorithms for the diagnosis of occlusal and approximal surface caries.⁷

Subtraction radiography is a type of digital radiography that uses computer software to display an image representing the difference between two images of the same object. With storage-phosphor digital radiography, it is possible to enhance small changes in density to facilitate their detection. Storage-phosphor images were found to be better than film for the detection of artificial recurrent caries, and false-positive scores were reduced.⁹ However, more studies exploring image enhancement and its effects on diagnosis are needed.⁷

One area that holds some promise is computer-aided diagnosis. ^7,10 On average, newer systems have been shown to perform as well as or better than human observers. However, before computer-aided diagnosis can be recommended for clinical use, its accuracy must be confirmed to be higher than that of trained observers. The suboptimal specificity and sensitivity of current receptors and film-based images limit the potential of computer-aided diagnosis since the data used to make the diagnosis is inadequate.⁷

Digital radiography use is more widespread in clinical practice in Europe but is increasing in the United States. There have been few published clinical studies using digital radiography for caries diagnosis. Therefore, many of the theoretical advantages of digital radiography over conventional film-based images have not yet been confirmed in clinical studies. Also, the economic benefits of such technology for the patient, the dentist, and society need to be determined⁷.

Quantitative laser or light fluorescence takes advantage of the natural luminescence exhibited by tooth tissue exposed to light at a wavelength of 488 m m. The two emerging methods for early caries detection that appear to hold the most promise for clinical practice are electrochemical impedance spectroscopy and quantitative laser (light) fluorescence.³ With quantitative light fluorescence a broad beam of diffuse monochromatic light within the blue-green region (wavelength of 488 m m) is produced by an argon laser source and applied to a tooth. This induces luminescence of the tooth. The natural fluorescence of enamel is in the yellow-green range and is observed through a high pass filter to exclude tooth-scattered blue laser light. Areas of demineralization appear as dark spots. In the quantitative method, the fluorescent light is detected by the instrument through the handpiece that delivers the light, and its intensity is quantified using a computer. The quantitative light fluorescence method has been validated; mineral loss was strongly correlated with a relative loss of fluorescence radiance. In one study, quantitative laser fluorescence allowed investigators to follow weekly changes in enamel during a five-week period. ^3,11

Recently, a small portable quantitative light fluorescence device suitable for intraoral use was described.¹² It uses a regular light source (quantitative light fluorescence method). It has been shown to sensitively and reproducibly quantify enamel lesions to a depth of 400 μm.¹² This light-induced fluorescence method is the only one that has been tested clinically.¹¹ Orthodontic patients at risk of developing buccal surface caries were studied for such changes. The results suggested that quantitative light fluorescence is appropriate for the in vivo monitoring of mineral changes in incipient enamel lesions. The investigators concluded that it may be useful for the evaluation of preventive measures in caries-susceptible individuals. ¹¹

DIAGNO-Dent is a similar instrument currently under validation and testing. It is commercially available in Canada (DIAGNODent; KaVo, Ontario, Canada).³ Its intended use is the quantification of caries on occlusal and smooth surfaces. A laser diode light source (similar to a laser pointer) produces light of a different wavelength than that produced by a fluorescing tooth and is delivered to the tooth by a fiber-optic handpiece. This fluorescent light is reflected back through the handpiece, where it is translated into an acoustic signal; and the wavelength is evaluated by the control unit, which displays a digital representation of the wavelength detected. Changes in wavelength are reportedly associated with the changes in mineralization of the enamel.

Recently, Angmar-Mansson³ described this technique that was introduced to dentistry in 1951. Its underlying principle is that the reduced mineral content of carious enamel increases its electrical conductivity, and this can be detected. The probe tip is placed in the occlusal fissure, and conductivity through the dental pulp to a handheld ground lead is measured. Studies have been done using the Vanguard and Cariesmeter L, devices no longer being manufactured. A newer instrument called the Electronic Caries Monitor (LODE Diagnostics, Groningen, Netherlands) is currently available. Studies on the two earlier models showed that electrical conductivity methods were superior to visual inspection, fiber-optic transillumination, and radiography for occlusal caries diagnosis. The recent revival of interest in fixed frequency electrical devices arises from their promise of the detection of occlusal lesions (which are proportionately more affected) and possibly approximal lesions.³

Electrical conductivity measurements can aid the detection of fissure caries in recently erupted molar teeth.³ Electrical conductivity measurements can be used to determine the probability that a sealant or a restoration is required within 18 to 24 months after eruption. However, because of the low specificity, electronic conductivity methods should not be used when one is deciding whether to treat a lesion operatively. Low specificity increases the chance of false-positive results and the likelihood of unnecessary invasive treatment. Therefore, electrical conductivity methods may be better used to detect early lesions and monitor lesion progression or arrest in sites where noninvasive intervention is indicated.¹¹ These lesions are often overlooked in conventional visual and radiographic examination .³

Alternating Current Impedance Spectroscopy Technique (ACIST) characterizes the electrical properties of a tooth and lesion to monitor and quantify change. If one applies an alternating current to a tooth bathed in saliva, an ionic current flow will be generated that will have a mean value of zero and will not polarize the electrolyte solution. Electrical impedance measurements reflect not only resistance but also other factors that hinder the flow of current. Ion motion is measured when electrical impedance spectroscopy is used to characterize dental hard tissues that have pores of ionic dimensions. These electrical impedance spectroscopy measurements reflect the current size of the pores. In carious hard dental tissues, the pores are larger than in sound tissue. This method was tested in a clinical study on root caries.¹⁴ It was concluded that repeated measurements over time could show whether the pores are getting bigger or smaller and subsequently whether caries is progressing or reversing.¹⁴

Greater resistance (impedance) measurements were found with small pore size, but there was overlap between experimental groups. Further testing should done to refine and validate this technique as a potential aid to diagnosis and monitoring of the caries process over time.¹⁴

Recent reviews of the utility of microbial tests (e.g., S. mutans, Lactobacillus sp. counts) to diagnose and predict caries activity have concluded that the tests have use as an adjunct to other methods of diagnosis in individual patients. They are probably most useful when measured initially to establish a baseline value for the patient, then measured periodically to detect change associated with an increased risk for caries. There are limitations to the accuracy of these tests that may not be correctable, even with further research, because of the inherent biology of these organisms and their relationships with the individual host.¹⁵

Verdenschocht and colleagues assessed developments in caries diagnosis and their relationship to treatment decisions.¹⁶ In the past when caries prevalence was higher, visual inspection and radiographic examination were probably adequate to diagnose caries requiring operative treatment. Now, with caries prevalence declining, these methods are no longer appropriate for all patients. For most patients in Western countries, caries detection is focused on finding small lesions. Verdenshocht and colleagues performed a meta-analysis on diagnostic tests of occlusal caries. They concluded that visual inspection performed the worst and electrical conductivity methods the best. Their meta-analysis for approximal caries showed that radiography was superior to fiber-optic transillumination. When histologic lesion depth or mineral loss was used for validation of the diagnostic method, the results were:

* For approximal lesions, quantitative fiber-optic transillumination had the highest correlation with lesion depth.

* For occlusal surfaces, visual inspection using a scoring system based on translucency and breakdown of enamel on air drying had the highest correlation with histology. This was followed by electrical conductivity methods.¹⁵

Verdenschocht and colleagues pointed out a study that illustrated that clinicians may improve their performance by training in using a detailed scoring system and taking the time to dry the field and conduct a thorough exam.¹⁵ Quantitative methods had the highest correlation with lesion depth and have more potential to monitor small changes over time.¹⁵

Because caries is a chronic, slowly progressive disease, its effects are not detected until years after its onset. Early detection of signs of caries in a young patient is time-consuming . It would be most productive to concentrate on finding early signs of caries in patients who have a high caries risk. The assessment of a patient’s caries risk should be the result of an intellectual process involving all information pertinent to the etiology of caries(e.g., diet, saliva flow, cariogenic microbes). Verdenschocht and colleagues showed that caries-related factors (dfms, DFMT, caries in first molars) performed best in predicting future caries onset. Multiple caries prediction tests (e.g., combinations of a mutans test, lactobacilli, saliva flow test, and plaque test) performed worse than existing caries as a predictor.¹⁵ Caries risk assessment has been reviewed elsewhere.⁵

Periodontal Disease

According to the Consensus Report for the 1996 World Workshop on Periodontics,¹⁷ it remains unclear as to how small changes in measurements from radiography and periodontal probing relate to the progression of periodontal disease over the long term. Digital subtraction radiography can provide an objective method to improve detection of bone changes too small to be seen by eye or by conventional interpretation of sequential radiographs.¹⁷ Controlled force periodontal probes have been found to underestimate probing depth compared to measurements taken with manual probes. The validity of the measurements of the controlled force probes is not known, and they cannot be recommended for clinical practice except for convenience. More research is needed to establish the validity of the measurements.¹⁷

The application of microbial diagnosis in periodontics is limited because of the inability of the tests to identify specific diseases or to predict disease progression. However, there is well-demonstrated validity for repeated microbial testing of patients who continue to experience disease progression in spite of excellent compliance and quality of periodontal care. It might also be indicated in high risk medically compromised patients where the dentist suspects that there is an unusual bacterial superinfection, or in the patient with early-onset periodontitis where Actinobacillus actinomycetemcomitans may be present. One can use the tests to demonstrate that this organism has been eradicated for successful treatment. These examples need to be proven in controlled clinical studies.¹⁵

Although there is evidence that some biochemical markers in gingival crevicular fluid correlate to clinical parameters, there is a wide range of activity that can be seen in patients with differing levels of health or disease. Therefore, there are no specific biochemical profiles that characterize specific periodontal disease. Some gingival crevicular fluid markers such as Prostaglandin E ₂, or interleukins IL-1A and IL-1B,¹⁸ may hold promise in regard to prediction of future disease progression or stability; but they have not been proved to be valid. More research is needed on effects of such tests on periodontal treatment planning before they can be recommended for routine clinical use.¹⁷

The Periodontal Susceptibility Test is a commercially available test that is used to identify carriers of specific genetic polymorphisms for Interleukins IL-1A and IL-1B in the presymptomatic testing of patients. This test was recently reviewed.^19,20 IL-1A and IL-1B are cytokines involved in the host response to bacterial challenge. Investigators have calculated the increased odds of developing severe periodontitis if certain genetic variants are present. Risk is defined as the probability of an adverse event occurring. The relationship between genotype and periodontal disease risk was examined by Kornman and colleagues.²⁰ They found that patients who were nonsmokers and genotype-positive for a specific variant had a six to eight times greater chance of developing severe periodontitis than those who did not have that genotype. Smoking negated the effect of the periodontal disease-susceptible genotype. Limitations of the study prevented the authors from calculating absolute risk, which is the data needed to provide valuable predictive information. Prospective studies need to look at absolute risk: How many genotype-positive individuals develop periodontitis?^19,20

Other possible genetic factors influencing the risk for periodontitis include Immunoglobulin G subclass 2 (IgG2) and its receptors, PMN chemotaxis (chemotactic agent receptors and intracellular signaling mechanisms), and PGE2 responses to bacterial lipopolysaccharide.²¹ All of these have a genetic component. However, because periodontitis is a multifactorial disease, environmental factors such as the presence of periodontopathic plaque must also be considered. Even if genetic factors are found to be predictive, there will always be the problem of false-positive and false-negative results because of incomplete penetrance and variable expressivity of genotypes. Ethical issues regarding when to test and what to do with the information are other concerns. For example, should this information be made available to insurance companies or family members? Is there any benefit to the patient in knowing his or her genotype, or is it a purely academic exercise? Patients must be provided with adequate and scientifically sound information regarding such tests in order to make appropriate decisions.¹⁹

It is likely that genotyping combined with assessment of other risk factors may be a strong predictor of periodontal disease. Genotyping is not a diagnosis but a risk assessment, similar to elevated serum cholesterol levels and the associated risk of cardiovascular disease. In the future, presymptomatic testing for periodontitis may become commonplace and molecular biomarkers may be incorporated into risk assessment. This could also lead to new treatment strategies. Because much needs to be understood about the implications of these tests, more research is needed before recommending them for daily periodontal practice. ¹⁷

Oral Cancer

Recent developments in the early diagnosis of oral cancer have been reviewed by Epstein and Scully.²² The present discussion will be limited to the use of toluidine blue as an aid to clinical examination in the early detection of oral malignancy, oral exfoliative cytology, and the use of DNA markers in the histopathologic diagnosis and prognosis of oral squamous cell carcinoma.

Several studies have shown that toluidine blue vital staining of suspicious oral epithelial lesions can help in the detection of oral squamous cell carcinoma , in high-risk populations, ^23-24 including patients with a history of a previous oral cancer²⁵ or a positive history of tobacco and/or alcohol use²³ and advanced age. Toluidine blue is used as a 1 percent or 2 percent oral rinse or application provided as a weak acid solution in water and is available in a ready-to-use kit (OraScan; Zila Industries, Phoenix, Ariz.). The kit consists of flavored solutions containing 1 percent toluidine blue as the staining rinse, and 1 percent acetic acid for use as both pre- and post-rinses. Toluidine blue is a basic metachromatic stain that has affinity for the perinuclear cisternae of DNA and RNA. Because cancer cells contain more DNA and RNA than normal epithelium, toluidine blue delineates areas of malignancy. Although most epithelial surfaces stain after an initial application with toluidine blue, only positive areas retain stain after rinsing with acetic acid.²⁵

There has been some concern about the potential carcinogenicity of toluidine blue. Both positive and negative results have been reported using toluidine blue in the Ames bacterial mutagenicity test.² However, animal studies suggest that toluidine blue itself is not carcinogenic, nor does it act as a co-carcinogen or promoter when given to an animal with a known carcinogen.²⁷ The meta-analysis of Rosenberg and Cretin²⁸ showed the effectiveness of toluidine blue in the identification of oral squamous cell carcinoma of the tongue. Sensitivity in the published data ranged from 93.5 percent to 97.8 percent, and the specificity ranged from 73.3 percent to 92.9 percent. Other studies have shown similar results.^{23, 25, 29} False-positive cases have been reported. Therefore, a return visit after 14 days is recommended for repeat of the procedure. Many of the cases of false-positives were inflamed or ulcerated lesions. Regardless of the interpretation of the staining results, if a lesion is clinically suspicious or if the patient is considered a poor risk for follow-up, biopsy should not be delayed.²²

Toluidine blue has been shown to be effective for the identification of malignancy. It has not been shown to be effective in the identification of dysplastic or premalignant lesions.²⁴ Toluidine blue is therefore recommended to be used as an adjunct to clinical examination in high-risk patients, especially those with a previous history of oral cancer.²⁴ For the general dental practice, toluidine blue can be used to aid in the decision to refer a patient to a specialist for management of a suspected malignancy.²²

Figure 6. Conventional cytologic smears may contain as many as 3,000 cells. Computer-aided cytology diagnostic systems help the cytopathologist by identifying groups of abnormal cells and displaying them in individual frames so that they are more easily and thoroughly assessed (Photo courtesy of Dr. Drore Eisen)

Scrapings of keratinized white lesions are of limited diagnostic value because they contain only superficial cells, with many false-negative results. Many carcinomas in situ in the oral cavity are red atrophic lesions, so cytology may be of some use in those cases. Cytologic smears of those lesions may show morphologic abnormalities in squamous cells diagnostic of cancer.^22,30 The technique involves the use of an oral biopsy brush (Oral Scan Laboratories Inc.; Suffern, N.Y.) that has been designed to obtain a complete transepithelial biopsy with minimal discomfort to the patient (Figures 3-5).

Figure 3. This leukoplakia located on the ventral surface of the tongue and anterior floor of the mouth is the type of clinically suspicious lesion that might initially be assessed using the oral brush biopsy technique

Figure 4. The specially designed brush for intraoral exfoliative cytology allows for a transepithelial sampling of the lesional tissue with minimal discomfort to the patient.

Figure 5. The brush is gently rotated along the lesion to sample as much of it as possible. There is minimal discomfort to the patient so local anesthesia is usually not indicated (Photo courtesy of Dr. Drore Eisen).

The diagnostic potential of cytology may be maximized by applying new molecular biological techniques and/or computer-aided diagnosis software³¹ (Figure 6). For example, in one study, cytologic smears of clinically normal epithelium were obtained from patients with carcinoma of the tongue and from normal controls. The cells from the cancer patients had more than a three-fold increased expression of cytokeratin 19. Cytokeratin 19 may be a diagnostic marker to monitor patients undergoing preventive chemotherapy.³² Proliferating cell nuclear antigen (PCNA) is a nuclear protein synthesized during cell replication that may serve as a marker of cell division in premalignant and malignant lesions since expression of PCNA has been shown to correlate with grade of dysplasia. This and other markers may be detectable in cells obtained from cytologic smears. The cytologic smear procedure is less invasive and faster than surgical biopsy. The diagnostic utility of exfoliative cytology and the testing for DNA markers or protein expression deserves further investigation.²²

Advances in molecular biology may provide more objective criteria for histopathologic diagnosis and information about the behavior and prognosis of specific tumor types. Included in these techniques are monoclonal antibodies; immunofluorescent procedures; and DNA, antigen and antibody studies. Certain cell-surface markers such as blood group antigens, histocompatibility group antigens, and squamous cell antigens have been identified and require further study. Oncogenes and tumor suppressor genes are currently receiving a lot of attention as potential markers. ²²

Carcinogenesis is a multistep process that involves a number of aberrant genetic events. Cancer is characterized by an accumulation of genetic mutations that allow for further genetic mutation. ³³ These mutations lead to increased cell replication and altered behavior. The p53 protein is responsible for maintenance of genomic integrity. It is a tumor suppressor. If a cell has a mutation, p53 arrests the cell cycle in G1(first gap phase) to prevent replication of damaged DNA and allow time for DNA repair prior to replication and chromosomal segregation.³⁴ Mutations in the p53 tumor suppressor gene are commonly seen in oral squamous cell carcinoma, and mutations in p53 have been associated with the use of tobacco products. In one study, p53 was detected by immunocytochemistry in 71 percent of squamous cell carcinomas and 56 percent of clinically normal mucosa in patients with head and neck carcinomas, but was absent in the control group and in heavy smokers without oral mucosal changes. ³⁵

In a more recent study, oral mucosal cells obtained by oral exfoliative cytology were examined in patients with oral squamous cell carcinomas and from healthy controls. The polymerase chain reaction was used to amplify the DNA obtained from these cells and restriction fragment length polymorphism analysis was performed on the DNA obtained in this way. With polymerase chain reaction, small samples of cells are adequate for analysis, unlike conventional techniques such as Southern Blot analysis. Loss of heterozygosity of tumor suppressor genes and loss of chromosomes are cytogenetic signs of malignancy. This study showed that 66 percent of oral cancers demonstrated loss of the p53 gene heterozygosity in one site and 50 percent showed loss of heterozygosity at another one. Huang and colleagues concluded that inactivation of p53 is involved in the development or progression of oral carcinogenesis. The oral exfoliative cytology technique used with polymerase chain reaction and restriction fragment length polymorphism analysis was simple and rapid and may be useful for preliminary screening of suspicious oral lesions. Additional analysis of genetic change in oral cancer may lead to a better understanding of pathogenesis.³⁶

Polymerase chain reaction and restriction fragment length polymorphism analysis was also used in another study looking at microsatellite markers, which are short repetitive sequences of DNA, in oral squamous cell carcinomas in adults younger than 40. ³⁷ Studies that have assessed other potential markers in oropharyngeal carcinoma disclosed their potential diagnostic and prognostic value: TGF-α, EGFR, c-erbB-2/neu and PCNA.³³

In summary, oral cancer detection may be improved through the use of computer-aided oral exfoliative cytology using a transepithelial sampling technique and toluidine blue for high-risk patients. Oral cancer diagnosis, prognosis, and treatment may be improved through the study and use of genetic markers identified both through immunohistochemistry techniques and polymerase chain reaction and other molecular biology procedures.

Saliva and Molecular Diagnostic Methods

Saliva is used to test for drugs, such as alcohol. Salivary cotinine levels are used to monitor compliance in tobacco cessation. Salivary estradiol levels are used to indicate preterm labor in high-risk women, and salivary cortisol levels are considered to be at least as good a measure of adrenal cortical function as serum cortisol.^38,39 Viral infections such HIV^40,41 and influenza can be detected in saliva.³⁹

Detection of tumor biomarkers secreted in saliva could be used to diagnose cancer recurrence.^42,43 For example, c-erbB-2 protein expression in breast tumor tissue correlates to levels in saliva. Preliminary results indicate that c-erbB-2 protein expression in saliva may be useful for measuring patient response to chemotherapy and/or surgical treatment for breast cancer. ⁴²

Salivary HIV tests are currently being used.^40,41 Compared to venipuncture, saliva collection is noninvasive; and there is potentially less risk of disease transmission to the health care worker. The sampling techniques are probably also more acceptable to the patient. ⁴⁰ In the past, saliva HIV tests were inadequate because they were not sensitive enough to detect low levels of immunoglobulin; but they have improved. Wisnom and colleagues⁴⁰ recently compared two commercially available salivary HIV antibody tests and serum tests for the same patients from either a high- or low-risk group. Reactive tests were confirmed using Western Blot analysis. Their results indicated that the serum and saliva tests were 99.8 percent and 100 percent specific, respectively; and both were 100 percent sensitive. Although the two systems are not currently available in the United States, they have great potential for future applications.⁴⁰

There is tremendous potential for saliva sampling to be used instead of serum for assessment of systemic disease. The Human Genome Project is expected to have the entire human genome mapped by the year 2003.² This project may provide DNA markers that may singly or in combination provide some indication of risk of various systemic and oral diseases. It is possible that many of these markers could be detected in exfoliated cells from saliva. Saliva testing could become a routine part of the dental and medical office visit.^38,39

The new DNA chip has the potential to provide fast analysis of the entire genome of a particular individual. The DNA chip combines the state of the art of both silicon chip technology and molecular biology. Photolithographic techniques are used to generate miniaturized arrays of densely packed oligonucleotide probes. These probe arrays can be applied to parallel DNA hybridization analysis to directly yield sequence information.⁴⁴ The chip is scanned by a laser and read by a computer. This technology has demonstrated speed and accuracy in detecting mutations in even large genes such as that for cystic fibrosis,⁴⁵ and BRCA 1 (a breast cancer gene).⁴⁶

It is conceivable that in the future a patient’s DNA could be sampled from exfoliated cells in saliva, or from a lesion via the brush biopsy technique, and then that DNA analyzed using oligonucleotide arrays (DNA chip technology). The information obtained regarding genetic polymorphisms or mutations in key genes such as p53 or interleukin 1 could be applied to the diagnosis of a suspicious lesion (p53 or cytokeratin 19) or reveal an underlying susceptibility to a disease such as oral squamous cell carcinoma (p53) or early onset periodontitis (interleukin1). DNA chip technology represents the union of digital and molecular technology and exemplifies the type of technology that may some day revolutionize oral diagnosis and oral medicine.

Conclusions

Advances in diagnostic procedures improve the chances of earlier detection of dental caries, periodontal disease and oral cancer. Risk assessment is an integral component of the diagnostic process, both from the standpoint that advanced testing should be reserved for those most at risk and because some of the "diagnostic tests" are actually risk assessment tools. Molecular diagnostics and computer-aided diagnosis increase the potential of oral exfoliative cytology and histopathologic examination. Saliva has tremendous potential as a source of biologic molecules to serve as indicators of past history of disease, current disease, and future risk of disease. It has the potential to replace serum in many analyses.

The common theme behind all these advances is that early detection and diagnosis of disease may be facilitated by the application of digital and molecular technologies. Dentists of the near future can be expected to be able to effectively prevent disease and promote health through these improved diagnostic techniques. In order for this to occur, dental schools need to prepare the dentists of the 21st century with the skills needed to assess new technologies and rationally implement into dental practice those that are scientifically sound.

Acknowledgements/

The author would like to thank Dr. James Geist and Dr. Drore Eisen for permission to use their photographs and to Dr. Sara Gordon for her assistance during the preparation of this manuscript.

Author/

Carol Anne Murdoch-Kinch, DDS, PhD, is an associate professor of diagnostic and surgical sciences at the University of Detroit Mercy School of Dentistry.

References

1. Loesche WJ, Schork A et al, Assessing the relationship between dental disease and coronary heart disease in elderly U.S. veterans. J Am Dent Assoc 129(3):301-11, 1998.

2. Collins FS, Shattuck Lecture -- Medical and societal consequences of the Human Genome Project. New Engl J Med 341(1):28-40, 1999.

3. Angmar-Mansson BE, AL-Khateeb S, and Tranaeus S, Caries diagnosis. J Dent Educ 62(10):771-9, 1998.

4. Douglass CW, Risk assessment in dentistry. J Dent Educ 62(10): 756-61, 1998.

5. Pitts NB, Risk assessment and caries prediction. J Dent Educ 62(10):762-70, 1998.

6. Papapanou P, Risk assessments in the diagnosis and treatment of periodontal diseases. J Dent Educ 62(10):822-52, 1998.

7. Wenzel A, Digital radiography and caries diagnosis. Dentomaxillofac Radiol 27:3-11, 1998.

8. Wenzel A, Hintze H, et al, Radiographic detection of occlusal caries in noncavitated teeth. A comparison of conventional film radiographs, digitized film radiographs, and RadioVisiography. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 72:621-6, 1991.

9. Nummikoski PV, Martinez TS, et al, Digital subtraction radiography in artificial caries recurrent caries detection. Dentomaxillofac Radiol 21:59-64, 1992.

10. van der Stelt PF, Computer assisted caries detection. Proceedings of the First Annual Indiana Conference on the Early Detection of Dental Caries, 1996. Indiana University School of Dentistry, Indianapolis, pp 253-64.

11. Al-Khateeb S, Forsberg CM, et al, A longitudinal laser fluorescence study of white spot lesions in orthodontic patients. Am J Orthod Dentofacial Orthop 113:595-602, 1998.

12. Al-Khateeb S, ten Cate JM, et al, Quantification of formation and remineralization of artificial enamel lesions with a new portable fluorescence device. Adv Dent Res 11(4):502-6, 1997.

13. Ricketts DNJ, Electrical conduction detection methods. Proceedings of the First Annual Indiana Conference on the Early Detection of Dental Caries, 1996. Indiana University School of Dentistry, Indianapolis, pp 67-80.

14. Levinkind M, Electrochemical impedance strategies for early caries detection. Proceedings of the First Annual Indiana Conference on the Early Detection of Dental Caries, 1996, Indiana University School of Dentistry, Indianapolis, pp 183-94.

15. Bowden GH, Does assessment of microbial composition of plaque/saliva allow for diagnosis of disease activity of individuals? Comm Dent Oral Epidemiol 25:76-81, 1997.

16. Verdenschocht EH, Angmar-Mansson B, et al, Developments in caries diagnosis and their relationship to treatment decisions and quality of care. Caries Res 33:32-40, 1999.

17. Genco RJ, Jeffcoat M, et al, Consensus Reports from the 1996 World Workshop in Periodontics. Section 1. Periodontal disease: epidemiology and diagnosis. J Am Dent Assoc 129:9S-14S, 1998.

18. Lerner UH, Modeer T, et al, Gingival crevicular fluid from patients with periodontitis contains bone resorbing activity. Eur J Oral Sci 106(3)778-87, 1998

19. Greenstein G, Understanding a commercially available genetic susceptibility test for periodontitis. Compend 20(4):301-12, 1999.

20. Kornman KS, Wang HY, et al, The interleukin-1 genotype as a severity factor in adult onset periodontal disease. J Clin Periodontol 24:72-7, 1997.

21. Schenkein HA, Inheritance as a determinant of susceptibility for periodontitis. J Dent Educ 62(10):840-51, 1998.

22. Epstein JB and Scully C, Assessing the patient at risk for oral squamous cell carcinoma. Spec Care Dent 17(4):120-8, 1997.

23. Warnakuasuriya KAAS, Johnson NW, Sensitivity and specificity of OraScan toluidine blue mouthrinse in the detection of oral cancer and precancer. J Oral Pathol Med 25:97-103, 1996.

24. Martin IC, Kerawala CJ, Reed M, The application of toluidine blue as a diagnostic adjunct in the detection of epithelial dysplasia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:444-6, 1998.

25. Epstein JB, Oakley C, et al, The utility of toluidine blue application as a diagnostic aid in patient previously treated for upper oropharyngeal carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 83:537-47, 1997.

26. Dunipace AJ, Beaven R, et al, Mutagenic potential of toluidine blue evaluated in the Ames test. Mut Res 279:255-9, 1992.

27. Redman RS, Krasnow SH, Sniffen RA, Evaluation of the carcinogenic potential of toluidine blue O in the hamster cheek pouch. Oral Surg Oral Med Oral Pathol 74:473-80, 1992.

28. Rosenberg D, Cretin S, Use of meta-analysis to evaluate polonium chloride in oral cancer screening. J Oral Surg 67:621-7, 1989.

29. Fellowman RS, Epstein J, et al, Toluidine blue O as an oral cancer diagnostic aid. Abstract 822. J Dent Res 78:208, 1999.

30. Ogden GR, Cowpe JG, Green MW, Detection of field change in oral cancer using oral exfoliative cytologic study. Cancer 68:1611-5, 1991.

31. Cowpe JG, Ogden GR, Green MW, Comparison of planimetry and image analysis for discrimination between normal and abnormal cells in cytological smears of suspicious lesions of the oral cavity. Cytopathology 4(1):26-35, 1993.

32. Ogden GR, McQueen S, et al, Keratin profiles of normal and malignant oral mucosa using exfoliative cytology. J Clin Pathol 46:352-6, 1993.

33. Ibrahim SO, Lillehaug JR, et al, Expression of biomarkers (p53, transforming growth factor alpha, epidermal growth factor receptor, c-erbB-2/neu and the proliferative cell nuclear antigen) in oropharyngeal squamous cell carcinomas. Oral Oncol 35:302-13, 1999.

34. Moll UM, Schramm LM, p53- An acrobat in tumorigenesis. Crit Rev Oral Biol Med 9(1):23-37, 1998.

35. Gallo O, Bianchi S, p53 expression: a potential biomarker for risk of multiple primary malignancies in the upper aerodigestive tract. Eur J Cancer (Part B, Oral Oncol) 310:53-7, 1995.

36. Huang M-F, Chang Y-C, et al Loss of heterozygosity of p53 gene of oral cancer detected by exfoliative cytology. Oral Oncol 35:296-301, 1999.

37. Jin Y-T, Myers J et al, Genetic alterations in oral squamous cell carcinoma of young adults. Oral Oncol 35:251-6, 1999.

38. Slavkin HC, Toward molecularly based diagnostics for the oral cavity. J Am Dent Assoc 129:1138-43, 1998.

39. Mandel ID, The diagnostic uses of saliva. J Oral Pathol Med 19:119-25, 1990.

40. Wisnom C, DePaola L, et al, Clinical applications of two detection systems for HIV using saliva. Oral Dis Suppl 1: 585-587, 1997.

41. Scully C, HIV topic update: salivary testing for antibodies. Oral Dis 3:212-5, 1997.

42. Achord AP, Bigler LG, et al, Use of saliva to detect recurrence of disease in high risk breast cancer patients. Abstract 1884. J Dent Res 78:341, 1999.

43. Copper MP, Braakhuis BJ, et al, A panel of biomarkers of carcinogenesis of the upper aerodigestive tract as potential intermediate endpoints in chemoprevention trials. Cancer 71:825-30,1993.

44. Pease AC, Solas D, et al, Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc Natl Acad Sci USA 91(11):5022-6, 1994.

45. Cronin MT, Fucini, et al, Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays. Hum Mutat 7(3):244-55, 1996.

46. Hacia JG, Brody LC, et al, Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour fluorescence analysis. Nat Genet 14(4):441-7, 1996.

To request a printed copy of this article, please contact/Carol Anne Murdoch-Kinch, DDS, PhD, Box 129, University of Detroit Mercy School of Dentistry, 8200 W. Outer Drive, P.O. Box 19900, Detroit, MI, 48219-0900.