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The goal of the differential diagnostic process is to identify the nature and location of a disease by analyzing the relevant signs and symptoms exhibited in the patient. Thus, it is based upon a carefully elicited history and detailed physical examination. While the diagnosis of some oral lesions can be made on the basis of the history and/or clinical findings, for others, the definitive diagnosis requires the aid of some tools that enable the clinician to take into consideration the histopathologic findings in addition to clinically relevant data.
General dental practitioners are often the first point of contact for patients with oral white and red lesions, which represent a wide spectrum of diagnoses of varying seriousness. Some clinical features are classical and others overlap between different diagnoses; they should be correlated with patient history and, sometimes, other investigations for diagnosis.
In a recent study of more than 17,000 people in the United States, oral white lesions were found in 27.9% of the group,1,2 thus representing a common clinical finding that makes clear the necessity for general dentist to be familiar with the differential diagnoses. Obviously, the goal is to achieve the correct diagnosis of each oral lesion, but for white and red lesions, it is of utmost importance to differentiate benign lesions from potentially malignant ones (dysplasia) or oral cancer (squamous cell carcinoma).
The clinical term leukoplakia is a diagnosis of exclusion with no specific histopathological connotation except the presence of dysplasia in a variable percentage of lesions; it has been redefined to describe a predominantly white lesion with premalignant potential.3,4
Oral erythroplakia, too, is a diagnosis of exclusion. Therefore, from a clinical point of view, some diseases of the oral mucosa with erythematous changes should be considered in the differential diagnosis; among them, erythematous candidiasis and atrophic oral lichen planus are the most important.5
Moreover, because persistent oral ulcers—which may be the final common manifestation—are sometimes clinically indistinguishable from a diverse spectrum of conditions, ranging from traumatic lesions, infectious diseases, systemic and local immune-mediated lesions, up to neoplasms,6 they require an accurate differential diagnosis.
As has been stated by numerous experts in the field, for all the above-mentioned oral lesions, biopsy is mandatory in cases of doubt.5
Biopsy and Histopathologic Examination
The skill required to perform a tissue biopsy, which is an important step in the diagnostic process for oral lesions, should be well within the capability of most practicing dentists. Scalpel or punch biopsy and histopathological examination represent the current gold-standard diagnostic test for potentially malignant oral mucosal lesions and oral squamous cell carcinoma (OSCC).7,8 However, biopsy is necessary not only to establish or exclude malignant disease in suspected lesions, but also to supplement the clinical diagnosis to establish a definitive one.
The accuracy of the diagnosis of oral lesions depends on the quality of the biopsy, adequate clinical information, and correct interpretation of the biopsy results.9 As an adequate and appropriate collection of tissue is essential for accurate examination, diagnosis, and ultimately treatment,10,11 an appropriate biopsy contains tissue that is representative of the lesion and mainly depends on the following three factors—selection of the biopsy site, the type of biopsy, and the submission of an adequate specimen to the laboratory.10,12
The two types of biopsy techniques to be considered are incisional versus excisional. In the excisional biopsy, all the margins of the lesion are entirely included in the surgical excision, while only a portion of the lesion is incised with incisional biopsy.
As in the case of large lesions, there may be discrepancies in the histological features found from one site within the lesion to another; therefore, clinicians should be aware that incisional biopsy could result in under-diagnosis. This is a particular concern in cases of ‘‘mixed" forms of OSCC and non-homogeneous lesions, in which there may be some areas that demonstrate obvious invasive disease, while others may indicate epithelial dysplasia.9,10,13 Hence, depending on the site where the biopsy is performed, false-negative results are still occasionally possible.9,14 Indeed, studies have shown that leukoplakia lesions, where dysplasia has been excluded by incisional biopsy, may prove to contain OSCC in up to 10% when wholly excised.7 To overcome these limitations, it has been proposed that a biopsy should include red rather than white areas, because the red areas are most likely to reveal the presence of dysplasia in the lesion.7 Furthermore, the use of toluidine blue staining and possibly other adjunctive tools can help to select the site for biopsy in large lesions;7 and the use of multiple biopsies reduces significantly both rates of under-diagnosis of carcinoma (29.5% vs. 11.9%) and undiagnosed carcinoma (12% vs. 2.4%).13
In any case, the site selected for a biopsy, particularly in larger lesions, must be representative of the overall pathology present in the lesion,10 and the specimens harvested must be of sufficient size and depth to include part of the advancing margin of tumor. Because some characteristics of the primary tumor—such as the grading of histological differentiation and depth of invasion—have been shown to have prognostic value in terms of tumor recurrence, lymph-node involvement, and cause-specific survival, ideally the deep margin should be included; however, if this is not possible, the peripheral margin is often sufficiently representative.9,15
Other investigators suggest avoiding excisional biopsy, even if it overcomes previous limitations. Because it is possible to perform histopathological examination of the entire clinically abnormal region,9 it carries both the risk of over treating benign lesions14 and incomplete treatment of malignant lesions when an insufficient margin of tissue is removed.7 If the former is ethically questionable, the latter is definitely more serious in that masking the clinical evidence of the site and the character of the lesion may limit the surgeon or radiotherapist.7
The traditional means of collecting tissue for histological examination is via scalpel biopsy of tissue. However, because oral biopsy specimens can be affected by a number of artifacts resulting from crushing, injection, or incorrect fixation,9 other surgical instruments have been proposed to avoid artifacts. A frequently used alternative to scalpel biopsies are punch biopsies, which are proven to be a useful and often easy biopsy method, depending on the type and the clinical access to the lesion.10 Disposable punch biopsies—which are readily available on the market—come in different sizes and produce few artifacts within the tissue.16 However, they are not free of artifacts and their benefits have not been definitively established.17
Other surgical instruments that have been proposed to obtain diagnostic biopsy specimens include removal of tissue by the use of a laser or electrosurgery.18 However, these techniques can induce tissue distortion or introduce thermal cytological artifacts including carbonization, nuclear elongation, and vacuolar degeneration of tissue. These artifacts—particularly in small specimens—not only may affect the ability of the pathologist to accurately assess the tissue, but, of particular concern when assessing dysplastic changes, thermal damage induced along the margins cut by the laser could simulate cytological atypia.9,10,18 Hence, it has been proposed that a better solution may be to excise with a scalpel and use electrosurgery or laser to control hemorrhage at the biopsy site.10
Finally, an important issue is correct specimen fixation after tissue removal, because incorrect handling can introduce artifacts and hinder the pathologist’s ability to accurately assess the tissue or can change it, thereby rendering the tissue non-diagnostic.10 For routine histological examination, 10% formalin is the fixative of choice. However, if other diagnostic techniques are required, other specific procedures may be performed. For instance, if a blistering disease is suspected, the specimen must be placed in cold physiologic solution so that direct immunofluorescence can be performed.
Autoimmune blistering diseases comprise a heterogeneous group of pathologies19 where the onset of clinical symptoms occurs after autoantibodies directed against structural components of the skin are produced and mucous membranes mediate the tissue damage.
Even if some typical clinical features represent part of the diagnostic criteria, an autoimmune bullous disease cannot be diagnosed by clinical signs alone and requires the detection of autoantibodies; examples would include tense blisters and severe skin pruritus for bullous pemphigoid; and flaccid blisters and a positive Nikolsky sign—the separation of epithelial layers caused by a mechanical insult to the apparently healthy mucosa—for pemphigus vulgaris.19 Indeed, immunofluorescent (IF) techniques increase the accuracy of diagnosis for dermatologic conditions of the oral mucosa,20 and direct immunofluorescence (DIF) microscopy is still recognized as the gold-standard19 diagnostic procedure in such pathologies because it permits the detection of autoantibodies in patients’ mucosal epithelium.
The link between autoantibodies and keratinocytes revealed by IF varies depending on the disease: in pemphigus, an intercellular binding of IgG and/or C3 is found in the epithelium—mostly in spinous layer—making a figure like a "fisherman’s net"; in pemphigoid diseases, linear staining of IgG and/or C3 is seen at the dermal-epidermal junction; and in patients with dermatitis herpetiformis, granular IgA deposits are typically observed at the dermal papillae.19
After removal, the specimen must be spliced into two pieces, one of which is placed in transport medium (Michel’s solution) for immunofluorescence and the other is placed in formalin for routine histological examination. Treating the specimens in this order reduces the risk of the ‘‘fresh’’ specimen becoming contaminated by formalin, thereby making immunofluorescence studies difficult.10 However, an alternative may be to collect two different specimens—a "lesional" specimen comprising all or part of the blister, and a "peri-lesional" specimen harvesting a piece of apparently healthy mucosa near the blister. The latter procedure may reduce patient compliance, increasing the discomfort due to two biopsy sites. However, it must be kept in mind that, when bisecting one specimen, peri-lesional mucosa should be included for DIF,19 especially if the roof of the blister is no longer present.
Exfoliative Cytology⁄Brush Biopsies
Exfoliative oral cytology—the study and interpretation of the characteristics of oral mucosa that naturally or artificially flakes off—is a non-invasive method that, in some situations, may be useful in the evaluation of mucosal pathology. Cutaneous or mucous cytodiagnosis—also known as the "Tzanck test" or "Tzanck smear" after Russian dermatologist Arnault Tzanck, who introduced it in 1947—was proposed as a diagnostic aid for bullous (pemphigus vulgaris and pemphigoids) and vesicular (herpetic infections) dermatoses, as well as certain skin tumors (basal cell and squamous cell carcinomas) in which neoplastic cells tend to exfoliate more easily because of their diminished cell cohesion.21 The latter, in particularly, represented the most interesting application evaluating superficial cellular characteristics of lesions for features of atypia, which may indicate malignancy. Because diagnosis based on examination of cytological features alone is not recommended for definitive diagnosis of malignant lesions,10 this technique has been largely replaced by more sophisticated and expensive methods. However, it is a quick, easy, noninvasive or minimally invasive, inexpensive procedure requiring little special equipment or training,21 and, thus, it may still be considered a useful first-level diagnostic test in oral infectious and immune diseases. A straight scalpel or a metal spatula may be used to take the smear from a recent lesion by gently scraping it. The material obtained is smeared onto microscope slides, allowed to air dry, and routinely stained with May Grünwald-Giemsa stain for 20 to 25 minutes.21
The diagnosis of herpes simplex virus (HSV), varicella, and herpes zoster is clinical in most cases. However, the presence of "ballooning cells"—multinucleated giant keratinocytes, with hyperbasophilic cytoplasm and nuclei exhibiting a blurry chromatin network—collected from the bottom of the lesions provide a rapid and reliable diagnosis of infection by the herpes virus group.22 Note that most conditions characterized by vesiculae or bullae usually appear as mouth mucosal ulcers⁄erosions due to oral traumatisms6 and due to the fact that rupture occurs rapidly because of the moist oral environment.
Several clinical and laboratory techniques are used to confirm a provisional diagnosis of oral candidiasis. In pseudomembranous clinical forms, the presence of Candida hyphae can be confirmed with periodic acid-Schiff (PAS) staining of a cytology smear of the pseudomembrane, thus allowing for a quick and accurate diagnosis of oral candidiasis.23
However, in other clinical forms of oral Candida infection (erythematous or atrophic) or associated lesions (eg, denture-related stomatitis), cytology smears usually fail to show any hyphal elements, but may reveal fungal spores. In these cases, cultures on specific media of swabs taken from the lesion may be useful to verify that a fungal infection is present.24
In the early stages of pemphigus vulgaris (oral pemphigus), a useful diagnostic finding in the exfoliative cytologic smear collected from the bottom of the lesions—usually painful erosions lacking a blister roof—is the presence of "mourning-edged" or Tzanck cells, which are acantholytic keratinocytes characterized by round shape, with a hypertrophic, often atypical neoplastic-like nucleus and a peripherally basophilic cytoplasm. 21
Since its introduction for cervical smears in gynecological lesions, the brush has proven to be a more convenient instrument to the examiner than the wooden or metal spatula when dealing with oral lesions.25 Moreover, in the last 2 decades, the accuracy of cytological analysis has improved with the advent of computer-assisted analysis, liquid-based cytology, and ancillary molecular analysis.25
Tolonium chloride or toluidine blue (TB) has been used for more than 40 years as a diagnostic aid for the detection of oral cancer. It is a simple and inexpensive diagnostic tool that uses a metachromatic nuclear vital blue dye, which may preferentially stain tissues undergoing rapid cell division (such as inflammatory, regenerative, and neoplastic tissue), to sites of nuclear material of malignant lesions and potentially malignant lesions (PML) or both but not normal mucosa. The binding results in the staining of abnormal tissue in contrast to adjacent normal mucosa.7-9
Among the different clinical protocols for TB staining applications, the most commonly followed comprises five steps 1.) patient rinses with 1% acetic acid for 20 seconds; 2.) patient uses a similar rinse with water twice for 20 seconds; 3.) patient rinses with 5 cc to 10 cc 1% TB solution while touching the lesion with a swab or gauges soaked in the solution; 4.) patient rinses with 1% acetic acid for about 1 minute; 5.) patient rinses with water.7
The sensitivities of TB as a diagnostic adjunct varied from 38% to 98% (median, 85%) and specificities varied from 9% to 93% (median, 67%),8 whereas others reported a higher sensitivity and specificity ranging from 93.5% to 97.8% and 73.3% to 92.9%, respectively.7,9,26 The PPVs ranged from 33% to 93% (median, 85%) and the NPVs from 22% to 92% (median, 83%).8
However, taking into account study biases, the evidence supports a recommendation for the use of TB as a surveillance or diagnostic adjunct in higher-risk populations, while there is insufficient evidence to determine whether the use of this or other adjunctive techniques will increase the detection of oral malignancies in community screening programs or in lower-risk populations such as patients in general dental practices.8
Analysis of current evidence suggests that TB is effective in the identification of SCCs and carcinoma in situ (CIS) and its sensitivity is significantly higher at detecting carcinomas, than in the detection of dysplasia;8,9,26 thus, the probability of a false-negative finding for invasive OSCC is low and the absolute number of false-positive tests is expected to be reduced.7
It has been proposed that a sequence of two applications—the first at baseline and the second at a follow-up visit approximately 2 weeks after the first application—will reduce false- positive findings secondary to inflammatory conditions in a general dental population, while eliminating potential causes such as minimizing frictional sources.8,27
It has also been suggested that TB may also help preoperatively by providing information on lesion margins—as even mucosa of a normal appearance might be expected to contain molecular changes of early carcinogenesis that would not have been addressed during conventional oral examination alone—and guide incisional biopsy site selection in the treatment of potentially malignant and malignant oral lesions.7,9,28
Optical Light-Based Detection Systems
Light-based detection systems represent techniques that are promoted or assessed to improve earlier detection and diagnosis of oral malignancy, operating on the theory that structural and metabolic changes take place in the mucosa during carcinogenesis, giving rise to distinct profiles of absorption and refraction when exposed to different types of light or energy.9,28 Such systems include: ViziLite® and ViziLite® Plus with TBlue (Zila Pharmaceuticals, www.zila.com); Microlux DL (AdDent, www.addent.com); Orascoptic DK (Orascoptic, a Kerr Company, www.orascoptic.com); and VELscope® (LED Dental, www.velscope.com ).
The ViziLite is the best-known system that uses tissue chemiluminescence. Neoplastic epithelial cells tend to have an altered nuclear-cytoplasmic ratio. Dehydration with acetic acid highlights this higher nuclear density and imparts an "acetowhite" appearance to tissues. This phenomenon can be further amplified by replacing conventional lighting with diffuse blue-white chemiluminescent illumination. The normal epithelium takes on a blue hue, while the "acetowhite" lesions appear distinctly white.29
The VELscope system is a multi-use device with a handheld scope through which the clinician can scan the mucosa visually for changes in tissue fluorescence.8
All tissues, due to the presence of naturally occurring fluorescent chromophores (fluorophores) within the cells, have a tendency to glow (fluoresce) in the dark, either spontaneously (auto-fluorescence) or if an external sensitizer is applied to the tissues. The reflective and absorptive pattern of oral mucosal tissue fluorescence is variable—including different sites in the mouth—and is affected by different conditions. Exposure to the blue light spectrum (400 nm to 420 nm) may maximize a loss of fluorescence visualization suggestive of PML or OSCC, and this may facilitate detection of lesions not detectable during oral examination under normal incandescent white light.7,8
Blood and Serological Tests
Other investigations such as blood and serological tests—in addition to oral and general clinical examinations—may be needed to correctly diagnose the cause of oral mucosal lesions. For instance, blood tests may be useful for excluding or confirming suspected underlying conditions as hematological disorders, HIV infection, or diabetes.
When an infectious cause is suspected, serological tests may be performed, although in oral mucosal lesions, microbiological smear tests are sometimes preferable. Indeed, confirmation of positivity to herpes simplex virus 1 in a patient with oral ulceration is not clinically useful in view of the fact that up to 90% of the US population has circulating antibodies against the virus, and 40% of the subjects who have been exposed to HSV will develop recurrent infections.30
Due to molecular identification of auto-antibodies target antigens made possible by novel test systems, in the great majority of patients, serological tests may help the diagnosis of oral blistering autoimmune diseases. Recombinant and cell-derived forms of the target antigens have been applied in the development of sensitive and specific ELISA assays for the detection of autoantibodies against desmoglein 1 in pemphigus foliaceus, desmoglein 3 in pemphigus vulgaris, BP180 in bullous pemphigoid, envoplakin in paraneoplastic pemphigus, and laminin 332 in mucous membrane pemphigoid.19 Hence, these ELISA systems are not only suitable for the diagnosis of blistering diseases, but they are also useful in monitoring serum autoantibody levels during the course of the disease because serum autoantibody levels in patients correlate with disease activity.19
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About the Authors
Andrea Santarelli, DDS
Department of Clinic Specialistic and Stomatological Sciences
Polytechnic University of Marche
Lorenzo Lo Muzio, DMD, PhD, MD
Department of Surgical Sciences
University of Foggia
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