Biopsy
Diagnosis and Management of Oral Soft-tissue Lesions: The Use of Biopsy, Toluidine Blue Staining, and Brush Biopsy
Janice P. Handlers, DDS
Copyright 2001 Journal of the California Dental Association.
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Upon discovery of a lesion after thorough oral soft-tissue examination, the dentist is confronted with the often-troublesome decision of how best to manage the patient. The management protocol must provide for early diagnosis in the case of oral cancer so as to reduce cancer morbidity and mortality. Options for management would include an observation period of some defined time, use of toluidine blue stain or oral brush biopsy to screen the lesion, or immediate biopsy and follow-up. This article reviews the assets and limitations of each and suggests a rational, science-based approach to diagnosis and management.
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Once a soft-tissue lesion has been discovered on clinical oral examination, how should the clinician proceed? The clinician, at this point has several options:
* Observe the lesion for a defined period and reassess;
* Screen the lesion before scalpel biopsy by the use of toluidine blue staining or computer-assisted oral brush biopsy analysis (OralCDx); or
* Perform a scalpel biopsy of the lesion.
Observation of Lesion
The decision to "watch" a lesion must be based on a clinical likelihood that the lesion is of reactive or inflammatory etiology. In the case of a white lesion, the presumed local irritating factors should be eliminated. In most instances, these lesions will resolve within two to four weeks. Ulcers and red lesions should heal within two weeks. Oral cancers do not disappear without definitive therapy and may take time to evolve to a more advanced stage that will be clinically obvious. Therefore, all lesions that fail to resolve in this period should have a biopsy performed.
Too often, early carcinoma appears as a painless lesion with minor local changes, and the clinician underestimates the clinical significance of the lesion. The patient is told to return only if there is any change in the lesion prior to the next regular recall examination, usually in six months. At this point, if the lesion has shown no change, the dentist feels validated in assuming that it is entirely innocuous and is comfortable in continuing the observation period, only to be surprised when sometime in the future the lesion does change and is finally diagnosed as squamous carcinoma. Sometimes, it changes in the six-month period and is diagnosed, but not until six months after the initial presentation.
There is sufficient evidence that visual inspection alone is not adequate to differentiate early oral cancer from benign lesions regardless of the expertise of the clinician. Sandler1 found that approximately 25 percent of 207 early stage oral cancers lacked any clinical features of malignancy. Dentists specializing in oral and maxillofacial pathology, oral medicine, and oral surgery clinically characterized 647 lesions as benign in a study by Sciubba and colleagues. 2 At least 29 of these were diagnosed malignant or dysplastic upon scalpel biopsy.
A professional delay is defined as the period from the patient’s first consultation with a health care professional to his or her first consultation with the treating specialist.3 Such professional delays in diagnosis result in increased risk for local extension and spread to distant sites.3-7 The prognosis of the patient relates directly to the stage at which the cancer is diagnosed. The five-year survival rate in the United States for a localized oral squamous carcinoma is 81 percent, 44 percent for patients with nodal metastases, and 21 percent for patients with distant spread.8 In addition, failure to pursue diagnosis of a suspicious lesion or improper delay in the initiation of the diagnostic process can be considered negligence, and the dentist can encounter legal difficulties. 9
Screening the Lesion
Screening procedures such as the use of toluidine blue staining and OralCDx are used to determine the significance of an oral lesion prior to scalpel biopsy. These tests supplement the clinical oral examination in the early detection of premalignant and malignant oral lesions, which are often quite innocuous in appearance.
Toluidine Blue Staining
Toluidine blue is a metachromatic dye that shows affinity for the perinuclear cisternae of DNA and RNA. Selective staining of malignant and dysplastic cells might be explained in that these cells contain quantitatively more nucleic acids than normal tissue. Toluidine blue staining, in vivo, by malignant and dysplastic cells might also be the result of the immediate binding by sulfated mucopolysaccharides, which are found in higher quantities in tissues that are actively growing, such as tumors and tissues that are healing.10
The staining technique involves applying a 1 percent aqueous toluidine blue dye to the suspicious lesion for approximately 30 seconds, followed by a tap water rinse. The lesion is then lightly blotted with 1 percent acetic acid to reduce the background level of staining. Only positive areas will retain stain after this decolorization process (Figures 1a and b).
The technique is highly efficient in detecting malignant disease with reported sensitivity of over 95 percent.11-14 It has significantly less usefulness in detecting premalignant lesions. Warnakulasuriya and colleagues14 found a false-negative rate for oral epithelial dysplasias of 22 percent. Martin and colleagues13 found false-negative staining rates for carcinoma in situ of 42 percent and 58 percent for moderate and severe dysplasia. False-positive results are quite common in ulcerated inflammatory or traumatic lesions.11-14
The data indicates that there is limited value in using toluidine blue stain to determine the significance of an oral lesion prior to biopsy. Positive staining may indicate either an inflammatory or malignant lesion. Negative staining may mislead the clinician into believing that the lesion is a benign process when it may indeed be a premalignant lesion; however, toluidine blue staining may be of use in helping the clinician in choosing incisional biopsy sites within suspicious lesions.
OralCDx Computer-Assisted Oral Brush Analysis Method
OralCDx (OralScan Laboratories, Suffern, N.Y.) is a computer-assisted method of analysis of the oral brush biopsy in the detection of precancerous and cancerous lesions of the oral mucosa. The kit consists of a glass slide, fixative, and an oral brush biopsy instrument that is used to obtain a transepithelial specimen. The collected sample is spread onto the glass slide and bathed with the liquid, ethanol-based fixative. The slide is sent to OralScan Laboratories where it is scanned by the OralCDx neural net computer system to detect potentially abnormal cells. The computer produces approximately 200 digitized images of the suspicious cells for review by specially trained pathologists. The test results are faxed to the referring doctor.
The seminal study by Sciubba and colleagues2 reports a high sensitivity and specificity for malignant and dysplastic lesions. In the study, 945 lesions were identified by dentists specializing in oral and maxillofacial pathology, oral medicine, and oral surgery and classified as Class I lesions, if clinically believed to be suspicious (298 lesions), and as Class II lesions, if clinically believed to be benign (647 lesions). A scalpel biopsy was performed for all the clinically suspicious lesions. The OralCDx brush biopsy did correctly classify all of the histologically confirmed malignant or dysplastic lesions as "positive" or "atypical" (131 cases). It also identified 29 clinically benign appearing lesions as "positive" or "atypical," which subsequently were histologically confirmed as malignant or dysplastic. The implication is that these patients may not have received a biopsy on initial examination, as these lesions were clinically perceived as benign.
The remaining 618 clinically benign appearing lesions, including 99 that were classified as "atypical" on OralCDx, were not confirmed with scalpel biopsy. It would be useful to know how many of these may have been histologically confirmed as premalignant or malignant since there remains a question as to whether the false-negative rate indicated in the paper is accurate. It should not be assumed that the 618 Class II lesions not subjected to biopsy would have produced results identical to those that were subjected to scalpel biopsy. In addition, the reasons given in the study for the lack of biopsy confirmation of the 99 lesions with "atypical" OralCDx results were that patients were lost to follow-up; and, in the majority of other instances, the investigator "determined clinically that the oral lesion was benign."2 The study itself confirms the difficulty that even specialty-trained clinicians have in clinically determining benignity, in that 29 lesions clinically perceived to be benign were actually premalignant or malignant. If these lesions did indeed clinically appear to represent pemphigus, lichen planus, or geographic tongue as indicated in the paper, perhaps OralCDx was not the appropriate initial test of choice.
This author’s experience at Oral Pathology Associates, Inc., has been fairly limited. The pathology group has been in receipt of 16 biopsies with previous OralCDx "atypical" results. Fifteen of the 16 cases were confirmed histologically as benign. One case was diagnosed as pemphigus vulgaris, one as exogenous pigmentation, one as lichen planus, two as lichenoid mucositis, one as pyogenic granuloma and epithelial hyperplasia, one as migratory glossitis, one as inflammatory fibroepithelial hyperplasia, and eight as benign keratosis. Only one of the 16 cases was confirmed as dysplastic. Although far from conclusive, this might suggest a higher false-positive rate than previously reported. Of the two biopsies received on lesions with "negative" OralCDx results, one was histologically confirmed as chronic granulomatous inflammation and one as benign keratosis and chronic mucositis.
It would seem that OralCDx might best be used in those instances where the lesion is thought clinically to be reactive or inflammatory (e.g., denture sore, ulcer, cheek-biting trauma). Rather than watch the lesion for two to four weeks, the clinician could use OralCDx immediately to help identify potentially harmful lesions. However, as recommended by the ADA Council on Scientific Affairs15 and by the manufacturer, all OralCDx "atypical" and "positive" results must be confirmed by incisional tissue biopsy followed by histologic examination to completely characterize the lesion. Persistent lesions, even with "negative" results, must receive adequate follow-up evaluation. At this point, scalpel biopsy should be performed to make a definitive diagnosis.
In cases where the lesion clinically appears suspicious, although OralCDx might be employed, it would seem that an immediate scalpel biopsy would be more definitive and therefore a better clinical choice. Certainly, if oral cancer is high on the list of differential diagnoses, a scalpel biopsy should immediately be pursued. As Dr. Sol Silverman, Jr., suggests, "There’s no point in using the brush biopsy technique on lesions so advanced you can see them from across the room."16
In that OralCDx only tests the overlying epithelium, it is not appropriate for lesions situated in the submucosa and covered by apparently normal mucosa such as fibromas, salivary gland lesions, or pigmented lesions. It is not for use on the vermilion border of the lip. It is also not appropriate for generalized white or vesiculoerosive lesions such as pemphigus vulgaris, mucous membrane pemphigoid, or lichen planus (reticular and erosive forms). These generalized conditions rarely imitate premalignant or malignant conditions; and regardless of what results are obtained via OralCDx, scalpel biopsy is required for definitive diagnosis. In summary, a certain amount of clinical acumen seems to be necessary in knowing when to use the OralCDx brush biopsy.
Scalpel Biopsy
The reason to perform a scalpel biopsy on a clinical lesion is to establish a definitive diagnosis. It is not "a test for cancer." While it is generally true that a cancer diagnosis must be confirmed by biopsy before treatment can be given, it is not a diagnostic tool for cancer only. Biopsy can and should be used routinely to diagnose the hundreds of benign conditions that occur in the oral and paraoral regions. If a biopsy is performed on representative tissue, it is the most reliable test and serves as the gold standard against which all other tests are judged.
There are several things to keep in mind when performing an oral biopsy that will contribute to the success of the procedure.
* Carefully select the biopsy site(s) -- The incisional biopsy should include the most representative portion(s) of the lesion (Figure 2). As indicated above, toluidine blue staining may be useful in determining the most suspicious area(s). There is little need to include a border of "normal" tissue. The exception to this rule is when an incisional biopsy of an ulcer or desquamative condition is performed. In this case, it is important to include a margin of normal tissue and ensure that mucosal sloughing does not occur during the biopsy procedure itself or during transport of tissue to the laboratory. This can be accomplished by passing a length of suture through the sloughing portion into submucosa and then out through the "normal" mucosa. The suture should not be tied or knotted. The specimen should then be placed with the connective tissue side down on a dry piece of paper (e.g., sterile insert from scalpel blade). The connective tissue will adhere to the dry paper. Paper and specimen should be put into the specimen bottle containing 10 percent neutral buffered formalin. This will ensure that the specimen fixes flat, without twisting, and that the surface mucosa will not be traumatized during transport.
* Remove sufficient tissue -- Generally, the larger the sample, the greater the chance of an accurate pathologic diagnosis. Fixation causes shrinkage, color changes and firming of the tissue. The greater size of the tissue biopsy allows for that shrinkage and permits the pathologist to better orient and cut the specimen, avoiding tangential sectioning, which can greatly hinder the definitive diagnosis.
* Use sharp instruments (scalpel, dermatologic punch, scissors) -- This is especially critical when performing an incisional biopsy or excising a small lesion. Do not use electrosurgical knives or a carbon dioxide laser as these instruments cause heat damage and produce distortion and artifact that can seriously interfere with microscopic evaluation.
* Handle tissues gently -- Crushing and squeezing the sample with forceps can interfere with diagnosis. Placing a suture through the specimen prior to removal will afford control and atraumatic handling. It will also make it less likely that the specimen will be dropped, sucked into an aspirator, or inadvertently swallowed.
* Place specimen immediately into fixative solution -- Fixation retards the normal enzymatic degradation and bacterial growth that occurs after tissue death. These biological processes will interfere with microscopic evaluation.
Management
Once the biopsy result has been received, the clinician determines the appropriate management of the patient based on the definitive diagnosis. The clinical scenario that seems to be the most problematic in developing a rational follow-up protocol is the white lesion that is diagnosed as "benign keratosis." Several studies have reported that these lesions do have a significant tendency to undergo malignant transformation, with rates varying from 0.13 percent to 15.7 percent.17-18 Risk factors that increase the likelihood of transformation include:
* The patient’s being a nonsmoker;
* The patient’s being female;
* The lesion’s having an erythematous component;
* The lesion’s having a clinical appearance of proliferative verrucous leukoplakia;
* Microscopic evidence of candidiasis;
* Location of the lesion on the tongue, floor of mouth, and soft palate; and
* The patient’s having pain or discomfort. 18-21
As it is not possible to distinguish clinically which lesion is undergoing malignant transformation, management of the patient will revolve around elimination of the lesion. The first step should be the elimination of any local irritating factors, including, but not limited to, the use of tobacco or alcohol, candida, and sources of trauma. If the lesion persists in spite of this conservative approach, surgical removal (excisional biopsy) with microscopic evaluation of the lesion is the most effective treatment. If the lesion is large, surgical removal may have to be accomplished in stages, or ablation with a carbon dioxide laser may be performed. For recurrent lesions, biopsy should be re-performed. Although chemoprevention has been used experimentally, its long-term effectiveness has not been established.17
Any lesion that is microscopically diagnosed as "dysplasia," must be removed in its entirety as these are considered premalignant lesions. Lumerman and colleagues22 retrospectively evaluated 44 of 308 cases of oral epithelial dysplasia diagnosed by their biopsy service. Thirty-four percent of patients (15) had a recurrence of the dysplasia and 16 percent (7) developed oral carcinomas seven to 78 months later (mean of 34 months). Silverman and colleagues18 reviewed follow-up data for 22 patients with oral leukoplakia and microscopic dysplasia reported in a prospective study of 257 oral leukoplakia patients. These patients were followed for a mean time of 8.1 years. Eight of the patients (36.4 percent) with epithelial dysplasia developed oral carcinomas.
Ideally, surgical excision should be performed with microscopic evaluation of margins. For large lesions, ablation with a carbon dioxide laser is effective; but, again, a biopsy should be performed on any recurrent lesions to rule out invasion. Any lesion microscopically diagnosed as malignant must be referred immediately for definitive therapy.
Conclusion
Biopsy remains the gold standard in the diagnosis of oral lesions. "Watch and wait" is only appropriate for lesions that are thought to be inflammatory (e.g., denture sores ulcers or cheek-biting trauma). The waiting period should be no longer than four weeks before definitive diagnosis by biopsy. An alternative to the observation period would be to immediately perform an OralCDx brush biopsy on the lesion and potentially identify lesions that require immediate biopsy ("atypical" or "positive" results). All persistent lesions that are diagnosed as "negative" on OralCDx should be definitively diagnosed by biopsy. Any lesion that is not thought to be inflammatory or is obviously suspicious for cancer should have an immediate biopsy. Toluidine blue staining may have value in helping the clinician to select the most representative areas for biopsy.
Early detection of oral cancer and removal of potentially malignant lesions are two weapons in the cancer control program. Early detection results in increased survival and reduced morbidity. Removal of lesions that may have malignant potential may prevent the occurrence of cancer.
Author
Janice P. Handlers, DDS, is the co-director of Oral Pathology Associates, Inc.
References
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To request a printed copy of the article, please contact/Janice P. Handlers, DDS, 11500 W. Olympic Blvd., Suite 390, Los Angeles, CA 90064 or jphandlers@msn.com.
Legends
Figure 1a. Squamous cell carcinoma of the right lateral border of the tongue and the floor of the mouth -- unstained.
Figure 1b. Positive royal blue staining of squamous cell carcinoma by toluidine blue.
Figure 2. Incisional biopsy of white lesion on buccal mucosa. It includes a good-sized portion of a representative area. No border of normal tissue is necessary.
