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The Toluidine Blue Test in Lesions of the Oral Cavity

Eugene N. Myers, M.D.

The use of toluidine blue as an ad junct in the diagnosis of epidermoid cancer of the oral cavity has been de scribed by several authors.1-4 This in vivo demonstration of oral cavity can cers is based upon the observation that the topical application of toluidine blue, an acidophilic metachromatic nuclear stain, will stain an area of carcinoma in situ or invasive carcinoma, whereas normal mucosa will not stain. This test is quite useful in several situations. In the large, far-advanced cancer of the oral cavity, the applica tions are rather limited, since mere in spection of the lesion may be sufficient for clinical diagnosis. (Fig. 1.) His tologic diagnosis is then made by direct biopsy. However, in less distinct areas where dysplasia or hyperkeratotic le sions are present, it is helpful to stain the lesion to differentiate it from a true carcinoma. The stain also may be used to help differentiate a traumatic or inflammatory ulcer from a cancer. Staining the lesion prior to excision is helpful in determining the margins of resection. In addition, staining can demonstrate a small second primary or a satellite lesion adjacent to a larger lesion.
Dr. Myera 18 from the Department of Otolaryn gology, Hospital of the University of Pennsylvania. Philadelphia, Pennsylvania.

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The earlier reports of Shedd, et al. and Strong, et al. have vividly demon strated the use of this stain in oral can cer.'4 Their observations were limited to epidermoid carcinoma, and the usual benign oral lesions such as hyperkera tosis, lymphoid hyperplasia, lichen pla nus, and traumatic ulcerations. We have had the opportunity to study 70 patients with lesions in the oral cavity with toluidine blue, and have demonstrated positive staining of mela noma, fibrosarcoma, and lymphosar coma in addition to epidermoid cancer. Staining of these lesions has not been reported in the earlier series. The ob servation that malignant lesions other than epidermoid cancer take the tolui dine blue stain prompted this report.
Technique of Staining

The same technique of staining was used in 70 patients with lesions of the oral cavity. The patient was instructed first to rinse the mouth with water, and to swallow several sips of water. Excess saliva was aspirated with a suction. Acetic acid one percent was then ap plied with a cotton applicator as a mucolytic agent. If there was a large deposit of fibrin or debris in an ulcer this was also removed by suction. A small amount of toluidine blue one per cent was then applied, with a cotton applicator, to the entire lesion and some of the surrounding mucosa. The patient then rinsed his mouth with wa ter to wash away the excess toluidine blue. Cases in which the toluidine blue test was positive were biopsied imme diately. In addition, many lesions which did not take up the stain were biopsied to document the nature of the lesion. Those negative lesions which were not biopsied were kept under close observa tion. The efficacy of this procedure was demonstrated in a series of 70 patients. There were no false positives. Those lesions which did not stain were proved
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by later biopsy or repeat clinical exam ination to be benign. Forty-seven patients had epidermoid carcinomas. Two patients had malig nant melanoma, one fibrosarcoma, and one lymphosarcoma. These tumors all dyed very vividly. One patient with a recurrent adenoid cystic carcinoma of the buccal mucosa which was not ul cerated did not take the stain. It is im portant to understand that only those lesions which have some ulceration of the mucosa will take the stain. Those which are completely mucosal-covered will not. Nine patients were seen with ulcera tions which were either radiation ul cers, inflammatory ulcers, or ulcers from a trauma in the buccal mucosa. These either stained weakly, spottedly or not at all. Ten other patients had benign lesions which did not stain, in cluding leukoplakia, hyperkeratosis, lichen planus, and median rhomboid glossitis. The two patients with melanoma in the mucosa of the hard palate were of exceptional interest. One was a recur rent melanoma which had been treated by electrodesiccation five years prior to staining. At the time of the test there was a 5 mm. lesion of recent origin in the anterior portion of the hard palate which was mucosa-covered except for a small linear ulceration in the center of the lesion. This was found to stain positive with toluidine blue. Biopsy revealed recurrent melanoma, and the patient was again treated. The second patient with melanoma had a large ul cerated lesion in the hard palate which was not pigmented and took up the stain vividly. (Fig. 2.) The biopsy was positive for malignant melanoma. This patient presented with two addi tional pigmented melanomas, one on the anterior and one on the lateral maxil lary alveolus, and both were positive for melanoma on biopsy. In addition to the three primary mucous membrane

one patient had a third primary appear after two other oral cavity lesions had been treated. The patient with a mu cous membrane melanoma described above presented with three distinct pri mary lesions widely separated in the mucous membrane. This series is not broken down into locations in the oral cavity although most of these were in the tongue and floor of the mouth. (Figs. 3 and 4.)

Fig. 2. Deeply ulcerated malignant melanoma. (Surrounding normal mucosa does not stain.)

melanomas, the patient presented with metastases to one side of the neck, and within three months developed metas tases to the other side of the neck. One patient had a fibrosarcoma in volving the soft and hard palate, the maxilla, the pharynx and the mandible. A large necrotic mass was present on the soft palate. This did not stain, but adjacent to this was a small ulcerated area in the hard palate near the maxil lary tubercle which did stain with to luidine blue. This proved to be spindle cell fibrosarcoma and a resection in cluding a partial mandibulectomy, par tial maxillectomy, and pharyngectomy was carried out followed by radiation therapy. This series included a patient with a lymphosarcoma of the tonsil which had a small ulceration in the center of the lesion. This lesion had the charac teristics of an epidermoid cancer. It took up the toluidine blue stain, but biopsy revealed lymphosarcoma. This was treated with radiation therapy. Two patients had second primary epidermoid carcinomas in various parts of the oral cavity at the time of their first examination. In addition, we saw another second primary which appeared well after treatment of the first, and

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ulcerated

squamous

cancer in floor of mouth with two small satellite areas posterior to the main tumor.

However, lesions were stained in areas such as hard and soft palate, tonsil, alveolar ridge, posterior pharyngeal wall, and buccal mucosa. (Fig. 5.) In the advanced lesions it is difficult to tell the exact primary sites since two, three or four anatomical structures
137

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Fig. 5. Sharply demarcated squamous cell car

cinoma in soft palate stains vividly.

may be involved. It is also interesting that benign and malignant lesions can coexist at one time. Figure 6 demon strates an epidermoid cancer in the floor of the mouth which had been treated with radiation therapy and had recurred. Adjacent to this is an area of leukoplakia which does not take the stain. In the crevices of this lesion, the stain does puddle and should not be misinterpreted as being a positive stain.
Discussion

can be avoided. The lack of false posi tives or negatives enhances its value as a screening test. The test itself is remarkably sim ple and requires only cleansing of the ulcer so that the stain will come in con tact with the surface of the ulcer. Earlier studies have shown that normal mucous membrane does not stain; therefore, one must not depend upon this test for those tumors which spread without involvement of overlying mu cous membrane. The description of positive staining by toluidine blue of ulcerated tumors such as melanoma, fibrosarcoma, and lymphosarcoma further extends the usefulness of this test, which hereto fore has been described only in epider moid cancer. The test is to be used as an adjunct and as a screening test, and is not a substitute for biopsy and close observation, which are the best way to deal with lesions of the oral cavity.
References
1. Niebel. H. H., and Chomet, B.: In vivo staining test for delineation of oral intraepithelial neo plastic change: preliminary report. J. Amer. Dent. Asen. 68: 801-806, 1964. 2. Shedd, D. P.; Hukill. vivo staining properties Surg. 110: 631.634, 1965. P. B., of oral and Bahn, S.: cancer. Amer. In J.

The use of toluidine blue as a screen ing test is a valuable adjunct to in spection and palpation of the oral cavity in the search for malignant lesions. It is of great use in follow-up after radiation therapy, or after other treatment for mucous membrane le sions since unnecessary biopsies for postradiation or traumatic ulcerations

3. Shedd, D. P., et al.: Further appraisal vivo staining properties of oral cancer. Surg. (Chicago) 95: 16-22. 1967.

of in Arch.

4. Strong, M. S.; Vaughan, C. W.. and Incze. J. S.: Toluidine blue in the management of carcinoma of the oral cavity, Arch. Otolaryng. (Chicago) 87: 527-531, 1968.

Fig. 6. Recurrent cancer (left) stains. In leuko

plakia (right) the toluidine blue puddles in

crevices. (This should not be misinterpreted a positive stain.)

as

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