False negative staining is very rarely observed in OSCC, particularly in modern series [9,12,76,77,79,82–84], in which improved study designs and pharmaceutical grade TBlue are used. Nevertheless, binding of TBlue to the nucleic acids may occur in mucosal ulcerations, granulation tissue [68,69,74,79,83,84], and in inflammatory lesions that can contribute to false positive outcomes [9,76,84,85]. However, unlike malignant lesions, the blue appearance of these traumatic/benign lesions may not persist as long in the tissue  and may localize at the periphery of the ulceration, presenting subjective differences in assessment of stain retention that may guide clinical impression . In order to reduce positive outcomes in inflammatory lesions, a 2-week review of lesions not felt to be at high risk of cancer at first evaluation is recommended [2,74]. On the contrary, TBlue does not stain all early stage dysplastic lesions , which suggests that some dysplastic lesions may not have the LOH associated with TBlue retention . Recent studies showed that histologically benign lesions that were TBlue positive have revealed molecular abnormalities and were therefore at increased risk of progression to cancer [52,77], suggesting that ‘false positive’ TBlue results may represent molecularly true positive lesions with high risk of progression to OSCC .
TBlue for topical application has historically been made by compounding laboratory grade powder [84,86]. In 2005, the US Food and Drug Administration (FDA) cleared the only pharmaceutical grade TBlue available for oral use in the United States. TBlue, as part of a light source examination kit (ViziLite Plus) indicated for use as a diagnostic auxiliary to conventional oral cancer screening, is produced by Zila Pharmaceuticals, Inc. (Phoenix, Arizona, USA). The TBlue is part of a swab system to be used at the discretion of the healthcare provider to physically mark oral mucosa lesions differentially identified during ViziLite examination. Since 2008, TBlue has been available in Canada, the United Kingdom, Ireland, France, Germany, Spain, Portugal, and Andorra with distribution agreements in place for Greece, Cyprus, Russia, and Belarus. This system is registered with the Medicines and Healthcare Products Regulatory Agency in the UK as a ‘Conformité Européenne’ marked that certifies that a product has met EU health, safety, and environmental requirements of a medical device enabling the company to marketing to all European Union (EU) member states. Introduction is planned in Italy in 2009. Regulatory approval is also being sought in China, Korea, India, and Australia.
Zila Pharmaceuticals, Inc., also holds licenses for marketing authorization in the United Kingdom, Belgium, Portugal, Luxembourg, Finland, the Netherlands, and Greece for a pharmaceutical grade TBlue product in a rinse form called OraTest. Distribution is planned in 2009. OraTest is a diagnostic kit, indicated as an adjunct method to clinical examination in the initial diagnosis and treatment of malignant lesions and conditions of the oral mucosa as well as previously treated OSCC patients.
The laboratory grade TBlue, unlike the pharmaceutical grade, may vary between batches, manufacturers, composition, stability, and purity with an unknown shelf life. These differences may be a reason for variability in findings in older studies. Taste is a greater issue with the rinse application than cotton swab application to localized areas.
As is the case in all diagnostic tools and adjuncts, sensitivity and specificity of oropharyngeal application of TBlue is impacted by the prevalence of disease in a population ; therefore, outcomes in high-risk patients may not be replicated in lower risk populations [72••]. Additionally, inclusion of equivocal stain results either as positive or negative also impacts the above-mentioned characteristics in some studies. In order to assess sensitivity and specificity of a diagnostic adjunct, all lesions at entry must be assessed using the gold standard test of biopsy. Some trials did not complete biopsies of TBlue negative tissue and, therefore, assessing the sensitivity and specificity of TBlue in these trials cannot be completed [72••,79,84,89]. Further, as histopathologic interpretation is subjective, a panel of blinded pathologists should provide outcome data. Studies that have used these methods provide more creditable data to guide clinical utilization [2,9,77,85,86,90] (Table 1).
A recent systematic review of adjunctive examination aids included 2400 lesions in studies varying from 18 to 1030 lesions, stained with TBlue with histologic outcomes [72••]. The sensitivity and specificity of TBlue varied from 38 to 98% (median 85%) and 9 to 93% (median 67%), respectively, whereas the positive predictive value (PPV) ranged from 33 to 93% (median 85%) and the negative predictive value (NPV) from 22 to 92% (median 83%) [72••].
The TBlue literature shows that it is a practical, rapid, inexpensive, and effective adjunct diagnostic tool in mucosal disease clinics and cancer centers with experienced providers in high-risk patients. TBlue may assist in detection of oral mucosa with molecular changes with or without phenotypic changes on biopsy that are associated with OPLs or OSCC [7,12,52,72••,75–77,83,90]. TBlue used in addition to clinical examination increased efficacy in detecting OSCC or premalignant lesions or both in high-risk clinics [64,76,85,91••]. In other prospective studies, the use of TBlue is reported to be more sensitive than clinical examination alone in high-risk clinics in detecting premalignant or malignant lesions in patients who had been previously treated for carcinoma of the upper aerodigestive tract and in identifying oral mucosal lesions with high-risk molecular features [76,77].
It is not known whether the more widespread use of TBlue in general practice as an examination adjunct will result in increased diagnosis of dysplasia and malignancy or will lead to increasing numbers of biopsies of benign mucosal changes. However, Epstein et al.[91••] showed use in high-risk patients examined by experienced providers reduced the number of biopsies of benign lesions by approximately 50% and identified all severe dysplasia and OSCC lesions would occur with TBlue as part of the clinical protocol. TBlue is recommended as an adjunct to the clinical examination of oral mucosal lesions, specifically in high-risk patients by expert providers. When TBlue is used in general practice and suspicious mucosal lesions are identified, referral to centers experienced in the diagnosis and treatment of OPLs and OSCC is recommended.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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