In two recent review articles on oral cancer diagnostic aids, Patton et al. [16] and Lingen et al. [17] concluded that based upon published studies, oral brush biopsy with computer-assisted analysis has been demonstrated to be valuable for detecting dysplasia and cancer when evaluating "clinically suspicious" lesions. Both groups suggested that the accuracy of the oral brush biopsy for testing "minimally suspicious" lesions has not yet been established. Our study indisputably demonstrates that the brush biopsy is as sensitive and specific for evaluating "minimally suspicious" lesions as well as "suspicious" lesions and makes it an effective test to evaluate the entire spectrum of lesions detected during an oral cancer screening examination.
The accuracy of the oral brush biopsy has been questioned by some authors who reported lower sensitivity and specificity results compared to those reported in our study [18–20] Purported oral brush biopsy "false-negative" or "false positives" cases in the literature are not a reliable comparison of the efficacy of the brush biopsy vs. the scalpel biopsy since these discrepant anecdotes, which have been quoted repeatedly in the literature [16, 17], are of questionable value. In almost all cases, discrepant results were reported from patients who had a scalpel biopsy and brush biopsy performed at widely different times- often months or over a year apart. Within a given oral lesion, dysplasia is multicentric and therefore, unless the 2 biopsies test the same part of the dysplastic lesion by chance, the results will often be discrepant. Furthermore, the biologic nature of a lesion may change over time as benign lesions may become dysplastic and dysplasia may also regress [21]. Finally, the histologic diagnosis of dysplasia is not easily reproduced amongst oral pathologists, with poor intraobserver and interobserver variability in the diagnosis of oral dysplasia [22, 23]; therefore, a discrepant result between brush biopsy and scalpel biopsy may also, in fact, represent a false negative or false positive scalpel biopsy result.
The limitations of comparing any two biopsy results performed at different times are highlighted in a study of 200 patients with leukoplakia [24]. In this study, scalpel biopsies were obtained from the same lesion at different times, and the results showed an agreement rate between two scalpel biopsies of only 56%. In another study by Holmstrup et al [25], the degree of dysplasia in biopsies from 101 oral lesions was compared with the results from the entire specimen which was examined in step sections, and the concurrent diagnosis was achieved in only 49% of cases. As others have noted, when comparisons are made between any two biopsy techniques (i.e. brush biopsy vs. scalpel biopsy or scalpel biopsy vs. scalpel biopsy), only studies comparing the results of both biopsies performed at the same time and from the same portion of the suspicious lesion should be considered valid [26, 27]. In the 3 studies in which an oral lesion was simultaneously tested with both a brush biopsy and scalpel biopsy, including our study, the oral brush biopsy with computer assistance has been shown to have a sensitivity and specificity well over 90%.
For purposes of determining specificity, it is assumed that when the brush biopsy detects dysplasia or cancer which is not found on the scalpel biopsy, such cases are classified as brush biopsy "false positives." Although histology is the standard for diagnosis, as with all anatomic pathology laboratories, histologic sampling is not without errors, and false positives as well as false negatives do occur [28]. In our study, there were 5 patients whose brush biopsies were abnormal and whose scalpel biopsies were negative. It is possible that several cases of dysplasia or cancer may have been missed with the scalpel biopsy. Under ideal circumstances, these patients would be recalled and a second scalpel biopsy would be performed.
In another study, brush biopsies and scalpel biopsies were performed concomitantly with the authors reporting several brush biopsy false negatives in identifying dysplasia, however, all cancers were identified with the brush biopsy [20]. As the authors in that study point out, confirmation of a brush biopsy false negative result requires a brush biopsy tissue defect to be present in the histological specimen of the scalpel biopsy to ensure that the same part of the lesion was sampled by both biopsies. Yet in that study, not a single example of matched brush and scalpel biopsies was presented for any of the purported discrepant results. Therefore, a likely conclusion is that different portions of the lesion may have been sampled. Furthermore, surprisingly, 43 of the 69 lesions in that study were presumably dysplastic and 15 were frank cancers, despite the fact that all of the lesions in the study were not clinically suspicious. Bukhardt has analysed their results and found some glaring problems supporting the notion that the authors' claim of finding dysplasia on brush biopsy negative specimens likely represents false positive histology results [29].
The high positive predictive value (PPV) of our study is in agreement with 5 other studies published [14, 15, 19, 28, 30] and contrasts with the results of a study by Bhoopathi et al. who reported a low PPV [31]. In their study, the authors claim that 3 "positive" brush biopsy specimens were negative on histology even though "positive" brush biopsy cases always display cellular features pathognomonic for dysplasia or carcinoma; therefore, it is doubtful that these cases represent actual false positive brush biopsy results.
The presence and number of basal cells in the brush biopsy specimen, as detected by both the computer and the examining pathologist, is the standard used by the laboratory to determine the adequacy of the sample. In other published clinical trials, incomplete brush biopsy results with an insufficient basal cell count have been reported between 2% and 7%. The incomplete rate in our study was 7% and within the reported range of other studies. Our rate of incompletes could be due to the fact that our study was conducted by residents-in-training. As their experience with the technique improved, the number of incomplete samples decreased.
The brush biopsy kits used in our study include both a proprietary brush and an analysis with neural network computer assistance of the specimens. Researchers, including the author's group, have attempted to use the brush biopsy instrument without computer assistance [32], the same brush in a liquid based preparation [33], a different brush with analysis of specimens using computer assistance [34], and a variety of so called instruments or "brush biopsies" that collect a complete epithelial sample. When subjected to studies, however, all of these proved to have an unacceptably poor sensitivity and specificity. As highlighted in many studies [14, 35, 36], the combination of a transepithelial brush specimen and neural network analysis of that specimen are necessary to ensure an accurate result.
The limitations of the current study include: (a) not being able to recall and re-biopsy patients with abnormal brush biopsies and negative scalpel biopsies to determine if these represented false negative scalpel biopsy results; and (b) "minimally suspicious" lesions which were included in the study are highly subjective, and what may appear to be suspicious to one observer may not be suspicious to others who examine the same lesion. Although we did not calibrate the examiners as to which lesions were minimally suspicious, the outcome of the study would not likely to have changed; (c) since this study was carried out in a location in India with a high prevalence of oral lesions, the results should be evaluated with care.