|Year : 2014 | Volume
| Issue : 2 | Page : 232-238
Clinical correlative study on early detection of oral cancer and precancerous lesions by modified oral brush biopsy and cytology followed by histopathology
Swati Gupta1, Jigna S Shah2, Shilpa Parikh2, Piyush Limbdiwala2, Sumit Goel1
1 Department of Oral Medicine and Radiology, Subharti Dental College, Subhartipuram, Meerut, India
2 Department of Oral Medicine and Radiology, Government Dental College and Hospital, Ahmedabad, Gujarat, India
|Date of Web Publication||14-Jul-2014|
B-202, Nandan Apartments, 31 A, Saket, Meerut - 250 002
Source of Support: None, Conflict of Interest: None
Background: Oral cancer accounts for 5% of all malignant tumors and 60% of these lesions are well advanced at the time of diagnosis. The early diagnosis could prevent a large number of deaths due to this disease.
Aims: This study aims to evaluate and compare the clinical usefulness of exfoliative cytology, modified brush biopsy (without computer assisted analysis), and biopsy in early detection of oral premalignant and malignant lesions.
Materials and Methods: Modified oral brush biopsy (using baby toothbrush) and exfoliative cytology was performed on 225 clinically diagnosed oral precancerous lesions (suspected) selected from among 1099 lesions in 877 patients. Scalpel or punch biopsy was performed based on clinic-cytological evidence (test result and/or clinical judgment) on only 117 lesions. All the specimens were analyzed manually in a double-blinded fashion.
Statistical Analysis Used: Results were analyzed using Statistical package for Social Sciences version 12 (SPSS Inc., Chicago, IL, USA) and subjected to Fischer exact test.
Results: Of 117 lesions that underwent all the three tests, modified oral brush biopsy showed a reasonably higher specificity (68.42%) and sensitivity (81.69%) when compared with specificity (86.48%) and sensitivity (48.57%) of cytology.
Conclusion: The results demonstrate that modified oral brush biopsy has higher efficacy than routine cytology and can be used as a potentially practical oral cancer screening tool in resource challenged settings. However, clinical judgment is of prime importance. Immediate biopsy is mandatory in highly suspicious lesions proposed under the diagnostic criteria of "clinically diagnosed carcinoma in situ".
Keywords: Cytology, early detection, oral brush biopsy, oral cancer, oral precancer
|How to cite this article:|
Gupta S, Shah JS, Parikh S, Limbdiwala P, Goel S. Clinical correlative study on early detection of oral cancer and precancerous lesions by modified oral brush biopsy and cytology followed by histopathology. J Can Res Ther 2014;10:232-8
|How to cite this URL:|
Gupta S, Shah JS, Parikh S, Limbdiwala P, Goel S. Clinical correlative study on early detection of oral cancer and precancerous lesions by modified oral brush biopsy and cytology followed by histopathology. J Can Res Ther [serial online] 2014 [cited 2017 Dec 13];10:232-8. Available from: http://www.cancerjournal.net/text.asp?2014/10/2/232/136539
| > Introduction|| |
Oral cancer is the most common cancer and constitutes a major health problem in developing countries, representing the leading cause of death. Although representing 2-4% of the malignancies in the West, squamous cell carcinoma accounts for almost 40% of all cancers in the Indian subcontinent.  In India, the age standardized incidence rate of oral cancer is 12.6/100,000 population.  Despite numerous advances in the treatment, the 5-year survival has remained approximately 50% for the last 50 years. Factors mitigating against early detection include lack of self-examination, patient fears, asymptomatic early lesions, and misdiagnosis. ,
Techniques that have been used to improve earlier detection and diagnosis of oral malignancy include exfoliative cytology, vital tissue staining (toluidine blue), visualization adjuncts (ViziLite Plus with TBlue, ViziLite, Microlux DL, Orascoptic DK, VELscope), and OralCDx brush biopsy in addition to histological examination of tissue. ,
Exfoliative cytology is the microscopic study of cell samples collected from the mucosal surface. 
Oral brush biopsy utilizes a brush to obtain a complete transepithelial biopsy specimen with cellular representation from each of the three layers of the lesion: The basal, intermediate, and superficial layers, unlike cytology instruments, which collect only exfoliated superficial cells. The patented brush biopsy tool, together with analysis of oral brush biopsies assisted with sophisticated computers make the OralCDx test (Suffern, New York) highly accurate. It became commercially available in 1999. Modified oral brush biopsy is a noncomputerized assisted analysis of brush biopsy sample obtained by a toothbrush.  This may have application in resource challenged areas and could be a risk free method of evaluating oral lesions. However, the gold standard diagnostic test for oral mucosal lesions that are suggestive of premalignancy or malignancy remains tissue biopsy and histopathological examination. 
In this study, our purpose is to compare the clinical usefulness and efficacy of exfoliative cytology, modified oral brush biopsy, and biopsy as diagnostic tools in early detection of oral cancer and precancerous lesions, while utilizing clinical knowledge.
| > Materials and methods|| |
This study was conducted among 202 patients having 225 clinically diagnosed oral precancerous lesions (suspected) selected from patients reporting with precancerous lesions (1099 lesions in 877 patients). The Institutional Ethical Committee approved the study. The patients were selected randomly irrespective of their age, sex, and site. The inclusion and exclusion criteria for patients enrolled in the study are summarized in [Table 1] and [Table 2]. Written informed consent was obtained from all patients enrolled in the study.
|Table 1: Inclusion and exclusion criteria for patients enrolled in the study|
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Clinical data collection
inclusive patients were interviewed for a thorough medical and dental history. Detailed history was taken and recorded. General examination was done to rule out any systemic diseases and vitals of the patient were recorded. Clinical examination included extraoral and intraoral examination. All data was recorded on a specially prepared proforma. Necessary investigations were conducted such as radiographic investigations and laboratory investigations. Specific investigations were done for every patient included in the study-exfoliative cytology, modified brush biopsy followed by punch/incisional biopsy based on clinical cytologic evidence.
Cytosmear was taken by scrapping the lesion using tongue blade. The material scrapped was spread on a clean dry glass slide and fixed using fixative 100% ethanol. The slide was then sent for examination to a qualified pathologist. These were first stained with modified papanicolaou's method and then examined under the microscope for dysplastic changes in the cells.
Modified oral brush biopsy procedure
A commercially available nylon baby toothbrush (Johnson and Johnson) was to be used to obtain a complete transepithelial biopsy with minimal discomfort. Using moderate pressure, the brush was repeatedly brushed in one direction over the entire lesion many times until pin point bleeding was obtained, signaling entry into lamina propria and thus obtaining epithelial cells through the full-thickness of the epithelium. The material from the brush was spread on the middle-third of clean dry glass slide. The smears were then fixed immediately with 100% ethanol for staining with the modified papanicolaou's method. The smears were sent for evaluation to a qualified pathologist.
As the cellularity from this type of brush biopsy was scant, only slides with at least 30 well preserved cells (not obscured by blood, exudates, or necrotic material) from deep epithelial cells were considered adequate.  Inadequate specimen was excluded from the data.
Scrapings for cytologic examination were taken from each abnormality, and biopsies were performed whenever warranted by clinical cytologic evidence. Biopsy was performed in all those patients who showed positive results in exfoliative and/or modified oral brush biopsy, and in those cases where in spite of negative results of above two procedures, the clinical judgment warranted the need for biopsy. A total of 117 lesions underwent biopsy.
After site selection, local anesthetic (2% lignocaine hydrochloride) was given by submucosal technique. Punch biopsy was performed using punch (number 5). In some cases, incisional biopsy was performed. The specimen collected was preserved in formalin. For histological diagnosis (after routine processing and paraffin embedding), several sections (3-4 μm) were cut from each specimen collected and stained with hematoxylin and eosin. All specimens were examined manually, independently by two different pathologists in a double-blind fashion. If there were any discrepancies, a third opinion was obtained to reach a final diagnosis.
Evaluation and interpretation of the specific laboratory investigations
Parameters that were analyzed in the smear (both cytosmear and modified brush biopsy samples) included enlarged nuclei, nuclear pleomorphism, nuclear borders, nucleocytoplasmic (N/C) ratio, number of nuclei, binucleation, keratinization, tadpole forms, hyperchromatism, chromatin pattern, and distribution and discrepancy in N/C maturation. The most important diagnostic feature of cancer cells are anisonucleosis, anisocytosis, abnormal nuclear texture and nuclear hyperchromasia and abnormal N/C ratio.
Based on above specified parameters cytologic specimens were classified as follows: ,,
- Class 0: Inadequate specimen
- Class 1: No/abnormal or atypical cells
- Class 2: Atypical cytology but no evidence of malignancy
- Class 3: Cytology suggestive of but not conclusive for, malignancy
- Class 4: Cytology strongly suggestive of malignancy
- Class 5: Cytology conclusive for malignancy.
For analysis, classes 3-5 were to be considered positive and classes 1 and 2 as negative [Figure 1].
|Figure 1: (a-e) Representative photomicrographs of cytology classes 1-5 respectively from amongst the study specimen (×400)|
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Modified brush biopsy specimen analysis
Based on above specified parameters brush biopsy specimens were classified as follows: 
- Type 0: Inadequate specimen
- Type 1: Benign: Those with no specific changes in the above parameters were to be labeled as benign
- Type 2: Dysplastic: Those exhibiting borderline changes were considered dysplastic
- Type 3: Malignant: Cells showing clear cut evidence of these changes as evidenced by necrotic and dysplastic changes.
For analysis, dysplastic, and malignant lesions (types 2 and 3) were to be considered positive and benign lesion (type 1) as negative [Figure 2].
|Figure 2: (a-c) Representative photomicrographs of modified oral brush biopsy types 1-3 respectively from among the study specimens (×400)|
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Biopsy specimen analysis
Specimen was analyzed for gross changes in the epithelium, connective tissue and for dysplastic features in the epithelium. Based on the degree of dysplasia or architectural loss, these biopsy specimens were classified as negative, mild dysplasia, mod dysplasia, severe dysplasia, well differentiated squamous cell carcinoma(scc),moderately differentiated squamous cell carcinoma, and poorly differentiated squamous cell carcinoma.
Specific therapy was instituted depending on the clinical diagnosis and regular follow was done. Complete excision was advised in all those lesions that were histopathologically diagnosed as carcinoma, or in which dysplastic changes were noted.
Statistical analysis used
All the results were analyzed using Statistical Package for Social Sciences (SPSS) Version 12.0 (SPSS, Chicago, IL, USA) and was subjected to Fischer exact test. Following formulas were used to analyze the efficacy of cytology and brush biopsy with biopsy as a gold standard:
- True positive (TP): Samples that were positive on both histology and brush biopsy or cytology
- True negative (TN): Samples that were negative on both histology and brush biopsy or cytology
- False positive (FP): Samples those were negative on histology and positive on brush biopsy or cytology
- False negative (FN): Samples that was positive on histology and negative on brush biopsy or cytology
- Sensitivity = TP/TP + FN
- Specificity = TN/TN + FP.
| > Results|| |
This study was conducted in 202 patients, having 225 lesions. All the lesions underwent conventional cytology and oral brush biopsy. Biopsy was not performed in every case. One hundred and seventeen lesions with abnormal cytology or brush biopsy were examined histopathologically.
During the study period, a total of 1099 lesions in 877 patients were recorded, out of which 225 lesions were included in the study. [Table 3] shows the frequency distribution of the sample (225 lesions) among the different precancerous lesions recorded (1099 lesions) during the study period. Important demographic information analyzed from the sample population is summarized in [Table 4].
|Table 3: Frequency distribution of all precancerous lesions recorded and/or included in the study (in 877 patients)|
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Only 117 lesions underwent histopathological diagnoses-43 (36.75%) were negative for malignancy, 14 were dysplastic, and 60 were diagnosed as squamous cell carcinoma. Among the 43 lesions tested negative for malignancy - mostly 18 (15.38%) were in association with submucous fibrosis, 10 were of speckled leukoplakia, 7 of carcinoma in situ, and 4 each of tobacco pouch lesion and verrucous leukoplakia. Of the 14 dysplastic lesions, 8 were clinically diagnosed carcinoma in situ (5 - mild, 1 - moderate, and 2 - severe), 4 of these were submucous fibrosis, and 1 each of tobacco pouch lesion and speckled leukoplakia, respectively. Sixty lesions were diagnosed as squamous cell carcinoma - 40 (34.19%) as moderately differentiated and 20 (17.09%) as well differentiated. Most of these were clinically diagnosed carcinoma in situ (32 [27.35%] among moderately differentiated, and 17 [14.53%] among well differentiated).
[Table 5] and [Table 6] respectively summarize the individual results of cytologic and brush biopsy analysis, which was done for all 225 lesions. Thirteen cytological smears and 12 brush biopsy specimens were unsatisfactory for evaluation. In our study, classes 3, 4, and 5 of cytology were considered as a positive test result. A total of 41 (18.22%) lesions tested positive on cytological examination, most of which were classed as 4 and 5, and were mostly (32 out of 80 cases) clinically diagnosed carcinoma in situ. Most of the lesions grouped as class 1 (negative) (161 lesions) belonged to submucous fibrosis (61 [27.1%]), speckled leukoplakia (52 [23.1%]), and clinically suspected carcinoma in situ (38 [16.9%]) [Table 5].
|Table 5: Correlating cytology class with the various clinically suspected lesions|
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|Table 6: Correlating brush biopsy type with the various clinically suspected lesions|
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In brush biopsy analysis [Table 6], types 2 and 3 were considered as a positive test result. A total of 74 (32.9%) lesions tested positive on brush biopsy evaluation. Most of the lesions that were grouped as type 3 (42 [18.7%]) were clinically diagnosed carcinoma in situ (34 [15.1%]). Most of the lesions grouped as type 1 (negative) (139 lesions) belonged to submucous fibrosis (54 [24%]), and speckled leukoplakia (50 [22.2%]).
[Table 7] shows TN, TP, FN, and FP for cytological examination and brush biopsy as compared with the gold standard histopathologic evaluation. Sensitivity based on the above values for cytology is 48.57% and specificity recorded as 86.48%. Sensitivity based on the above values for brush biopsy is 81.69% and specificity recorded as 68.42%.
| > Discussion|| |
Early detection of a premalignant or cancerous oral lesion promises to improve the survival and the morbidity of patients suffering from these conditions. Cytological study of oral cells and oral brush biopsy are nonaggressive techniques that are well-accepted by the patient, and have broad potential to fill the diagnostic gap that currently challenges the early detection of oral cancer and precancer including epithelial atypia and squamous cell carcinoma. 
In this study, out of 202 patients, most of the patients fall in 31-45 years of age group and next common age group was 46-60 years with strong male predominance, which was noted in all premalignant lesions. According to the literature, oral malignancies occur in about twice as many as men, and 95% are found in persons older than 40 years of age. , The most common premalignant lesions and condition found was oral submucous fibrosis (OSMF) followed by tobacco pouch lesion, which is seen mostly in association with submucous fibrosis as a result of placement and chewing of smokeless form of tobacco. There is high reported use of smokeless form of tobacco in Indian subcontinent. , Out of 225 lesions included in the present study, the most common site involved was buccal mucosa, followed by tongue and buccal vestibule as these are the most common site of occurrence of precancerous lesions in general. In contrast, the tongue and floor of mouth are the more commonly involved sites in the West. , These regional differences may be attributed to the extensive use of chewing tobacco in the Indian subcontinent compared to smoking in the West.  Our results were consistent with the study by Mehrotra et al. 
Our study strengthens the view that clinical examination plays a very important role in early detection and treatment of these premalignant and malignant lesions rather than relying solely on any diagnostic aids. 
It was observed that out of 72 clinically suspected lesions (traumatic ulcer/growth) in association with submucous fibrosis, only seven were diagnosed histopathologically as squamous cell carcinoma. All these seven cases had the following common characteristics - chronic ulcer with indurated borders and bleeding tendency associated with moderate to severe OSMF (≤20 mm mouth opening). The prognosis of submucous fibrosis is thus dependent on the severity.  Most of them were negative on cytological examination and brush biopsy. This could be attributed to the fact that most of the lesions in association with submucous fibrosis were due to chronic trauma from partially/buccally erupted third molar or sharp cusps. Mild dysplasia was reported in four of them - one of which turned out to be well differentiated squamous cell carcinoma. Rest three of them showed good clinical response once traumatic factor was removed.
In speckled leukoplakia and verrucous leukoplakia, most of the lesions tested were negative for cytology and brush biopsy. This could be attributed to the thickened surface keratin interferes with access to the cells of the lower epithelial layers.  The high malignant transformation rate for verrucous leukoplakia makes biopsy mandatory and avoidance of unnecessary delays. 
In tobacco pouch lesion, cytology and brush biopsy results were negative in 5 out of 7 cases, only in two cases both cytology and brush biopsy gave positive test result; both of these cases presented clinically as thick white nodular plaque like lesion surrounded by erythematous area and turned out to be squamous papilloma histopathologically which is a benign lesion. In general, these lesions have a favorable prognosis. 
Malignant transformation of oral lichen planus has been reported in the literature as has been witnessed in our study also.  Positive test result of brush biopsy was seen in this case diagnosed histopathologically as squamous cell carcinoma moderately differentiated while cytological findings were negative.
In this study, many lesions have been clinically characterized as carcinoma in situ based on a set of clinical criteria. At the end of this study, this criterion was reevaluated. It has been observed that these lesions rarely occur alone without the presence of any other precancerous lesion or condition like submucous fibrosis in 56 patients and leukoplakia in 7. This was in accordance with the prevalence of precancerous lesions in southeast Asia. 
In those patients with dysplastic changes (eight lesions) reported on histopathologic examination, three lesions were verrucous hyperplasia with mild dysplasia. Most of these lesions were associated with proliferative growth pattern and with minute papillary projections, and others were mixed red and white lesion. Brush biopsy was positive for malignancy in 5 of the 6 cases that were satisfactory on evaluation. Cytology was positive for malignancy in only two of these cases. Thus, brush biopsy has an added advantage over cytology in these cases. Those that were histologically diagnosed as squamous cell carcinoma well differentiated (17) - Eight of these lesions tested negative for cytology, while brush biopsy results could be relied upon as only one lesion was negative for brush biopsy. Thirty-two histopathologically diagnosed moderately differentiated squamous cell carcinoma - 21 lesions gave positive cytology results as compared to 26 positive brush biopsy results. Brush biopsy is slightly better than cytology as a diagnostic aid in these lesions. However, the clinical examination plays an important role.
Technically, in the present study, a total of 13 (5.8%) and 12 (5.3%) of the 225 lesions were unsatisfactory on evaluation by cytology and brush biopsy, which was comparable to other studies. This was due to drying artifacts resulting from the slightest delay in fixing and in a few cases due to obscuring by blood. ,
Our study results [Table 7] were consistent with reviewed literature on brush biopsy and cytology which suggests a high rate of FNs on cytology as compared with brush biopsy; and higher chances of FP on brush biopsy.  Reasons for the occurrence of FNs on cytology or brush biopsy evaluation included inadequate sampling, technical error, misinterpretation of the findings and bias, topographic error between the site of brush and scalpel biopsy, time delay between both. , The poor results are due in part to the fact that cytology instruments do not obtain a sample from the deepest layers of the oral lesions while oral brush biopsy samples transepithelial specimen. , The FP results of the oral brush biopsy (as seen in OralCDx) are possible in other oral lesions with a certain grade of epithelial atypia, for example, inflammatory conditions. If the brush biopsies would be used regardless of the clinical aspect of the lesions, the rate of FP results should markedly increase. Other reason for such FP result could be improper fixation or air drying. Hence, proper technique is a requisite to obtain good results. ,,
Correlation between the results of brush biopsy and cytology, showed that 134 lesions were negative both on brush biopsy and cytological examination both; 26 lesions that were negative on cytological examination were type 2 (17 lesions) and type 3 (9 lesions) on brush biopsy evaluation. This brush biopsy technique could possibly be a check on FN cytological examination.
Results of our study also showed a positive correlation among cytology classes 4 and 5 (strongly suggestive and conclusive for malignancy) and type 3 (positive for malignancy) of modified oral brush biopsy, except one which fell in the dysplastic category (type 2) of modified oral brush biopsy. Most of these lesions were found to be squamous cell carcinoma, with three of them dysplastic, histopathologically. Only three FP cases were also reported among these. More discrepancy was seen in the results of cytology and modified oral brush biopsy among the cytology classes 1, 2, and 3 when compared to types 1 and 2 of modified oral brush biopsy. This discrepancy in the results was due to deeper penetration of brush in the lesion epithelium, so that some of the basal cells could also be examined where dysplastic changes are first evident, whereas cytological assessment is based on only superficial exfoliated cells, which are evident only in cases of obvious malignancy.
Reviewed literature suggests that sensitivity of cytological diagnosis in a meta-analysis of 1306 cases from 14 studies showed an average of only 87.4% (ranging from 73.8% to 100%), , and that of OralCDx has a sensitivity of >95% and specificity over 90%, , while the statistical sensitivity of the modified oral brush biopsy using baby toothbrush was >76.8% and statistical specificity was >93.3%.  The sensitivity in our study was comparatively less 81.69% due to lack of use of a specially designed brush, and the specimens analyzed without computer assistance. The specificity in our study is 68.42%, which is far lower than the 93% reported in the US collaborative first detailed study of the brush biopsy technique.  However, in interpreting the specificity it is important to appreciate that, in the present study, only a relatively small number of lesions reported as negative by modified brush biopsy, subsequently underwent incisional biopsy, and those that did were either with atypical result for cytology and/or brush biopsy, or clinically suspicious lesions.
The oral brush biopsy has been criticized for adding time and cost to the diagnosis of oral lesions without additional benefit to the patient. Because the brush biopsy detects only cellular atypia, positive oral brush biopsy results must be confirmed with a scalpel biopsy for definitive diagnosis. The need to perform two procedures may significantly delay diagnosis.  The cost has been brought down in our study as compared to that in case of computerized assisted oral brush biopsy by using a regular baby toothbrush for easy accessibility (even in case of those with restricted mouth opening as is seen in submucous fibrosis) instead of specially designed brush biopsy kit; along with saving the cost of using computerized neural networks which has limited centers by a thorough evaluation by an experienced pathologist.
| > Conclusion|| |
Exfoliative cytology and modified brush biopsy are noninvasive, painless outpatient-based procedure. These are advisable in routine treatment planning and follow-up of patients with oral precancerous lesions. For confirmation of the diagnosis, biopsy is must. Modified oral brush biopsy shows a reasonable specificity (68.42%) and sensitivity (81.69%) that makes it a potentially practical tool in resource challenged settings. However, clinical judgment of the dentist is of prime importance not only to reduce the number of FP results in brush biopsy, but also to identify all highly suspicious lesions following the diagnostic criteria of clinically diagnosed carcinoma in situ that require immediate biopsy. Modified brush biopsy can be used as a screening tool in all clinically innocuous lesions with higher efficacy than cytology.
| > Acknowledgments|| |
We sincerely thank our pathologists Dr. Sagrika Tripathi and Dr. Jigar Suthar for their help and support in analyzing the specimens. We also extend our thanks to our statisticians G.C. Patel and Roopesh Tiwari.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]