Evaluation of the Role of Toluidine Blue Paint as an Adjunctive Method to Biopsy in Suspicious Oral Lesion: A Hospital Based Study

Abstract

Early detection is a major step in the success of cancer therapy. Histopathology report is considered as the gold standard in the formulation of management protocol of any malignancy worldwide. But unfortunately, there is a delay in the detection of oral cancer very often due to inconclusive histopathology reports. The main reason behind it is obtaining a biopsy specimen from the non-representative area of the lesion. A hospital-based evaluation of the role of Toluidine Blue dye, used as an adjunctive method prior to biopsy was conducted in a tertiary care hospital on 200 patients presenting with oral lesions persistent for more than 3 weeks. The participants were divided into two equal groups by alternate sampling. In one group biopsy was taken by clinical judgment and in others, Toluidine Blue was used prior to obtaining a biopsy to decide the area to be biopsied. Data was collected using a predesigned proforma and was analyzed with the help of SPSS version 20. Results in two groups were compared with respect to sensitivity, specificity, positive and negative predictive values, false positive and false negative percentages. The Sensitivity, Specificity, Positive Predictive Value and Negative Predictive Value of wedge biopsy without staining were 73.68, 58.14, 70.00, and 62.50% respectively. These values were 95.08, 82.05, 89.23, and 91.43% respectively when Toluidine Blue staining was done as an adjunctive before the biopsy procedure. These results indicate the promising role of Toluidine blue staining before the biopsy to diagnose oral malignancy more efficiently than obtaining biopsy specimens on clinical assessment only and in avoiding the delay in initiating the treatment in case of oral malignant lesions.

Introduction

Cancer is the silent epidemic of modern civilization. Tobacco, beetle chewing, and alcohol consumption are the most important risk factors of developing oral pre-malignant as well as malignant lesions [1]. In India, squamous cell carcinoma of the oral cavity (OSCC) is the commonest and the third most common cancer in males and females, respectively [2] [3]. While 3,00,000 new patients are annually diagnosed with oral cancer worldwide, in India it constitutes about 30–40% of the overall cancer load [4]. Over 5 people die every hour, suffering from oral cancer in India [5]. Such huge morbidity and mortality affect the quality of life, financial and emotional aspect of not only the patient but of the entire nation too.

Globocan 2012 showed that, contrary to the survival rate of 27% in advanced OSCC, early-stage OSCC has an 82% survival rate [6]. But, in reality, approximately two-thirds of oral squamous cell carcinomas (OSCC) are diagnosed at advanced stages (stage 3 or 4),[7] leading to only a 40–50% 5 year survival rate in spite of advancement in treatment modalities.[8] Literature throughout the world has widely evaluated visual screening of oral cancer and its efficacy and cost-effectiveness in reducing oral cancer mortality [9] [10]. Though, spreading awareness in the form of primary prevention is important, but secondary prevention in the form of early diagnosis and treatment can also reduce the morbidity and mortality to a significant extent. In spite of that, in 2012, worldwide 145,000 deaths occurred of which 77% were in the less developed regions [11].

The reason for this high figure among underdeveloped countries may be a lack of vigorous screening in a high-risk group and lack of clinical knowledge and proper training amongst health workers. Oral pre-malignant lesions and malignant lesions, at times, are very innocuous in appearance. They can even be asymptomatic and impalpable and can present only in the form of a mucosal lesion; [12, 13] making them difficult to be differentiated from reactive and inflammatory lesions even with an expert clinical eye [14]. About 16% of the cases that appear to be leukoplakia clinically show malignancy on histopathology [15]. Conventional oral examination and biopsy from the edge of the suspicious lesion along with normal tissue followed by histopathological examination is the standard technique of diagnosis [1]. But the technical problem lies in confirming the representative area for taking a biopsy in cases of suspected lesions [14].

These challenges have led to further research and advent of various non-invasive adjunctive tools starting from painting the area with dye to identify the accurate area for biopsy, to use of advanced gadgets like Optical Coherence Tomography or Contact Endoscopy for early diagnosis of oral lesions [16] [17]. Toluidine Blue (dye) is one such tool, which is rapid in action, cheap, and easy to perform. The present study was contemplated with the objective of assessing the effectiveness of Toluidine Blue to increase the accuracy in the diagnosis of oral malignant and pre-malignant lesions as an adjunct to conventional oral examination and histopathological examination.

Materials and Methods

A hospital-based evaluation of diagnostic method was conducted on 200 patients presenting with oral lesions persistent for more than 3 weeks, in spite of removal, treatment of possible causative agents (sharp teeth, ill-fitting denture, infection, etc.) if any,[18] in Out-patients’ Department of a tertiary care hospital, from August 2017 to October 2019. All the 200 patients, irrespective of age, gender, socioeconomic and demographic characteristics, were allotted in Group A (100 patients), and Group B (100 patients) by alternate sampling method and informed written consent was taken prior to any intervention. The patients having any systemic disease with contraindication of oral biopsy, patients undergoing any orthodontic procedure that may interfere with the examination, and post-operated and post-radiotherapy patients with suspicion of recurrence or residual lesions were excluded from the study. Relevant history, epidemiological data, with special emphasis on age, duration of disease, and exposure to possible carcinogenic substances were recorded in a detailed structured questionnaire. The clinical examination was done by a single expert otorhinolaryngologist for proper assessment and to eliminate bias. On local examination by inspection and palpation, the location, size of the lesion, the extent of local infiltration, oral hygiene, and cervical lymph node status were assessed. Wedge biopsy and histopathological analysis under local anesthesia were done by a single surgeon in all cases, from the edge of the lesion without staining in Group A (conventional method) and after staining with Toluidine Blue in Group B (method under evaluation).

One% Toluidine Blue solution was prepared from 1 gm Toluidine Blue dye powder, 10 ml 1% Acetic Acid, 4.19 ml Absolute Alcohol, and 86 ml of Distilled Water [12]. Firstly, the mouth was rinsed with water to remove all the debris. After that 1% Acetic Acid rinse was given for 20 s to remove saliva, and the area was dried with gauze. Then the dye was applied over the oral lesion, with a cotton swab, very gently, for 1 min, covering approximately 2 cm margin all around the lesion. 1% Acetic Acid rinse was given thereafter for 1 min and the dye uptake pattern was observed under proper light and exposure [12]. None of the patients complained about any adverse reaction after the application of Toluidine Blue.

A stain was considered as positive for malignancy if the lesion diffusely or partly stained dark blue (royal or navy blue), or had stippled appearance (Fig. 1a). Benign lesions stain lightly and mostly limited to margins (Fig. 1b). A negative stain means no absorption of the dye. The areas which retained the stain but not considered positive were regular papillar crevices on the dorsum of the tongue, dental plaques, gingival margins around each tooth, diffuse stain of soft palate transferred from the retained stain on the dorsum of the tongue. Gentle swabbing with 1% Acetic Acid was done to remove excess stain. All equivocal lesions were considered positively stained unless proved otherwise.

Fig. 1.

Appearances of different lesions after staining with toluidine blue a suspected malignant lesion, b suspected benign

The biopsy was done from the area stained maximum with Toluidine Blue in Group B, whereas in Group A, biopsies were taken from the area with most clinical suspicion (by inspection and palpation) of the pre-malignant or malignant lesion (wedge including the margins of the lesions). Formalin-preserved tissues were sent for histopathological examination. The pathologists examining the biopsy specimens were not informed regarding the staining information of the samples. Repeat biopsy was done after an interval of 3 weeks whenever the histopathology report was inconclusive.

For the statistical analysis, we used histopathological reports as the gold standard to compare two groups, clinical suspicion (Group-A) and Toluidine Blue staining results (Group-B). The collected data was entered through the SPSS version 20 software. Sensitivity, Specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV), False-positive percentage, and False-negative percentages were calculated for both groups.

Results and Analysis

Among all 200 patients, 63 (31.5%) pre-malignant, 55 (27.5%) malignant and 82 (41%) benign cases were diagnosed in histopathology (Fig. 2a). Among the 82 benign lesions, 43.9% hyperkeratosis, 34.1% hyperparakeratosis, and 22% papillomatosis were diagnosed (Fig. 2b). The pre-malignant lesions were mild (19%), moderate (33.3%) and severe (30.2%) grades of dysplasia and carcinoma-in-situ (17.5%) (Fig. 2c). All the malignant lesions were invasive squamous cell carcinoma with various degrees of differentiation, i.e. well-differentiated (38.2%), moderately differentiated (34.5%), and poorly differentiated (27.3%).

Fig. 2.

Distribution of all the cases according to Histopathology

Wedge biopsies had been done in Group A without any prior staining procedure, depending upon the clinical suspicion. The histopathological outcome has been summarized in Fig. 3. If we compare the outcome of clinical suspicion with the findings of the histopathological analysis, among total 60 cases that were clinically suspected to be premalignant or malignant, 42 cases corroborated in histopathology, whereas it is 25 histopathology negative cases out of 40 cases with negative clinical suspicion initially. (Table 1) The Sensitivity, Specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of wedge biopsy without staining were 73.68, 58.14, 70.00, and 62.50% respectively (Table 1 and 3).

Fig. 3.

Outcome of all the patients in Group A

Table 1.

2X2 Table for Group A

Clinical Suspicion of pre-malignant or Malignant Lesion	Histopathology proved	Histopathology proved
	Yes	No
Yes	42(a)	18(b)
No	15(c)	25(d)

Table 3.

Comparative evaluation between groups

Parameters	Group A (without staining) (n1 = 100)	Group B (toludine blue staining) (n2 = 100)
True positive (a)	42	58
True negative (d)	25	32
False positive (b) [% = b/(b + d) × 100]	18 (41.86%)	7 (17.95%)
False negative (c) [% = c/(a + c) × 100]	15 (26.32%)	3 (4.92%)
Sensitivity [ a/(a + c) × 100]	73.68%	95.08%
Specificity [ d/(b + d) × 100]	58.14%	82.05%
Positive predictive value [a/(a + b) × 100]	70.00%	89.23%
Negative predictive value [d/(c + d) × 100]	62.50%	91.43%

In Group B, the 100 patients in whom oral lesions were stained prior to taking a biopsy, the sensitivity was 95.08% in detecting premalignant and malignant lesion (58 stain positives out of 61 histopathology positives) and 82.05% specificity in declaring lesions negative (32 stain negatives out of 39 histopathology negatives) (Fig. 4). Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were 89.23 and 91.43% respectively, for Toluidine Blue staining (Table 2 and 3).

Fig. 4.

Outcome of all the patients in Group B

Table 2.

2X2 Table for Group B

Toluidine Blue Staining	Histopathology proved	Histopathology proved
	Yes	No
Positive	58(a)	7(b)
Negative	3(c)	32(d)

Also, in comparison to wedge biopsy with only clinical suspicion, the staining with Toluidine Blue has significantly reduced the incidence of false positives (41.86% in Group A vs 17.95% in Group B). In Group B, there are only 3 false-negative cases due to the staining of inflammatory and traumatic lesions with Toluidine Blue. Comparison of Sensitivity, Specificity, Positive predictive value, and Negative Predictive values between the two groups is depicted in Table 3.

Discussion

Oral carcinogenesis is a multi-step procedure, starting from a focal clonal proliferation of altered stem cells near the basement membrane and moving upwards in form of various grades of dysplasia, then carcinoma-in-situ and finally, invasive carcinoma [19]. This progression is due to progressive accumulation of genetic alterations, which is in majority loss of heterozygosity (LOH), and these genotypic changes, in time, give rise to subsequent phenotypic, histologic, and clinical profile changes [13]. ‘Field cancerization’ is another concept that proposes the presence of genetic aberrations required for carcinogenesis throughout the upper aero-digestive tract of the high-risk populations [20]. that can lead to the second primary tumor, and this concept is important in oral malignancies, too.

The difference in 5 years survival rates of early stages (Stage 1 or 2) vs late stages (Stage 3 or 4), as stated earlier, clearly proves it. For early diagnosis of the malignant and premalignant conditions of the oral cavity, a timely tissue biopsy with the conclusive report is essential. But unfortunately, many of the histopathology reports remain either inconclusive or not consistent with clinical assessment, which requires repeat biopsy. The main cause of this is obtaining a biopsy sample from the non-representative area of the lesion. In the time period between the first presentation of the patient and obtaining a conclusive histopathology report, the disease may be upstaged. Here is the need for a procedure that can increase the possibility of obtaining a conclusive report in the first instant.

Vital staining is the process of dyeing living cells or tissues, which reveals unapparent cytological details [17]. Toluidine Blue was first used as a vital stain by Richart in 1963 for staining carcinoma in situ of the uterine cervix [21]. Its use in oral cancer diagnosis has been first described by Niebel and Chomet in 1964 [22].

Toluidine blue is a basic thiazine metachromatic dye with a high affinity for acidic tissue components, thereby staining tissues rich in DNA and RNA [23]. It preferentially stains acidic cellular components like DNA and RNA and can be retained in the intercellular spaces also [13]. As the rapidly dividing malignant as well as dysplastic cells contain more nucleic acid concentration, Toluidine Blue stains them more than the normal epithelial cells [17]. Also, malignant cells have leaky cell membranes and wider intracellular canals facilitating dye penetration and the haphazard arrangement of tumor cells causes more dye accumulation in intercellular spaces [24]. Loss of heterozygosity at 3p14, 9p21, 17p13, and presence of more than one site of loss was reported more frequently in toluidine blue positive samples and correlated with the risk of progression to invasive carcinoma [13]. Genotypic changes often proceed with clinical changes in oral carcinogenesis, therefore, Toluidine Blue, as a vital stain, highlights oral pre-malignant and malignant lesions even if they are missed out in a thorough clinical examination. That is why it is very effective in the early diagnosis of oral pre-malignant and malignant lesions. None of the existing literature reported any significant adverse reaction to the Toluidine Blue application in the detection of oral malignant lesions including the present study.

The present study has effectively found the sensitivity and specificity of Toluidine Blue use, 95.08, and 82.05% respectively, which corroborates with existing literature. According to Pallagatti et al. [8], [8] the sensitivity, specificity, PPV, and NPV of Toluidine Blue staining was 95, 71.45, 84.6, and 90.9%, respectively. Again, Allegra et al. conducted a similar study on toluidine blue and reported a sensitivity of 96.2% and a specificity of 77.7% [14].

A Toluidine stain positive case, therefore, can be considered as pre-malignant or malignant unless proven otherwise by histopathology. Also, a negative stain favors the diagnosis of a benign lesion. So, every lesion stained with Toluidine Blue should undergo wedge biopsy and histopathological examination for confirmation of diagnosis and initiation of appropriate treatment. Toluidine Blue also determines the appropriate area of biopsy and eliminates the possibilities of biopsy from the non-representative area. The reduced number of false positives with the use of Toluidine Blue dye in the present study (only 17.95%) denotes that, Toluidine Blue staining is more important in predicting the presence of oral pre-malignant and malignant lesions in clinically non-suspicious cases, thereby eliminating the tendency of under-diagnosis by clinical examination alone, which is of utmost importance.

Toluidine Blue stains only surface epithelium, 2–10 cell layers, [25] so, cannot tell about the depth of lesion and staging. So, it cannot be a conclusive diagnostic tool, the histopathological confirmation still holds the prime importance in staging and formulation of the management protocol. But it is a practical, rapid, inexpensive, non-invasive, easy to perform and effective tool [13], which can be used even at the primary health care level for the screening of any suspicious oral mucosal lesion. It is also a safe method in the dose it is used in the present study and others reported in the available literature [25]. Oral examination and Toluidine Blue screening can therefore also be recommended for every patient with high-risk factors for oral cancer at least once a year or more frequently according to the doctor’s consultation.

Toluidine Blue application also helps in localizing occult second primary tumor within the oral cavity and oropharynx, usually sharing the same field of molecular change with the primary tumor [26] as explained by the theory of field cancerization [20]. These second primary tumors are often inapparent and missed clinically and are of great concern regarding the establishment of the appropriate treatment protocol. The diagnosis of satellite lesions of oral cancers is also possible due to the genetic level mechanism of Toluidine Blue. Toluidine rinse is more effective rather than only cotton swab application in these cases [27].

Clinical detection of recurrent oral cancer is more difficult in previously treated cancer patients due to post-treatment mucosal changes resulting from radiation and surgery. Toluidine Blue can be a useful tool for diagnosis by colour change, specifically Toluidine Blue mouth rinse [12]. The use of toluidine blue is reported to be more sensitive than clinical examination alone in post-treatment high-risk patients and lesions with high-risk molecular features [28] [29]. So, follow-up of these post-treatment oral cancer patients routinely with Toluidine Blue staining will be a drastic step forward.

There are certain disadvantages of toluidine blue, false positive are one of them, due to marginal activity and dye uptake in traumatic and inflammatory lesions. It can be controlled by 2–3 weeks conservative approach in every lesion suspected to be benign. Toluidine Blue use can decrease biopsy from benign lesion by at least 50%, [30] if done in a proper way by eliminating false positives.

The staining pattern is till now a matter of debate and no universal guideline is available regarding when to consider it positive and when negative. There is also debate regarding the relationship between the increased intensity of stain and an increase in the severity of dysplasia [13]. There is no fixed staining method described till date, but mouth rinse is preferred in case of detection of second primary lesions [28]. A thick layer of keratin over any oral lesion prevents dye penetration, the same as saliva or discharge. That is why bare mucosal ulcers stain better than any ulcer covered with a scab, non-homogenous leukoplakia stains better than homogenous leukoplakia [17].

Limitations of the Study and Further Scope

The study was conducted in a hospital setting with the patients attended the otolaryngology outpatient department. Alternate sampling was done to allocate the patients into two groups, but as such no proper randomization method was adopted. Therefore the comparison of baseline characteristics of the two groups was not relevant, which decreased the strength of the study. There is a scope for a better designed randomized control trial (RCT) to better establish the role of Toluidine Blue as an adjunctive tool to obtain more conclusive histopathology reports. Population-based studies can also be planned to evaluate the role of it as a screening tool for early detection of oral cancer.

Conclusion

The multistep oral carcinogenesis is the reason for varied clinical presentations of oral pre-malignant and malignant lesions, which can often be missed or creates confusion during a clinical examination. The burden of morbidity and mortality associated with oral pre-malignant and malignant lesions can effectively be reduced by early diagnosis and appropriate treatment. Toluidine blue, as a vital stain, can aid in the diagnosis of oral premalignant and malignant lesions in adjunct with clinical examination and even without visible clinical changes by working at the genetic level. Although histopathology examination still has its role in the confirmation of diagnosis, the staging of oral cancers, and formulation of the treatment protocol, the role of toluidine blue is of special importance in increasing the possibility to obtain conclusive histopathology report by guiding the surgeon to obtain biopsy specimen from representative area. It can also be helpful in the determination of second primary and satellite lesions and recurrence in post-treatment cases. The excellent sensitivity and specificity of using this stain points towards considering it solely as a universal screening tool in diagnosing suspicious oral lesions.

Compliance with Ethical Standards

Conflict of interest

There is no conflict of interest among the authors. Necessary permission was obtained from the institutional ethics committee.

Informed Consent

Informed and written consent was taken from every study participant.