A Study of Toluidine Blue Staining in Suspected Oral Malignancies in Patients Presenting to Tertiary Care Hospital in Central India

Abstract

Oral malignancies are a major cause of morbidity and mortality in worldwide and especially in Indian subcontinent. India stands at first position in total number of oral cancers in worldwide. Hence it is the need of hour to have safe and reliable screening test for early diagnosis of oral malignancies. Toluidine blue (TB) staining is an ideal screening test with high sensitivity and specificity. This study carried out in our department with objective to study efficacy of TB in oral malignancies and potential oral malignant lesions and also to evaluate and compare TB staining with histopathological examination of lesion. This prospective study conducted on 183 patients who attended ENT department. Out of 183 patients 146 (79.8%) were positive with TB and 37 (20.2%) were not stained with TB. Out of 146 positive patients 134 were turned out to be positive for malignancy in histopathological examination. Out of 37 negative patients 5 were positive for malignancy in histopathological examination and 32 were negative. Out of 183 patients 139 (76%) were positive whereas 44 (24%) were found negative for malignancy. In present study clinical examination of oral cavity reveal most of the patients had leukoplakis (37.7%) followed by swelling (26.8%), ulcer (26.2%). Positive and negative predictive value of TB was 91.8% and 86.5% respectively whereas positive and negative predictive value of histopathology was 96.4% and 72.7% respectively. TB stain is cheap, quick and easily available readymade kit and no side effects and so have excellent patient compliance so it can be best utilized as a premier tool in initial detection of suspected oral malignancy.

Introduction

India stands at first position in number and incidence of the total number of oral cancers worldwide where it is responsible for more than 70% shares of oral cancers and in India oral malignancies are 1/3rd of all malignancies [1]. There are a number of factors responsible for the high occurrence, as to Lung cancer in males and breast cancer in female is the commonest all over the world [2].

A white patch or a simple non healing ulcer could be a first sign of an oral malignancy. A red lesion represents a little more severe threat. Local tobacco abuse habits have a large role in site of occurance of oral premalignant lesion, in Indian scenario is it buccal mucosa in at least half of the cases. Rural population is largely forgotten in our public health system and when symptoms are negligible, they often delays seeking a quality medical care and similar symptoms of premalignant or malignant lesions with benign lesion aggravates the helpless scenario [3]. Hence it is high time we found a safe and reliable way to differentiate between them. There are different diagnostic tools and dyes available for initial detection of suspected oral malignancies and a few of them are available in ready to use kit form also [4, 5]. They can be utilized as a screening test. An ideal screening test will have very high sensitivity and minimal false positive and negative predictive values [6].

Toluidine blue (TB) is an organic compound chemically named as Tolonium chloride. Metachromasia is the property of tissues, in which they stain with a dye and change its color compared to living tissue, where it will be easy to differentiate. Toluidine blue is a basic dye which belongs to Thiazine group and when in solution form becomes blue-violet in colour. It has acidophilic properties.

The silver line between the clouds is that, if we found Oral lesions in first stage, the 5 year survival rate is appreciably good, reaching up to 80% in good centres [7]. In fact there is no universally accepted consensus for the correlation of a clinical examination finding and the success rate of picking a pre malignant or malignant lesions.

Whenever a cancerous growth is present, it need to have manifold increase in synthesis of DNA and RNA at local level producing growth factors and thus necessitating a high blood flow and higher number of new capillaries near it [8]. DNA and RNA being Nucleic acids possess acidic nature and with toluidine blue it makes a covalent bond, which is the basis of metachromasia. The loose binding of tissues and dishonouring of boundaries by cancerous growth add to the penetration of toluidine blue into the local environment and enhance the detection of a stained tissue. Many authors have studied the efficacy of TB in detection of suspected cases, but in last 3 decades there is lot of disparities and discrepancies about the data [9]. Thus our study becomes the need of the hour and substantiate our effort.

Objectives of Study

1. The main objective of this study was to determine whether TB application would aid in the diagnosis of oral malignancies and suspected premalignant lesions.

2. To study the efficacy of 1% TB in the identification of oral malignancies and potentially malignant oral lesions and also to correlate between clinical examination and TB staining in oral cancers 3. Compare, clinical examination, TB staining and histopathological examination (HPE) of lesion (Fig. 1).

Fig. 1.

a Showing suspected symptomatic lesion, but not visible in clinical examination, i.e. naked eyes. b Lesion gets stained with Toluidine blue dye and representative area was biopisied

Materials and Methods

Study Area

The present study was carried out in the Department of Otorhinolaryngology in centrally located teaching hospital.

Study Design

Hospital based prospective study.

Inclusion Criteria for Study Participants

Individual of all age group and gender presenting with signs or symptoms suggestive of suspected oral malignancies coming to the department of ENT, in our institute.

Exclusion Criteria

1. Those who have biopsy proven diagnosis of oral malignancies.

2. Those who are having suspected allergy to dyes and such chemicals.

3. Those patients already on treatment—for oral malignancies.

Study Period

The total duration of study was about 18 months from 01 February, 2017 to 15th July, 2018.

Sampling

Sample Size All the patients coming to out-patients department and admitted in the inpatient wards of the department of Otorhinolaryngology in our institute.

Sampling Technique/Procedure All patients coming to department of Otorhinolaryngology were attended by physicians and those found to have symptoms suggestive of suspected oral malignancies were further investigated.

Diagnosis Definitive diagnosis of Oral malignancy involved demonstration of lesion with respect to histopathological methods using recommended procedure.

All the patients assessed on the basis of socio demographic profile, behavioral habits, clinical and physical examination, TB staining of lesion, histopathological examination.

Technique of Staining

TB can be used in two forms. It is either applied to the site of the lesion with a cotton applicator or it is used as mouth rinse. The procedure of staining is as follows.

Oral Examination

• Rinsing the mouth twice with water for 20 s to remove the debris.

• Application of 1% acetic acid for 20 s to remove any ropey saliva.

• Application of 1% toluidine blue solution for 20 s either with cotton swab when a mucosal lesion is seen or given as a rinse when no obvious lesion is detected.

• Application of 1% acetic acid to reduce the extent of mechanically retained stain.

• Rinsing oral cavity with water.

Lesions shown DARK BLUE areas are considered test positive for premalignant an/malignant lesion.

Data Collection/Interview

Before starting interview all participants were explained the purpose and objective of study in local language. There after study participants were asked for the written informed consent. Facts related to our interview questions were cross checked with available medical record, bills and other document if available with the study participants.

Data Analysis

Before data entry responses were coded and there after data entry was done using Microsoft Excel version 2013. At a later stage all the data was transferred to SPSS version 22.0 for statistical analysis. Descriptive summary using frequencies, proportions, graphs and cross tabs were used to display study results. Probability (p) was calculated to test for statistical significance at 5% level of significance (p value < 0.05). Association between various factors was using appropriate statistical tests.

Observations and Results

Present study titled “A study of toluidine blue staining in suspected oral malignancies” was performed on 183 Subjects at Department of Otorhinolaryngology and head and neck surgery in centrally located teaching hospital during February 2017 to July 2018 after fully explaining them about the procedure in their own language and obtaining consent and the following observations were made;

Mean age of study cohort is 51.03 ± 14.01 years. Maximum patients belong to age group of > 60 years [52 (28.4%)] followed by 51–60 years [44 (24%)] and 41–50 years [38 (20.8%)]. In present study, maximum patients were male [119 (65%)] followed by female [64 (35%)]. In present study, out of 183 patients, 143 (78.1%) were found positive for tobacco addiction, 46 (25.1%) were found positive for alcohol addiction, 37 (20.21%) were addicted to both tobacco and alcohol. Analysis of site of lesion distribution revealed that maximum patients had lesion at buccal mucosa [109 (59.6%)] followed by tongue lateral [42 (23%)] and soft palate [11 (6%)] (Table 1). In present study local examination revealed that most of the patients had leukoplakia [69 (37.7%)] followed by lump [49 (26.8%)] and ulcer [48 (26.2%)] (Table 2). In present study TB stain results showed that, out of 183 patients’ 146 (79.8%) patients were found to be positive for TB staining (Table 2). In histopathology, out of 183 patients, 139 (76%) were found positive whereas 44 (24%) were found negative. In present study, we compared the age group with TB stain results, which revealed that staining results were equally distributed among different age groups (p = 0.199). Out of total 146 TB staining positive patients histopathology has shown 134 (91.78) positive for malignancy. Out of total 37 TB staining negative results, histopathology has shown 5 (13.51%) positive results. In present study, Positive and negative predictive value of toluidine blue staining was 91.8% and 86.5% respectively. Whereas Positive and negative predictive value of histopathology was 96.4% and 72.7% respectively.

Table 1.

Site of lesion distribution

Site of lesion	No. of patients	Percentage
Buccal mucosa	109	59.6
Gingivo buccal sulcus	3	1.6
Hard palate	4	2.2
Lips	4	2.2
Retromolar trigone	10	5.5
Soft palate	11	6.0
Tongue lateral	42	23.0
Total	183	100.0

Table 2.

Toluidine blue stain results

Toluidine blue stain	No of patients	Percentage
Negative	37	20.2
Positive	146	79.8
Total	183	100.0

Comparing Histopathology with toluidine blue staining revealed that out of 139 patients in which histopathology was positive, toluidine blue staining was able to detect 134 (96.40%) positive cases where as out of total 44 negative cases reported in histopathology, toluidine blue staining detected 12 (27.27%) positive cases (Table 3).

Table 3.

Comparing histopathology with toluidine blue staining results

Toluidine blue staining	Results	Histopathology		Total	P value
		Positive	Negative
Positive	Count	134	12	146	< 0.001
	% Within toluidine blue stain	91.8	8.2	100.0
	% Within histopathology	96.4	27.3	79.8
Negative	Count	5	32	37
	% Within toluidine blue stain	13.5	86.5	100.0
	% Within histopathology	3.6	72.7	20.2
Total	Count	139	44	183
	% Within toluidine blue stain	76.0	24.0	100.0
	% Within histopathology	100.0	100.0	100.0

Discussion

In India, among the top 5 cancers, oral cancer has a third place, which is common problem in entire Indian subcontinent itself [10] There are many schemed etiologies for oral cancer, of which tobacco abuse is reason numero uno. A basic metachromatic dye, TB is utilized and it stains the nuclear matter of malignant lesions, but not that of a normal mucosa. The nuclei of cancer cells show increased rate of synthesis of DNA, resulting in heightened level of pickup of TB. A toluidine blue rinse may be employed in screening to include all high-risk sites. If we follow proper steps, those lesions not detected during a visual inspection may be seen divulged by the stain [10].

In the present study, out of 183 patients, 146 patients were found positive with toluidine blue staining and 37 were not stained with the TB dye. Out of the 146 patients who were positive for TB, 134 patients were turned out to be positive in Histopathological examination. Out of the 37 patients who were not stained with TB dye, 32 were tested Negative in Histopathology examination.

Further we found that Mean age of study cohort is 51.03 ± 14.01 years. Maximum patients belong to age group of > 60 years [52 (28.4%)]. maximum patients were male [119 (65%)] followed by female [64 (35%)]. In a similar study was done by Cancela-Rodríguez et al. including 160 patients with oral mucosal disorders. Increase in life expectancy is thought to be a risk factor for development of cancer. Cancela-Rodríguez et al. [11] reported almost similar mean age of 55.3 ± 16.1 years almost comparable result with present study. A study from Haryana, India done by Pallagatti et al. [12] which focused on 40 oral mucosal lesions in 32 patients and reported that male predominance (90.62%). Widespread tobacco usage is prevalent in females belonging to Western world and hence this could be a reason for the contradiction to present study, reported female predominance (51.87%) [11] by Cancela-Rodríguez et al. in which he studied 160 patients; with oral mucosal diseases which included suspicious or malignant lesions detected at clinical/visual examination, later confirmed by histopathological evaluation.

Analysis of site of lesion distribution revealed that maximum patients had lesion at buccal mucosa [109 (59.6%)] followed by tongue lateral [42 (23%)] and soft palate [11 (6%)] and lips 5 (2.87%). In agreement to present study Pallagatti et al. which included 32 patients reported that most common site was buccal mucosa in 57.5% of the total cases (23 lesions). It was followed by labial vestibule 27.5% (11 lesions) and buccal vestibule and commissural areas equally contributed the next 2 spots 7.5% (3 cases each) [12]. But in contrast to present study Crăcană et al. [13], in his 2016 study found that one of the common location was lip (32.67%). It may be due to smoking is more prevalent in western countries as in our country tobacco is consumed in quid form and also due to difference in color of skin. The location of the suspected lesion is closely related to the local tobacco habits. In some areas of South East Asia especially, in Andhra Pradesh state—there is a reverse smoking practice where burning end of cigar is kept inside the oral cavity and in these population hard and soft palate carcinoma is prevalent (Fig. 2).

Fig. 2.

Suspected lesion on lateral border of tongue

In present study TB stain results showed that, out of 183 patients’ 146 (79.8%) patients were found to be positive for TB staining. Toluidine blue in known to detect relative, rather than absolute differences between normal and malignant cells and tissue. A study from UK by Cancela-Rodríguez et al. included 160 patients with oral mucosal disorders that included suspicious or malignant lesions detected at clinical naked eye examination, later confirmed by histopathological evaluation. Cancela-Rodríguez et al. [11] found that only 54 patients were positive for TB where as a large portion 106 were Negative for the same. Pallagatti et al., the Indian author found that out of the 40 numbered study group, 29 were positive for TB (69.5%) and rest were Negative; and this result is summarized in the form a table and given below [4].

Out of total 146 toluidine blue staining positive results, histopathology has shown 134 (91.78%) positive and 12 (8.21%) negative results. Out of total, 37 TB staining negative results, histopathology has shown 5 (13.51%) positive results. Comparing Histopathology with TB staining revealed that out of 139 patients in which histopathology was positive, TB staining was able to detect 134 (96.40%) positive cases where as out of total 44 negative cases reported in Histopathology, TB staining was able to detect 12 (27.27%) positive cases. Cancela-Rodríguez et al. revealed that comparison of the TB positivity and histopathology revealed 19 test results were true positive (TP) and 35 were false positive. In Cancela-Rodríguez et al. the HPE reported a total of 29 confirmed or borderline lesions. Nineteen of these lesions were stain-positive so were true positive, while the remaining 10 were false negative. Therefore, TB was able to detect 65% of the malignant/dysplastic lesions in the study [11] (Table 4).

Table 4.

Comparison of percentage of TB staining with other studies and present study

S. nos.	Study	Total no. patients	T.B. positive	% of T.B. positive
1.	Warna Kulasuriya	145	102	70.3
2.	Ram et al.	46	31	67.3
3.	Allegra et al.	45	26	57.7
4.	Pallegatti	40	29	69.5
5.	Sinha et al.	51	46	90.1
6.	Present study	183	146	79.8

Positive and negative predictive value of toluidine blue staining was 91.8% and 86.5% respectively. Whereas Positive and negative predictive value of histopathology was 96.4% and 72.7% respectively. In agreement to present study Allegra et al. reported that there is a 53.9% probability that clinically negative specimens could be histologically positive and a 6.7% probability that a toluidine blue negative sample could be histologically positive [14]. In agreement to present study Cancela-Rodríguez et al. in a similar study reported positive predictive value of 35.2%, and a negative predictive value of 90.6%, (Table 5) in other words. TB should support us in decreasing the number of biopsies to a significant equable while identifying all lesions picturing severe dysplasia and OSCC.

Table 5.

Comparison of positive and negative predictive values of other studies with present studies

Study	Positive predictive value	Negative predicted value
Allegra et al. [14]	53.9	6.7%
Cancela-Rodríguez et al. [11]	35.2	90.6
Pallagatti et al. [12]	84.6	90.9
Present study 2018	91.8	86.5

There are many advantages and a few disadvantages for screening with TB. It offers a fast, safe and low cost method to screen the ongoing suspected oral malignancies and prevent them from further deliration and the disadvantage may be a psychological trauma once test is positive, but an inevitable part of treatment procedure. A method to avoid false positive results was offered by Mashberg, in his classical study done in 1980, which advocated a second evaluation after 10–14 days so that the inflammatory lesion may heal in this time and hence with this way, he reduced the false positive rate to less than 8% [15]. Allegra et al. studied 45 oral mucosal lesions in 32 patients (13 female, 19 male) and concluded that TB stain has been shown to be a reliable aid when clinical examination is unable to determine lesions at high risk of progression and then it improves rate of early diagnosis for oral cavity cancer [14].

Conclusion

Patients with oral malignancies form a major stake in the total cancer burden of the society which again causing tremendous worry in the health sector of our country itself. The mortality and morbidity associated are high owing to the late diagnosis often a predilection with rural India. Ministry of Health and welfare is spending a lot of resources in prevention of oral malignancies and its early detection. Early detection not only increase the 5 year survival rate, but also helps to alleviate the morbidity, which is of great concern. Strengthening of the primary health care system & referral system is of utmost importance when coming to early detection of oral lesions.

Compliance with Ethical Standard

Conflict of interest

The authors declare that they have no conflict of interest.

Informed Consent

Informed consent was obtained by all individual participating in this study.